Physiol Rev 87: 825– 872, 2007;
doi:10.1152/physrev.00030.2006.
Regulatory Binding Partners and Complexes of NHE3
MARK DONOWITZ AND XUHANG LI
Departments of Medicine and Physiology, GI Division, The Johns Hopkins University
School of Medicine, Baltimore, Maryland
I. Overview
II. Mechanisms of Acute Regulation of NHE3
A. NHE3 phosphorylation
B. Acute regulation of NHE3 can occur by changes in trafficking or changes in turnover number
III. Intracellular NHE3 Activity
IV. NHE3 COOH-Terminal Regulatory Domain and NHE3 Complexes
A. Regulatory domain
B. NHE3 complexes
C. Intramolecular interactions
D. Dynamic aspects with signaling
V. NHE3 COOH-Terminal Binding Partners
A. COOH-terminal domain 1, amino acids 455–585: CHP, ezrin, and phosphoinositol phosphates
B. COOH-terminal domain 2, amino acids 586 – 660: calmodulin, calmodulin kinase II,
and NHERF family
C. COOH-terminal domain 3, amino acids 661–756
D. COOH-terminal domain 4, amino acids 757– 832: DPPIV(?), PP2A, and megalin
VI. Functional Interactions of NHE3 With Proteins Not Known to Directly Bind NHE3
A. PEPT1/PEPT2
B. NHE1
C. SLC26A3 (DRA) and SLC26A6 (PAT1)
D. CFTR
825
827
829
832
834
834
835
836
837
838
838
838
842
854
854
856
856
856
856
857
Donowitz M, Li X. Regulatory Binding Partners and Complexes of NHE3. Physiol Rev 87: 825– 872, 2007;
doi:10.1152/physrev.00030.2006.—NHE3 is the brush-border (BB) Na⫹/H⫹ exchanger of small intestine, colon, and
renal proximal tubule which is involved in large amounts of neutral Na⫹ absorption. NHE3 is a highly regulated
transporter, being both stimulated and inhibited by signaling that mimics the postprandial state. It also undergoes
downregulation in diarrheal diseases as well as changes in renal disorders. For this regulation, NHE3 exists in large,
multiprotein complexes in which it associates with at least nine other proteins. This review deals with short-term
regulation of NHE3 and the identity and function of its recognized interacting partners and the multiprotein
complexes in which NHE3 functions.
I. OVERVIEW
NHE3 (SLC26A3) is one of nine isoforms of the mammalian Na⫹/H⫹ exchanger (NHE) gene family (60, 62, 121,
357, 358, 476, 537). The mammalian NHEs consist of
isoforms that occur primarily in the plasma membrane
and those that appear to primarily reside in intracellular
organelles (60). However, it may be that all NHEs have at
least transient plasma membrane localization (62). The
evolutionary history of the NHEs showed that the intracellular NHEs arose in a setting in which their transport
was driven by H⫹ gradients generated by V-ATPases (62,
182, 342, 353, 354). The earliest evolving plasma memwww.prv.org
brane NHEs exist both in the plasma membrane and on
the membranes of intracellular organelles (22, 33, 48, 255,
359, 448). These NHEs traffick continually between the
plasma membrane and intracellular organelles (62, 226,
255, 434). They arose at a time of evolutionary appearance
of the Na⫹-K⫹-ATPase, which creates a Na⫹ gradient and
provides the energy for their exchange of extracellular
Na⫹ for intracellular H⫹. The most recent NHEs to evolve
appear to be the NHEs that are present predominantly on
the plasma membrane (359, 401, 450, 451). The plasma
membrane and intracellular NHEs can be separated by
differences in the amino acid residues in their membranespanning domains, especially the putative P-loop (244,
0031-9333/07 $18.00 Copyright © 2007 the American Physiological Society
825
826
MARK DONOWITZ AND XUHANG LI
335, 339, 417, 475). NHE1-5 are the plasma membrane
NHEs. Of these, NHE3 and NHE5 traffic between the
recycling endosomes and the plasma membrane under
basal conditions, while NHEs 1, 2, 4 appear to be statically
present in the plasma membrane. The intracellular NHEs
include NHE6, -7, and -9. It is not known whether the
intracellular NHE isoforms exist statically in specific organelles or whether individual intracellular NHEs exist in
multiple organelles (328, 342, 505). NHE8 resembles the
intracellular NHEs based on amino acid composition but
appears to be present primarily on the plasma membrane,
being found in the brush border of the renal proximal
tubule and small intestine (20, 181, 182, 513). The driving
force for the NHEs on the plasma membrane is the Na⫹
gradient generated by Na⫹-K⫹-ATPase (123). For resident
organellar NHEs, which appear to generally exchange
cytosolic K⫹ (perhaps also Na⫹) for intraorganellar H⫹,
the driving force is primarily the H⫹ gradient generated by
the organellar V-ATPase (61, 465). These NHEs transport
H⫹ out of the compartment in exchange for cytosolic K⫹
or Na⫹ and thus help set the organellar pH (61, 465).
NHE3 and NHE5 also appear to function on intracellular
organelles (8, 134, 163, 434). In a subset of early endosomes, extracellular Na⫹ appears to provide the driving
force for acidification (162–165). All plasma membrane
members of the NHE gene family transport Na⫹ and H⫹
with a stoichiometry of 1:1 (20, 21, 476). The relative
affinity for transport of Na⫹ versus K⫹ by the organellar
NHEs in exchange for intraorganellar H⫹ is not known
(60, 61). Please note, a distantly related sperm transporter
is not considered a member of the SLC26A family. Also, a
proposed colonic Cl⫺-dependent NHE isoform (384, 385,
400) is believed to be a cloning artifact (403), and rather,
the suggestive physiological studies are suspected of representing the now recognized Cl⫺ dependence of multiple
NHE isoforms (4).
NHE3 is present in the brush border (BB) of small
intestine (duodenum, jejunum, ileum), colon, gallbladder,
parietal cells of some species but not others, cholangiocytes, some pancreatic duct cells, proximal tubule of the
kidney, thick ascending limb of Henle and proximal portion of the long descending thin limb of Henle, a few
neurons in the cerebellum and respiratory neurons of the
ventrolateral medulla (where it is suggested as being involved in respiratory rhythm generation, neuronal chemosensitivity, and the central CO2 respiratory response), in
perineuronal cells (not nerves) of the laryngeal nerve,
chondrocytes, parotid duct, and epidydimus duct (1–3, 20,
24, 25, 30, 47, 48, 56, 58, 60, 62, 64, 98, 99, 101, 138, 154,
159, 200, 207, 210, 226, 236, 254, 255, 259, 282, 291, 295,
313, 326, 345, 365, 375, 378, 383, 389, 394, 395, 414, 424,
438, 508, 517). In addition, NHE3 is present in gills of fish
(86, 141, 509) and is involved in mouse blastocyst formation (237).
Physiol Rev • VOL
The major recognized functions of NHE3 are in the
apical membrane of the proximal tubule and small intestine and colon (20, 121, 160, 403, 452, 511, 537). NHE3 is
responsible for the majority of NaCl absorption in the
intestine and kidney, acting predominantly in their proximal portions (explaining proximal tubule and small intestinal linked Na⫹ and Cl⫺ absorption). In addition, it
accounts for the majority of HCO⫺
3 absorption in the
kidney since the amount of NHE3 appears to exceed the
amount of apical anion exchanger, with the residual
Na⫹/H⫹ exchange activity being equivalent to HCO⫺
3 absorption (89, 121, 123, 184, 313, 314, 330, 512, 537). At least
in the gastrointestinal (GI) tract and kidney, NHE3 is
functionally linked to a BB Cl⫺/HCO⫺
3 exchanger and
takes part in neutral NaCl absorption (123, 259, 260, 275).
In this process one molecule of Na⫹ and one molecule of
Cl⫺ are absorbed together with no net movement of charge.
This process depends on intracellular carbonic anhydrase to
⫺
⫺
generate the H⫹ and HCO⫺
3 (123). The Cl /HCO3 exchanger
involved probably varies among intestinal segments and
appears to include PAT1 (putative anion transporter or
SLC26A6) or DRA (downregulated in adenoma, SLC26A3)
(20, 35, 159, 223, 268, 275, 406, 482, 483). The current
suggestion is that PAT1 may dominate in Na⫹ absorptive
cells in the duodenum and in jejunum, while DRA is
involved in ileum and colon, although this appears to be
species dependent. For instance, in some species, DRA is
the duodenal villus apical anion exchanger. In addition,
some tissues and even some cells may have both DRA and
PAT1; for instance, in mouse duodenum, DRA is in crypt
cells and PAT1 is in the villus cells (415). In the kidney,
the involved anion exchanger also appears to be PAT1,
although SLC26A7, another anion exchanger, is also reported to be in the proximal tubule BB apical membrane.
The neutral NaCl absorptive process is highly regulated (121, 123, 537). It is both rapidly (over minutes)
stimulated and/or inhibited as part of digestion in the GI
tract and in response to neurohormal stimulation in both
GI tract and kidney proximal tubule. NHE3 is the part of
neutral NaCl absorption that has been shown to be directly regulated, although similar detailed studies have
not been reported for the BB Cl⫺/HCO⫺
3 exchanger. In
fact, in preliminary studies, the apical membrane anion
exchange may be inhibited by protein kinase C (397, 398).
In colon, in addition to the NHE3/PAT1 or DRA-linked
NaCl absorption, there is an additional neutral Na⫹-linked
anion absorptive process in which NHE3 or NHE2 is
linked to a short-chain fatty acid/anion exchanger (Fig. 1).
(176, 267, 466, 467). A congenital diarrheal disease due to
lack of BB NHE activity (almost certainly NHE3) occurs
(55, 238, 336). It is not due to a structural or localization
abnormality in NHE3.
In the kidney and intestine and in all cell culture
models in which NHE3 is expressed endogenously or
exogenously, NHE3 cycles between the plasma mem-
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
827
endocytosis and/or exocytosis, and changes in NHE3 halflife (50, 70, 97, 121, 130, 212, 226, 330, 358, 537). We have
found that NHE3 simultaneously exists in multiple large
multiprotein complexes particularly on the plasma membrane under basal conditions and that the NHE3 complexes change as part of some NHE3 regulation in terms
of complex size and associating proteins (8, 71, 296, 298,
340). There appears to be coordinated regulation of NHE3
and anion secretion in murine jejunum, which would
allow coordinated movement of intestinal Na⫹, Cl⫺,
HCO⫺
3 , and water transport (158, 159).
This review examines the current understanding of
the mechanisms of NHE3 regulation, the identified NHE3
complexes, and the proteins which directly and/or indirectly interact with NHE3, with an emphasis on the NHE3
binding partners and their role in acute NHE3 regulation.
⫹
FIG. 1. Two forms of neutral Na anion absorption in mammalian
colon. The neutral NaCl absorptive processes involve NHE3 linked to
either SLC26A3 or -6 based on cells involved (A) and the short-chain
fatty acid (SCFA)/anion exchanger linked to either NHE2 or NHE3, with
NHE2-SCFA uptake active even in the presence of cAMP, which inhibits
NHE3 (B).
brane or BB and the recycling compartments under basal
conditions. The compartment involved has not been characterized biochemically, but is assumed to be early endosomes perhaps before incorporation of the V-ATPase (8,
134, 162–165, 481). NHE3 functions to acidify the early
endosomes. The driving force in this vesicular acidification appears to be the concentration gradient of Na⫹,
which depends on extracellular Na⫹ that is trapped in the
vesicle as it is endocytosed from the plasma membrane
(134). In several cell types, including fibroblasts and the
opossum kidney (OK) renal proximal tubule cell line, as
first shown by D’Souza et al. (134), NHE3 functions to
acidify early endosomes (134). This acidification has been
suggested by Geckle and co-workers (162, 163) to be
necessary for receptor-mediated endocytosis of albumin
in renal proximal tubule cells. The effect of the NHE3
inhibitor S3226 on receptor-mediated/clathrin-dependent
endocytosis is additive with inhibition of endocytosis
caused by knock out of ClC-5 (404, 481). This suggests
two separate uptake processes are involved in proximal
tubule endocytosis (481). Whether this also occurs in the
intestine or is relevant to intestinal function is not known,
nor is it known what replaces this process in cells lacking
NHE3.
Short-term regulation of NHE3 mimics digestive and
renal physiology or regulation by changes in the dietary or
neurohumoral environment of the intestine and kidney
and also models pathological states, including diarrhea
(13, 121, 123, 330, 537). The mechanisms involved in
short-term NHE regulation include changes in plasma
membrane turnover number, changes in amount of NHE3
expressed on the plasma membrane by altering rates of
Physiol Rev • VOL
II. MECHANISMS OF ACUTE REGULATION
OF NHE3
NHE3 is one of the most regulated transport proteins.
It is both rapidly stimulated and inhibited as part of
normal digestive physiology, and it contributes to multiple pathophysiological states when it is downregulated
for a prolonged period (121, 130, 330, 537). Rapid regulation of NHE3 occurs over minutes in the intestine as part
of digestion either in response to change in luminal end
products of digestion (at least, generation of D-glucose
and short-chain fatty acids) or to changes in the neurohumoral mileu of the intestine. Changes in NHE3 activity
in the kidney occur with a somewhat slower time frame
(many minutes to a few hours) in response to drugs,
dietary changes (especially change in Na or K intake), or
changes in blood pressure/volume (13, 321). While the
agonists that alter NHE3 in the GI tract and kidney are
somewhat different, the response of NHE3 to changes in
second messengers is the same, i.e., NHE3 inhibition induced by parathyroid hormone (PTH) may occur over
many minutes in the kidney proximal tubule via activation
of the adenylate cyclase/cAMP/protein kinase A (PKA) II
plus protein kinase C (PKC) systems, while similar effects
on NHE3 activity occur in the small intestine over a few
minutes in response to elevation of vasoactive intestinal
polypeptide (VIP), secretin, or cholingergic agonists and
accompanying changes in cAMP or Ca2⫹.
Transport kinetics for NHEs, including NHE3, include Michaelis-Menton kinetics for extracellular Na⫹ uptake (kinetics of K⫹ transport by intracellular organellar
NHEs has not been defined as yet), with 1:1 exchange of
extracellular Na⫹ for H⫹ (20, 21, 252, 358, 449, 452).
However, intracellular H⫹ export is more complex, with
H⫹ both being transported and activating the exchanger
allosterically through the so-called H⫹ modifier site (21,
198, 422, 471, 472, 476). This leads to the K⬘(H⫹)i being a
87 • JULY 2007 •
www.prv.org
828
MARK DONOWITZ AND XUHANG LI
complex term, with a Hill coefficient of ⬃2 (21). Regulation of NHE3 generally involves changes in NHE3 Vmax
and often, but not always, changes in K⬘(H⫹)i (121, 357,
537). However, usually this is not associated with changes
in the NHE3 Km(Na) extracellular. Also, occasionally
there are changes in the NHE3 set point (73). Two basic
regulatory mechanisms are involved in short-term NHE3
regulation: 1) changes in turnover number with no
changes in trafficking; and 2) changes in trafficking (121,
330, 537). The latter involves changes in regulated exocytosis and/or endocytosis, which can include effects in
both by the same agonist.
Some of the agonists that rapidly regulate NHE3 in
the intestine or renal proximal tubule are listed in Table 1.
Categories of ligands include neurohumoral substances
produced in the GI tract or systemically, neural mediators
most made in the GI tract, kinase regulators, phosphatase
inhibitors, changes in osmolarity, and end products of
digestion.
The stimulation of NHE3 by an end product of digestion was identified by Turner and co-workers (Fig. 2)
when they showed that NHE3 was rapidly stimulated in
TABLE
1.
response to luminal D-glucose by a process involving
SGLT1 (213, 410, 458 – 460, 542). This occurred by activation of an apical membrane initiated linear signaling pathway that includes early activation of p38 mitogen-activated protein (MAP) kinase, followed by activation of
MAPKAPK2 (MAP kinase activated kinase 2), phosphatidylinositol 3-kinase (PI 3-K), Akt2, and ezrin, which lead
to stimulation of NHE3 activity due to increased exocytosis. The model used to identify this pathway is the Na⫹
absorptive colon cancer cell line Caco-2, which is a small
intestinal Na⫹ absorptive cell model. The importance of
the identification of this pathway is that it demonstrates
that NHE3 is an intermediate in the increased Na⫹ absorption that occurs in the postprandial state by two
mechanisms. 1) It is an intermediate in end product of
digestion initiated stimulation of intestinal Na⫹ absorption, where it couples to other mechanisms of stimulated
postprandial Na⫹ absorption. However, it is not known
how much of this NHE3 activation accounts for D-glucosedriven jejunal Na⫹ absorption versus the solvent drag that
was thought to be the magnifying effect to Na⫹/
D-glucose uptake across the jejunal apical membrane
Short-term regulation of NHE3
Agonist
NHE3
Adenosine (31, 114–117, 416)
Aldosterone (108, 220)
ATP depletion (4, 65, 68, 73, 155, 234, 292)
Albumin (292)
Angiotensin II (118, 136, 203, 249, 272, 288, 289, 374, 453, 456, 457, 514)
ANP (281)
cAMP (201, 209, 232, 271, 277, 281, 282, 323, 331, 338, 497–500, 503, 504, 532, 533, 543)
cGMP (72, 142, 228)
Clostridium difficile toxin (197)
Dopamine (10, 29, 175, 212, 299, 368–370, 514)
EGF (130, 246, 298)
Elevated [Ca2⫹]i, PKC (14, 95, 96, 122, 124, 127, 132, 144, 232, 235, 250, 286, 298, 320, 391, 439, 490, 507, 536, 545)
Endothelin-1 (273, 274, 301, 371, 372)
Epinephrine (17, 139, 295, 305, 429)
FGF (224)
Glucagon (16)
Glucocorticoids (13, 15, 37, 50, 77, 79, 85, 183, 187, 188, 231, 247, 278, 435, 477, 528–531)
Hyperosmolarity (12, 120, 157, 234, 346, 419)
Hypertension (519–522, 526, 539, 540)
Hyposmolarity (173, 234, 489)
Insulin (256, 257)
Interferon-␥ (312, 390, 510)
LPA (87, 285)
Nitric oxide (167)
PTH (23, 97, 153, 290, 315–317, 334, 521, 541)
Okadaic acid (292)
Opiates (214)
Serotonin (125, 168)
Short-chain fatty acid (52, 53, 176, 267, 323, 466, 467)
Somatostatin (305)
Squalamine (9)
Thrombin (292)
Vitamin D (166)
S,I*
S
I
S
S
I
I
I
I
S or I based on cell type†
S
I
S
S
S
I
S
I
I
S
S
I
S
I
I
S
S
I
S
S (may be indirect)
I
S
S
S, stimulate; I, inhibit. * Adenosine has been shown to exert concentration-dependent effects on NHE3 activity. The adenosine analog
N(6)-cyclopentyladenosine stimulates NHE3 at concentrations ⬍10⫺8 M and inhibits at concentrations ⬎10⫺8 M. † Mostly inhibitory. Reference
numbers are given in parentheses.
Physiol Rev • VOL
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
FIG. 2. Putative signaling pathway by which luminal D-glucose acts
via SGLT1 to stimulate NHE3 by increasing its exocytosis. RE, recycling
endosomes. [Adapted from work of Turner and co-workers (213, 410,
458 – 460, 542).]
(149, 309, 310, 460). 2) Regulation of NHE3 is also initiated by neurohumoral ligands that are released during
digestion, as described below. Important unanswered
questions about the linkage of NHE3 stimulation to end
products of digestion in the lumen of the small intestine
include the following: 1) What is the initial intracellular
signal? Does it occur in intact intestine in addition to in a
cell culture model, and if so, is the signal transduction
involved the same as in cell culture models and how is it
altered by changes in the digestion-induced alteration in
the neurohumoral milieu? This is important given the
recognition that even major diarrhea-related secretagogues, such as cholera toxin, which alter ion transport in
epithelial cells that can be examined in cell culture models, exert approximately half of their effects in intact
intestine via neurally mediated mechanisms that involve
the enteric nervous system (308). 2) Do other end products of digestion, such as L-amino acids and di- and tripeptides, have a similar role? If so, is it via the same
signaling pathway as D-glucose? 3) How far inside the cell
away from the apical domain are signals from this pathway activated and do they change signaling in the entire
cell?
Short-chain fatty acids take part in linked neutral
Na⫹ absorption in the colon (Fig. 1) (176, 267, 466, 467).
NHE3 is the BB NHE linked to BB Cl⫺/HCO⫺
3 exchange;
however, there is an additional neutral Na⫹ absorptive
process linked to a BB short-chain fatty acid/anion exchange (Fig. 1). The latter leads to neutral Na⫹/shortchain fatty acid uptake. Both NHE3 and NHE2 are able to
link to colonic short-chain fatty acid uptake (467). The
Physiol Rev • VOL
829
regulation of this process is assumed to be driven by the
high luminal concentration of short-chain fatty acids, plus
the Na⫹ gradient. This probably accounts for some basal
colonic Na⫹ absorption given the high luminal concentration of short-chain fatty acids in the normal colon. cAMP
elevation only inhibits NHE3 and thus blocks the neutral
NaCl absorptive process but does not inhibit the NHE2linked SCFA absorptive process. Of note, this process
may be stimulated since cAMP stimulates NHE2 (54, 232,
323, 347). This is the theoretical basis for the development
of an oral rehydration solution (ORS) that increases colonic luminal short-chain fatty acid concentration by using relatively amylase-resistant carbohydrates, such as
corn starch (386) In an early report, this ORS shortened
the duration and volume of cholera diarrhea, the first ORS
reported to have both these effects (386).
As examples of the rapid neurohumoral ligand regulation of NHE3 (Table 1), epidermal growth factor and
␣2-adrenergic agonists stimulate ileal Na⫹ absorption,
and lysophosphatidic acid increases NHE3 in OK renal
proximal tubule cell line. Stimulation of renal proximal
tubule BB NHE3 also occurs with endothelin-1, angiotensin, and exposure to acidosis. Most of these agonists
stimulate NHE3 exocytosis without changing the rate of
endocytosis. These agonists act to increase BB NHE3
amount, with comparable increases in NHE3 activity and
percent of NHE3 on the apical membrane (285, 295).
cAMP, cGMP, and elevated intracellular Ca2⫹ and the
neurohumoral substances and bacterial toxins that cause
these second messengers to increase all inhibit neutral
NaCl absorption and its component BB NHE3 (121, 197,
330, 537). This occurs in both the GI tract and renal
proximal tubule. The regulated mechanisms vary widely.
At one extreme, cAMP inhibits NHE3 in PS120 fibroblasts
by changing the NHE3 turnover number without affecting
the amount of plasma membrane NHE3 (130, 277). In
contrast, in OK cells, cAMP initially decreases the turnover number, but in ⬃30 min, it is associated with an
increase in rates of endocytosis, followed by inhibition of
exocytosis (60 min) and a decrease in total NHE3 half-life
(506, 543). This demonstrates highly coordinated NHE3
regulation in response to a single second messenger.
A. NHE3 Phosphorylation
NHE3 is phosphorylated under basal conditions
(525), and that phosphorylation is often affected as part of
acute NHE3 regulation (97, 115, 212, 271, 330, 331, 372,
453, 507, 547). Changes in NHE3 phosphorylation are
necessary for some acute regulation of NHE3 but apparently not for others. There are examples of NHE3 regulation by both changes in trafficking and not involving
trafficking that are dependent on changes in NHE3 phosphorylation. This basic regulatory mechanism is partially
87 • JULY 2007 •
www.prv.org
830
MARK DONOWITZ AND XUHANG LI
understood with identification of some phosphorylation
sites of NHE3, some roles of phosphatases in NHE3 regulation, the role of kinase localization to specific subcellular domains, and involvement of NHE3 complexes all
being partially defined. In contrast, further NHE3 phosphorylation sites, additional roles of phosphatases, and
restricted spatial and temporal phosphorylation of NHE3
as part of regulation are all predicted, and cAMP has even
been shown to act by a non-PKA-dependent mechanism
that inhibits NHE3 in some cells without causing a change
in NHE3 phosphorylation (EPAC, exchange protein directly activated by cAMP) (209). This area was reviewed
with great insight by Moe (330), whose laboratory has
provided many of the studies in this area.
NHE3 is phosphorylated under basal conditions in
several cell culture models as well as in intact tissue.
These include fibroblasts, OK proximal tubule, and
Caco-2 cells as well as in renal proximal tubule and ileal
BB. The great majority of basal and stimulated phosphorylation is on Ser with minor phosphorylation of Thr (212,
232, 330, 331, 372, 507, 525, 547). Tyr phosphorylation of
NHE3 has not been identified. Of interest is that at least
one Tyr residue (Y323 of rabbit NHE3) is predicted to be
phosphorylated with a 95% probability by Netphos2.0. The
phosphorylated sites have only been partially defined, and
use of mass spectroscopic approaches has revealed previously unidentified phosphorylated residues of NHE3
(X. Li, A. Pandey, and M. Donowitz, unpublished data).
The role of basal phosphorylation is unclear since mutagenesis which eliminated up to six putative PKC phosphorylation sites and sites phosphorylated by cAMP and
other putative Ser phosphorylation sites did not alter
basal NHE3 activity (507). This led to the conclusion that
some basal phosphorylation does not have a major role in
basal NHE3 activty. In contrast, the protein phosphatase
inhibitor okadaic acid acutely stimulated NHE3 activity,
demonstrating that at least some kinase(s) stimulate
NHE3 activity under what is considered to be basal conditions (292).
In analyzing the role of stimulated phosphorylation
of NHE3, Moe (330) appropriately pointed out that most
rapid stimulation and inhibition of NHE3 are coupled to
protein kinases, and pharmacological inhibitors of kinases prevent many examples of NHE3 regulation. He
categorized the questions related to the role of phosphorylation of NHE3 in its regulation into 1) Is the site phosphorylated in vivo? 2) Is phosphorylation of a site of
NHE3 necessary for a regulatory function? 3) Is phosphorylation of a site of NHE3 sufficient for a regulatory
function? The overview, not surprisingly, given that complexes and associated regulatory proteins are known to
be involved in NHE3 regulation and that the endocytic
machinery itself is known to be regulated by phosphorylation, is that while phosphorylation of NHE3 appears to
be necessary for some but not all its acute regulation,
Physiol Rev • VOL
NHE3 phosphorylation may not be sufficient for either
stimulation or inhibition (330, 507, 525). Interestingly,
Netphos 2.0 software predicted 19 COOH-terminal Ser/
Thr phosphorylation sites of rabbit NHE3 with probabilities of phosphorylation of ⬎92%. All currently known
phosphorylated residues of NHE3 are among the predicted list. This suggests that the role of Ser/Thr phosphorylation of NHE3 is only partially defined.
1. Evidence that NHE3 is phosphorylated as part of its
acute regulation
A) CAMP. In vitro, purified cAMP-dependent protein
kinases phosphorylate NHE3 in the COOH terminus. More
importantly, in intact cells exposed to elevated cAMP,
immunoprecipitated NHE3 showed increased phosphorylation by specific phosphopeptide mapping, direct
tandem mass spectroscopic analysis, and anti-phospho
antibodies to specific NHE3 amino acids (97, 212, 264,
271, 330, 506, 525, 547). Moreover, a back-phosphorylation approach showed that PKA elevation in vivo led to
occupation of PKA sites in NHE3 and inhibited subsequent in vitro phosphorylation (497, 500). The exact
amino acid residues that are phosphorylated by cAMP
elevation remain somewhat controversial. The most definitive studies showed that cAMP caused concentrationdependent in vivo phosphorylation of rat NHE3 Ser605 and
Ser552, which are in PKA consensus sequences (330, 543).
Both were necessary for cAMP to maximally inhibit
NHE3, since mutation of these Ser individually decreasing
and both together preventing NHE3 inhibition by cAMP.
In contrast, Kurashima et al. (271) showed that NHE3
truncated to amino acid (aa) 585 lost all cAMP inhibition
and that Ser605 and Ser634 of rat NHE3 were necessary for
NHE3 inhibition by cAMP (271). They showed that the
Ser605 was phosphorylated in the presence of PKA. However, although Ser634 was not phosphorylated (either
basal or with cAMP), it was also necessary for cAMP to
fully inhibit NHE3. Each of these Ser contributed ⬃50% to
cAMP inhibition of NHE3, with mutation of both totally
preventing cAMP-induced changes in NHE3 activity in
fibroblasts. In these studies, Ser552 was not phosphorylated. The reason for the discrepancy is unclear, although
phosphopeptide mapping demonstrates that NHE3 that is
immunoprecipitated from cells with elevated cAMP has
changes in multiple phosphopeptides, suggesting changes
in phosphorylation at multiple sites (272, 525, 547). It is of
note that mutation of Ser from other putative PKA phosphorylation sites in the NHE3 COOH terminus did not
alter cAMP effects on NHE3 activity (Ser-330, -514, -576,
-662, -691, -692, -805). It remains to be determined whether
phosphorylation of the Ser residues occurs as part of
acute NHE3 regulation by mechanisms that do not involve
changes in cAMP. As described in more detail below,
cAMP-induced phosphorylation of NHE3 is necessary for
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
all functional effects of cAMP on NHE3 described. Moreover, in OK renal proximal tubule cells, elevating cAMP
with dopamine inhibited NHE3 over the same time and
concentration range that increased NHE3 phosphorylation (212).
B) CGMP. cGMP kinase II (cGKII) is a BB protein that is
involved in acute NHE3 inhibition in response to the
luminally released gut humoral substances guanylin and
uroguanlyin. Guanylin is released as part of digestion.
Heat-stable Escherichia coli enterotoxin is similar structurally to guanylin/uroguanylin and binds to the same BB
receptor, guanylate cyclase C. It increases the cGMP content in the cell at the apical domain and activates cGKII.
This activation leads to acute inhibition of NHE3 by a
process in which NHE3 is phosphorylated (B. Y. Cha, H.
Kochinsky, P. Aronson, H. de Jonge, and M. Donowitz,
unpublished data).
2⫹
C) ELEVATED CALCIUM. Elevated Ca
inhibits NHE3 by a
PKC-␣-dependent process (250, 286). PKC-␣ becomes
part of the NHE3 complexes with elevated Ca2⫹ in both
PS120 cells and ileal Na⫹ absorptive cell BB (286, 298).
However, PKC phosphorylation of NHE3 could not be
demonstrated in PS120 cells or in ileal BB, and mutagenesis studies of NHE3 suggest that PKC phosphorylation of
NHE3 does not correlate with NHE3 regulation (507, 525).
Wiederkehr et al. (507) showed that purified PKC phosphorylated similar phosphopeptides in the NHE3 COOH
terminus in vitro that occurred with phorbol ester exposure to intact cells in vivo. However, Yip et al. (526) did
not see consistent changes caused by phorbol ester exposure on NHE3 phosphopeptides from NHE3 transfected PS120 cells [although in retrospect this may not
have occurred due to lack of expression of sufficient
Na⫹/H⫹ exchanger regulatory factor (NHERF) proteins in
these cells; see below]. In detailed studies using AP-1
cells, mutation of six Ser in the NHE3 COOH terminus
which were in putative PKC consenses sequences prevented phorbol ester regulation of NHE3 in vivo (Ser-13,
-552, -575, -661, -690, -804) (507). However, PKC has been
associated with NHE3 stimulation, inhibition, or no effect
depending on the cell model studied. Also, Wiederkehr
and Moe and co-workers (507) importantly showed that
when NHE3 was expressed in AP-1 cells and individual
clones selected, phorbol esters stimulated, inhibited, or
had no effect on NHE3 activity depending on the specific
clone studied. In spite of this, phorbol esters stimulated
NHE3 phosphorylation with identical phosphopeptide
maps, and this was regardless of whether phorbol esters
stimulated, inhibited, or had no effect on NHE3 activity
(507). Thus identical changes in NHE3 phosphorylation
were associated with the full spectrum of changes in
NHE3 activity. Moreover, in preliminary studies of effects
of elevated Ca2⫹ on NHE3 regulation in PS120 cells and
OK cells in which NHERF2 complexes are required for
inhibition, we could not demonstrate changes in phosPhysiol Rev • VOL
831
phorylation of IP NHE3. Thus the conclusion for Ca2⫹/
PKC inhibition of NHE3 is that while PKC (PKC-␣) is
involved and joins the NHE3 complexes, the PKC phosphorylated substrate does not appear to be NHE3, although at least one of the Ser that can be phosphorylated
by PKC is necessary for the inhibition of NHE3.
That NHE3 phosphorylation is necessary for some
acute regulation but not others eliminates the possibility
that phosphorylation is sufficient for all NHE3 regulation.
Even for cAMP inhibition, phosphorylation is not sufficient. For instance, in OK cells, dopamine elevated cAMP
content acting both by DA-1 and DA-2 receptors, with the
effects on NHE3 being synergistic. DA-1 inhibited NHE3
activity, while DA-2 itself had no effect on NHE3 activity
but DA-1 plus DA-2 caused a greater effect than DA-1
alone. In spite of this, DA-1 and DA-2 individually increased phosphorylation of NHE3 on the same sites as
when they were studied together (212, 330). These findings, plus the failure of PKC phosphorylation to consistently affect NHE3 activity (507), demonstrate that phosphorylation of NHE3 is not sufficient to explain NHE3
regulation. In addition, the quantitative role of NHE3
phosphorylation in NHE3 regulation needs further examination.
In spite of the correlation of phosphorylation of specific amino acids of NHE3 as part of its regulation with
changes in turnover number, for instance, with cAMP
inhibition, the series of molecular events that lead to the
regulation downstream from the changes in phosphorylation are not understood. The changes in NHE3 trafficking
are partially understood at the level of changes in associating proteins and changes in associating with some parts
of the trafficking machinery, although again molecular
details are lacking (see below).
D) OTHER KINASES. Several other kinases also either
associate with or regulate NHE3 activity. PI 3-K, a major
NHE3 regulating enzyme, is discussed below. A kinase
which binds directly to NHE3 is calmodulin (CaM) kinase
II (545), while other kinases which join NHE3 are part of
complexes involved in regulation. These include PKA,
PKG, and PKC, all of which associate with NHE3 via BB
PDZ proteins of the NHERF family. Understanding how
kinases regulate proteins now includes understanding
how kinases are activated locally in cells with the consequence that specific signaling pathways appear to involve
a few substrates, rather than all putative cell substrates.
This specificity is attributed, at least partially, to activation of kinases at specific locations in the cells as part of
specific receptor/signaling pathways. The location of
NHE3 complexes with BB PDZ proteins and their association with kinases may partially explain this phenomenon
for NHE3 at the BB. In addition, multiple other kinases
are involved in acute NHE3 regulation, although apparently several steps away from NHE3. For instance, already mentioned is that D-glucose activates Caco-2 BB
87 • JULY 2007 •
www.prv.org
832
MARK DONOWITZ AND XUHANG LI
NHE3 by a process that involves the p38 MAP kinase,
MAPAKP2, PI 3-K, and Akt2. Also, endothelin both stimulates and inhibits NHE3 in a dose-dependent manner by
a process that involves the Ca2⫹-sensitive kinase PYK2
(274). Thus phosphorylation of NHE3 by kinases that
associate with it via its signaling complexes or serve as
intermediates in the signaling networks are necessary for
some but not all aspects of acute NHE3 regulation both
via changes in turnover number and by trafficking. This
incompletely defined area requires much further study to
define other associating kinases and phosphatases and
how changes in phosphorylation leads to changes in
NHE3 complex formation, trafficking, turnover number,
as well as how the NHE3 signal complexes are involved in
controlling NHE3 phosphorylation as well as in phosphorylation of the NHE3 regulatory machinery. NHE3 stimulation by SGK1 is described in section VB8CVI.
E) SER/THR PHOSPHATASES. Another understudied area is
the role of regulated phosphatase activity in NHE3 regulation. Protein phosphatase (PP) 2A was reported as associating with NHE3 when cAMP was elevated acutely
(380). The role of this association has not been defined.
However, the PP1 and PP2A inhibitor okadaic acid stimulates NHE3 activity (292). This suggests that basal phosphatase activity inhibits NHE3 activity, although it was
not established whether the phosphatase involved physically associated with NHE3 or whether it was changes in
NHE3 phosphorylation or in NHE3 regulatory proteins
that were responsible for changes in NHE3 activity. Given
the large number of kinases shown to associate with
NHE1, at least calls for further examination of kinase and
phosphatase binding partners of NHE3, both directly and
as part of its signaling complexes.
In addition, not yet explored is the role of localization
of NHE3 into lipid rafts (LR) in the BB in determining the
state of NHE3 phosphorylation under basal or stimulated
conditions. Some NHE3 is in LR and is involved in basal
cycling and endocytosis and growth factor stimulation of
NHE3 (295, 339). While NHERF1 and NHERF2 in the BB
do not appear to be LR associated (295), the state of the
other NHERF family members is not known, nor is it
known whether the kinases involved with NHE3 regulation are in apical membrane LR.
B. Acute Regulation of NHE3 Can Occur
by Changes in Trafficking or Changes
in Turnover Number
In all cells and tissues in which NHE3 has been
studied, NHE3 stimulation and inhibition are at least partially due to changes in trafficking, with separate endocytic and exocytic processes that can be regulated independently (88, 97, 121, 226, 270, 537). In many cases there
is also regulation by changes in NHE3 turnover number,
Physiol Rev • VOL
which in some cases occurs faster than changes in rates
of trafficking (121, 537).
In evaluating mechanisms of regulation of NHE3, the
model used determines some of the results. Of the models
used, changes in turnover number as well as regulated
endocytosis and exocytosis have been demonstrated in
the renal proximal tubule cell line, OK cells, the intestinal
Na⫹ absorptive cell line, Caco-2 cells, intact small intestine, and NHE3 null fibroblasts transfected with NHE3. In
all cells/tissues, NHE3 exists both on the plasma membrane and in intracellular vesicles, which are probably
endosomes, under basal conditions.
The tissue which NHE3 seems to be differently regulated from others is intact renal proximal tubule (45,
288 –290, 321, 322, 519, 521, 522, 526, 539 –541). This in
spite of the fact there is an intracellular pool of NHE3,
similar to other cells, which is probably endosomal, as has
been demonstrated by electron microscopy (47, 48). In
intact proximal tubule, NHE3 seems to cycle predominantly between microvilli and intervillus areas. In the
apical membrane of proximal tubule cells, Biemesderfer
et al. (45) showed that NHE3 exists in two pools, thought
to be in the microvilli and in the intervillus spaces (45). In
the former, NHE3 is in lighter complexes (9. 2s), does not
associate with megalin, and appears to be active. In contrast, in the latter, NHE3 is in heavier complexes (18s),
associates with megalin, and appears to be less active or
inactive. As part of signaling, NHE3 in proximal tubule
partially moves from a lighter distribution on sucrose
density gradients that includes apical membrane markers,
to heavier fractions which have more markers of the
intervillus compartment but have some overlap with both
BB and intracellular endosomal markers. In these density
gradient studies initiated by Mircheff and pursued by
McDonough, NHE3 was shown to be present in three
fractions. The debate is what they represent and how
distinct they are: alkaline phosphatase is enriched in the
microvillus fraction, galactosyltransferases are enriched
in microvillus clefts, and acid phosphatase is enriched in
endosomes. As an example, with PTH treatment, ⬃20% of
total NHE3 shifts from low-density membranes enriched
in microvillar markers (541) to mid-density membranes
enriched in intermicrovillar cleft markers and later to
heavier density membrane with some endosomal markers. Models used to evaluate changes in NHE3 trafficking
in proximal tubule have included acute hypertension-induced natriuresis, PTH, and an angiotensin-converting
enzyme inhibitor, all of which caused rapid and reversible
inhibition of NHE3 activity and redistribution of NHE3 in
the sucrose gradients from lighter to heavier density fractions interpreted as away from BB to intervillus clefts.
McDonough and co-workers (519, 526, 539, 540) showed
that 20 min of acute hypertension led to redistribution of
NHE3 from BB to the base of the BB, while a control
protein, NaPi2a, internalized into endosomes. However,
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
how much of each fraction is BB versus microvillus clefts
versus endosomes was not resolved.
Of importance, the Biemesderfer studies characterized the Triton X-100 soluble pools of NHE3 (45, 46). Only
⬃50% of total proximal tubule NHE3 was Triton X-100
soluble. Thus this model does not consider ⬃50% of BB
NHE3. They also demonstrated that anti-NHE3 antibodies
did not recognize the entire NHE3 pool and showed that
at least part of what was not recognized was bound to
megalin (46). The role of megalin specifically in NHE3
regulation has not been resolved (see below).
In ileal Na⫹ absorptive cells, epidermal growth
factor (EGF) and clonidine stimulated active NaCl absorption and BB NHE3 activity, which correlated with
the increase in BB NHE3 amount (130, 246, 295). This
stimulation was LR dependent, being inhibited by disrupting LR with methyl-␤-cyclodextrin (M␤CD) (295,
340). Basal endocytosis was also LR dependent with the
amount of NHE3 coimmunopurified with EEA1 containing vesicles decreasing with M␤CD and with cytoskeleton disruption with cytochalasin D (295, 340). Thus, in
ileum, unless antibodies are blocked from recognizing
large populations of NHE3, the addition of NHE3 to the
BB as part of rapid stimulation is not associated with
shifts within the BB of NHE3 pools characterized by
differences in detergent solubility. Whether Biemesderfer’s and McDonough’s careful studies in proximal tubule and our ileal studies of the EGF and clonidine
induced increases in BB NHE3 are describing comparable pools of NHE3 is not known. However, the current view is that ileal NHE3 but not renal proximal
tubule NHE3 traffics between endosomes and BB. It is
also not known whether ileal NHE3 exists in a large
intermicrovillar membrane pool, as occurs in proximal
tubule. In addition, since a significant portion of renal
NHE3 is Triton X-100 insoluble, the role of LR in renal
NHE3 trafficking should be determined.
Oppositely, carbachol rapidly decreased the amount
of NHE3 in the BB and increased the amount coimmunopurified with EEA1, suggesting regulation by trafficking
(298). However, whether the regulated process is endocytosis, exocytosis, or both in intact tissue is still not
established. Also, in rabbit ileum, the amount of NHE3 in
BB increases over minutes with EGF and clonidine and
decreases with carbachol (295, 298). This supports trafficking of NHE3 as a mechanism involved with acute
NHE3 regulation. This is specific for ileum, and mechanisms in the ileum and proximal tubule could be separate.
A portion of ileal Na⫹ absorptive cell NHE3 is in the same
fractions of sucrose density or OptiPrep gradients as
EEA1 (30%), and some NHE3 can be coimmunopurified
with endosomal vesicles by using EEA1 antibody coupled
to beads (295). The amount of NHE3 copurified with
EEA1 vesicles based on immunopurification and colocalized with EEA1 based on sucrose density gradient was
Physiol Rev • VOL
833
reduced by cytochalasin D exposure. These results
strongly indicate that there is an early endosomal pool of
NHE3.
In epithelial cell culture models, basal NHE3 activity
is regulated by a balance of basal endocytosis and exocytosis. A major difference that has been recognized among
cell and tissue models of NHE3 is the percent of NHE3 on
the plasma membrane under basal conditions. This includes ⬃15% in fibroblasts (PS120 and AP-1 cells), 15–50%
in OK cells based on the application of both cell surface
biotinylation and confocal microscopy, ⬃80% in Caco-2
cells measured both by cell surface biotinylation and
confocal microscopy, ⬃80% in renal proximal tubule, and
⬃80% in rabbit ileal BB (7, 8, 116, 197, 371, 523). In
OK cells, NHE3 is in BB and an intracellular pool that
colocalizes with Rab 11 (8). In BB, the amount of NHE3 in
the central portion of BB over the recycling compartment
may be increased compared with in other parts of the
apical membrane (8). Based on fluorescence recovery
after photobleaching (FRAP) and use of cross-linkers, it
appears that basal trafficking is preferentially to this area
of the BB where NHE3 density is increased (71). This
suggests the presence of a targeting signal for NHE3 in
this localized domain in the BB.
Acute regulation of NHE3 in OK cells initially involves changes in turnover number or amount based on
the stimulus followed by changes in trafficking. Endothelin and metabolic acidosis increased the surface amount
of NHE3 and in parallel increased NHE3 activity (371,
523). PTH inhibited NHE3 acting by cAMP. At 30 min
NHE3 activity was decreased, but surface amount of
NHE3 was not affected. Amount of BB NHE3 was reduced
4 –12 h after PTH by a mechanism that involves increased
endocytosis and no change in exocytosis (97). This process was dynamin dependent, being inhibited by a dominant negative dynamin mutant. Increased endocytosis of
NHE3 was associated with increased association of NHE3
with clathrin and AP-2. How this time-dependent regulation of NHE3 occurs is postulated to be either initial
inhibition of all the affected NHE3 molecules followed by
their removal from the BB versus initial inactivation of
NHE3 with subsequent removal of only some of the BB
NHE3 and reactivation of the remaining. Unknown also is
whether NHE3 is active or not at the site of removal and
intracellularly. There is evidence that some of the recycling NHE3 is functionally active (8, 134, 481), but it is not
clear if this represents the pool removed from the BB.
Thus, in cell culture models, both changes in turnover
number and trafficking on and off the BB has been shown
to contribute to NHE3 regulation in multiple cell types
from different tissues, using different ligands, and by
different techniques.
A myriad of signaling molecules are linked to plasma
membrane receptors, the activation of which affect NHE3
activity. In addition, however, there are signaling mole-
87 • JULY 2007 •
www.prv.org
834
MARK DONOWITZ AND XUHANG LI
cules that are involved with NHE3 regulation at or close
to the apical membrane. Most clearly identified as having
a role in basal NHE3 activity is PI 3-K (78, 134, 136, 178,
224, 246, 270, 284, 295, 296). In all cells and most tissue
models of NHE3, basal NHE3 activity is under control of
PI 3-K, with PI 3-K inhibitors decreasing basal transport
rate and percent of NHE3 on the plasma membrane. In
rabbit ileum, wortmannin did not alter basal NaCl absorption, which must be confirmed as this is the only example
of PI 3-K not being involved in basal regulation of NHE3
(246). In contrast, in NHE null fibroblasts in which NHE3
is expressed (PS120 and AP-1), Caco-2 cells, and OK cells,
wortmannin inhibits basal NHE3 activity by ⬃50% (8, 130,
224, 270). PI 3-K is also involved in stimulated NHE3
activity. Constitutive activation of PI 3-K or Akt stimulates
NHE3 activity and increases plasma membrane amount in
fibroblasts and OK cells, while growth factor stimulation
of NHE3 in fibroblasts is blocked by ⬃50% by PI 3-K
inhibitors (224, 284), and lysophosphatidic acid (LPA)
stimulation of NHE3 in OK cells is 100% blocked by
wortmannin (285).
Comparison of NHE3 trafficking mechanisms should
be made with GLUT4, the best studied transporter that
trafficks to the plasma membrane, which occurs through
insulin-stimulated exocytosis (80, 222, 233, 420, 461, 486,
487, 544). The GLUT4 studies are reviewed to serve as
background to consider the various pathways and regulatory proteins known to be involved in regulation of
trafficking of a transport protein, although it must be
pointed out that NHE3 trafficking between the plasma
membrane and intracellular pools is much less dramatic
than regulated GLUT4 trafficking. A minimal amount of
GLUT-4 is on the plasma membrane under basal conditions, with minimal basal exocytosis from the recycling
compartment. In contrast, after insulin stimulation,
GLUT-4 traffics via both a storage vesicular pool and
increased exocytosis from the recycling compartment. In
NHE3, Alexander and Grinstein (11) performed a detailed
kinetic analysis of NHE3 expressed exogenously in the
distal renal tubule cell line MDCK (11). They identified
four pools of NHE3: 1) a mobile apical membrane population, the trafficking of which could be rapidly stimulated; 2) an immobile apical membrane pool that binds
the cytoskeleton by interaction with Rho GTPases; and 3
and 4) two intracellular pools with a fast and less rapid
exchange rate with the apical membrane. The relevance
of this distal tubule model to normal physiology of the
proximal tubule is unknown. Also unknown is how similar is the mechanism of regulation of exocytosis of NHE3
to the GLUT-4 model, which predicts a storage pool that
as yet is not well characterized biochemically and the
recycling endosomes as distinct, although perhaps interacting pools.
Physiol Rev • VOL
III. INTRACELLULAR NHE3 ACTIVITY
Does intracellular NHE3 transport Na⫹ and H⫹? Our
interpretation is that the answer is yes, although intracellular NHE3 function and its regulation have not been
studied in detail. Some of the reasons to suspect that this
pool of NHE3 is functional include the following. 1) In
several cell culture models, inhibition of NHE3 alkalinizes
an intracellular compartment thought to be early endosomes (8, 134, 163, 164). This indicates NHE3 normally
acidifies this compartment. 2) In proximal tubule of wildtype mice, NHE3 inhibition by cAMP increased early endosomal pH within 1 min, as marked by FITC-BSA, implying cAMP inhibits both BB and intracellular NHE3
activity and demonstrates that intracellular NHE3 is active. Note this effect is too fast to be explained by changes
in trafficking (164). 3) In NHE3 knock-out mouse, there is
reduced renal proximal tubule albumin uptake (165).
Whether this involves intracellular NHE3 is not known.
4) In proximal tubule of ClC-5 knock-out mouse, NHE3
has primarily an intracellular location (373), which has
been assumed to be in some pool of endosomes and does
not resemble the distribution of Golgi. This intracellular
pool is still able to allow albumin endocytosis, which is
strong evidence that intracellular NHE3 functions.
IV. NHE3 COOH-TERMINAL REGULATORY
DOMAIN AND NHE3 COMPLEXES
The NHE3 COOH terminus is necessary for all identified examples of acute NHE3 regulation (293, 534). This
generally has been examined by truncation or point mutants of the NHE3 COOH terminus. Truncation of the
NHE3 COOH terminus alters basal as well as regulated
NHE activity. Truncation to aa 756 increases NHE3 activity twofold, while truncation to aa 690 increases it to
sixfold of basal (7). This is accompanied by similar increases in the amount of NHE3 on the plasma membrane
in PS120 fibroblasts (2 ⫻ for 756, 6 ⫻ for 690) with the
transport per plasma membrane exchanger remaining
constant. Further truncating NHE3 to aa 585 did not
further alter the percent of NHE3 on the plasma membrane or the activity per plasma membrane exchanger,
although total expression was diminished. This suggested
that there are two endocytosis signals in the NHE3 COOH
terminus, one between aa 756 – 832 and the other dominant signal between aa 690 and 756. Additional truncation
of NHE3 to aa 509, 475, and 455 decreased NHE3 activity,
with NHE3 445 activity minimally detectable in the absence of stimulation with serum. As shown in Figure 3,
study of truncation mutants demonstrated that the multiple acute regulators of NHE3 activity require specific
domains of the COOH terminus to act. The sole exception
identified in any mammalian NHE is the domain required
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
for NHE response to hyperosmolarity. While not studied
in NHE3, the effect of hyperosmolarity to stimulate NHE1
and inhibit NHE2 was shown to involve the first extracellular loop between aa 41–53 of NHE1 (aa 31– 43 of NHE2)
(422). This is an area of great divergence among the
NHEs. This domain is believed to represent the first extracellular loop for NHE1, which is not believed to contain a cleavable signal peptide, while we found that NHE3
did have a cleavable signal peptide, thus making the initial
expressed part of NHE3 extracellular (475, 546). Similar
results were found for Nhx1, the yeast intracellular NHE
(505). Wakabayashi and co-workers also reported similar
suggestive evidence for a cleaved signal peptide in NHE6,
although his other evidence does not support this view
(328, 475). This hyperosmolar response of NHE1/NHE2 is
the only NHE regulation that does not depend entirely on
the intracellular COOH-terminal domain (422). Not only is
the NHE3 COOH terminus necessary for rapid regulation
of NHE3, but it defines the nature of the regulation.
Swapping the COOH terminus of NHE3 with that of NHE1
or NHE2 led to regulation consistent with the NHE providing the COOH terminus (534).
A. Regulatory Domain
The NHE3 (rabbit) COOH terminus is predicted to
begin at aa 455 based on the assumption of similarity in
structure to the crystallized bacterial Na⫹/H⫹ exchanger,
835
NhaA, which lacks any significant intracellular COOHterminal domain (216). The NHE3 COOH terminus from
aa 455– 832 (378 aa) almost certainly has tertiary structure, although this has not been determined using physical techniques. Programs that predict secondary structure
indicate that the first ⬃130 aa after the transport domain
is predominantly ␣-helical (74). In contrast, the NHE1
COOH terminus between aa 503–545 was crystallized
(cocrystallized with its binding partner CHP2) and shown
to be ␣-helical as was predicted from secondary structure
predicting programs (18, 40).
The NHE COOH terminus has multiple functional
subdomains. Initial hints of the existence of functional
subdomains in the COOH terminus come from COOHterminal truncation studies of NHE1. Based on the intracellular pH (pHi) sensitivity and changes in acute NHE1
regulation, Ideka et al. (219) divided the NHE1 COOH
terminus into four functional domains, and these are discussed on the assumption that all NHEs probably have
some similarities in the organization of their intrinsic
regulatory mechanisms (219). These four domains of
NHE1 are as follows: 1) aa 515–595, 2) aa 596 – 635, 3) aa
636 – 659, and 4) aa 660 – 815 (219). Subdomain 1 had a pHi
maintenance function, preserving pHi sensitivity in the
physiological range, whereas subdomain 2, overlapped
with the high-affinity CaM-binding site in subdomain 3,
and exhibited an autoinhibitory function. Subdomains 2
and 4 had no function for pHi sensitivity. Deletion of
FIG. 3. A: NHE3 organization including COOH-terminal domains involved in acute regulation and binding
partners. The NHE3 NH2 and COOH termini are illustrated. Shown above the
COOH terminus in blue are sites of action
of regulatory stimuli defined by the truncations which abolish the specific regulatory effects. The direction of the arrow
indicates acute stimulation (1) or inhibition (2) of NHE3 activity. Below the
COOH terminus in yellow are listed associating proteins and the areas of their
binding sites on NHE3. B: classification
of NHE3 COOH terminus into 4 domains
for discussion of binding partners.
Physiol Rev • VOL
87 • JULY 2007 •
www.prv.org
836
MARK DONOWITZ AND XUHANG LI
subdomain 1 abolished the decrease of pHi sensitivity
induced by cellular ATP depletion, suggesting that this
subdomain is involved in ATP regulation of NHE1. Deletion of subdomain 3 rendered the inhibition by ATP depletion less efficiently, suggesting a possible functional
interaction between subdomains 1 and 3. However, deletion of subdomain 2 partially abolished the inhibitory
effect of subdomain 3. Therefore, subdomain 2 was proposed to function as a mobile “flexible loop,” permitting
the CaM-binding subdomain in subdomain 3 to exert its
normal function. These findings further showed that the
COOH-terminal domain of the NHEs plays a key role in
regulating NHE activity. Fliegel and associates (294, 297)
examined the structure of NHE1 aa 516 – 815 by circular
dichroism spectroscopy and concluded that it was 35%
␣-helix, 17% ␤-turn, and 48% random coil. They concluded
that NHE1 was largely ␣-helical near the NH2-terminal
transport domain (aa 516 – 632), while aa 633– 815 was
largely ␤-sheet. Moreover, elevating Ca2⫹ decreased the
amount of ␣-helix and increased the amount of ␤-sheets in
the COOH terminus.
The COOH terminal 377 aa of NHE3 are intracellular,
based on accessibility to antibodies in the unpermeabilized
compared with permeabilized state. A single study, using
monoclonal antibodies, suggested that some COOH-terminal
epitopes were accessible to extracellular antibodies, and by
implication that this part of NHE3 contained extracellular
epitopes (48). There has been no explanation provided for
this finding. The cytosolic NHE3 COOH terminus interacts
with proteins, which play essential roles in regulating NHE3
activity, trafficking, and phosphorylation. For NHE1, at least
for CHP, this association is complicated and involves a
structural contribution that is necessary for NHE1 to have
any transport activity (see below). Several NHE3 COOHterminal interacting proteins have been reported, and domains required for specific regulatory effects are beginning
to be defined (Fig. 3B). Four subdomains of the NHE3
COOH terminus are arbitrarily defined in Figure 3. Detailed
description of NHE3 associating proteins that interact with
each subdomain are described later.
B. NHE3 Complexes
What are the mechanisms by which the NHE3 COOH
terminus takes part in the acute regulation of NHE3? The
identified mechanisms include 1) acting as a kinase substrate and undergoing changes in Ser/The phosphorylation, 2) interacting with the NH2 terminus, and 3) interacting with other proteins or lipids to change the location
or signaling molecules associating with NHE3. This section deals with NHE3 interacting proteins and putative
lipid interactions. Consistent with its association with
multiple binding partners, the estimated size of NHE3 on
density gradient centrifugation (OptiPrep or sucrose denPhysiol Rev • VOL
sity gradients) indicates that NHE3 rarely exists as a
monomer or dimer (⬃90 or ⬃180 kDa), but after solubilization in 1% Triton X-100 exists simultaneously in complexes between 350 and at least 1,000 kDa (Fig. 4A) (8,
295, 296, 298, 340). This observation was made in multiple
intact tissue types and cell culture models, including
mouse and rabbit small intestine, mouse proximal tubule,
the intestinal epithelial cell line Caco-2, the proximal
tubule cell line OK, and PS120 fibroblasts. Several characteristics of these complexes have been established. 1) In
multiple cell types, the size of NHE3 complexes has been
compared on the apical surface, identified by cell surface
biotinylation versus the total cell pool (8, 298; Li and Donowitz, unpublished data). The plasma membrane NHE3
complexes were larger than the total pool in OK cells (the
majority of the total NHE3 in OK cells is intracellular with
estimates from 50 to 85% of NHE3 being intracellular) (8,
196) and PS120 cells (⬃85% of total NHE3 is intracellular).
2) These complexes are dynamic with acute regulation of
NHE3 (298). Evidence that the NHE3 complexes were dynamic comes from studies in which ileum was exposed in
vitro to carbachol, which inhibited NHE3 by elevating intracellular Ca2⫹ and stimulated endocytosis, and in which OK
cells were exposed to LPA, which stimulated NHE3 by increasing exocytosis. BB membranes were prepared from
ileal Na⫹ absorptive cells, and total membrane was prepared
from the OK cells; in both cases, NHE3 shifted into larger
complexes as part of acute regulation of NHE3 (see Fig. 4B
for carbachol).
Thus it appears that NHE3 exists simultaneously as
multiprotein complexes in both the apical plasma membrane domain and intracellularly. The size of these complexes is larger on the plasma membrane than those
inside the cell. This indicates that the complexes are not
all formed in the endoplasmic reticulum/Golgi but probably are modified on arriving at or leaving the apical domain under basal and regulated conditions. Moreover,
these complexes are dynamic and are further changed as
part of signal transduction that acutely regulates NHE3
activity. It should be considered whether the association
or dissociation of NHE3-interacting proteins, which determine the size of NHE3 complexes, may define the specificity of the different modes of NHE3 regulation described
above. Of interest is that NHE1 and NHE2 also form
complexes (38, 378). With the use of identical conditions
to compare complexes of NHE1, -2, and -3 expressed in
PS120 cells, NHE1 and -2 formed much smaller complexes with smaller ranges in size than did NHE3 (Li and
Donowitz, unpublished data).
1. How many proteins are there in the NHE3 complexes?
With the assumption that the average size of an
NHE3-interacting protein is 70 kDa, the number of NHE3
proteins associating with a dimer of NHE3 (NHE3 like all
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
837
FIG. 4. A: NHE3 is present simultaneously in large, multiprotein complexes. Triton X-100 solubilized cells were separated on discontinuous sucrase gradients with molecular
weight standards run on parallel gradients, as shown below.
B: carbachol increases the size of NHE3 complexes in the ileal
brush border. Ileum was exposed to carbachol (1 ␮M) for 10
min. Brush border was prepared and studied as in A. [From Li
et al. (298), with permission from Blackwell Publishing.]
mammalian NHEs characterized exists as a dimer under
basal conditions) at any point in time is estimated to be
⬃3 to ⬃10 (based on complex sizes ranging from 350 to
1,000 kDa minus the size of a dimer of NHE3 divided by 70
kDa). It is still to be determined why NHE3 complexes
exhibit such a large range in size, the meaning of simultaneous isolation of many sized complexes containing
NHE3, and how these complexes are regulated. Our current view is that different combinations of known and yet
to be identified NHE3-interacting proteins exist simultaneously in NHE3 complexes producing different sizes of
complexes. In fact, given the possibility of some complex
degeneration during processing, we hypothesize that even
more proteins associate with NHE3.
C. Intramolecular Interactions
Does dimerization or oligomerization of NHE3 occur in
vivo and contribute to the formation of the large complexes
of NHE3? Disuccinimidyl suberate (DSS) cross-linking
showed that homodimerization exists for NHE1 and NHE3
(146). The NH2-terminal transmembrane domain is sufficient
for this dimerization since deletion of the 300 aa COOH
terminus of NHE1 did not alter dimerization. However, the
Physiol Rev • VOL
COOH terminus appears to take part in dimerization. Domain 1 of the NHE1 COOH terminus, aa 562–579, was identified as the “dimerization domain” (208). Deletion of this
region disrupted disulfide cross-linking between the COOH
termini and functionally markedly reduced the intracellular
pH sensitivity of the exchanger, suggesting that this aspect
of the dimer might be necessary for the pH-dependent regulation of NHE1 (208). Multimerization of NHE3 was also
reported when NHE3 was analyzed by SDS-PAGE and Western blotting under denaturing conditions (250); however,
whether this represents a true picture of in vivo complex
formation is not known.
Using cross-linking reagents such as the Ca2⫹
phenanthroline or the bifunctional sulfhydryl reagent
methanethiosulfonate, Wakabayashi et al. (208) suggested
that NHE1 forms a dimer between the COOH-terminal
domain intrinsic Cys794 and Cys561. This was the first
evidence of the existence of intermolecular interactions
between the COOH terminus of NHE1. However, because
NHE3 does not contain Cys residues corresponding to
794
Cys and Cys561 of NHE1, similar intramolecular interactions are not expected to occur in NHE3. If there were a
Cys-mediated COOH-terminal dimerization in NHE3, the
only potential residue would be the sole Cys595 in the
87 • JULY 2007 •
www.prv.org
838
MARK DONOWITZ AND XUHANG LI
COOH terminus of NHE3. It is conserved in NHE3 and
NHE5 across mammalian species including human, rat,
and rabbit. Whether intermolecular interaction of the
NHE3 COOH terminus occurs by other mechanisms
should be considered as well. Oligomerization of NHE3
could occur indirectly via its COOH-terminal binding proteins. Since the NHE3 COOH terminus interacts with PDZ
domain containing proteins, including NHERF1, NHERF2,
NHERF3, and NHERF4, and each has two to four PDZ
domains, NHE3 could oligomerize indirectly through different PDZ domains bound to its COOH terminus. It remains to be determined whether such oligomerization of
NHE3 occurs in cells or intact tissue models.
D. Dynamic Aspects With Signaling
Our previous studies in rabbit ileal villus cells demonstrated that the NHE3 complex sizes were dynamically
regulated (298). Exposure to carbachol, which inhibits
NHE3 activity and LPA, which stimulates NHE3, both
resulted in an increase in sizes of NHE3 complexes compared with control. We have to emphasize that, when no
change in complex size is observed upon specific treatments, it does not necessarily mean there is no change in
the number or composition of proteins in the complexes.
The sucrose gradient fractionation approach can only
reliably detect relatively large changes in complex size
that resist exposure to 1% Triton X-100 (ⱖ100 kDa), not
the subtle changes that may result from addition and/or
subtraction of small proteins or change in protein composition (i.e., addition of one protein and dissociation of
another with similar molecular weight). We hypothesize
that 1) the dynamic nature of NHE3 complexes is consistent with NHE3 trafficking under basal and stimulated/
inhibited conditions that occur by different mechanisms,
with separate signaling events. 2) The large range in
complex sizes may reflect dynamic NHE3 cellular distributions, where the number and composition of NHE3interacting proteins in one location (such as the plasma
membrane) is different from those in other subcellular
compartments (such as recycling endosomes). An example is the difference in complex size of NHE3 on the apical
membrane of OK cells (larger) compared with that intracellularly, suggesting different NHE3 interacting proteins
are involved in NHE3 regulation in those two domains (8).
3) Upon changes in signaling, the number/composition of
NHE3-interacting proteins changes, and such changes
may lead to either changes in surface amount of NHE3 or
direct modulation of the transport domain, thereby resulting in the changes in transport activity.
V. NHE3 COOH-TERMINAL BINDING PARTNERS
To allow easier molecular manipulation, we divided
the NHE3 COOH-terminal domain into four subdomains
Physiol Rev • VOL
and will describe interacting proteins with each (using
rabbit NHE3 as an example): 1) aa 455–585 (131 aa), 2) aa
586 – 660 (75 aa), 3) aa 661–756 (96 aa), and 4) aa 757– 832
(76 aa). These four NHE3 COOH-terminal subdomains
were not selected by function or structure, although there
are some relationships with the subdomains of NHE1
characterized by Wakabayashi and co-workers (219).
Study of the regulation of NHE3 mutants with serial
COOH-terminal truncations defined some of the roles of
NHE3 COOH-terminal subdomains in regulating NHE3
stimulation and inhibition (7, 293). There appears to be at
least three stimulatory domains in the COOH terminus of
NHE3, including aa 455– 475 for fetal bovine serum (FBS),
aa 475–509 for fibroblast growth factor (FGF), and aa
509 –543 for okadaic acid (OA) (293). There are also two
inhibitory domains, including aa 585– 689 for PKA and
PKC, and aa 661–757 for CaM kinase II (271, 292, 545).
Although it is not yet known how these examples of
regulation are executed at a molecular level, we hypothesize that they may be exerted through regulatory proteins
that interact with the corresponding regions. Such NHE3
regulatory proteins have been demonstrated by several
groups to bind specific COOH-terminal subdomains and
are either required for basal or regulated NHE3 activity.
Caution has to be taken to interpret the results of studies
on COOH-terminal truncation or deletion mutants since it
is possible that a deleted region may be required for the
integrity of a tertiary structure that supports a particular
regulatory function but is not itself physically involved in
such regulation.
So far, at least nine different proteins have been
reported to directly interact with the NHE3 COOH terminus. These include CHP, ezrin, CaM, CaM kinase II,
NHERF family members, dipeptidyl peptidase IV (DPPIV)
(may be indirect), PP2A, megalin, and Shank2. The list is
expected to grow. Also, it has been suggested that NHE3
binds to phosphoinositol phosphates.
A. COOH-Terminal Domain 1, Amino Acids
455–585: CHP, Ezrin, and
Phosphoinositol Phosphates
This domain is important for NHE3 basal function,
since multiple point mutations in this domain decrease
NHE3 function. The identified binding partners for NHE3
in this part of the molecule include CHP and ezrin. In
addition, it has been suggested that signaling phosphatidylinositides bind here as well (5). This NHE3 COOHterminal domain is a highly hydrophobic region and contains multiple areas predicted to be ␣-helical, including a
comparable area to NHE1 aa 503–545, which was shown
to be ␣-helical by crystallization to 2.7 Å when in a complex with CHP2 (18, 40). This domain has been shown to
be necessary for NHE3 regulation by FBS, FGF, and OA
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
based on truncation studies (293) and ATP (108)
(Fig. 3A).
1. CHP
CHP, calcineurin homologous protein (p22), is a calcium binding, multi-EF hands domain containing protein
that binds to and regulates trafficking and function of
multiple classes of proteins including NHE1 to NHE5
(304, 318, 344, 363). CHP binding to NHE3 is necessary for
NHE activity. There are three CHP isoforms (CHP1 to
CHP3, with CHP3 also called tescalcin) (318, 344). CHP1
and CHP2 have been crystallized and have an ␣-helical
structure with 4-EF hand domains and resemble the calcineurin regulatory subunit (18, 40, 344). EF hands domain 1/2 and 3/4 pair with each other, although only EF
hands 3 and 4 bind calcium via a hydrophobic pocket on
the opposite side of the molecule from that which binds
ligands (18, 40).
Association of NHE1 with CHP1 is needed for maintenance of the pHi sensitivity of NHE1 (362–364). This led
to the suggestion that CHP with tightly bound Ca2⫹ may
serve as an important structural element in the “H⫹ modifier” of NHE1 (40, 362). The “H⫹ modifier,” a putative
allosteric NHE regulatory site, was proposed to contain
two binding sites for internal H⫹ based on kinetic studies
of renal BB NHE and is suggested to include NHE1 aa
433– 450 [intracellular loop 5⬘ (IL5)] and aa 451– 470
[transmembrane domain 11 (TM11)] of the NHE1 NH2
terminus (363, 471, 472, 473). Extensive mutational analysis showed that mutation of Arg440 (R440D) in IL5
caused a large acidic shift of the pHi profile for 22Na⫹
efflux. The equivalent “H⫹ modifier” in NHE3 is located at
aa 389 – 405 (IL5) and aa 406 – 425 (TM11), with 41 and 62%
aa identity, respectively (Fig. 5). This suggests the “H⫹
modifier” is structurally and functionally very similar in
NHE1 and NHE3. Both CHP1 and CHP2 bind in this area
to NHE1–NHE5. NHE3 or NHE1 mutants, in which hydrophobic residues were altered to disrupt NHE3-CHP interaction, have little Na⫹/H⫹ exchange activity, suggesting
839
the CHP binding is required for basal NHE activity (304,
362). It is suggested that CHP1 always contains two Ca2⫹
with high affinity (Kd ⫽ 90 nM) when associated with
NHE1 in cells. Overexpression of green fluorescent protein (GFP)-tagged CHP1 with mutations in EF3 or EF4
that reduce Ca2⫹ binding significantly reduced the exchange activity in the neutral pHi range and partly
impaired the activation of NHE1 in response to various
stimuli, such as growth factors and osmotic stress
(362).
These studies were supported and extended by structural studies. CHP2-NHE1 binding has been characterized
by crystallization by Wakabayashi’s group (18, 40). The
CHP binding domain of NHE1 is located at aa 515–542, a
hydrophobic aa-enriched area. A similar CHP-binding domain also exists in NHE2, -3, -4, and -5, with corresponding areas of aa 495–522 (for NHE2), aa 473–500 (for
NHE3), aa 486 –513 (for NHE4), and aa 465– 492 (for
NHE5) (18). The His6-tagged human CHP2 (aa 1–196)
complexed with its binding domain-containing sequence
of NHE1 (aa 503–545) was crystallized in the presence of
yttrium (Ca2⫹-bound state), with a resolution of 2.7 Å (18,
40). The NHE1-binding sequence of CHP2 is folded into
two globular domains (N- and C-lobes) consisting of 12
␣-helices and four ␤-sheets. The CHP-interacting domain
of NHE1 forms a ␣-helix, which is oriented into the
hydrophobic cleft encompassing the N- and C-lobes of
CHP2. The stability of the complex is maintained by a
large area of hydrophobic interactions between NHE1
and CHP, including amino acids of NHE1: Ile-518, Ile-522,
Phe-526, Leu-527, Leu-530, Leu-531, Ile-534, and Ile-527.
There is also potential interaction between a unique region of CHP2 (aa 94 –104) and IL5 of NHE1. From the
crystal structure, the CHP2-unique region (the 7th
␣-helix) is oriented far outside of the hydrophobic cleft,
leading to the possibility that this ␣-helix interacts directly with IL5 of NHE1. Indeed, it was observed that
NHE1 and CHP cross-linked with each other through IL5
of NHE1 and the unique region of CHP2. Furthermore,
FIG. 5. Sequence alignment of NHE1 and
NHE3 in putative H⫹ modifier site and CHPbinding domains.
Physiol Rev • VOL
87 • JULY 2007 •
www.prv.org
840
MARK DONOWITZ AND XUHANG LI
either deletion of the CHP-unique region (18) or mutation
of Arg-440 in IL5 of NHE1 (472) resulted in similar large
acidic shifts of the pHi-dependent Na⫹ uptake and efflux.
On the basis of these observations, two important roles of
CHP1/2 in NHE1 function were proposed (18). 1) CHP
functions as an obligatory subunit (of NHE1), which is
tightly associated with the COOH terminus of NHE1 and
is essential for NHE1 activity in both Vmax and H⫹ affinity.
2) CHP may interact with intracellular H⫹ regulatory sites
of NHE1 (such as IL5 or other ILs), through the CHPunique region, and is involved in modulation of the H⫹
affinity, but not Vmax.
Recently, using nonoverexpressing NHE1 cell lines,
CHP1 was shown to have an additional and perhaps primary role in stabilizing NHE1 on the plasma membrane
(320a). We speculate that the role of CHP in the function
of NHE3 is likely to be similar to that of NHE1 based on
the following analysis. First, among the eight hydrophobic
residues (7 Leu/Ile and 1 Phe of the NHE1 COOH terminus
as discussed above between aa 515–542) that are thought
to be involved in the hydrophobic interactions between
NHE1 and CHP, seven are present in similar locations in
the CHP-binding domain of the NHE3 COOH terminus.
Second, IL5 of NHE3 is highly homologous to that of
NHE1 (75%) and contains Arg-396 that is equivalent to
Arg-440 of NHE1. Thus we suggest that IL5 of NHE3 is
potentially a H⫹ modifier site that interacts with the CHPunique region to modulate the H⫹ affinity.
The relationship between CHP1, CHP2, and CHP3 in
NHE function is unknown. CHP1 is ubiquitous. In contrast, CHP2 is present mostly in apical domain of intestinal epithelial cells and has minimal expression in kidney
(221). This suggests that 1) CHP2 is the primary isoform
for intestinal NHE function, while 2) CHP1 is a major
isoform for the function of kidney-expressed NHEs. CHP2
interestingly was also expressed in some tumor cells including hepatoma and cervical cancer, tissues that normally do not express it (364). CHP2 protects cells from
serum deprivation-related cell death by increasing pHi,
essentially mimicking the phenotype of malignant cells.
The role of this function in physiology/pathophysiology is
not understood.
Tescalcin/CHP3, a 24-kDa Ca2⫹ and Mg2⫹ binding
proteins with four EF hand domains which bind one
molecule of Ca2⫹, has been called CHP3 based on structural similarities to CHP1 and -2 (CHP1 and -2 are ⬃70%
identical and CHP1 and -2 and tescalcin are ⬃28% identical at amino acid level) (18, 297). There are no studies of
the association of tescalcin and NHE3, but Mailander
et al. (318) reported the tescalcin binds to the COOHterminal NHE1, and Fliegel and co-workers (297) then
showed this was to the COOH-terminal 182 aa, specifically
to His182. That is, it binds the COOH terminus of NHE1 at
a site separate from that which binds CHP1 and -2. This
part of NHE1 has minimal ␣-helical structure, and Ca2⫹
Physiol Rev • VOL
causes a shift to an even more B-sheet structure. The
NHE1/tescalcin association was predicted to influence
the pH sensitivity of NHE. An interesting but untested
possibility is that CHP1 and CHP3 binding sites might
interact to explain effects on pH sensitivity of NHE1.
CHP was also reported to be involved in acute ligandinitiated regulation of NHE3. The specific agonist involved was adenosine A(1) receptors in opossum kidney
(OK) and Xenopus laevis uroepithelial (A6) cells (116).
Activation of adenosine A(1) receptor [A(1)R] by N6-cyclopentyladenosine (CPA) inhibits the NHE3 via a phospholipase C/Ca2⫹ PKC signaling pathway. Mutation of
PKC phosphorylation sites on NHE3 does not affect regulation of NHE3 by CPA (consistent with PKC regulation
of NHE3 not involving phosphorylation of NHE3 by PKC),
but amino acid residues 462 and 552 are essential for
A(1)R-dependent control of NHE3 activity. Yeast twohybrid assay identified a minimal interacting region with
CHP localized to the COOH-terminal juxtamembrane region of NHE3 (amino acids 462–552 of opossum NHE3).
CPA (10⫺6 M) increased the CHP-NHE3 interaction by
30 – 60%. Overexpression of the polypeptide from the CHP
binding region (aa 462–552) interfered with the ability of
CPA to inhibit NHE3 activity and to increase the interaction between CHP and full-length NHE3, while decreasing
native CHP expression by siRNA abolished the ability of
CPA to inhibit NHE3 activity. Therefore, it was concluded
that CHP-NHE3 interaction was regulated by A(1)R activation, and this interaction is a necessary and integral
part of the signaling pathway between adenosine and
NHE3.
2. Direct binding of ERM proteins
The ERM proteins ezrin, radixin, and moesin bind to
NHE3 both directly (74) and indirectly via the NHERF
family (the latter interactions were the first functional
effects on regulation of NHE activity recognized and are
discussed below in sect. VB). Thus NHE3 associates with
the cytoskeleton using at least two COOH-terminal domains. Of note, some regulation of NHE3 which involves
the cytoskeleton has been identified but not attributed to
either of these interactions (11, 71, 75, 135, 155, 197, 269,
431– 433, 488). This represents an area requiring future
study.
ERMs are present in the ileal and proximal tubule BB
(59). These proteins link the actin cytoskeleton to intrinsic membrane proteins that have cytoplasmic domains.
The ERM proteins bind multiple plasma membrane NHEs
with direct physical interactions with NHE1 identified
first (112, 527). These occur via two positively charged
amino acid clusters in the most NH2-terminal first subdomain of the NHE1 COOH terminus. These appear to be the
same RK clusters that are involved in phosphatidylinositol 4,5-bisphosphate (PIP2) binding to NHE1. As elegantly
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
described by Barber and colleagues (38, 110 –112, 300),
NHE1 binding to the ERM proteins in fibroblasts is necessary to locate NHE1 in lamellipodia. Importantly, ERM
binding to NHE1 appears to be involved in organizing the
cytoskeleton in symmetrical cells. This NHE1-ezrin interaction involves NHE1 acting as an anchor in the lamellipodia for actin via ERM binding. The fibroblast functions
dependent on this NHE1 binding include localization of
ERM proteins and determination of fibroblast mobility
and polarity of migration as well as coordination of motility in a manner that is independent of the transport
activity of NHE1. Structurally, disruption of NHE1-ERM
binding is associated with abnormal stress fibers and
focal adhesions and results in lack of polar migration of
fibroblasts which are attempting to fill in a wound. This
striking abnormality in fibroblasts is in contrast to NHE1
knockout mice, which not only are viable, but have a
phenotype that includes seizures (39, 100). This suggests
redundancy in the function of this NHE1-ERM interaction.
The NHE1 knockout mice appear to have normal intestinal
and renal morphology and have decreased salivary
secretion.
The direct NHE3-ERM interaction appears quite separate from the type of interaction described for NHE1,
although whether, similarly to the role of NHE1, NHE3
binding to ERM proteins is important for the organization
of the BB is unknown. Direct ERM binding to NHE3
occurs in domain 1 of the COOH terminus (aa 515–595)
and involves a positive cluster of amino acids that include
R520, R527, and possibly K516 in an area of high pI
(estimated to be 12.1) (74). Ezrin binding to this area is
further evidence (along with programs predictive of secondary structure) that this area of NHE3 is ␣-helical, as
this is the only way that these positive amino acids would
form a cluster, which is needed for ezrin binding. The
ezrin domain involved in NHE3 binding is the third “clover
leaf” of the NH2-terminal FERM domain (59, 140, 193, 253,
367, 401, 440, 441, 527). This is the same part of the ERM
protein involved in binding to the NHERF family (74, 366,
402, 418). Functionally, direct ERM binding to NHE3 is
necessary for NHE3 trafficking (74). When these positive
amino acid mutants were expressed in PS120 fibroblasts
and OK cells (R520F, R527F referred to as NHE3 F1D),
there was a marked reduction in the transport activity of
NHE3, due to much less NHE3 on the plasma membrane.
The specific aspects of BB trafficking that depend on
direct ERM binding to NHE3 and thus are inhibited by
these mutations include endocytic recycling (exocytosis
from the recycling system) and delivery to the plasma
membrane of newly synthesized NHE3. Initial rates of
endocytosis were less affected, being only slightly reduced (note this would increase the amount of BB NHE3).
In addition to inhibited basal NHE3 activity, Ca2⫹ inhibition of NHE3 was reduced in the ezrin binding mutant.
Since Ca2⫹ inhibits NHE3 by stimulating endocytosis,
Physiol Rev • VOL
841
what explains this effect? The possible explanation
comes from 1) NHE3 plasma membrane half-life studies
demonstrating a normal initial decrease in plasma membrane NHE3 followed by a prolonged plasma membrane
presence, plus 2) FRAP studies with confocal microscopy
examining OK cell apical membranes of wild-type and
ezrin binding mutant NHE3, which showed that the mutant had a markedly reduced mobile fraction. We suggest
that Ca2⫹ inhibition of NHE3 normally involves complex
formation followed by endocytosis of NHE3. The ezrin
mutant has a normal rate of endocytosis of the NHE3
present under basal conditions in the vicinity of the intervillus clefts, where endocytosis initiates. However, we
speculate that direct ezrin binding to NHE3 is necessary
to move microvillus NHE3 to the intervillus cleft area for
endocytosis, and this is defective in the ERM binding
mutant (74). Whether the increased NHE3 complex formation and its subsequent endocytosis that occurs with
elevated Ca2⫹ regulation of NHE3 is affected in the direct
ezrin binding mutant has not been examined experimentally. Thus NHE3 is one of a growing family of ERM
binding proteins in which one molecule of the protein
binds two ERM molecules, one direct and one indirect via
NHERF family members (402, 484). Ezrin is now recognized to bind to proteins either directly or indirectly via
NHERF proteins. Substrates that only directly bind ezrin
include intracellular adhesion molecule (ICAM)-1,
ICAM-2, ICAM-3, CD43, CD44, CD95, syndecan2, and
NHE1 (110, 202, 306, 454, 527). Indirect associations have
been described with the ␤2-adrenergic receptor, cystic
fibrosis transmembrane regulator (CFTR), podocalyxin,
and now NHE3 (191, 324, 428, 454, 479). The renal podocyte plasma membrane protein podocalyxin appears most
similar to NHE3 in ERM binding (402). Podocalyxin binds
ezrin both directly and indirectly via NHERF1 or -2; however, the function of the direct ezrin binding has not been
discovered. Interestingly, it appears that podocalyxin may
directly bind ezrin using an ␣-helical structure since there
is not three or more consecutive positively charged amino
acids in the domain of podocalyxin in which it binds ezrin.
3. Phosphoinositol phosphates and ATP
All NHEs require ATP for function, although the
mechanism is not clear (4, 5, 65, 68, 73, 108, 155, 234, 292).
Domain 1 in the COOH terminus is the region responsible
for the cellular ATP dependency of NHE activity (5, 65,
108). Although ATP is not directly involved in the Na⫹/H⫹
exchange, cellular ATP has been known to be necessary
for NHE activity since 1986 (68). Subsequently, this phenomenon was observed in several different cells, including type II alveolar epithelial cells (64); PS120 cells transfected with NHE1 (292); AP1 cells transfected with NHE1,
NHE2, and NHE3 (234); and red blood cells (108). In
NHE1, the 80-aa subdomain 1 region (aa 515–595) adja-
87 • JULY 2007 •
www.prv.org
842
MARK DONOWITZ AND XUHANG LI
cent to the last transmembrane domain is involved in ATP
depletion-induced acidic shift of pHi-dependent activity. It
was proposed that this domain of NHE1 might interact
with the H⫹-modifier site, leading to its increased affinity
to H⫹ and that ATP depletion might prevent such an
interaction (3). The mechanism for the ATP dependency
was further elucidated by Aharonovitz et al. (5), who
showed that PIP2 might be directly involved in the ATPdependent NHE activity. PIP2 was shown to bind to the
positive charged amino acids (Arg or Lys) at aa 513–564 in
NHE1. Mutant NHE1 lacking the putative PIP2 binding
motif had greatly reduced transport capability, implying
that association with PIP2 is required for optimal activity.
On the basis of these experiments, it was proposed that
ATP depletion diminished the cellular PIP2 content and
thereby inhibited NHE activity (5). More recently, roles of
phospholipids in regulating NHE activity were further
demonstrated by Fuster et al. (155) through measuring
extracellular protein (H⫹) gradients during whole cell
patch-clamp studies with perfusion of the cell interior in
NHE1- or NHE3-expressing cells. In their experiments,
NHE1 activity is stable with perfusion of nonhydrolyzable
ATP [adenosine 5⬘-(␤,␥-imido)triphosphate], suggesting
Na⫹/H⫹ exchange does not require direct ATP hydrolysis.
However, NHE1 activity was abolished by ATP depletion
(2-deoxy-D-glucose with oligomycin or perfusion of
apyrase) but could be restored with PIP2 and was unaffected by actin cytoskeleton disruption. These data support 1) the previous reports that cellular ATP is required
to generate sufficient PIP2 for NHE activity and 2) the
PIP2 effect on NHE activity is directly on NHE1 itself
(through direct NHE binding). Importantly, NHE3 activity
was not affected by intracellular perfusion with PIP2
under conditions that activated NHE1. However, NHE3
was reversibly activated by phosphatidylinositol 3,4,5trisphosphate, suggesting that the role of phospholipids in
modulating NHE activity occurs in an NHE isoform-specific fashion.
B. COOH-Terminal Domain 2, Amino Acids
586 – 660: Calmodulin, Calmodulin Kinase II,
and NHERF Family
1. CaM and CaM kinase II
Ca2⫹-dependent activation of NHE1 is well documented (470, 473, 474, 476). NHE1 contains a CaM-binding
domain (CBD) at aa 637– 656. Deletion or mutation in this
area induces a constitutive activation of NHE1, resulting
in an increase in the affinity for intracellular H⫹ (an
alkaline shift of pHi-dependent exchange activity) and
loss of responsiveness to a rise in intracellular Ca2⫹ concentration (41, 219, 470, 476). Therefore, it was concluded
that the CBD reduces the affinity of NHE1 for intracellular
H⫹ by exerting an autoinhibitory function in quiescent
Physiol Rev • VOL
cells (473, 474, 476). This effect was replicated in an
NHE1 chimera, in which the CBD of NHE1 was replaced
with the corresponding domain in NHE2 (but not NHE4),
suggesting that both NHE1 and NHE2 contain a functionally similar CaM-binding domain (473). Moreover, photoaffinity cross-linking between immunoaffinity-purified
NHE1 and 125I-labeled CBD of NHE1 showed that the
CBD was efficiently cross-linked to NHE1, suggesting that
CBD physically interacts with an unspecified domain(s)
of NHE1 (473). Whatever the unspecified domain(s) might
be, its interaction with the autoinhibitory domain (CBD)
might be to alter the H⫹ affinity of the H⫹-modifier site
either directly or indirectly through a conformational
change of NHE1. Also, CaM is involved as an intermediate
in regulatory volume increase (RVI) stimulation of NHE1
activity, which occurs downstream of Janus kinase 2. The
latter phosphorylates CaM (157).
Fliegel et al. (294) recently showed that optimal CaM
binding to NHE1 requires a COOH-terminal conformation
that appears to be maintained at least in part by a stretch
of six or seven acidic residues (aa 753–759). Mutation of
these resulted in diminished and slower NHE1 activity
and ⬎50% reduction of CaM-NHE1 binding without significantly affecting the total and surface expression of
NHE1. These data suggest that a proper conformation of
the cytosolic COOH terminus of NHE is necessary for a
fully functional NHE.
CaM and CaM kinase II are present in the ileal and
renal proximal tubule BB and regulate neutral NaCl absorption in rabbit ileum (95, 128, 132, 195, 300, 545). Basal
NaCl absorption and BB NHE3 activity are inhibited by
CaM and CaM kinase II since 1) inhibiting CaM (with
W13) or CaM kinase (with H-7) stimulates neutral NaCl
absorption and 2) including CaM and CaM kinase II in the
presence of Ca2⫹ in ileal BB vesicles inhibited BB NHE3
activity (95, 122, 132, 144, 391, 545). In PS120 fibroblasts,
both CaM and CaM kinase II directly bind to the NHE3
COOH-terminal domain between aa 586 – 660 (545). Similar to ileal Na⫹ absorptive cells, a CaM kinase II inhibitor,
KN-62, stimulates basal NHE3 activity in PS120 fibroblasts
(545), by a NHERF2-dependent mechanism. Thus CaM
kinase II binds NHE3 directly and inhibits NHE3 activity
under basal Ca2⫹ conditions. CaM binds to the COOH
termini of NHE1, -2, and -3 and is involved in basal regulation in these three isoforms. While the mechanism of
this regulation is unknown, NHE3 is phosphorylated under basal conditions by CaM kinase II (M. Zizak and M.
Donowitz, unpublished data).
2. NHERF family of multiple PDZ domain proteins
Multi-PDZ domain containing proteins bind to NHE3
at domain 2 and are involved in NHE3 regulation. There
are multiple controversial aspects of these observations,
and this is an area of active study. A role for PDZ domain-
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
containing proteins in NHE3 regulation was first established by observations in PS120 fibroblasts that elevated
cAMP failed to inhibit NHE3 (534). These cells lack endogenous NHERF2 and have only a small amount of
NHERF1 (529, 532, 534). However, when these cells stably
expressed NHERF1 or NHERF2, cAMP inhibited NHE3,
as occurs in epithelial cells (277, 532, 534). Since this
observation, multiple aspects of stimulation and inhibition of NHE3 activity have been shown to depend on the
presence of PDZ domain containing proteins. Similar observations were subsequently established for CFTR and
other transport proteins. Not known is whether all acute
stimulation and/or inhibition of NHE3 requires or is influenced by NHERF family binding.
3. Overview of PDZ domain proteins (49, 63, 113,
156, 196, 215, 248, 352, 411, 455, 538)
PDZ domains (named after the three initially identified proteins which contained these domains, PSD-95,
Discs large, and ZO1) are ⬃90-aa modular protein-protein
interacting domains. Structurally they are made up of six
␤-sheets and two ␣-helices, with ligand binding in a linear
grove between ␤-sheet 2 and ␣-helix 2. PDZ domains are
commonly used in the human genome for protein-protein
interactions, and currently ⬎600 PDZ domains in human
proteins have been identified. Since many proteins contain multiple PDZ domains, ⬃400 mammalian proteins
have been identified as containing PDZ domains. PDZ
domain containing proteins often have more than a single
PDZ domain (as well as other protein interacting domains), with the structural relationship among the PDZ
domains fixed, i.e., the PDZ domains are not organized
like their stick diagrams, but their relationships among
themselves confers structure to their binding partners.
PDZ domain interactions with their ligands generally
occur at the ligand COOH terminus and primarily involve
amino acids at the 0 and ⫺2 positions, although up to at
least the last six amino acids can affect binding. Similar
internal sequences, called ␤-fingers, have been identified
in nNOS, PSD-95, syntrophin, and NHE3 and are used to
bind PDZ domains. There are several categories of PDZ
binding domains based on the ligand binding sites. Class
I consists of X S/T X hydrophobic, with I/V/L/M reported
at aa 0. Class II consists of X hydrophic, X hydrophobic.
Class III consists of X E/D X hydrophobic. The reported
affinities of PDZ domain-ligand interactions appear low, 1
nM-10 ␮M, which seems appropriate for dynamic interactions (196).
PDZ domain containing proteins are generally cytoplasmic and connect the plasma membrane to the cytoskeleton via binding to the cytoplasmic tails of membrane proteins. Identified binding partners are of many
categories and include growth factor receptors (EGFR,
platelet-derived growth factor receptor), G protein-couPhysiol Rev • VOL
843
pled receptors, neurotransmitter receptors, transport proteins, adhesion molecules, cytoskeletal elements, transcription factors, and PIP2 (248). Understanding functions
of PDZ domain proteins must consider not only their
ligands but also their location, which may be dynamic.
Their location is generally at specific membrane domains
(196). In epithelial cells there are separate PDZ domain
proteins restricted to the BB, basolateral (BLM) and tight
junctions (TJ), as well as cell-cell and cell-matrix contacts
(63); and in polarized neuronal cells, PDZ proteins are at
multiple depths from the surface (156, 455).
The major function of PDZ domains is as scaffolds,
using their ability to homo- or heteromultimerize and
form cysteine bonds (156, 428, 455). While defining functions of PDZ proteins has been complicated by redundant
expression of multiple family members in the same location at the same time, specific functions identified include
1) complex formation, in most cases on the plasma membrane but also at intracellular sites. The site of assembly
of these complexes is not known, although for NHE3 the
complexes on the BB and plasma membrane are larger
than intracellular forms, suggesting at least part of the
formation is at the plasma membrane (8, 298). 2) They
organize plasma membrane domains, including LR. This is
critical for cell signaling, in which certain interacting
signaling molecules are included, while others are excluded. This allows increased efficiency, speed, and specificity in signaling (248). 3) They establish and maintain
polarity in embryos as well as epithelial and neuronal
cells (49). 4) They regulate trafficking-sorting of proteins,
moving from TGN to the BB, BLM, or TJ, and endocytic
recycling versus lysosomal targeting (185, 248). 5) They
cluster or retain proteins in the plasma membrane. For
instance, PSD-95 clusters ion channels and InaD retains
TRP channels, PKC, and PLC in the rhabdomere (455).
Also, PDZ proteins have been suggested as retaining
NaPi2a and CFTR in the BB rather than the BLM (43,
186, 205, 206, 276, 409). The latter is controversial (418).
6) They affect ion channel gating. CFTR gating is altered
by binding to tandem PDZ domains of NHERF1 or PDZK1,
although the mechanism is not understood (381, 480).
Understanding the roles of PDZ domain proteins in
polarized cells has evolved from the initial perception that
they served a strictly scaffolding role to allow complexes
to form. As recently reviewed by Sheng and co-workers
(215, 248), these proteins are abundant, often an order of
magnitude greater than the transporters they help regulate. It appears that these proteins may have some highly
cell specific effects. Their recognized epithelial actions
include (63) 1) assembly of specific proteins into large
static molecular complexes at specific locations in a cell.
This includes forming clusters of transporters at the
plasma membrane. Both plasma membrane and cytoplasmic proteins can be brought into the same complex,
including signaling molecules (36, 190). 2) They allow
87 • JULY 2007 •
www.prv.org
844
MARK DONOWITZ AND XUHANG LI
dynamic trafficking of proteins by bringing molecular motors (for instance, bringing both kinesins and myosins,
including myosin VI) into apical membrane complexes
(430, 522). The complexes can move around the cell as
preassembled packages or from one site such as the
apical membrane to others. Both microtubule tracks and
actin-myosin motors are used. PDZ proteins on the surface of the cargo vesicles can act as receptors for motors
and lead to movement to targeted sites. 3) They deliver
and/or stabilize plasma membrane proteins including
transport proteins. This includes regulation of exocytosis,
endocytosis, subcellular localization, determination of
subunits present, and control of intrinsic activity.
The PDZ proteins are often also highly regulated (43,
248). They bind to multimers of themselves and other PDZ
proteins. Myristoylation or palmitoylation are used to link
the PDZ proteins to plasma membrane or intracellular
sites, and binding to tetraspannin proteins can be involved in surface expression. There is generally dynamic
regulation of the PDZ proteins and thus of their complexes, which involve changes in subcellular distribution,
phosphorylation, and degradation of both the PDZ domain protein and the PDZ binding ligand (3, 91, 92, 192,
265, 280, 348, 444, 463). The regulation of actions of PDZ
proteins can be affected by phosphorylation of the ligand
COOH-terminal amino acids, especially the S/T at the ⫺2
position. Most commonly, disassembly of complexes occurs with PDZ ligand phosphorylation. For instance,
Weinman showed that ␤2-adrenergic receptor binding to
NHERF1 was decreased by receptor phosphorylation (unpublished data).
4. Epithelial apical membrane PDZ domain proteins
relevant to NHE3 regulation
The BB of mammalian small intestine and renal proximal tubule and cell culture models of intestinal Na⫹
absorptive cells (Caco-2, HT29, T84 cells) and renal proximal tubule cells (OK cells) contain varying amounts
(from none to amounts up to 0.1% of the total cell protein)
of four related PDZ domain proteins in or near the apical
membrane which are members of a gene family, called the
NHERF family (126, 205, 276, 405, 442, 492). There are
additional unrelated PDZ domain proteins in the BB
which affect NHE3, such as Shank2 (194). These NHERF
family members include NHERF1 [which is also called
NHERF or EBP50 ezrin binding protein 50 kDa in size
(EBP50)], NHERF2 [also called NHE3 kinase A regulatory
protein (E3KARP), SIP-1, TKA-1], NHERF3 or PDZK1
(also called clamp, CaP70, diphor-1 or NaPi-cap1), and
NHERF4 or IKEPP (intestinal and kidney enriched PDZ
domain protein, also called PDZK2 diphor-2 and NaPicap2) (Fig. 6). These four proteins are scaffolds and consist of multiple PDZ domains (2 for NHERF1 and -2 and 4
for NHERF3/PDZK1 and NHERF4/IKEPP). The only other
Physiol Rev • VOL
FIG. 6. NHERF family members showing number of PDZ domains,
other protein-protein interacting domains, and number of amino acids.
recognized domain that any of the four proteins contain is
an ERM (ezrin, radixin, moesin) binding domain at the
COOH terminus of NHERF1 and -2 (533).
These four members of the NHERF family are closely
related. Mammalian PDZ proteins originated in worms
and flies rather than in bacteria, plants, or yeast (126,
442). In fact, bacteria, plants, and yeast lack true PDZ
proteins in which the ligand binding grove is linear and
rather have related proteins that have a circular binding
cleft. NHERF family origins can be traced to C. elegans
and D. melanogaster (126, 442; A. Sharma, C. Brett, and
M. Donowitz, unpublished data). Blast analysis across
species of human NHERF1 identified 400 related proteins.
Clustal W analysis was used to align these sequences, and
they were organized into a Claudogram using PAUP v4.1,
which showed that these four PDZ proteins were the most
related proteins in multiple genomes. The 12 individual
PDZ domains from these 4 proteins, as defined by Scansite (PFAM), had their origins traced to either worms or
flies. Eleven of 12 NHERF family PDZ domains appear to
have evolved from 2 worm proteins that are probably
related to each other by gene duplication. Similarly, two
fly PDZ domain-containing proteins are highly related to
the human NHERF family PDZ domains and have a closer
similarity than with the worm proteins for some of these
PDZ domains but with no relationship with others of
those PDZ domains. However, one of these fly proteins
has homology to a single NHERF family PDZ domain that
lacks homology to the worm proteins (126). Thus we
suggested that the NHERF family evolved from gene duplication in the worm, which evolved into a protein in fly
which also evolved into a separate PDZ domain that via
evolution produced the human NHERF family.
Why there are multiple similar, related PDZ domain
proteins in the apical domain in the same epithelial cell is
not understood, although some hints have emerged recently. Two areas of divergence have been identified
among the four NHERF family members: A) subtle differences in location in the apical surface of kidney and
intestine (469, 537) and B) somewhat different binding
partners among proteins including those that have been
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
shown to be involved in NHE3 regulation (72, 87, 217, 229,
230, 250, 355, 402, 423).
A) APICAL SURFACE DISTRIBUTION OF NHERF FAMILY IN ILEUM
AND RENAL PROXIMAL TUBULE.
All NHERF family proteins are
in or near the BB. Wade and Weinman (469) demonstrated
in mouse renal proximal tubule that NHERF1 was in the
BB while NHERF2 was also in the BB but located mostly
just below the BB in an area consistent with the intervillus
clefts (where clathrin-coated pits form) (469). Murer and
co-workers (311) further demonstrated that NHERF3/
PDZK1 had a similar distribution to NHERF1 in the renal
proximal tubule BB, while NHERF4/IKEPP was apical
plus subapical (172, 467). Our results in mouse ileum and
Caco-2 cells expressing HA-NHE3 support this differential
distribution. In both mouse ileal Na⫹ absorptive cells and
Caco-2 cells, NHERF1 and NHERF3/PDZK1 are largely in
the BB, with NHERF1 also appearing to be present in
goblet cells (535). NHERF2 and NHERF4/IKEPP are distributed in the BB and subapically, although NHERF4/
IKEPP also is in the supranuclear cytosolic area. Thus BB
location of these proteins appears similar under basal
conditions in ileum and renal proximal tubule although
NHERF2 is also in the ileal microvilli, perhaps to a greater
extent than in the proximal tubule. How the apical membrane and intracellular localization of these proteins
change with conditions which regulate NHE3 is not
known.
Also, not all species have all NHERF family members
or have them in the same locations. For instance, while
human, rabbit and mouse renal proximal tubules contain
NHERF2, it appears to be lacking in rat proximal tubules
(469). Moreover, different epithelial cell culture models
express different amounts of each NHERF family member. For instance, the renal proximal tubule cell line OK
has NHERF1, but generally lacks or has low expression of
NHERF2. Caco-2 cells have little NHERF2 while another
colon cancer cell lines HT-29 contains an abundant
amount of NHERF2 (529).
B) NHERF FAMILY BINDING PARTNERS. These are only partially known, although ⬃40 binding partners have already
been identified (see recent review in Ref. 408). Moreover,
there are few organized comparisons reported of whether
binding partners of one member of this family also bind
other members, and some studies are contradictory,
which is not unexpected given their generally low binding
affinity. Common binding partners of NHERF1 and
NHERF2 (listed by category of ligand) include the following (109, 408): tyr kinase receptors (PDGFR), G proteincoupled receptors (␤2-adrenergic receptors, PTH1R),
transporters (NHE3, CFTR, Na⫹-K⫹-ATPase), enzymes
(PLC-␤1 and -␤2, PKC-␣, EP164), and cytoskeleton linkers
(ezrin). A recent review listed ligands of NHERF1 that do
not bind NHERF2. These include TRP4 and -5, NBC3,
H⫹-ATPase, Kir1.1, ␬-opiod receptor, LPA2R, GRK6A, inducible nitric oxide synthase, and YAP65 (192, 214, 332,
Physiol Rev • VOL
845
377, 408, 437), although most studies were not performed
in detail enough to be definitive. Ligands that appear to
bind NHERF2 and not NHERF1 include TAZ (a transcription factor) (230), ␣-actinin-4 (250), adenosine 2␤ receptor (416), PLC-␤3 (217), DRA (275), SKG1 (529), podocalyxin (data contradictory; Refs. 300, 402, 436), SRY-1,
cGKII (72) TRPV5 (143), Ca2⫹-ATPase 2b (107), and ClC-5
(211). No specificity in binding has been established as yet
for NHERF3/PDZK1 (binds NHE3, CFTR, NaPi2a, ezrin,
DRA, D-AKAP2 and ␣-actinin-4) (173, 174, 205, 393, 408,
479) and NHERF4/IKEPP (binds guanylate cyclase C,
NHE3, TRPV5, and TRPV6) (405, 442, 464). While these
studies are generally preliminary, the concept of specificity among binding partners of the NHERF family is established.
5. “Hand-off” hypothesis
The possible role of these multiple NHERF family
proteins may relate to the “hand-off hypothesis” first suggested by the Murer group (27, 42, 43, 204, 206) and
supported by the studies of Guggino and co-workers for
CFTR (81– 83). Under basal conditions in Caco-2 cells,
NHE3 and NHERF3/PDZK1 overlap in the BB (535). We
found that with elevated intracellular Ca2⫹, NHERF3/
PDZK1 stays in the BB but forms aggregates and separates from NHE3, which in 10 min appeared to move to
the area of the intervillus clefts (536). We suggest that this
may represent separation of BB NHE3 from NHERF3/
PDZK1 before initiation of endocytosis. Studies with
CFTR and a Golgi-resident single PDZ domain-containing
protein called Cal supports this concept. Cal associates
with CFTR under basal conditions intracellularly, but under conditions in which CFTR trafficks to the apical membrane, there is less Cal-CFTR association and more
NHERF1-CFTR association (81– 83). This supports sequential interaction of transport proteins with individual
NHERF family members or other intracellular PDZ domain containing proteins, based on states of regulated
stimulation versus inhibition. However, support for this
hypothesis is very preliminary, and details of transfer and
reasons for changes in NHE3/individual NHERF protein
interactions are unknown.
6. Model systems used to understand the cell specific
role for NHERF proteins in NHE3 regulation
The roles of each NHERF family member in NHE3
regulation have been approached at multiple levels, although conclusions are still preliminary. These approaches include 1) expression of individual NHERF family proteins in null simple cell lines, for instance, PS120
fibroblasts. The strength of this approach is that these
cells largely lack NHERFs (529, 533), although small
amounts of NHERF1 have been reported (6) and our
laboratory also is able to identify small amounts of
87 • JULY 2007 •
www.prv.org
846
MARK DONOWITZ AND XUHANG LI
NHERF2 in these cells using more sensitive antibodies
than initially reported. 2) There is expression in polarized
epithelial cell lines either that lack a specific NHERF
family protein (i.e., OK cells contain NHERF1 but generally lack or have very low levels of NHERF2) (529) or use
of siRNAi in these cell lines. 3) Knockout mice models
have been reported for NHERF1 (93, 102–104, 243, 276,
333, 409, 495, 500) and NHERF3/PDZK1 (67, 93, 262, 263,
413, 445, 478).
Most relevant is understanding of how the NHERF
family proteins take part in NHE3 regulation in intact
intestine. To date, only a few studies have been reported.
The conclusions from studies of the renal proximal tubule
and intestinal knockout mouse models that have emerged
are that multiple NHERF family members are involved in
cAMP, cGMP, and Ca2⫹ inhibition of NHE3. These results
are strongly suggestive that interactions among the
NHERF family members are probably involved. Also, it
appears that there may be major differences in the role of
these proteins in renal proximal tubule and intestine (338,
409). It remains unknown whether all acute regulation of
NHE3 by extracellular ligands requires the NHERF family.
There are no studies indicating a role for the NHERF
family in long-term regulation of NHE3. A single report of
abnormal phosphate handling in NHERF1 knockout
mouse supports a role of NHERF1 in chronic phosphate
transport (495). This complexity in intact tissue, already
recognized, supports the study of simpler systems to identify potential roles for each member of the NHERF family
in NHE3 regulation. However, while reductionist studies
must be used to understand how the NHERF proteins can
carry out specific regulatory functions, they must be studied together in the context of native intestine and proximal tubule in NHE3 regulation to allow dissection of the
complexity that almost certainly occurs in intact tissue.
A) NHE NULL FIBROBLASTS: EXPRESSION IN PS120 FIBROBLASTS
INDIVIDUALLY OF NHERF1, NHERF2, NHERF3/PDZK1, AND NHERF4/IKEPP.
Regulation of NHE3 by cAMP, cGMP, or elevated Ca2⫹
has been characterized based on inhibition of NHE3 by all
three second messengers in ileum and colon as well as in
renal proximal tubule (Table 2). NHERF1 reconstitutes
only cAMP inhibition of NHE3 and is not able to reconstitute cGMP (in presence of cGKII transfected into the
cells) or elevated Ca2⫹ inhibition of NHE3 (72, 250, 533).
2. NHERF family dependence of second
messenger inhibition of NHE3 in PS120/NHE3 cells
TABLE
NHERF1
NHERF2
PDZK1
IKEPP
cAMP
cGMP
Elevated Ca2⫹
⫹
⫹
⫹
NS
⫺
⫹
⫺
⫺
⫺
⫹
⫹
Stimulates
⫹, Reconstitutes inhibition of NHE3; ⫺, has no effect on NHE3
activity; NS, not studied.
Physiol Rev • VOL
NHERF2 reconstitutes cAMP, cGMP, and elevated Ca2⫹
inhibition of NHE3 (72, 250, 533). PDZK1 reconstitutes
cAMP and Ca2⫹ but not cGMP inhibition of NHE3 (535,
536; N. Zachos and M. Donowitz, unpublished data).
There are only preliminary studies of IKEPP, but it seems
to mediate stimulation of NHE3 by elevated Ca2⫹. This is
not what occurs in intact intestinal and renal epithelial
cells but might mimic part of stimulatory mechanisms of
NHE3 that are Ca2⫹ dependent. cGMP inhibition of NHE3 is
not reconstituted by IKEPP expression (Zachos and Donowitz, unpublished data).
B) EPITHELIAL CELL CULTURE MODELS: EXPRESSION OF NHERF
FAMILY MEMBERS IN EPITHELIAL CELLS, EITHER ENDOGENOUSLY OR
EXOGENOUSLY. OK cells express NHERF1 and NHERF3 endogenously but have none or very low amounts of
NHERF2 (211, 529, 533). cAMP inhibits NHE3 in OK cells
by a complicated stepwise process with rapid inhibition
which is not associated with changes in surface amount
(turnover number change), which by 30 min is associated
with increased endocytosis and decreased surface NHE3
amount. By 60 min it is also associated with decreased
exocytosis. All aspects require cAMP phosphorylation of
NHE3 (28, 97, 212, 330, 331, 380, 506, 543). In OK cells,
elevating cGMP did not affect NHE3 activity (72). However, in OK cells expressing NHERF2, cGMP inhibited
NHE3, an effect that was dependent on presence of both
NHERF2 and cGKII (latter transfected in but not expressed endogenously) (72). This demonstrated that
NHERF2 was necessary for cGMP regulation in polarized
epithelial cells as well as in fibroblasts.
In addition to being needed for acute inhibition of
NHE3, NHERF2 but not NHERF1 is necessary for LPA stimulation of NHE3 (285). This is discussed below.
C) NHERF1 FAMILY KNOCKOUT MICE. These mice have been
created by homologous recombination and characterized
by Shenilokar, Weinman and colleagues primarily for effects on renal function and structure (93, 102–104, 243,
276, 409, 491, 495, 500). A second NHERF1 knockout
model has been reported by Georgescu and co-workers
(333). Structurally, the BB of renal proximal tubule cells
and the small intestine of the Shenilokar/Weinman mouse
are normal by electron microscopy (409; Hubbard, Murtazina, Kovbasnjuk, and Donowitz, unpublished data).
However, in the Georgescu NHERF1 knockout mouse,
small intestine was reported to have abnormal ileal villi
with less and shorter microvilli and thickened and disorganized terminal webs (333). What explains this difference is not known, and both NHERF1 knockout mice
were on the same C57Bl/6 background. Functionally,
basal transport of NHE3 was similar in wild-type and
NHERF1 knockout proximal tubule based on studies of
BB vesicles, proximal tubule cells in primary culture, and
two-photon microscopy of kidney cortical slices (338,
409, 500). However, cAMP failed to alter NHE3 activity in
proximal tubule characterized by all three approaches
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
(338, 409, 500). In contrast, in ileum, basal transport was
also normal, but cAMP inhibited NHE3 at 30 min by ⬃50%,
which is similar to what occurs in wild-type NHE3 (338).
What explains these differences is not clear, although
cAMP failed to increase NHE3 phosphorylation in proximal tubule (409, 500), which had been shown to be necessary for cAMP inhibition of NHE3 in both fibroblasts
and proximal tubule cell models (OK cells). Biochemical
studies in kidney showed that amounts of NHE3,
NHERF2, and NHERF3/PDZK1 were normal (increased
NHERF2 in one of two knockout mouse models). In contrast, the amount of BB ezrin and phosphoezrin were
decreased, suggesting that failure to form the NHE3/
NHERF1/ezrin/PKAII complex might explain the lack of
effect of cAMP in kidney (333). What the differences in
the NHE3 complexes are that allow cAMP to inhibit NHE3
in the ileum are not understood, although it is possible
that NHERF2 has different functions (or locations) in
proximal tubule and ileum.
In more preliminary studies of mouse colon using the
Ussing chamber/voltage-clamp technique, comparing
wild-type to NHERF1, NHERF2, and NHERF3/PDZK1
knockouts, all three NHERF knockouts affected regulation of Na⫹ absorption that was attributed to NHE3 (93).
Most significantly, NHERF3/PDZK1 knockout had markedly reduced basal NHE3 activity and lacked cAMP,
cGMP, or Ca2⫹ inhibition of NHE3. NHERF2 knockout
had reduced basal Na⫹ absorption and absent Ca2⫹ inhibition of NHE3. NHERF1 knockouts had the least effect
with slightly reduced basal cAMP, cGMP, and Ca2⫹ inhibition of NHE3.
7. NHERF-interacting domains of NHE3
NHE3 and NHERF1– 4 directly interact. In multiple
cell types, NHE3 interacts with NHERF1, NHERF2, and
NHERF3/PDZK1 based on yeast two-hybrid assays (173,
174, 205, 277, 407, 532, 536). This is true when NHE3 and
the NHERF proteins are both expressed in fibroblasts, OK
cells stably transfected with NHERF2, Caco-2 cells,
mouse ileum, and rabbit ileal BB. Thus they are in the
same complex, although the amount of coprecipitation is
relatively small and surprisingly variable. Whether this is
due to the detergents used for solubilization or reflects
the low affinities of the interactions is not known. That at
least some of these interactions are direct has been established using fusion proteins of the NHE3 COOH terminus and recombinant NHERF proteins. Direct proteinprotein interactions of the NHE3 COOH terminus with
each member of the NHERF family has been demonstrated (445, 532, 533, 536; and Thelin, Hodson, Zachos,
Donowitz, and Milgram, unpublished data). NHE3 binds
via an internal sequence to NHERF proteins (532, 533).
The domain in the NHE3 COOH terminus which binds to
the NHERFs is between aa 586 – 660. In multiple pulldown
Physiol Rev • VOL
847
and/or overlay studies, rabbit NHE3 only bound to intact
NHERF1– 4 via this internal domain, specifically not binding with its very COOH terminus, that includes aa 757– 832
(532, 533). When recombinant NHERF2 was overlaid with
35
S-NHE3 COOH-terminal constructs, binding occurred
with full-length NHE3 COOH terminus (aa 475– 832), aa
475–711, and aa 475– 660 but not to aa 475–585 (532). This
also suggested that binding occurred between aa 585– 660
in the NHE3 COOH terminus. IP of NHE3/585 (NHE3
truncated at the COOH terminus to aa 585) failed to
coprecipitate NHERF1 or NHERF2, and vice versa, when
both were expressed in PS120 cells, supporting that aa
between 585– 660 were necessary for the NHE3-NHERF
interactions. In contrast, NHERF3 was reported to bind
in vitro to rabbit NHE3 COOH-terminal 131 aa but not
when the four COOH-terminal amino acids of this construct were removed (445). Is there further evidence that
the COOH-terminal four amino acids of NHE3 are involved in binding to NHERF members? The data are
inconsistent. On initial yeast two-hybrid screening, truncating the NHE3 COOH terminus to examine binding to
NHERF1 and NHERF2 resulted in autoactivation, and
thus the question could not be addressed (533). This was
repeated by Weinman (504) with mutations of the last
four amino acids with changes in the 0 and ⫺2 and ⫺3
positions eliminating binding (504). In addition, using a
membrane-friendly variant of the yeast two-hybrid system
based on ubiquitin, NHERF1 and NHERF2 binding to the
NHE3 COOH terminus was also demonstrated with evidence that in addition to binding the last four amino acids,
NHERF1 but not NHERF2 bound NHE3 internally (Murer,
Gisler, Moe, and Biber, unpublished data). Of note, while
these yeast interactions suggested COOH-terminal binding, they are prone to false positives. Moreover, when the
COOH-terminal truncations of NHE3 were examined for
physical interactions with NHERF1 by pull-down assays,
the COOH-terminal four amino acids were not necessary
for the physical association (504). Thus the bulk of the
evidence supports that NHE3 binds NHERF1, NHERF2,
NHERF3/PDKZ1, and NHERF4/IKEPP in a NHE3 COOHterminal domain between aa 586 – 660. The interaction
with the COOH-terminal four amino acids of NHE3 in
mammalian cells is not proven, though still possible, even
though the last four amino acids of NHE3 are a classic
COOH-terminal class I PDZ binding domain (STHM). We
speculate that NHE3 does not bind the NHERFs with its
last 4 aa because they are buried in its tertiary structure.
8. Role of NHERF family members in NHE3
regulation (compared with functions
of other PDZ domain containing proteins in
polarized epithelial cells)
A) MAINTENANCE OF POLARITY OF EPITHELIAL CELLS. While
multiple PDZ domain proteins are present at the tight
87 • JULY 2007 •
www.prv.org
848
MARK DONOWITZ AND XUHANG LI
junctional complexes and are necessary for establishment
of epithelial polarity (these include mammalian Scrib-1,
PAR-3, and Pals-1) (63), this is not a role of NHERF
proteins, and these proteins are not junctional.
B) DELIVER AND MAINTAIN NHE3 IN THE MEMBRANE UNDER BASAL
CONDITIONS.
The NHERF family is not involved in determining the percent of NHE3 on the plasma membrane (71,
126, 250, 537). In NHE3 truncation mutants which have
had the NHERF binding domains removed, the same percent of NHE3 is present on the plasma membrane or BB
as that in wild-type NHE3 and in NHE3 truncations which
bind the NHERF members. Moreover, PS120 cells, which
contain no NHERF proteins (no endogenous proteins and
use of NHERF1 siRNA to remove the small amount of
endogenous NHERF1 expression), have the same plasma
membrane percentage of NHE3 as cells in which multiple
NHERF proteins were expressed (M. Cavet, R. Sharker,
and M. Donowitz, unpublished data). Moreover, in
NHERF1 null mice, NHE3 is present in a normal amount
and location in the renal proximal tubule (409). This
makes it unlikely that there is a role for NHERF members
in basal trafficking of NHE3 to the plasma membrane
and/or membrane retention, at least in PS120 fibroblasts.
Of interest, NHERF3/PDZK1 requires the presence of a
membrane-associated protein MAP17 for it to be in the BB
of OK cells (261, 376, 413). In contrast to changes in
NHERF members in which NHERF1/2 bind to actin via
ezrin, disruption of microtubules with colchicine in rat
renal proximal tubule led to NHE3 and NHERF1 appearing on the BLM in addition to the BB (396).
C) RECONSTITUTION OF REGULATED RAPID CHANGES IN NHE3
ACTIVITY BY ACTING AS SCAFFOLDS FOR NHE3 COMPLEX FORMATION.
I) cAMP inhibition of NHE3 activity. This was the initial
model developed in which NHERF1 or NHERF2 appeared
necessary for cAMP to inhibit NHE3 activity. These studies were based on models of cholera toxin inhibition of
small intestinal NaCl absorption by elevation of cAMP.
Similar inhibition by elevated cAMP occurred in renal
proximal tubule of BB Na⫹/H⫹ exchange activity, and this
was shown by Weinman, Shenolikar, and co-workers to
require a soluble 55-kDa protein which was separate from
the Na⫹/H⫹ exchangers (493, 496, 498, 499, 501, 503).
They named this protein NHERF (now called NHERF1)
and cloned it by initial partial purification via column
chromatography, determination of a partial amino acid
sequence, design of an oligonucleotide probe based on
this sequence, and screening of a rabbit renal library
(501). That NHERF1 was necessary for cAMP inhibition
of NHE3 was initially demonstrated in PS120 fibroblasts
(533). These cells lack endogenous NHERF proteins, but
when stably transfected with NHE3, it was assumed they
would respond to elevated cAMP with NHE3 inhibition
and serve as a model for cAMP regulation of NHE3.
However, acute elevation of cAMP in these cells had only
very small effects on NHE3 activity until these cells were
Physiol Rev • VOL
transfected with either NHERF1 or NHERF2 (533).
NHERF1 or NHERF2 was necessary to allow cAMP to
phosphorylate NHE3 (547). In fact, all described aspects
of cAMP inhibition of NHE3 require NHE3 phosphorylation (330, 493, 494, 496, 500, 506, 543). PKA II was the
kinase involved (277). PKAs exert many of their effects by
binding to A kinase anchoring proteins (AKAPs), which
place the kinase at specific cell locations close to PKA
substrates and also to other proteins involved in regulation of the ligand. The nature of the NHE3 complex involved in this cAMP effect evolved from recognition by
Goldenring and associates (133, 387) that NHERF1 was a
human ezrin binding protein (also called EBP50). Ezrin
was shown to be a low-affinity AKAP (133). NHERF2 was
also shown to bind ezrin, while neither NHERF1 nor
NHERF2 were AKAPs (277, 532). The model that has
evolved and stood up to multiple tests indicated that there
was a NHE3 complex involved that consisted of NHE3
bound to the PDZ domain proteins NHERF1 or NHERF2.
These NHERFs also bound to ezrin, which acted as an
AKAP to allow cAMP-activated catalytic subunit of PKA II
to be freed up close to NHE3, which was then phosphorylated to lead to NHE3 inhibition (Fig. 7) (121, 126, 205,
492, 494, 497, 498, 502, 537, 547). Thus the role of
NHERF1/NHERF2 was suggested as being to form the
NHE3 complex by binding to both NHE3 and ezrin and in
this way exposing NHE3 to activated PKA II catalytic
subunit. That this binding of NHERF1 to ezrin was required for cAMP inhibition of NHE3 was demonstrated in
PS120 and OK cells. In OK cells, in which wild-type
NHERF1 was present, expression of NHERF1 lacking the
COOH-terminal 30 amino acids did not reconstitute cAMP
inhibition of NHE3 or NHE3 phosphorylation, acting in a
dominant negative manner to prevent cAMP inhibition of
FIG. 7. Model of putative NHE3 complexes containing NHERF1 or
NHERF2 in epithelial cell brush border involved in acute cAMP inhibition of NHE3. The essential features are a 5-component complex that
leads to PKA phosphorylation of NHE3. The proteins involved are NHE3,
NHERF1 or -2, ezrin, actin, and PKAII.
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
NHE3 and phosphorylation of NHE3 (497, 502). The domains of NHERF1/NHERF2 involved in this complex formation are the PDZ2-COOH terminus or a somewhat extended PDZ2 which binds the NHE3 COOH terminus plus
the COOH-terminal ERM binding domain (502, 532). The
ERM binding domain of NHERF1 includes the COOHterminal 23 amino acids which is predicted to be ␣-helical
and contains 4 positively charged amino acids on side of
the ␣-helix. When three of these positive charged amino
acids are mutated, NHERF1 is unable to reconstitute
cAMP inhibition of NHE3 and fails to bind ezrin (532).
NHERF2 has a similar structure with its COOH-terminal
30 amino aicds being ␣-helical with four grouped positively charged amino acids on one side of the helix with
mutation of three amino acids blocking ezrin binding and
reconstitution of cAMP inhibition of NHE3 (B.Y. Cha, C.
Yun, and M. Donowitz, unpublished data).
There are many aspects of understanding this NHE3
complex involved in cAMP inhibition of NHE3, which are
only partially defined, in addition to the specific site of
NHE3 involved in NHERF binding described above.
1) Blocking the NHE3 COOH terminus has been reported to reduce the cAMP inhibition by ⬃25%, although
we have not found any significant effect (504). Basal and
growth factor stimulation of NHE3 activity and percent on
the plasma membrane do not appear affected.
2) Whether the NHE3/NHERF1 or NHERF2/ezrin/
PKAII complex (Fig. 7) is static has not been definitively
established. Studies in epithelial cells are required to
address this issue, since trafficking is only part of NHE3
regulation by cAMP in a polarized epithelial cell model
and does not contribute at all in PS120 fibroblasts. In
fibroblasts, Yun et al. (532) showed that NHERF2 and
NHE3 colocalize in the plasma membrane similarly
both under basal conditions and after cAMP exposure
but do not colocalize in the recycling juxtanuclear compartment under either basal conditions or after cAMP
exposure.
3) Given that direct ezrin binding to NHE3 occurs,
why does this not appear to allow cAMP phosphorylation
and regulation of NHE3 (74)? It is possible that the location of the direct ezrin/PKAII is not correct to allow the
catalytic subunit of PKA II to phosphorylate NHE3. However, given that it is freed up catalytic subunit that is
involved, this does not appear to be a likely explanation.
4) Does the regulation of NHE3 via BB PDZ proteins
apply to NHE2, which is also present in the small intestinal and colonic BB? While studies have not been done in
adequate detail with NHE2 to comment definitively, there
is no evidence of an interaction with NHERF proteins.
Also, in Caco-2 cells and PS120 cells, cAMP stimulates
rather than inhibits NHE2 (52, 53, 232, 292, 323, 347).
Moreover, NHE2 appears to be present mostly in the
plasma membrane, does not appear to traffick on and off
the plasma membrane under basal conditions, is not afPhysiol Rev • VOL
849
fected by inhibitors of PI 3-K under basal conditions, and
does not appear to bind NHERF1 or NHERF2, all characteristics different from NHE3 (69, 70, 347).
5) Do these cell culture models of cAMP inhibition of
NHE3 apply to intact models of renal proximal tubule and
small intestine/colon? These questions have begun to be
addressed in knockout models of NHERF1. It must be
remembered that the initial suggestion for involvement of
an accessory regulatory protein in BB Na⫹/H⫹ exchange
activity came from studies in BB of renal proximal tubule.
In NHERF1 knockout mice, cAMP (true for forskolin,
cAMP, and PTH) failed to inhibit BB NHE3 in the renal
proximal tubule. This was due to failure of cAMP to
increase phosphorylation of NHE3 (409, 500). The failure
of cAMP to inhibit NHE3 in NHERF1 knockout mice was
true in BB vesicles made from kidney cortex, primary
cultures of proximal tubules, and renal cortical slices
acutely studied using a two-photon microscope using
SNARF-4F to measure intracellular pH (338). The explanation is only partially determined, but in a second
NHERF1 knockout mouse in the same C57Bl/6 background, changes in proteins involved in the NHE3 complexes identified in PS120 cells occurred (333). Specifically, NHERF1 knockout mice had less renal proximal
tubule BB membrane ezrin and p-ezrin and increased
NHERF2 (changes in NHERF2 and ezrin in renal proximal
tubule BB were not seen in the Shenilokar and Weinman
knockout mouse). The explanation for the failure for
renal proximal tubule BB NHERF2 to reconstitute cAMP
inhibition of NHE3 is not known, but we hypothesize that
it is the lack of phosphorylated ezrin in the BB that
explains lack of cAMP effect due to failure to provide
AKAP function for NHE3 phosphorylation. This suggests
that other AKAPs that are present in the BB cannot substitute for ezrin, perhaps because of failure to closely
associate with NHE3 via NHERF family binding. Another
suggested possibility is that NHERF2 may not directly
bind NHE3 with sufficient affinity to allow NHE3 phosphorylation or may be in too low a concentration at the
microvilli to allow this to occur. In contrast, in ileum of
NHERF1 knockout mice, cAMP inhibits NHE3 similarly to
wild-type mouse (338). Interestingly, ezrin and p-ezrin
were also decreased in the ileal BB, suggesting that in
ileum another AKAP is able to allow cAMP to inhibit
NHE3. There are multiple AKAPs in the ileal BB and at
least NHERF3/PDKZ1 is known to associate with
D-AKAP2 (173, 174).
Regulation of NHE3 has been described in small
intestine of the NHERF3/PDZK1 knockout mouse in preliminary form (93, 276). Amounts of NHERF1 and NHE3
in the small intestinal BB were normal as was the apparent NHE3 location. The kidney of these NHERF3/PDZK1
knockout mice has a normal-appearing BB, and the
amount of NHERF1 is normal or there may be a small
increase in NHERF1 amount when the animals are fed a
87 • JULY 2007 •
www.prv.org
850
MARK DONOWITZ AND XUHANG LI
high-protein diet (67). In contrast, in colon of NHERF3/
PDZK1 knockout mice, not only is NHE3 activity decreased to 33% of wild type, but cAMP, cGMP, and elevated Ca2⫹ fail to affect colonic NHE3 activity (93). This
identifies a major role for NHERF3/PDZK1 in NHE3 regulation in mouse colon, although mechanistic understanding is lacking.
6) Regulation of NHE3 by cAMP should be analyzed
in the presence of additional proteins that also bind
NHERF proteins, including DRA and CFTR? This is discussed in section VI.
II) cGMP inhibition of NHE3 activity. cGMP amount
in small intestinal Na⫹ absorptive cells is increased by
luminal exposure to the intrinsic gut peptides guanylin or
uroguanylin or the heat-stable E. coli enterotoxin by binding to their common receptor, the BB guanylate cyclase C
(121, 123, 405). This is associated with inhibition of small
intestinal NaCl absorption. Unexpectedly, in PS120 fibroblasts and OK cells, cGMP failed to affect NHE3 activity
(72). Most cells in culture lack cGKII, which is normally
present in the BB of epithelial cells (319, 462). However,
cGMP failed to affect NHE3 even in cells transfected with
cGKII (72). However, NHERF2 expression in both PS120
and OK cells reconstituted cGMP inhibition of NHE3 (72).
NHERF1 or NHERF3/PDZK1 was unable to reconstitute
cGMP inhibition of NHE3 (72; N. Zachos and M. Donowitz, unpublished data). NHERF2 and cGKII directly interacted as shown by in vitro pull-down/overlay assays and
coprecipitated when both were expressed in fibroblasts
and from ileal BB in which they were expressed endogenously (72). The specificity of NHERF2 but not NHERF1
to reconstitute cGMP inhibition of NHE3 may be due to
failure of NHERF1 to bind cGKII as studied by in vitro
overlay and pull-down assays (72). The recognized mechanism of the NHERF2 reconstitution of cGMP inhibition
FIG. 8. Model of putative NHE3 complexes involving NHERF2 in
epithelial cell brush border involved in acute cGMP inhibition of NHE3
and NHERF4/IKEPP in inhibition of guanylate cyclase C (gcc). The
essential features are a 3-component complex that leads to PKG phosphorylation of NHE3. The proteins involved are NHE3, NHERF2, and
cGKII. Ezrin is not necessary for this process, perhaps because the
complex is further fixed to the BB by myristoylation of cGKII.
Physiol Rev • VOL
of NHE3 (Fig. 8) included that NHERF2 acted as a cGMP
kinase anchoring protein (GKAP) by binding cGKII (72).
In the NHE3/NHERF2/cGKII complex, which was involved in cGMP inhibition of NHE3, cGKII had to be
anchored in the BB by both binding to the PDZ2-COOH
terminus of NHERF2 and directly to the BB by myristoylation. In preliminary studies, cGMP inhibition of NHE3
was associated with cGMP-dependent phosphorylation of
NHE3 (B.Y. Cha, H. Kocinsky, P. Aronson, and M. Donowitz, unpublished data). The source of elevated cGMP at
the BB in intestine and kidney is BB guanylate cyclase,
which is membrane anchored by binding to IKEPP (405,
442). A function of NHERF4/IKEPP appears to be to
reduce the level of generated BB guanylate cyclase (405).
Unknown is whether the entire signaling process (shown
in Fig. 8) is part of a single NHERF family scaffolded
complex.
III) Elevated Ca2⫹ inhibition of NHE3 activity.
2⫹
Ca is acutely elevated in intestinal Na⫹ absorptive cells
by the cholinergic agonist carbachol, exposure to which
mimics part of digestive physiology (129, 388). Similar
elevation of intracellular Ca2⫹ in these cells occurs with
serotonin, mimicking part of the pathophysiology of the
diarrhea of carcinoid syndrome (168). Ca2⫹-dependent
inhibition of NHE3 has been demonstrated in PS120 fibroblasts and rabbit ileal Na⫹ absorptive cell BB to involve a two-step process in which NHE3 complexes rapidly form at least partially on the plasma membrane,
enlarge, and then internalize (Fig. 9) (250, 286, 298). This
decreases the amount of plasma membrane NHE3, with
inhibition of NHE3 activity being due to the decrease in
amount of surface NHE3 (250, 286). In PS120 cells, elevation of Ca2⫹ failed to affect NHE3 activity unless the cells
were transfected with NHERF2 but not NHERF1 (250).
Ca2⫹ elevation in NHERF2-containing cells was associated with formation of complexes that included NHE3,
␣-actinin-4 (non-muscle actinin), and PKC-␣, with effects
on NHE3 activity and on complex formation occurring
within minutes of initial Ca2⫹ elevation (250, 286, 298).
Actinin-4 involvement was determined using a proteomic
approach in which glutathione-S-transferase (GST)NHERF2 but not GST-NHERF1 pull-downs of ileal lysate
isolated a protein of ⬃100 kDa which was identified by
mass spectroscopy as ␣-actinin-4 (250). Ability of
NHERF2 but not NHERF1 to bind actinin-4 appears to
explain the specificity of NHERF2 in reconstituting Ca2⫹
inhibition of NHE3 (250). NHERF2 coprecipitated NHE3
and actinin-4 under basal and elevated Ca2⫹ conditions.
While the association of NHERF2 and actinin-4 increased
with elevated Ca2⫹, that with NHE3 was Ca2⫹ independent. Oppositely, IP actinin-4, coimmunoprecipitated
NHERF2 and NHE3 in a Ca2⫹-dependent manner. The
explanation appears to be that, with elevated Ca2⫹, the
association of actinin-4 with NHERF2 was increased, and
since the ratio of NHERF2/NHE3 binding was Ca2⫹ inde-
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
FIG. 9. Model of putative NHE3 complexes involving NHERF2 in
elevated Ca2⫹ inhibition of NHE3. Complexes initially form consisting of
NHE3, NHERF2, ␣-actinin-4, and protein kinase C (PKC)-␣. NHE3 is
endocytosed. It is not known whether NHE3 is endocytosed with the
entire complex or with any of its associating proteins. The essential
features are a 4-component complex that leads to Ca2⫹-dependent/PKCdependent inhibition of NHE3. The proteins involved are NHE3,
NHERF2, ␣-actinin-4, and PKC-␣.
pendent, this led to more NHE3 coprecipitation as well.
That actinin-4 was involved in Ca2⫹ inhibition of NHE3
was demonstrated by expressing the different actinin-4
domains separately in cells containing NHE3 and
NHERF2 and searching for dominant negative effects
(250). Actinin-4 is made up of actin binding, spectrin
homology, and EF hand domains. The actinin-4 construct
containing the actin binding plus spectin homology domains but lacking the EF hands domains prevented elevated Ca2⫹ from inhibiting NHE3, while the other actinin-4 constructs, including full length, increased the Ca2⫹
inhibition of NHE3. It was concluded that not only did
␣-actinin-4 join NHE3 complexes with elevated Ca2⫹, but
was necessary for Ca2⫹ inhibition of NHE3 via its EF hand
domain. We speculate that local Ca2⫹ elevation involving
the EF hand domains of actinin-4 is involved in NHE3
inhibition, although the specific step involved is unknown. The Ca2⫹ inhibition of NHE3 was associated with
NHE3 entering larger complexes, which was both observed on SDS-PAGE gels and visualized as clumping in
PS120 cells by confocal light microscopy. These complexes contained at least NHE3, NHERF2, actinin-4, and
PKC-␣ (Fig. 9). On the basis of cell surface biotinylation,
some of these complexes could be seen forming on the
plasma membrane, but it is not known whether they also
form intracellularly (250). Temporally, cell surface NHE3
complexes formed before an increase in intracellular
NHE3 was demonstrable, suggesting that complex formation preceeded internalization (250). That over time intracellular clumps containing NHERF2, NHE3, actinin-4, and
PKC-␣ were visible suggested that the complexes were
taken up together or formed intracellularly. However,
trafficking of the complexes as a unit has not been demPhysiol Rev • VOL
851
onstrated, and it is not known whether the large complexes are present intracellularly in epithelial cells of
intact ileum or proximal tubules. PKC is involved in elevated Ca2⫹ inhibition of NHE3 in intact rabbit ileal Na
absorptive, but neither calmodulin nor calmodulin kinase
appears to be involved (96, 121, 123, 125, 127, 128, 131).
This was also the case in PS120 cells expressing NHE3
and NHERF2. Based on biochemical and inhibitor studies
(isoform specific pseudosubstrate inhibitors), PKC-␣ was
shown to be the PKC isoform that joined the NHE3 complexes and was necessary for Ca2⫹ inhibition of NHE3
(286). PKC-␣ directly bound NHERF2 based on pull-down
assays. PKC-␣ bound to PDZ domain 1 of NHERF2 coprecipitated with NHERF2 in a Ca2⫹-dependent manner and
colocalized with NHE3, NHERF2, and actinin-4, based on
confocal microscopy, after Ca2⫹ elevation both on the
plasma membrane and within the cell (PS120 cells). The
specificity of NHERF2 in Ca2⫹ inhibition of NHE3 was not
related to PKC-␣ binding, since PKC bound both to
NHERF1 and NHERF2 in a Ca2⫹-dependent manner (286).
The role of PKC was not in formation of the NHE3 complexes, but rather in internalization of NHE3. PKC did not
appear to affect the phosphorylation status of NHE3,
suggesting that its control of endocytosis was via phosphorylation of components of the endocytosis machinery,
as has been demonstrated previously (286). Of interest is
that the PKC appears to be able to affect phosphorylation
of the endocytosis machinery from the cytosolic face.
Many aspects of Ca2⫹ inhibition of NHE3 in overexpressing PS120 fibroblasts were duplicated by studies in
rabbit ileal Na⫹ absorptive cell BB and mouse ileal villus
Na⫹ absorptive cells (93, 122, 123, 125, 128, 298; R. Murtazina, O. Kovbasnjuk, and M. Donowitz, unpublished
data). Acute Ca2⫹ elevation with carbachol or serotonin
inhibited BB NHE3 activity and surface amount to similar
degrees. This was associated with an increase in amount
of NHE3 in endosomes, consistent with increased endocytosis, although a role for decreased exocytosis has not
been excluded (298). This inhibition of NHE3 activity was
associated with an increase in size of NHE3 complexes in
the ileal BB based on sucrose density gradient fractionation (298). Actinin-4 similarly shifted into larger complexes of similar size to those of NHE3 (298). In addition,
after Ca2⫹ elevation, the same proteins that appeared to
form NHE3 complexes in PS120 cells also were found in
NHE3 complexes in ileal BB. Immunoprecipitated
NHERF2, coprecipitated actinin-4, PKC-␣, and activated
PKC with increased association with Ca2⫹ elevation
(298). The amount of actinin-4 that was immunoprecipitation from the BB was increased with elevated Ca2⫹
consistent with the freed up actinin-4 in PS120 cells.
However, different sources are likely, with actinin-4 moving from the adhesion plaques to the NHE3 complexes in
PS120 cells, while it probably is mobilized from the tight
junctions in ileal Na⫹ absorptive cells. In ileal Na⫹ ab-
87 • JULY 2007 •
www.prv.org
852
MARK DONOWITZ AND XUHANG LI
sorptive cells, the carbachol effects on Na⫹ absorption
were c-Src dependent, with carbachol stimulating c-Src
phosphorylation and the Src family blocker PP2 preventing carbachol inhibition of Na⫹ absorption and the increase in size of complexes containing NHE3 and actinin-4 (298). In preliminary studies, in mouse ileum, serotonin inhibition of NHE3 is NHERF2 dependent (R.
Murtazina, O. Kovbasnjuk, R. Sharker, H. deJonge, B.
Hogema, U. Seidler, E. Weinman, and M. Donowitz, unpublished data).
IV) LPA stimulation of NHE3. In addition to second
messenger inhibition of NHE3, an example of acute stimulation that is also dependent on NHERF2 but not
NHERF1 was demonstrated (87, 285). LPA, considered a
mediator of restitutive aspects of inflammation, stimulates NHE3 activity, as studied in the apical membrane of
polarized OK cells. LPA has no effect on NHE3 activity in
these cells, which lack endogenous NHERF2, unless cells
transfected with NHERF2 are studied (285). This stimulation of NHE3 activity was associated with and was
equivalent in magnitude to the increase in amount of BB
NHE3 and was due to an increase in rate of exocytosis.
There was no change in the rate of endocytosis of surface
NHE3. The LPA stimulation of NHE3 was PI 3-K dependent (285). The mechanism of the NHERF2 dependence
of this LPA effect was not in the degree of PI 3-K activation, which was similar in OK cells that contained or
lacked NHERF2. Surprisingly, the mechanism appears to
involve the previously reported stimulation of intracellular Ca2⫹ in response to LPA (119). LPA increased intracellular Ca2⫹ in OK cells, and the magnitude and time of
elevation was increased in OK cells expressing NHERF2
(87). Moreover, the LPA stimulation of NHE3 was Ca2⫹
dependent, being prevented by BAPTA-AM (87). This effect further involved phospholipase C (PLC) activity,
since the PLC inhibitor U73122 prevented LPA stimulation of NHE3, with likely involvement of PLC-␤3, the only
PLC-␤ isoform expressed in these cells (87). In the presence of NHERF2, LPA caused a larger and more prolonged elevation of intracellular [Ca2⫹] (87, 423). Thus
increased PLC activity in OK cells occurred only with
NHERF2 and not NHERF1, which is consistent with the
previous demonstration that NHERF2 but not NHERF1
binds PLC-␤3, although both bind the B1 and B2 isoforms (356). In contrast to the role of elevated Ca2⫹ in
inhibition of NHE3, which is mediated via PKC-␣, a
general PKC inhibitor did not alter LPA stimulation of
NHE3. The role of the elevated intracellular Ca2⫹ in
LPA stimulation of NHE3 is not known, although the
final steps of exocytosis and Golgi plasma membrane
trafficking, including V and T SNARE function are Ca2⫹
dependent, including the very distal step in exocytosis
that involves synaptotagmin.
While the involvement of elevated Ca2⫹ in NHE3
stimulation via exocytosis initially was not expected,
Physiol Rev • VOL
given the above description of the role of elevated Ca2⫹
acting via PKC in NHE3 inhibition by carbachol and serotonin, another almost identical example of stimulation
of NHE3 by a PLC-dependent mechanism has been reported. Adenosine acting via A1 receptors at a low concentration (⬃10⫺9 M) rapidly stimulates NHE3 activity (15
min), while at higher concentrations (maximum effect at
10⫺6 M) it inhibits NHE3 (114, 117). The adenosine stimulation of NHE3 was Ca2⫹ dependent, being prevented by
BAPTA-AM, involved PLC, since the PLC inhibitor U73122
inhibited the effect, was not dependent on PKC activity,
and appeared dependent on an increase of Ca2⫹ from
intracellular stores (117). The mechanism of the elevated
Ca2⫹ stimulation of NHE3 in OK cells was postulated to
involve calcineurin homologous protein and also inhibition of adenylate cyclase (115, 117). This was based on the
adenosine agonist CPA opposing the effects on NHE3
activity of 8-bromo-cAMP (117). However, no biochemistry was provided, and the effects of CPA and cAMP were
additive, consistent with separate mechanisms rather
than CPA acting by inhibiting adenylate cyclase to decrease the cAMP content. Thus the paradox of elevated
Ca2⫹ under some circumstances stimulating NHE3 occurs
with 1) LPA exposure (87, 285), 2) low concentrations of
adenosine (114, 117), and 3) IKEPP expression in PS120
cells expressing NHE3 is associated with ionomycin
(Ca2⫹)-induced stimulation of NHE3 (N. Zachos and M.
Donowitz, unpublished data). We hypothesize that the
more common response of Ca2⫹-induced NHE3 inhibition by stimulated endocytosis of NHE3 and consequent inhibition of activity requires formation of a
NHERF family complex that involves NHE3 and leads
to changes in trafficking. However, in the absence of
any necessary component of the complex, the Ca2⫹
stimulation of exocytosis that occurs in every cell and
appears to involve synaptotagmin as a final step that is
responsive to elevated Ca2⫹ (51, 349) is unmasked. We
suggest that this functions even during the Ca2⫹ inhibition, since exocytosis continues.
V) Luminal PTH receptor-NHERF complex as a
molecular switch. BB NHE3 is inhibited by both elevation
in cAMP and Ca2⫹, although the NHE3 complexes involved appear to be distinct. This concept is supported by
additivity of NHE3 inhibition with elevation of cAMP plus
Ca2⫹ in PS120/NHE3 cells expressing NHERF2 (B.Y. Cha
and M. Donowitz, unpublished data). This interaction of
cAMP and Ca2⫹ is relevant for PTH regulation of NHE3.
The PTH receptor has a COOH-terminal class I PDZ domain binding motif which is necessary for its binding to
NHERF2 (153, 316). In PS120 cells expressing the PTH
receptor and NHERF2, PTH increased inositol phosphates 20- to 25-fold and had a minimal effect (2-fold) on
cAMP content, while in similar cells lacking NHERF2,
PTH increased cAMP 10- to 15-fold and minimally increased inositol phosphates (2– 4 times) (316). Pertussis
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
toxin treatment both inhibited PTH activation of PLC and
also partially restored PTH activation of adenylate cyclase. Thus it was suggested that NHERF2 and perhaps
NHERF1 (426) act as a molecular switch allowing PTH to
activate a PLC and to inhibit activation of adenylate cyclase to increase cAMP (316). NHERF2 not only is involved with activation of a PLC, but is associated with
inhibition of adenylate cyclase probably via activation of
a Gi/o. NHERF2 binds PLC-␤3 at its in PDZ domain 1,
while it interacts with the PTH receptor with its PDZ2COOH terminus, suggesting that even though PTH added
to the apical surface inhibited NHE3, NHE3 and the PTH
receptor were probably not physically associated with the
same NHERF2 molecule. It is not known whether they
were in the same large complexes. This model held true in
an epithelial cell (315, 317). In OK cells, NHERF1 assembles an apical signaling complex that includes the PTH1
receptor PLC-␤3, NHE3, and actin. Also, in mouse proximal tubule from NHERF1 knockout versus wild-type
mice, PTH receptor coupling to PLC activity was NHERF1
dependent (66). In contrast, activation of Gi/o to inhibit
adenylate cyclase activity did not occur. This leaves open
the question of how widespread is the action of NHERF
members as a molecular switch between adenylyl cyclase
and Ca2⫹ signaling.
VI) Glucocorticoids and serum and glucocorticoidinducible kinase 1. Glucocorticoids stimulate intestinal
and renal proximal tubule NaCl absorption (13, 15, 37, 50,
77, 79, 85, 188, 231, 247, 360, 361, 399, 435, 528 –531), an
effect previously thought to start in ⬃18 h, but recently
shown to occur even faster (477, 528, 529). Until recently,
the mechanism was unclear with stimulation of Na⫹-K⫹ATPase and transcriptional stimulation of NHE3 through
increased NHE3 mRNA (NHE3 has a glucocorticoid response element in the 5⬘-flanking region), previously
shown to be involved, although what percent of this response they provided was unknown. Recently, Yun, Lang,
and co-workers (477, 528, 529) showed that glucocorticoids also stimulated NHE3 activity within 3– 4 h by a
process that did not require stimulation of transcription,
although some of the stimulation of NHE3 activity may
have been transcription dependent. This process required
NHERF2 but not NHERF1 and was associated with binding of serum- and glucocorticoid-inducible kinase 1
(SGK1) to NHERF2 but not to NHERF1 in vivo and in
vitro (529). SGK1 is an Akt-related, Ser/Thr kinase. With
the use of in vitro interaction assays, it was suggested that
the NHERF2-SGK1 interactions predominantly used the
NHERF2 PDZ-2 domain, although data were contradictory (90). Glucocorticoids increased the amount of SGK1
by transcription activation, providing a mechanism by
which glucocorticoids could affect NHE3 activity separately from the documented increase in NHE3 mRNA
that occurs with a slightly later time course (531).
Glucocorticoid stimulation of NHE3 activity was PI 3-K
Physiol Rev • VOL
853
dependent, being inhibited by the PI 3-K inhibitor
LY294002, and also required PDZK1 (399, 477, 529). The
3-phosphoinositide-dependent protein kinase 1 (PDK1),
which phosphorylates and activates SGK1, bound the
COOH terminus of NHERF2 which contained a PIF
sequence (FXXF⫽FSNF). PIF sequences have been
shown to bind PDK1. In fact, PDK1 and NHERF2 coprecipitated, indicating that they interacted in vivo.
That SGK1 (but not SGK2 or SGK3) was involved with
stimulation of NHE3 activity was demonstrated by
studies using kinase dead SGK1 as a dominant negative
inhibitor of glucocorticoid stimulation of NHE3 activity. PDK1 hypoexpressing mice had marked reduction
of glucocorticoid stimulation of NHE3 (399)
The mechanism of the NHERF2 effect in SGK1-dependent stimulation of NHE3 in epithelial cells in response to glucocorticoids is not known, although it is
assumed that it involves anchoring of SGK1 to the BB in
close apposition to PDZK1 or NHE3 and perhaps provides
the PIF sequence to place PDK1 near SGK1 so that the
phosphorylated and activated SGK1 can activate NHE3.
SGK1 phosphorylates NHE3 at S663, mutation of which
blocks the glucocorticoid stimulation of NHE3 (477).
Also, SGK1 was shown to phosphorylate T102 of NHERF2
in vitro, the first suggestion that NHERF2 is phosphorylated (529). However, whether this occurs in vivo or is an
in vitro artifact is not known, and NHERF2 has not been
shown to be phosphorylated under any conditions in vivo.
Thus glucocorticoid stimulation of NHE3 activity is a
complex combination of transcriptional stimulation of
NHE3 and SGK1, with SGK1 binding to NHERF2 where it
is activated by PDK1 to stimulate NHE3 separately from
transcription by a PI 3-K-dependent process which would
be predicted to involve increased NHE3 exocytosis and
requires phosphorylation of NHE3.
9. Other BB PDZ proteins in NHE3 regulation
In addition to the four NHERF family proteins,
Shank2 is another BB PDZ domain containing protein
involved in NHE3 regulation, although where it binds
NHE3 is not known (194). Shank2, a single PDZ domain
containing protein, is present in the BB of some epithelial
cells, including Caco-2, T84, and rat colon surface
and crypt cells. Shank2 or CortBP (SH3 and ankyrin repeats containing protein 2/cortactin binding protein) is a
member of another gene family of PDZ proteins with
three members (Shank 1–3). The role of Shank2 in regulation of transporters includes the following: 1) binds
NHE3, CFTR, and NaPiIIa in a yeast two-hybrid screen
(251) and 2) decreases the effect on NHE3 of cAMP when
studied in fibroblasts. Thus Shank2 has an effect different
from the NHERF family and suggests that up- and downregulation of NHE3, which is part of physiological regulation of NHE3, involves different classes of PDZ domain
87 • JULY 2007 •
www.prv.org
854
MARK DONOWITZ AND XUHANG LI
proteins. 3) Shank2 has a similar function in CFTR regulation, opposing the NHERF stimulation of CFTR phosphorylation by cAMP (251) with effects on both basal and
cAMP stimulation. Lack of ability to quantitate extent of
association of Shank2 with substrates has limited most
studies of NHERF family and Shank2 interaction, although NHE3 and Shank2 have been shown to coprecipitate (194). Shank2 was expressed in PS120 cells with
NHE3; Shank 2 increased the membrane expression and
basal activity of NHE3 but prevented the cAMP inhibition
of NHE3. Oppositely, knockdown of endogenous Shank2
in Caco-2 cells decreased the expression of NHE3 protein
with an accompanying decrease in NHE3 activity and an
increased cAMP inhibitory response (194). 4) Thus, in
addition to the apical membrane NHERF family, an additional apical membrane PDZ protein is involved in regulation of regulated NHE3 activity and amount. Where
NHE3 binds Shank2 and how Shank2 interacts with
NHERF family proteins and coordinates their effects in
NHE3 regulation in intact tissue is an unknown but important area for future study.
10. Regulation of NHERF proteins
Thus scaffolding by multiple NHERF family members
of NHE3 complexes is involved in rapid changes in NHE3
activity. Regulation of the NHERF proteins also occurs
but is only beginning to be understood. This involves at
least agonist binding-related multimerization and phosphorylation (150, 192, 199, 501, 547). Binding to substrate
by the NHERFs is thought to be constitutive, although
with low affinities. Interaction with ligands in some cases
depends on activation of the ligand (191, 343). For instance, NHERF1 only binds activated ␤2-adrenergic receptor (191). The Ser/Thr kinase GRK-5 phosphorylates
the ␤2-adrenergic receptor ⫺2 position Ser, which leads
to uncoupling from NHERF1 binding. However, while not
well defined, some regulatory functions of NHERF1 do
not depend on changes in its phosphorylation (277,
501, 547).
NHERF1 (106, 150, 199, 277, 280, 386, 547) and
NHERF3/PDZK1 (106, 205, 206) are phosphoproteins
under basal conditions, while NHERF2 has not been
shown to be phosphorylated in vivo under basal or any
regulated condition. However, all the NHERF proteins,
including NHERF2, have phosphorylation consensus
sequences (Scansite). Of importance, NHERF1 is phosphorylated by multiple kinase including GRK-6A (S289),
which increases its dimerization (192). It is also phosphorylated by cyclin-dependent kinase II and dephosphorylated by PP1 and PP2A at S279 and S301 (199).
cAMP inhibition of NHE3 is not associated with
changes in NHERF1 phosphorylation (277). Moreover,
mutation of a phosphorylated amino acid of NHERF1
did not alter cAMP regulation of NHE3, demonstrating
Physiol Rev • VOL
that the NHERF1 phosphorylation was not necessary
for cAMP regulation of NHE3 or coprecipitation with
NHE3 (277, 547). This is based on studies in PS120
cells, and it is not established whether this holds true
for polarized cells. PKC also phosphorylates NHERF1
in its PDZ2 domain (150). This is associated with a
lowering of the affinity of NHERF1 for CFTR and disrupting NHERF1-CFTR binding and the NHERF1 increase in CFTR-related current (103, 302, 381). PDZK1
phosphorylation at S509 has been demonstrated, and
there is suggestion of stimulation of the extent of
PDZK1 phosphorylation in response to glucagon in
hepatocytes (343).
11. Homo- and heteromultimerization
of NHERF family
Multimerization of NHERF1, NHERF2, and NHERF3/
PDZK1 all occur mostly based on in vitro studies. However, reports of whether these interactions depend on the
phosphorylation status of the NHERF protein and which
of their PDZ domains are involved are not consistent from
study to study. The NHERF protein multimerization suggests that all (or most) members of this family might be
included in the same NHE3 complexes and bring different
ligands into the complexes, akin to the large complexes at
different distances from the plasma membrane that occurs with the postsynaptic density complexes scaffolded
by INAD (455).
C. COOH-Terminal Domain 3, Amino Acids
661–756
There are no identified binding partners.
D. COOH-Terminal Domain 4, Amino Acids
757– 832: DPPIV(?), PP2A, and Megalin
A review of phosphatase regulation of NHE3 activity
serves as an introduction. NHE3 is phosphorylated under
basal conditions in all cells examined. Okadaic acid, a
PP2A inhibitor, stimulates activity of rabbit NHE3 expressed in fibroblasts (293). It requires aa 509 –585 in the
NHE3 COOH terminus. This indicates that PP2A under
basal conditions inhibits NHE3 activity (293) and that an
unknown kinase(s) stimulates NHE3 activity under basal
conditions. In contrast, the tyrosine kinase inhibitor
genistein inhibits NHE3 activity, and this effect requires
the COOH-terminal 96 aa of NHE3 (245, 246; Donowitz,
unpublished data). This is further evidence for a role for
a balance of kinases/phosphatases in NHE3 regulation.
However, that NHE3 is not phosphorylated on Tyr (525)
suggests this effect occurs via an intermediate which is
Tyr phosphorylated.
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
1. DPPIV
DPPIV (also called CD26) is a BB serine protease
which cleaves NH2-terminal dipepides from peptides with
a penultimate Pro or Ala residue. It is present in large
amounts in the BB of renal proximal tubule and small
intestine (241). The recent demonstration that the epithelial Na⫹ channel ENaC is regulated by an extracellular
protease has increased interest in regulation of transport
protein function by proteases (392). The functional association of DPPIV with NHE3 was examined after demonstration that NHE3 physically interacted with DPPIV
based on use of monoclonal antibodies to immunoprecipitate NHE3 from OK cells and search for interacting proteins (170). The association involves microvillus NHE3
but appears to be indirect. DPPIV effects on NHE3 require
aa 702–756, although there is no evidence that this is the
site at which DPPIV binds NHE3 (170, 171). DPPIV activity was necessary for NHE3 activity, since DPPIV inhibitors inhibited NHE3 activity (171). This inhibition was not
associated with a change in the amount of BB NHE3 nor
with a change in the size of NHE3 complexes. Furthermore, the DPPIV inhibitor effect was not altered by the
PKA inhibitor H-89, but was inhibited by the tyrosine
kinase inhibitor genistein. Not only did genistein inhibit
basal NHE3 activity, as we previously reported, but the
effects of the DPPIV inhibitor and genistein were not
additive (171). Thus it appears that a tyrosine kinase
pathway is involved in the steps in signal transduction
that are affected by DPPIV which are involved in NHE3
stimulation. A protein of 212 kDa changed in Tyr phosphorylation in parallel with the effects of genistein and
the DPPIV inhibitor, suggesting that it might be involved
in regulation of NHE3 (171). The mechanism of DPPIV in
regulated NHE3 activity as well as the signaling pathways
involved in its effects are unknown.
A recent study has called into question the specificity
of the DPPIV-NHE3 interactions (177). DPPIV is a receptor in the invasive human prostate tumor cell line 1-LN for
plasminogen type II as well as angiostatin. These cells
were suggested as expressing NHE1, -2, and -3. Plasminogen type II alkalinized these cells by an amiloride-sensitive process, which also inhibited their invasiveness, suggesting involvement of an NHE. Moreover, NHE1, -2, and -3
all coprecipitated DPPIV and plasminogen II. While
these studies require confirmation, especially the specificity of the NHE involved, a strong functional link
between DPPIV and Na⫹/H⫹ exchange activity is
supported (177).
2. PP2A
It was expected that phosphatases might directly
bind to NHE3 since 1) phosphorylation is important for
regulation of NHE3; 2) kinases have been shown to associate with multiple NHE isoforms, including NHE1 and
Physiol Rev • VOL
855
NHE3; and 3) kinase inhibitors and phosphatase inhibitors alter NHE3 regulation. Indeed, PP2A binds to the
COOH-terminal 50 aa of NHE3 (aa 783– 832) following
dopamine exposure, presumably via the increased cAMP
(380), which inhibits NHE3 activity. This binding was
demonstrated in vivo based on coprecipitation studies in
OK renal proximal tubule cells and was direct based on
yeast two-hybrid and in vitro (pull-down) studies. The
PP2A-B isoform, ␥-subunit was involved. Surprisingly,
okadaic acid blocked the dopamine effect on NHE3 activity. This indicated that the PP2A effect on dopamine
inhibition of NHE3 was due to effects of PP2A separate
from dephosphorylation of the increases in NHE3 phosphorylation that occurs with dopamine activation by PKA.
Moreover, the demonstration that the domain of the
NHE3 COOH terminus to which PP2A binds is not necessary for effects of okadaic acid on basal NHE3 activity
shows that another pool of PP2A is involved with regulation of basal NHE3 activity.
While not shown for NHE2, PP1 was demonstrated to
be involved in NHE1 regulation by Fliegel and co-workers
(327). NHE1 was dephosphorylated in vitro by PP1. Overexpression of PP1 decreased NHE1 activity, while PP1
inhibitors increased NHE1 activity. PP1 binds NHE1 both
in vivo and in vitro.
3. Megalin
Megalin binds NHE3 and may be involved in its regulation (45, 46). Megalin is a large protein (gp330) and a
member of the low-density lipoprotein receptor gene family (242), as reviewed in Reference 44. It is thought to be
the major endocytic receptor of renal proximal tubule and
binds albumin, lipoproteins, vitamin binding proteins including vitamin D binding protein, insulin, and drugs as
well as NHE3 (44). In addition to its role in endocytosis,
knockout of megalin in mice reduced the proximal tubule
BB size and led to low-molecular-weight proteinuria (44,
287). Studies have largely been based in the renal proximal tubule, in which megalin is present in large amounts.
Megalin also is present in the distal half of the rat small
intestine and colon but not the proximal small intestine,
and even in the distal small intestine the amount of message and protein present is a small fraction of that present
in rat renal cortex (518). Megalin is also expressed in the
mouse gallbladder (145). The proximal tubule and ileum also contain cubulin, which binds to and interacts
with megalin (145). Cubulin is the intrinsic factor receptor. No studies have been reported of megalin interaction with NHE3 in the ileum. In the rabbit renal
proximal tubule, NHE3 tightly binds megalin, and NHE3
exists as megalin-bound and megalin-free forms (45,
46). This was reviewed under trafficking of NHE3
above. The interrelationships among these processes
have important implications. For instance, decreased
87 • JULY 2007 •
www.prv.org
856
MARK DONOWITZ AND XUHANG LI
albumin uptake in proximal tubule activates NHE3 production, among other genes (256), and increased distal
kidney albumin uptake causes renal damage (44). Also,
megalin knockout mice have abnormal NaPi2a trafficking and increased amounts of NaPi2a in the proximal
tubule BB (26). As recently determined by Biemesderfer (44), megalin undergoes regulated intramembrane
proteolysis, perhaps similar to notch, and is implicated
in a PKC-metalloprotease-␥-secretase process which is involved in transcriptional regulation of additional proteins.
This is a fertile area for study because of the implications
that the loss of BB megalin in knockout mice might lead
to changes in gene transcription, as well as abnormalities
in NHE3 endocytosis. Neither of these has been adequately studied as yet.
VI. FUNCTIONAL INTERACTIONS OF NHE3
WITH PROTEINS NOT KNOWN TO
DIRECTLY BIND NHE3
A. PEPT1/PEPT2
The BB di/tripeptide transporter PEPT1 (SLC15A1) is
present primarily in small intestinal BB, and to a lesser
extent in proximal tubule BB (152, 351). It is H⫹ coupled,
being stimulated by a transapical membrane H⫹ gradient
(usually outside acidic corresponding to the luminal acid
microclimate, although luminal acidity is not necessary),
but is Na⫹ independent. This H⫹ gradient normally depends on BB NHE activity in small intestine and Caco-2
cells, requires extracellular Na⫹, is inhibited by a specific
NHE3 inhibitor (S1611) and by elevating cAMP (which
also inhibits NHE3), and is calmodulin dependent (19, 57,
148, 239, 240, 337, 446, 447, 485). Increase of Gly-Sar
uptake via PEPT1 was proportional to NHE3 expression
in a transfection system, showing NHE3 can drive PEPT1
function (485).
These effects are consistent with the involvement of
NHE3 and not NHE2, since NHE3 is inhibited and NHE2
stimulated by cAMP. The dependence of hPEPT1 transport activity on NHE3 is consistent with direct or indirect
linkage of BB NHE3 activity with PEPTI transport of
di/tripeptides across the small intestinal BB. It is unknown whether the linkage of NHE3 and hPEPT1 involves other than the apically generated acid microclimate. The implications of this linkage are that in diarrheal
diseases, in which there is inhibition of NHE3 activity
(elevated cAMP, cGMP, Ca2⫹), it would be predicted that
there would be inhibition of di/tripeptide uptake by
PEPT1, although Na⫹-dependent L-amino acid uptake
should be intact.
Similar to PEPT1, PEPT2 (SLC15A2) is also a renal
proximal tubule apical membrane electrogenic H⫹-coupled peptide cotransporter that unlike PEPT1 is a highPhysiol Rev • VOL
affinity, low-capacity transporter (445, 446). It also uses
an inward-directed H⫹ gradient to energize uptake of diand tripeptides. It can couple to apical membrane NHEs,
using their locally generated apical H⫹ microclimate and
creating uptake equivalent to Na⫹/di-tripeptide cotransport. NHERF3 (PDZK1) binds PEPT2 in the proximal
tubule apical membrane and increases its surface expression and its activity (351). It is not known whether this
represents increased trafficking to the membrane or stabilization in the membrane. There is no evidence of direct
binding of either PEPT1 or PEPT2 to NHE3.
B. NHE1
In polarized epithelial cells, NHE1 is often on the
BLM and NHE3 on the apical surface, and thus there is no
question of physical association. Is there any evidence of
functional association? In the rat renal medullary thick
ascending limb, inhibiting NHE1 by amiloride, Na⫹ removal, or nerve growth factor (NGF) inhibits apical NHE3
activity (179, 488). This inhibitory effect on NHE3 did not
occur in NHE1 knockout mice. This demonstrates the
dependence of this interaction on NHE1 (180). The NHE1
inhibition-related downregulation of NHE3 was dependent on an intact cytoskeleton and was inhibited by both
jasplakinolide and latrunculin B (488). That latrunculin
disrupts F-actin while jasplakinolide stabilizes and prevents actin remodeling indicates that this cytoskeletal
linkage of NHE1 activity with BB NHE3 activity is complicated, and no mechanisms for the linkage have been
established. Similar inhibition of BB NHE3 by inhibition
of BLM NHE1 has not been reported in either small
intestine or proximal tubule.
C. SLC26A3 (DRA) and SLC26A6 (PAT1)
In small intestine and colon, part of intestinal Na⫹
absorption is via functionally linked Na⫹/H⫹ exchange
and Cl⫺/HCO⫺
3 exchange (20, 260). As elegantly shown, in
BB vesicle studies, linkage was by intravesicular pH (259,
260). However, because BB vesicles do not contain all
components of the apical signaling/regulatory mechanism, how linkage of Na⫹/H⫹ and the BB Cl⫺/HCO⫺
3
exchangers (DRA and/or PAT1 as reviewed sect. II) occur
in intact intestine is not known. Both DRA (EKF) and
PAT1 (TRL) end in amino acids resembling type 1 PDZ
domain binding motifs. It has recently been shown that
DRA like NHE3 bind to the second PDZ domain of
NHERF2 and PDZ domains 2 and 3 of NHERF3/PDZK1,
while PAT1 binds in vitro to NHERF3?PDZK1 PDZ domains 1 and 2 (275, 276). It is not known whether NHE3
and DRA or PAT1 are physically linked or whether they
are present in the same BB complexes. However, some
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
coordinated regulation is indicated by upregulation of
DRA mRNA in NHE3 knockout colon (325).
SLC26A6 functions as the small intestinal Cl⫺/oxalate
transporter (151, 227). Mice lacking SLC26A6 develop Ca
oxalate kidney stones, apparently due to inability to secrete oxalate in ileum; effects on Na⫹ absorption in this
model have not been described. In congenital chloridorrhea, in addition to diarrhea since birth, there is decreased fertility. In the efferent duct of the epidydimus,
NHE3, CFTR, and SLC26A3 normally are all present, but
in congenital diarrhea, there is absence of CFTR in addition to SLC26A3 (207). The association of the same three
proteins as is present in some intestinal Na⫹ absorptive
cells suggests a functional interaction preserved across
tissues as well as a role in male reproduction.
D. CFTR
In small intestine and colon, neutral NaCl absorption,
which occurs largely in the villus Na⫹ absorptive cells,
occurs in parallel with Cl⫺ secretion, which occurs largely
in the crypt. In the Cl⫺ secretory cell, apical Cl⫺ channels
include CFTR and ClC-2. Given that the GI tract is involved in volume homeostasis and attempts to minimize
loss of stool water and Na⫹, coordinated regulation of
Na⫹ absorption and Cl⫺ secretion has been predicted.
That this occurs both by changes in acute signaling and
control at the transport protein level has been indicated
by recent studies using knockout mice (159). In small
intestine of NHE3 knockout mice, maximal cAMP-induced anion secretion was decreased, and this was associated with a decreased amount of CFTR protein and
mRNA expression. This result was unexpected, since
cAMP inhibits NHE3 in cells that lack CFTR (533). In
small intestine of CFTR knockout mice, Na⫹ absorption
was reduced in parallel with a decrease in NHE3 protein
expression (94, 158, 159). The mechanism of coordinated
regulation is not known. Of note, the opposite regulation
of Na⫹ absorption with increased secretion occurred as
well. In a mouse model of osmotic diarrhea, expression of
both NHE3 and DRA mRNA increased (421).
Is there further evidence for coordinated control of
these two regulated processes, and do NHE3 and CFTR
physically interact? In the intestine, CFTR and NHE3 are
mostly not in the same cells, although some CFTR appears to be in some villus Na⫹ absorptive cells (159).
However, in pancreatic duct cells, which contain both
NHE3 and CFTR, and in PS120 fibroblasts stably transfected with NHE3 and CFTR, there was physical association of NHE3 and CFTR based on coprecipitation (6).
This required the COOH-terminal four amino acids (PDZ
binding domain) of CFTR. In the pancreatic duct, there
was similar parallel regulation of NHE3 and CFTR as
occurred in mouse intestine, with the ⌬508 CFTR mutant
Physiol Rev • VOL
857
associated with decreased NHE3 amount and activity.
Similarly in PS120 cells, the presence of CFTR but not
CFTR lacking the COOH-terminal four amino acids increased the magnitude of cAMP inhibition of NHE3 (6).
CFTR without the COOH-terminal four amino acids had
no change in Cl⫺ channel activity but eliminated coprecipitation of NHE3 and reduced cAMP inhibition of NHE3.
Thus CFTR regulates NHE3 activity, which appears to be
another example of the regulatory function of CFTR,
altering activity and/or amount of an associated transporter. In this case, CFTR appears to stabilize NHE3 in the
brush border or plasma membrane and acutely increases
the cAMP inhibition of NHE3. How much of the effects of
⌬508 CFTR and reduced NHE3 expression in parallel in
pancreatic duct are due to effects during development is
unknown.
What about the converse, Does NHE3 regulate
CFTR? In a renal cell model (A6 cell) which expressed
endogenous CFTR and NHERF1, and in which NHE3 was
stably expressed, the magnitude of PKA-dependent inhibition of CFTR was decreased with no change in CFTR
amount (32). This effect required PKA increased phosphorylation at NHE3 Ser552 or Ser605 and required an
AKAP, since it was inhibited by the AKAP competitive
inhibitor S-HT31. Also, antisense suppression of CFTR
resulted in less cAMP inhibition of NHE3. Thus CFTR and
NHE3 physically associate with each other, at least when
exogenously expressed in the same cell, by a complex
that includes a NHERF family member, and they affect
each other’s regulation. A recent observation suggested
that the NHE3/CFTR interaction (147) involving NHERF2
was similar to the ␤2-adrenergic receptor/NHE3 involving
NHERF1 (191) in that the NHERF amount seems to be
rate limiting. When NHE3 was expressed in cells that
contain CFTR, it reduced PKA-dependent apical CFTR
expression/activation by a process that required NHE3 be
occupying its NHERF binding domain. This suggests a
competition for locally expressed NHERF. However, the
importance of this effect is not established physiologically, primarily because NHE3 and CFTR are usually not
in the same cell. Concerning other potential mechanisms
of linkage of CFTR/NHE3 regulation, changes in cell volume have been suggested (158). In the mouse jejunal mid
villus area, both NHE3 and CFTR are present in the same
cells (158). Here cAMP-activated anion secretion leads to
cell shrinkage, with cell shrinkage known to inhibit NHE3
activity. This also indicates why lack of CFTR prevents
cAMP effects on NHE3 activity, although it would not
explain why cAMP phosphorylation of NHE3 is required
(since it is not needed for shrinkage-induced inhibition of
NHE3 activity). Our interpretation is that the data favor a
functional linkage rather than a physical linkage of CFTR
and NHE3, although the mechanism(s) for interactions
are not adequately understood as yet.
87 • JULY 2007 •
www.prv.org
858
MARK DONOWITZ AND XUHANG LI
ACKNOWLEDGMENTS
We thank Peter Aronson, Orson Moe, and Ursula Seidler
for access to data prior to appearance in print.
We acknowledge the expert editorial assistance of H.
McCann.
Address for correspondence: M. Donowitz, Ross 925, Johns
Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205 (e-mail: mdonowit@jhmi.edu).
GRANTS
This work was supported in part by National Institute of
Diabetes and Digestive and Kidney Diseases Grants RO1-DK26523, RO1-DK-61765, KO1-DK-62264, PO1-DK-44484, PO1-DK72084, and R24-DK64388 (The Hopkins Basic Research Digestive
Diseases Development Core Center) and by the Hopkins Center
for Epithelial Disorders.
REFERENCES
1. Abedin MZ, Giurgiu DI, Abedin ZR, Peck EA, Su X, Smith PR.
Characterization of Na⫹/H⫹ exchanger isoform (NHE1, NHE2 and
NHE3) expression in prairie dog gallbladder. J Membr Biol 182:
123–134, 2001.
2. Abu-Shaweesh JM, Dreshaj IA, Martin RJ, Wirth KJ, Heinelt
U, Haxhiu MA. Inhibition of Na(⫹)/H(⫹) exchanger type 3 reduces duration of apnea induced by laryngeal stimulation in piglets.
Pediatr Res 52: 459 – 464, 2002.
3. Adamsky K, Arnold K, Sabanay H, Peles E. Junctional protein
MAGI-3 interacts with receptor tyrosine phosphatase beta (RPTP
beta) and tyrosine-phosphorylated proteins. J Cell Sci 116: 1279 –
1289, 2003.
4. Aharonovitz O, Kapus A, Szaszi K, Coady-Osberg N,
Jancelewicz T, Orlowski J, Grinstein S. Modulation of Na⫹/H⫹
exchange activity by Cl. Am J Physiol Cell Physiol 281: C133–C141,
2001.
5. Aharonovitz O, Zaun HC, Balla T, York JD, Orlowski J, Grinstein S. Intracellular pH regulation by Na(⫹)/H(⫹) exchange requires phosphatidylinositol 4,5-bisphosphate. J Cell Biol 150: 213–
224, 2000.
6. Ahn W, Kim KH, Lee JA, Kim JY, Choi JY, Moe OW, Milgram
SL, Muallem S, Lee MG. Regulatory interaction between the
cystic fibrosis transmembrane conductance regulator and HCO⫺
3
salvage mechanisms in model systems and the mouse pancreatic
duct. J Biol Chem 276: 17236 –17243, 2001.
7. Akhter S, Cavet ME, Tse CM, Donowitz M. COOH terminal
domains of Na(⫹)/H(⫹) exchanger isoform 3 are involved in the
basal and serum-stimulated membrane trafficking of the exchanger. Biochemistry 39: 1990 –2000, 2000.
8. Akhter S, Kovbasnjuk O, Li X, Cavet M, Noel J, Arpin M,
Hubbard AL, Donowitz M. Na⫹/H⫹ exchanger 3 is in large complexes in the center of the apical surface of proximal tubulederived OK cells. Am J Physiol Cell Physiol 283: C927–C940, 2002.
9. Akhter S, Nath SK, Tse CM, Williams J, Zasloff M, Donowitz
M. Squalamine, a novel cationic steroid, specifically inhibits the
brush-border Na⫹/H⫹ exchanger isoform NHE3. Am J Physiol Cell
Physiol 276: C136 –C144, 1999.
10. Albrecht FE, Xu J, Moe OW, Hopfer U, Simonds WF, Orlowski
J, Jose PA. Regulation of NHE3 activity by G protein subunits in
renal brush-border membranes. Am J Physiol Regul Integr Comp
Physiol 278: R1064 –R1073, 2000.
11. Alexander RT, Furuya W, Szaszi K, Orlowski J, Grinstein S.
Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in
epithelia: role in apical retention and targeting. Proc Natl Acad Sci
USA 102: 12253–12258, 2005.
12. Alexander RT, Grinstein S. Na⫹/H⫹ exchangers and the regulation of volume. Acta Physiol 187: 159 –167, 2006.
13. Alpern RJ, Yamaji Y, Cano A, Horie S, Miller RT, Moe OW,
Preisig PA. Chronic regulation of the Na/H antiporter. J Lab Clin
Med 122: 137–140, 1993.
Physiol Rev • VOL
14. Alrefai WA, Scaglione-Sewell B, Tyagi S, Wartman L, Brasitus
TA, Ramaswamy K, Dudeja PK. Differential regulation of the
expression of Na⫹/H⫹ exchanger isoform NHE3 by PKC-alpha in
Caco-2 cells. Am J Physiol Cell Physiol 281: C1551–C1558, 2001.
15. Ambuhl PM, Yang X, Peng Y, Preisig PA, Moe OW, Alpern RJ.
Glucocorticoids enhance acid activation of the Na⫹/H⫹ exchanger
3 (NHE3). J Clin Invest 103: 429 – 435, 1999.
16. Amemiya M, Kusano E, Muto S, Tabei K, Ando Y, Alpern RJ,
Asano Y. Glucagon acutely inhibits but chronically activates
Na(⫹)/H(⫹) antiporter 3 activity in OKP cells. Exp Nephrol 10:
26 –33, 2002.
17. Amemiya M, Mori H, Imamura S, Toyoda A, Funayama I,
Asano Y, Kusano E, Tabei K. Stimulation of NHE3 in OKP cells
by an autocrine mechanism. Nephron Exp Nephrol 96: e23–30,
2004.
18. Ammar YB, Takeda S, Hisamitsu T, Mori H, Wakabayashi S.
Crystal structure of CHP2 complexed with NHE1-cytosolic region
and an implication for pH regulation. EMBO J 25: 2315–2325, 2006.
19. Anderson CM, Thwaites DT. Indirect regulation of the intestinal
H⫹-coupled amino acid transporter hPAT1 (SLC36A1). J Cell
Physiol 204: 604 – 613, 2005.
⫺
20. Aronson PS. Ion exchangers mediating Na⫹, HCO⫺
3 and Cl transport in the renal proximal tubule. J Nephrol 19 Suppl 9: S3–S10,
2006.
21. Aronson PS, Nee J, Suhm MA. Modifier role of internal H⫹ in
activating the Na⫹-H⫹ exchanger in renal microvillus membrane
vesicles. Nature 299: 161–163, 1982.
22. Attaphitaya S, Park K, Melvin JE. Molecular cloning and functional expression of a rat Na⫹/H⫹ exchanger (NHE5) highly expressed in brain. J Biol Chem 274: 4383– 4388, 1999.
23. Azarani A, Goltzman D, Orlowski J. Structurally diverse Nterminal peptides of parathyroid hormone (PTH) and PTH-related
peptide (PTHRP) inhibit the Na⫹/H⫹ exchanger NHE3 isoform by
binding to the PTH/PTHRP receptor type I and activating distinct
signaling pathways. J Biol Chem 271: 14931–14936, 1996.
24. Azuma KK, Balkovetz DF, Magyar CE, Lescale-Matys L,
Zhang Y, Chambrey R, Warnock DG, McDonough AA. Renal
Na⫹/H⫹ exchanger isoforms and their regulation by thyroid hormone. Am J Physiol Cell Physiol 270: C585–C592, 1996.
25. Bachmann O, Riederer B, Rossmann H, Groos S, Schultheis
PJ, Shull GE, Gregor M, Manns MP, Seidler U. The Na⫹/H⫹
exchanger isoform 2 is the predominant NHE isoform in murine
colonic crypts and its lack causes NHE3 upregulation. Am J
Physiol Gastrointest Liver Physiol 287: G125–G133, 2004.
26. Bachmann S, Schlichting U, Geist B, Mutig K, Petsch T, Bacic
D, Wagner CA, Kaissling B, Biber J, Murer H, Willnow TE.
Kidney-specific inactivation of the megalin gene impairs trafficking
of renal inorganic sodium phosphate cotransporter (NaPi-IIa).
J Am Soc Nephrol 15: 892–900, 2004.
27. Bacic D, Capuano P, Baum M, Zhang J, Stange G, Biber J,
Kaissling B, Moe OW, Wagner CA, Murer H. Activation of
dopamine D1-like receptors induces acute internalization of the
renal Na⫹/phosphate cotransporter NaPi-IIa in mouse kidney and
OK cells. Am J Physiol Renal Physiol 288: F740 –F747, 2005.
28. Bacic D, Kaissling B, McLeroy P, Zou L, Baum M, Moe OW.
Dopamine acutely decreases apical membrane Na/H exchanger
NHE3 protein in mouse renal proximal tubule. Kidney Int 64:
2133–2141, 2003.
29. Bacic D, Wagner CA, Hernando N, Kaissling B, Biber J, Murer
H. Novel aspects in regulated expression of the renal type IIa
Na/Pi-cotransporter. Kidney Int Suppl: S5–S12, 2004.
30. Bagnis C, Marsolais M, Biemesderfer D, Laprade R, Breton S.
Na⫹/H⫹-exchange activity and immunolocalization of NHE3 in rat
epididymis. Am J Physiol Renal Physiol 280: F426 –F436, 2001.
31. Bagorda A, Guerra L, Di Sole F, Helmle-Kolb C, Favia M,
Jacobson KA, Casavola V, Reshkin SJ. Extracellular adenine
nucleotides regulate Na⫹/H⫹ exchanger NHE3 activity in A6-NHE3
transfectants by a cAMP/PKA-dependent mechanism. J Membr Biol
188: 249 –259, 2002.
32. Bagorda A, Guerra L, Di Sole F, Hemle-Kolb C, Cardone RA,
Fanelli T, Reshkin SJ, Gisler SM, Murer H, Casavola V. Reciprocal protein kinase A regulatory interactions between cystic
fibrosis transmembrane conductance regulator and Na⫹/H⫹ ex-
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
changer isoform 3 in a renal polarized epithelial cell model. J Biol
Chem 277: 21480 –21488, 2002.
Baird NR, Orlowski J, Szabo EZ, Zaun HC, Schultheis PJ,
Menon AG, Shull GE. Molecular cloning, genomic organization,
functional expression of Na⫹/H⫹ exchanger isoform 5 (NHE5)
from human brain. J Biol Chem 274: 4377– 4382, 1999.
Barmeyer C, Harren M, Schmitz H, Heinzel-Pleines U, Mankertz J, Seidler U, Horak I, Wiedenmann B, Fromm M, Schulzke JD. Mechanisms of diarrhea in the interleukin-2-deficient
mouse model of colonic inflammation. Am J Physiol Gastrointest
Liver Physiol 286: G244 –G252, 2004.
Barone S, Amlal H, Xu J, Kujala M, Kere J, Petrovic S,
Soleimani M. Differential regulation of basolateral Cl⫺/HCO⫺
3 exchangers SLC26A7 and AE1 in kidney outer medullary collecting
duct. J Am Soc Nephrol 15: 2002–2011, 2004.
Bates IR, Hebert B, Luo Y, Liao J, Bachir AI, Kolin DL,
Wiseman PW, Hanrahan JW. Membrane lateral diffusion and
capture of CFTR within transient confinement zones. Biophys J 91:
1046 –1058, 2006.
Baum M, Biemesderfer D, Gentry D, Aronson PS. Ontogeny of
rabbit renal cortical NHE3 and NHE1: effect of glucocorticoids.
Am J Physiol Renal Fluid Electrolyte Physiol 268: F815–F820,
1995.
Baumgartner M, Patel H, Barber DL. Na(⫹)/H(⫹) exchanger
NHE1 as plasma membrane scaffold in the assembly of signaling
complexes. Am J Physiol Cell Physiol 287: C844 –C850, 2004.
Bell SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL,
Vorhees CV, Shull GE, Scott WJ. Targeted disruption of the
murine Nhe1 locus induces ataxia, growth retardation, seizures.
Am J Physiol Cell Physiol 276: C788 –C795, 1999.
Ben Ammar Y, Takeda S, Sugawara M, Miyano M, Mori H,
Wakabayashi S. Crystallization and preliminary crystallographic
analysis of the human calcineurin homologous protein CHP2
bound to the cytoplasmic region of the Na⫹/H⫹ exchanger NHE1.
Acta Crystallograph Sect F Struct Biol Cryst Commun 61: 956 –
958, 2005.
Bertrand B, Wakabayashi S, Ikeda T, Pouyssegur J,
Shigekawa M. The Na⫹/H⫹ exchanger isoform 1 (NHE1) is a novel
member of the calmodulin-binding proteins. Identification and
characterization of calmodulin-binding sites. J Biol Chem 269:
13703–13709, 1994.
Biber J, Gisler SM, Hernando N, Murer H. Protein/protein
interactions (PDZ) in proximal tubules. J Membr Biol 203: 111–118,
2005.
Biber J, Gisler SM, Hernando N, Wagner CA, Murer H. PDZ
interactions and proximal tubular phosphate reabsorption. Am J
Physiol Renal Physiol 287: F871–F875, 2004.
Biemesderfer D. Regulated intramembrane proteolysis of megalin: linking urinary protein and gene regulation in proximal tubule?
Kidney Int 69: 1717–1721, 2006.
Biemesderfer D, DeGray B, Aronson PS. Active (96 s) and
inactive (21 s) oligomers of NHE3 in microdomains of the renal
brush border. J Biol Chem 276: 10161–10167, 2001.
Biemesderfer D, Nagy T, DeGray B, Aronson PS. Specific association of megalin and the Na⫹/H⫹ exchanger isoform NHE3 in
the proximal tubule. J Biol Chem 274: 17518 –17524, 1999.
Biemesderfer D, Pizzonia J, Abu-Alfa A, Exner M, Reilly R,
Igarashi P, Aronson PS. NHE3: a Na⫹/H⫹ exchanger isoform of
renal brush border. Am J Physiol Renal Fluid Electrolyte Physiol
265: F736 –F742, 1993.
Biemesderfer D, Rutherford PA, Nagy T, Pizzonia JH, AbuAlfa AK, Aronson PS. Monoclonal antibodies for high-resolution
localization of NHE3 in adult and neonatal rat kidney. Am J Physiol
Renal Physiol 273: F289 –F299, 1997.
Bilder D. PDZ proteins and polarity: functions from the fly. Trends
Genet 17: 511–519, 2001.
Bobulescu IA, Dwarakanath V, Zou L, Zhang J, Baum M, Moe
OW. Glucocorticoids acutely increase cell surface Na⫹/H⫹ exchanger-3 (NHE3) by activation of NHE3 exocytosis. Am J Physiol
Renal Physiol 289: F685–F691, 2005.
Bommert K, Charlton MP, DeBello WM, Chin GJ, Betz H,
Augustine GJ. Inhibition of neurotransmitter release by C2-doPhysiol Rev • VOL
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
859
main peptides implicates synaptotagmin in exocytosis. Nature 363:
163–165, 1993.
Bookstein C, DePaoli AM, Xie Y, Niu P, Musch MW, Rao MC,
Chang EB. Na⫹/H⫹ exchangers, NHE-1 and NHE-3, of rat intestine.
Expression and localization. J Clin Invest 93: 106 –113, 1994.
Bookstein C, Musch MW, Xie Y, Rao MC, Chang EB. Regulation
of intestinal epithelial brush border Na(⫹)/H(⫹) exchanger isoforms, NHE2 and NHE3, in C2bbe cells. J Membr Biol 171: 87–95,
1999.
Bookstein C, Xie Y, Rabenau K, Musch MW, McSwine RL, Rao
MC, Chang EB. Tissue distribution of Na⫹/H⫹ exchanger isoforms
NHE2 and NHE4 in rat intestine and kidney. Am J Physiol Cell
Physiol 273: C1496 –C1505, 1997.
Booth IW, Stange G, Murer H, Fenton TR, Milla PJ. Defective
jejunal brush-border Na⫹/H⫹ exchange: a cause of congenital secretory diarrhoea. Lancet 1: 1066 –1069, 1985.
Borensztein P, Laghmani K, Froissard M, Paillard M. [Molecular aspects of Na⫹/H⫹ exchange in the renal tubule: localization
and adaptation to the acid-base state]. Nephrologie 17: 377–381,
1996.
Brandsch M, Ganapathy V, Leibach FH. H⫹-peptide cotransport
in Madin-Darby canine kidney cells: expression and calmodulindependent regulation. Am J Physiol Renal Fluid Electrolyte
Physiol 268: F391–F397, 1995.
Brant SR, Yun CH, Donowitz M, Tse CM. Cloning, tissue distribution, functional analysis of the human Na⫹/H⫹ exchanger isoform, NHE3. Am J Physiol Cell Physiol 269: C198 –C206, 1995.
Bretscher A, Chambers D, Nguyen R, Reczek D. ERM-Merlin
and EBP50 protein families in plasma membrane organization and
function. Annu Rev Cell Dev Biol 16: 113–143, 2000.
Brett CL, Donowitz M, Rao R. Evolutionary origins of eukaryotic
sodium/proton exchangers. Am J Physiol Cell Physiol 288: C223–
C239, 2005.
Brett CL, Tukaye DN, Mukherjee S, Rao R. The yeast endosomal Na⫹K⫹/H⫹ exchanger Nhx1 regulates cellular pH to control
vesicle trafficking. Mol Biol Cell 16: 1396 –1405, 2005.
Brett CL, Wei Y, Donowitz M, Rao R. Human Na⫹/H⫹ exchanger
isoform 6 is found in recycling endosomes of cells, not in mitochondria. Am J Physiol Cell Physiol 282: C1031–C1041, 2002.
Brone B, Eggermont J. PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes. Am J
Physiol Cell Physiol 288: C20 –C29, 2005.
Brown SE, Heming TA, Benedict CR, Bidani A. ATP-sensitive
Na⫹-H⫹ antiport in type II alveolar epithelial cells. Am J Physiol
Cell Physiol 261: C954 –C963, 1991.
Cabado AG, Yu FH, Kapus A, Lukacs G, Grinstein S, Orlowski
J. Distinct structural domains confer cAMP sensitivity and ATP
dependence to the Na⫹/H⫹ exchanger NHE3 isoform. J Biol Chem
271: 3590 –3599, 1996.
Capuano P, Bacic D, Roos M, Gisler SM, Stange G, Biber J,
Kaissling B, Weinman EJ, Shenolikar S, Wagner CA, Murer H.
Defective coupling of apical PTH-receptors to phospholipase C
prevents internalization of the Na⫹/phosphate cotransporter NAPiIIa in NHERF1 deficient mice. Am J Physiol Cell Physiol 292:
C927–C934, 2007.
Capuano P, Bacic D, Stange G, Hernando N, Kaissling B, Pal
R, Kocher O, Biber J, Wagner CA, Murer H. Expression and
regulation of the renal Na/phosphate cotransporter NaPi-IIa in a
mouse model deficient for the PDZ protein PDZK1. Pflügers Arch
449: 392– 402, 2005.
Cassel D, Katz M, Rotman M. Depletion of cellular ATP inhibits
Na⫹/H⫹ antiport in cultured human cells. Modulation of the regulatory effect of intracellular protons on the antiporter activity.
J Biol Chem 261: 5460 –5466, 1986.
Cavet ME, Akhter S, de Medina FS, Donowitz M, Tse CM.
Na⫹/H⫹ exchangers (NHE1-3) have similar turnover numbers but
different percentages on the cell surface. Am J Physiol Cell Physiol
277: C1111–C1121, 1999.
Cavet ME, Akhter S, Murtazina R, Sanchez de Medina F, Tse
CM, Donowitz M. Half-lives of plasma membrane Na⫹/H⫹ exchangers NHE1-3: plasma membrane NHE2 has a rapid rate of
degradation. Am J Physiol Cell Physiol 281: C2039 –C2048, 2001.
87 • JULY 2007 •
www.prv.org
860
MARK DONOWITZ AND XUHANG LI
71. Cha B, Kenworthy A, Murtazina R, Donowitz M. The lateral
mobility of NHE3 on the apical membrane of renal epithelial OK
cells is limited by the PDZ domain proteins NHERF1/2, but is
dependent on an intact actin cytoskeleton as determined by FRAP.
J Cell Sci 117: 3353–3365, 2004.
72. Cha B, Kim JH, Hut H, Hogema BM, Nadarja J, Zizak M, Cavet
M, Lee-Kwon W, Lohmann SM, Smolenski A, Tse CM, Yun C,
de Jonge HR, Donowitz M. cGMP inhibition of Na⫹/H⫹ antiporter
3 (NHE3) requires PDZ domain adapter NHERF2, a broad specificity protein kinase G-anchoring protein. J Biol Chem 280: 16642–
16650, 2005.
73. Cha B, Oh S, Shanmugaratnam J, Donowitz M, Yun CC. Two
histidine residues in the juxta-membrane cytoplasmic domain of
Na⫹/H⫹ exchanger isoform 3 (NHE3) determine the set point. J
Membr Biol 191: 49 –58, 2003.
74. Cha B, Tse M, Yun C, Kovbasnjuk O, Mohan S, Hubbard A,
Arpin M, Donowitz M. The NHE3 juxtamembrane cytoplasmic
domain directly binds ezrin: dual role in NHE3 trafficking and
mobility in the brush border. Mol Biol Cell 17: 2661–2673, 2006.
75. Chalumeau C, du Cheyron D, Defontaine N, Kellermann O,
Paillard M, Poggioli J. NHE3 activity and trafficking depend on
the state of actin organization in proximal tubule. Am J Physiol
Renal Physiol 280: F283–F290, 2001.
76. Chang EB, Field M, Miller RJ. Enterocyte alpha 2-adrenergic
receptors: yohimbine and p-aminoclonidine binding relative to ion
transport. Am J Physiol Gastrointest Liver Physiol 244: G76 –G82,
1983.
77. Charney AN, Donowitz M. Prevention and reversal of cholera
enterotoxin-induced intestinal secretion by methylprednisolone induction of Na⫹-K⫹-ATPase. J Clin Invest 57: 1590 –1599, 1976.
78. Charney AN, Egnor RW, Cassai N, Sidhu GS. Carbon dioxide
affects rat colonic Na⫹ absorption by modulating vesicular traffic.
Gastroenterology 122: 318 –330, 2002.
79. Charney AN, Kinsey MD, Myers L, Gainnella RA, Gots RE.
Na⫹-K⫹-activated adenosine triphosphatase and intestinal electrolyte transport. Effect of adrenal steroids. J Clin Invest 56: 653– 660,
1975.
80. Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn
CR. Phosphatidylinositol 3-kinase activation is required for insulin
stimulation of pp70 S6 kinase, DNA synthesis, glucose transporter
translocation. Mol Cell Biol 14: 4902– 4911, 1994.
81. Cheng J, Moyer BD, Milewski M, Loffing J, Ikeda M, Mickle
JE, Cutting GR, Li M, Stanton BA, Guggino WB. A Golgiassociated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression. J Biol Chem
277: 3520 –3529, 2002.
82. Cheng J, Wang H, Guggino WB. Modulation of mature cystic
fibrosis transmembrane regulator protein by the PDZ domain protein CAL. J Biol Chem 279: 1892–1898, 2004.
83. Cheng J, Wang H, Guggino WB. Regulation of cystic fibrosis
transmembrane regulator trafficking and protein expression by a
Rho family small GTPase TC10. J Biol Chem 280: 3731–3739, 2005.
84. Cho JH, Musch MW, Bookstein CM, McSwine RL, Rabenau K,
Chang EB. Aldosterone stimulates intestinal Na⫹ absorption in
rats by increasing NHE3 expression of the proximal colon. Am J
Physiol Cell Physiol 274: C586 –C594, 1998.
85. Cho JH, Musch MW, DePaoli AM, Bookstein CM, Xie Y,
Burant CF, Rao MC, Chang EB. Glucocorticoids regulate
Na⫹/H⫹ exchange expression and activity in region- and tissuespecific manner. Am J Physiol Cell Physiol 267: C796 –C803, 1994.
86. Choe KP, Kato A, Hirose S, Plata C, Sindic A, Romero MF,
Claiborne JB, Evans DH. NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, function in gills. Am J
Physiol Regul Integr Comp Physiol 289: R1520 –R1534, 2005.
87. Choi JW, Lee-Kwon W, Jeon ES, Kang YJ, Kawano K, Kim HS,
Suh PG, Donowitz M, Kim JH. Lysophosphatidic acid induces
exocytic trafficking of Na(⫹)/H(⫹) exchanger 3 by E3KARP-dependent activation of phospholipase C. Biochim Biophys Acta 1683:
59 – 68, 2004.
88. Chow CW, Khurana S, Woodside M, Grinstein S, Orlowski J.
The epithelial Na(⫹)/H(⫹) exchanger, NHE3, is internalized
through a clathrin-mediated pathway. J Biol Chem 274: 37551–
37558, 1999.
Physiol Rev • VOL
89. Chu J, Chu S, Montrose MH. Apical Na⫹/H⫹ exchange near the
base of mouse colonic crypts. Am J Physiol Cell Physiol 283:
C358 –C372, 2002.
90. Chun J, Kwon T, Lee E, Suh PG, Choi EJ, Sun Kang S. The
Na(⫹)/H(⫹) exchanger regulatory factor 2 mediates phosphorylation of serum- and glucocorticoid-induced protein kinase 1 by
3-phosphoinositide-dependent protein kinase 1. Biochem Biophys
Res Commun 298: 207–215, 2002.
91. Chung HJ, Huang YH, Lau LF, Huganir RL. Regulation of the
NMDA receptor complex and trafficking by activity-dependent
phosphorylation of the NR2B subunit PDZ ligand. J Neurosci 24:
10248 –10259, 2004.
92. Chung HJ, Xia J, Scannevin RH, Zhang X, Huganir RL. Phosphorylation of the AMPA receptor subunit GluR2 differentially
regulates its interaction with PDZ domain-containing proteins.
J Neurosci 20: 7258 –7267, 2000.
93. Cinar A, Chen Riederer M, B, Hogema B, de Jonge H, Donowitz M, Weinman E, Kocher O, Seidler U. Differential effects of
PDZ-adapter protein NHERF1, E3KARP and PDZK1 knockout on
the regulation of NHE3 transport activity in native murine colonic
epithelium (Abstract). Gastroenterology 130: A99, 2006.
94. Clarke LL, Harline MC. CFTR is required for cAMP inhibition of
intestinal Na⫹ absorption in a cystic fibrosis mouse model. Am J
Physiol Gastrointest Liver Physiol 270: G259 –G267, 1996.
95. Cohen ME, Reinlib L, Watson AJ, Gorelick F, Rys-Sikora K,
Tse M, Rood RP, Czernik AJ, Sharp GW, Donowitz M. Rabbit
ileal villus cell brush border Na⫹/H⫹ exchange is regulated by
Ca2⫹/calmodulin-dependent protein kinase II, a brush border membrane protein. Proc Natl Acad Sci USA 87: 8990 – 8994, 1990.
96. Cohen ME, Wesolek J, McCullen J, Rys-Sikora K, Pandol S,
Rood RP, Sharp GW, Donowitz M. Carbachol- and elevated
Ca(2⫹)-induced translocation of functionally active protein kinase
C to the brush border of rabbit ileal Na⫹ absorbing cells. J Clin
Invest 88: 855– 863, 1991.
97. Collazo R, Fan L, Hu MC, Zhao H, Wiederkehr MR, Moe OW.
Acute regulation of Na⫹/H⫹ exchanger NHE3 by parathyroid hormone via NHE3 phosphorylation and dynamin-dependent endocytosis. J Biol Chem 275: 31601–31608, 2000.
98. Collins JF, Xu H, Kiela PR, Zeng J, Ghishan FK. Functional and
molecular characterization of NHE3 expression during ontogeny in
rat jejunal epithelium. Am J Physiol Cell Physiol 273: C1937–
C1946, 1997.
99. Colombani V, Silviani V, Marteau C, Lerique B, Cartouzou G,
Gerolami A. Presence of the NHE3 isoform of the Na⫹/H⫹ exchanger in human gallbladder. Clin Sci 91: 209 –212, 1996.
100. Cox GA, Lutz CM, Yang CL, Biemesderfer D, Bronson RT, Fu
A, Aronson PS, Noebels JL, Frankel WN. Sodium/hydrogen
exchanger gene defect in slow-wave epilepsy mutant mice. Cell 91:
139 –148, 1997.
101. Cremaschi D, Porta C, Botta G, Meyer G. Nature of the neutral
Na⫹-Cl⫺ coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na⫹/H⫹, Cl⫺/HCO⫺
3 double exchange, hydrochlorothiazide-sensitive Na⫹-Cl⫺ symport and Na⫹-K⫹-2Cl⫺ cotransport are all involved. J Membr Biol 129: 221–235, 1992.
102. Cunningham R, Xiaofei E, Steplock D, Shenolikar S, Weinman
EJ. Defective PTH regulation of sodium-dependent phosphate
transport in NHERF-1⫺/⫺ renal proximal tubule cells and wildtype cells adapted to low-phosphate media. Am J Physiol Renal
Physiol 289: F933–F938, 2005.
103. Cunningham R, Steplock D, Xiaofei E, Biswas R, Wang FS,
Shenolikar S, Weinman EJ. Adenoviral expression of NHERF-1
in NHERF-1 null mouse renal proximal tubule cells restores Npt2a
regulation by low phosphate media and parathyroid hormone.
Am J Physiol Renal Physiol 291: F896 –F901, 2006.
104. Cunningham R, Steplock D, Wang F, Huang H, Xiaofei E,
Shenolikar S, Weinman EJ. Defective parathyroid hormone regulation of NHE3 activity and phosphate adaptation in cultured
NHERF-1⫺/⫺ renal proximal tubule cells. J Biol Chem 279: 37815–
37821, 2004.
105. De Silva MG, Elliott K, Dahl HH, Fitzpatrick E, Wilcox S,
Delatycki M, Williamson R, Efron D, Lynch M, Forrest S.
Disruption of a novel member of a sodium/hydrogen exchanger
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
family and DOCK3 is associated with an attention deficit hyperactivity disorder-like phenotype. J Med Genet 40: 733–740, 2003.
Deliot N, Hernando N, Horst-Liu Z, Gisler SM, Capuano P,
Wagner CA, Bacic D, O’Brien S, Biber J, Murer H. Parathyroid
hormone treatment induces dissociation of type IIa Na⫹-Pi cotransporter-Na⫹/H⫹ exchanger regulatory factor-1 complexes. Am J
Physiol Cell Physiol 289: C159 –C167, 2005.
DeMarco SJ, Chicka MC, Strehler EE. Plasma membrane Ca2⫹
ATPase isoform 2b interacts preferentially with Na⫹/H⫹ exchanger
regulatory factor 2 in apical plasma membranes. J Biol Chem 277:
10506 –10511, 2002.
Demaurex N, Romanek RR, Orlowski J, Grinstein S. ATP
dependence of Na⫹/H⫹ exchange. Nucleotide specificity and assessment of the role of phospholipids. J Gen Physiol 109: 117–128,
1997.
Demoulin JB, Seo JK, Ekman S, Grapengiesser E, Hellman U,
Ronnstrand L, Heldin CH. Ligand-induced recruitment of Na⫹/
H⫹-exchanger regulatory factor to the PDGF (platelet-derived
growth factor) receptor regulates actin cytoskeleton reorganization by PDGF. Biochem J 376: 505–510, 2003.
Denker SP, Barber DL. Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1.
J Cell Biol 159: 1087–1096, 2002.
Denker SP, Barber DL. Ion transport proteins anchor and regulate the cytoskeleton. Curr Opin Cell Biol 14: 214 –220, 2002.
Denker SP, Huang DC, Orlowski J, Furthmayr H, Barber DL.
Direct binding of the Na-H exchanger NHE1 to ERM proteins
regulates the cortical cytoskeleton and cell shape independently of
H(⫹) translocation. Mol Cell 6: 1425–1436, 2000.
Dev KK. Making protein interactions druggable: targeting PDZ
domains. Nat Rev Drug Discov 3: 1047–1056, 2004.
Di Sole F, Casavola V, Mastroberardino L, Verrey F, Moe OW,
Burckhardt G, Murer H, Helmle-Kolb C. Adenosine inhibits the
transfected Na⫹-H⫹ exchanger NHE3 in Xenopus laevis renal epithelial cells. J Physiol 515: 829 – 842, 1999.
Di Sole F, Cerull R, Babich V, Quinones H, Gisler SM, Biber J,
Murer H, Burckhardt G, Helmle-Kolb C, Moe OW. Acute regulation of Na/H exchanger NHE3 by adenosine A(1) receptors is
mediated by calcineurin homologous protein. J Biol Chem 279:
2962–2974, 2004.
Di Sole F, Cerull R, Casavola V, Moe OW, Burckhardt G,
Helmle-Kolb C. Molecular aspects of acute inhibition of Na(⫹)H(⫹) exchanger NHE3 by A(2)-adenosine receptor agonists.
J Physiol 541: 529 –543, 2002.
Di Sole F, Cerull R, Petzke S, Casavola V, Burckhardt G,
Helmle-Kolb C. Bimodal acute effects of A1 adenosine receptor
activation on Na⫹/H⫹ exchanger 3 in opossum kidney cells. J Am
Soc Nephrol 14: 1720 –1730, 2003.
Dixit MP, Xu L, Xu H, Bai L, Collins JF, Ghishan FK. Effect of
angiotensin-II on renal Na⫹/H⫹ exchanger-NHE3 and NHE2. Biochim Biophys Acta 1664: 38 – 44, 2004.
Dixon RJ, Young K, Brunskill NJ. Lysophosphatidic acid-induced calcium mobilization and proliferation in kidney proximal
tubular cells. Am J Physiol Renal Physiol 276: F191–F198, 1999.
Doble MA, Tola VB, Cima RR, Zinner MJ, Klein MA, Soybel
DI. Luminal osmolarity downregulates gene expression of Na⫹/H⫹
exchanger (NHE3) in rat colon mucosa. J Gastrointest Surg 4:
531–535, 2000.
Donowitz M, Tse CM. Molecular physiology of mammalian epithelial Na/H exchangers NHE2 and NHE3. Curr Topics Membr 50:
437– 498, 2001.
Donowitz M. Ca2⫹ in the control of active intestinal Na and Cl
transport: involvement in neurohumoral action. Am J Physiol Gastrointest Liver Physiol 245: G165–G177, 1983.
Donowitz M, Welsh M. Regulation of mammalian small intestine
electrolyte secretion. In: Physiology of the Gastrointestinal Tract,
edited by Johnson L. New York: Raven, 1987, p. 1351–1388.
Donowitz M, Asarkof N. Calcium dependence of basal electrolyte
transport in rabbit ileum. Am J Physiol Gastrointest Liver Physiol
243: G28 –G35, 1982.
Donowitz M, Asarkof N, Pike G. Calcium dependence of serotonin-induced changes in rabbit ileal electrolyte transport. J Clin
Invest 66: 341–352, 1980.
Physiol Rev • VOL
861
126. Donowitz M, Cha B, Zachos NC, Brett CL, Sharma A, Tse CM,
Li X. NHERF family and NHE3 regulation. J Physiol 567: 3–11,
2005.
127. Donowitz M, Cohen ME, Gould M, Sharp GW. Elevated intracellular Ca2⫹ acts through protein kinase C to regulate rabbit ileal
NaCl absorption. Evidence for sequential control by Ca2⫹/calmodulin and protein kinase C. J Clin Invest 83: 1953–1962, 1989.
128. Donowitz M, Cohen ME, Gudewich R, Taylor L, Sharp GW.
Ca2⫹-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microvillus membranes of rabbit
ileum. Biochem J 219: 573–581, 1984.
129. Donowitz M, Fogel R, Battisti L, Asarkof N. The neurohumoral
secretagogues carbachol, substance P and neurotensin increase
Ca2⫹ influx and calcium content in rabbit ileum. Life Sci 31:
1929 –1937, 1982.
130. Donowitz M, Janecki A, Akhter S, Cavet ME, Sanchez F,
Lamprecht G, Zizak M, Kwon WL, Khurana S, Yun CH, Tse
CM. Short-term regulation of NHE3 by EGF and protein kinase C
but not protein kinase A involves vesicle trafficking in epithelial
cells and fibroblasts. Ann NY Acad Sci 915: 30 – 42, 2000.
131. Donowitz M, Madara JL. Effect of extracellular calcium depletion on epithelial structure and function in rabbit ileum: a model for
selective crypt or villus epithelial cell damage and suggestion of
secretion by villus epithelial cells. Gastroenterology 83: 1231–1243,
1982.
132. Donowitz M, Wicks J, Madara JL, Sharp GW. Studies on role of
calmodulin in Ca2⫹ regulation of rabbit ileal Na and Cl transport.
Am J Physiol Gastrointest Liver Physiol 248: G726 –G740, 1985.
133. Dransfield DT, Bradford AJ, Smith J, Martin M, Roy C,
Mangeat PH, Goldenring JR. Ezrin is a cyclic AMP-dependent
protein kinase anchoring protein. EMBO J 16: 35– 43, 1997.
134. D’Souza S, Garcia-Cabado A, Yu F, Teter K, Lukacs G,
Skorecki K, Moore HP, Orlowski J, Grinstein S. The epithelial
sodium-hydrogen antiporter Na⫹/H⫹ exchanger 3 accumulates and
is functional in recycling endosomes. J Biol Chem 273: 2035–2043,
1998.
135. Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T.
Axial flow modulates proximal tubule NHE3 and H-ATPase activities by changing microvillus bending moments. Am J Physiol Renal
Physiol 290: F289 –F296, 2006.
136. Du Cheyron D, Chalumeau C, Defontaine N, Klein C, Kellermann O, Paillard M, Poggioli J. Angiotensin II stimulates NHE3
activity by exocytic insertion of the transporter: role of PI 3-kinase.
Kidney Int 64: 939 –949, 2003.
137. Dudas PL, Mentone S, Greineder CF, Biemesderfer D, Aronson PS. Immunolocalization of anion transporter Slc26a7 in mouse
kidney. Am J Physiol Renal Physiol 290: F937–F945, 2006.
138. Dudeja PK, Rao DD, Syed I, Joshi V, Dahdal RY, Gardner C,
Risk MC, Schmidt L, Bavishi D, Kim KE, Harig JM, Goldstein
JL, Layden TJ, Ramaswamy K. Intestinal distribution of human
Na⫹/H⫹ exchanger isoforms NHE-1, NHE-2, NHE-3 mRNA. Am J
Physiol Gastrointest Liver Physiol 271: G483–G493, 1996.
139. Durbin T, Rosenthal L, McArthur K, Anderson D, Dharmsathaphorn K. Clonidine and lidamidine (WHR-1142) stimulate
sodium and chloride absorption in the rabbit intestine. Gastroenterology 82: 1352–1358, 1982.
140. Edwards SD, Keep NH. The 2.7 A crystal structure of the activated FERM domain of moesin: an analysis of structural changes
on activation. Biochemistry 40: 7061–7068, 2001.
141. Edwards SL, Donald JA, Toop T, Donowitz M, Tse CM. Immunolocalisation of sodium/proton exchanger-like proteins in the gills
of elasmobranchs. Comp Biochem Physiol A Mol Integr Physiol
131: 257–265, 2002.
142. Elitsur N, Lorenz JN, Hawkins JA, Rudolph JA, Witte D, Yang
LE, McDonough AA, Cohen MB. The proximal convoluted tubule
is a target for the uroguanylin-regulated natriuretic response.
J Pediatr Gastroenterol Nutr 43: S74 –S81, 2006.
143. Embark HM, Setiawan I, Poppendieck S, van de Graaf SF,
Boehmer C, Palmada M, Wieder T, Gerstberger R, Cohen P,
Yun CC, Bindels RJ, Lang F. Regulation of the epithelial Ca2⫹
channel TRPV5 by the NHE regulating factor NHERF2 and the
serum and glucocorticoid inducible kinase isoforms SGK1 and
87 • JULY 2007 •
www.prv.org
862
144.
145.
146.
147.
148.
149.
150.
151.
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
MARK DONOWITZ AND XUHANG LI
SGK3 expressed in Xenopus oocytes. Cell Physiol Biochem 14:
203–212, 2004.
Emmer E, Rood RP, Wesolek JH, Cohen ME, Braithwaite RS,
Sharp GW, Murer H, Donowitz M. Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane
Na⫹/H⫹ antiporter. J Membr Biol 108: 207–215, 1989.
Erranz B, Miquel JF, Argraves WS, Barth JL, Pimentel F,
Marzolo MP. Megalin and cubilin expression in gallbladder epithelium and regulation by bile acids. J Lipid Res 45: 2185–2198,
2004.
Fafournoux P, Noel J, Pouyssegur J. Evidence that Na⫹/H⫹
exchanger isoforms NHE1 and NHE3 exist as stable dimers in
membranes with a high degree of specificity for homodimers.
J Biol Chem 269: 2589 –2596, 1994.
Favia M, Fanelli T, Bagorda A, Di Sole F, Reshkin SJ, Suh PG,
Guerra L, Casavola V. NHE3 inhibits PKA-dependent functional
expression of CFTR by NHERF2 PDZ interactions. Biochem Biophys Res Commun 347: 452– 459, 2006.
Fei YJ, Kanai Y, Nussberger S, Ganapathy V, Leibach FH,
Romero MF, Singh SK, Boron WF, Hediger MA. Expression
cloning of a mammalian proton-coupled oligopeptide transporter.
Nature 368: 563–566, 1994.
Fordtran JS, Rector FC Jr, Carter NW. The mechanisms of
sodium absorption in the human small intestine. J Clin Invest 47:
884 –900, 1968.
Fouassier L, Nichols MT, Gidey E, McWilliams RR, Robin H,
Finnigan C, Howell KE, Housset C, Doctor RB. Protein kinase
C regulates the phosphorylation and oligomerization of ERM binding phosphoprotein 50. Exp Cell Res 306: 264 –273, 2005.
Freel RW, Hatch M, Green M, Soleimani M. Ileal oxalate absorption and urinary oxalate excretion are enhanced in Slc26a6
null mice. Am J Physiol Gastrointest Liver Physiol 290: G719 –
G728, 2006.
Freeman TC, Bentsen BS, Thwaites DT, Simmons NL. H⫹/
di-tripeptide transporter (PepT1) expression in the rabbit intestine.
Pflügers Arch 430: 394 – 400, 1995.
Friedman PA. PTH revisited. Kidney Int Suppl: S13–S19, 2004.
Furukawa O, Bi LC, Guth PH, Engel E, Hirokawa M, Kaunitz
JD. NHE3 inhibition activates duodenal bicarbonate secretion in
the rat. Am J Physiol Gastrointest Liver Physiol 286: G102–G109,
2004.
Fuster D, Moe OW, Hilgemann DW. Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical
methods. Proc Natl Acad Sci USA 101: 10482–10487, 2004.
Garner CC, Nash J, Huganir RL. PDZ domains in synapse assembly and signalling. Trends Cell Biol 10: 274 –280, 2000.
Garnovskaya MN, Mukhin YV, Vlasova TM, Raymond JR. Hypertonicity activates Na⫹/H⫹ exchange through Janus kinase 2 and
calmodulin. J Biol Chem 278: 16908 –16915, 2003.
Gawenis LR, Franklin CL, Simpson JE, Palmer BA, Walker
NM, Wiggins TM, Clarke LL. cAMP inhibition of murine intestinal Na/H exchange requires CFTR-mediated cell shrinkage of villus
epithelium. Gastroenterology 125: 1148 –1163, 2003.
Gawenis LR, Hut H, Bot AG, Shull GE, de Jonge HR, Stien X,
Miller ML, Clarke LL. Electroneutral sodium absorption and
electrogenic anion secretion across murine small intestine are
regulated in parallel. Am J Physiol Gastrointest Liver Physiol 287:
G1140 –G1149, 2004.
Gawenis LR, Stien X, Shull GE, Schultheis PJ, Woo AL,
Walker NM, Clarke LL. Intestinal NaCl transport in NHE2 and
NHE3 knockout mice. Am J Physiol Gastrointest Liver Physiol
282: G776 –G784, 2002.
Gebreselassie D, Rajarathnam K, Fliegel L. Expression, purification, characterization of the carboxyl-terminal region of the
Na⫹/H⫹ exchanger. Biochem Cell Biol 76: 837– 842, 1998.
Gekle M, Drumm K, Mildenberger S, Freudinger R, Gassner
B, Silbernagl S. Inhibition of Na⫹-H⫹ exchange impairs receptormediated albumin endocytosis in renal proximal tubule-derived
epithelial cells from opossum. J Physiol 520: 709 –721, 1999.
Gekle M, Freudinger R, Mildenberger S. Inhibition of Na⫹-H⫹
exchanger-3 interferes with apical receptor-mediated endocytosis
via vesicle fusion. J Physiol 531: 619 – 629, 2001.
Physiol Rev • VOL
164. Gekle M, Serrano OK, Drumm K, Mildenberger S, Freudinger
R, Gassner B, Jansen HW, Christensen EI. NHE3 serves as a
molecular tool for cAMP-mediated regulation of receptor-mediated
endocytosis. Am J Physiol Renal Physiol 283: F549 –F558, 2002.
165. Gekle M, Volker K, Mildenberger S, Freudinger R, Shull GE,
Wiemann M. NHE3 Na⫹/H⫹ exchanger supports proximal tubular
protein reabsorption in vivo. Am J Physiol Renal Physiol 287:
F469 –F473, 2004.
166. Gill R, Nazir TM, Wali R, Sitrin M, Brasitus TA, Ramaswamy
K, Dudeja PK. Regulation of rat ileal NHE3 by 1,25(OH)2-vitamin D3.
Dig Dis Sci 47: 1169 –1174, 2002.
167. Gill RK, Saksena S, Syed IA, Tyagi S, Alrefai WA, Malakooti
J, Ramaswamy K, Dudeja PK. Regulation of NHE3 by nitric oxide
in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 283:
G747–G756, 2002.
168. Gill RK, Saksena S, Tyagi S, Alrefai WA, Malakooti J, Sarwar
Z, Turner JR, Ramaswamy K, Dudeja PK. Serotonin inhibits
Na⫹/H⫹ exchange activity via 5-HT4 receptors and activation of
PKC alpha in human intestinal epithelial cells. Gastroenterology
128: 962–974, 2005.
169. Girardi AC, Thomson RB, Aronson PS. Identification of domain
of Na/H exchanger NHE3 mediating association with dipeptidyl
peptidase IV (DPPIV) (Abstract). FASEB J 19: A140, 2005.
170. Girardi AC, Degray BC, Nagy T, Biemesderfer D, Aronson PS.
Association of Na(⫹)-H(⫹) exchanger isoform NHE3 and dipeptidyl peptidase IV in the renal proximal tubule. J Biol Chem 276:
46671– 46677, 2001.
171. Girardi AC, Knauf F, Demuth HU, Aronson PS. Role of dipeptidyl peptidase IV in regulating activity of Na⫹/H⫹ exchanger isoform NHE3 in proximal tubule cells. Am J Physiol Cell Physiol 287:
C1238 –C1245, 2004.
172. Gisler SM, Stagljar I, Traebert M, Bacic D, Biber J, Murer H.
Interaction of the type II Na/Pi cotransporter with PDZ proteins.
J Biol Chem 276: 9206 –9213, 2001.
173. Gisler SM, Madjdpour C, Bacic D, Pribanic S, Taylor SS, Biber
J, Murer H. PDZK1. II. An anchoring site for the PKA-binding
protein D-AKAP2 in renal proximal tubular cells. Kidney Int 64:
1746 –1754, 2003.
174. Gisler SM, Pribanic S, Bacic D, Forrer P, Gantenbein A,
Sabourin LA, Tsuji A, Zhao ZS, Manser E, Biber J, Murer H.
PDZK1. I. A major scaffolder in brush borders of proximal tubular
cells. Kidney Int 64: 1733–1745, 2003.
175. Gomes P, Soares-da-Silva P. Dopamine acutely decreases type 3
Na(⫹)/H(⫹) exchanger activity in renal OK cells through the activation of protein kinases A and C signalling cascades. Eur J Pharmacol 488: 51–59, 2004.
176. Gonda T, Maouyo D, Rees SE, Montrose MH. Regulation of
intracellular pH gradients by identified Na/H exchanger isoforms
and a short-chain fatty acid. Am J Physiol Gastrointest Liver
Physiol 276: G259 –G270, 1999.
177. Gonzalez-Gronow M, Misra UK, Gawdi G, Pizzo SV. Association of plasminogen with dipeptidyl peptidase IV and Na⫹/H⫹
exchanger isoform NHE3 regulates invasion of human 1-LN prostate tumor cells. J Biol Chem 280: 27173–27178, 2005.
178. Good DW, Di Mari JF, Watts BA III. Hyposmolality stimulates
Na⫹/H⫹ exchange and HCO⫺
3 absorption in thick ascending limb
via PI 3-kinase. Am J Physiol Cell Physiol 279: C1443–C1454, 2000.
179. Good DW, George T, Watts BA III. Basolateral membrane
Na⫹/H⫹ exchange enhances HCO⫺
3 absorption in rat medullary
thick ascending limb: evidence for functional coupling between
basolateral and apical membrane Na⫹/H⫹ exchangers. Proc Natl
Acad Sci USA 92: 12525–12529, 1995.
180. Good DW, Watts BA III, George T, Meyer JW, Shull GE.
Transepithelial HCO⫺
3 absorption is defective in renal thick ascending limbs from Na⫹/H⫹ exchanger NHE1 null mutant mice. Am J
Physiol Renal Physiol 287: F1244 –F1249, 2004.
181. Goyal S, Mentone S, Aronson PS. Immunolocalization of NHE8
in rat kidney. Am J Physiol Renal Physiol 288: F530 –F538, 2005.
182. Goyal S, Vanden Heuvel G, Aronson PS. Renal expression of
novel Na⫹/H⫹ exchanger isoform NHE8. Am J Physiol Renal
Physiol 284: F467–F473, 2003.
183. Grahammer F, Henke G, Sandu C, Rexhepaj R, Hussain A,
Friedrich B, Risler T, Metzger M, Just L, Skutella T, Wulff P,
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
195.
196.
197.
198.
199.
200.
201.
Kuhl D, Lang F. Intestinal function of gene-targeted mice lacking
serum- and glucocorticoid-inducible kinase 1. Am J Physiol Gastrointest Liver Physiol 290: G1114 –G1123, 2006.
Guan Y, Dong J, Tackett L, Meyer JW, Shull GE, Montrose
MH. NHE2 is the main apical Na⫹/H⫹ exchanger in mouse colonic
crypts but an alternative Na⫹-dependent acid extrusion mechanism
is upregulated in NHE2-null mice. Am J Physiol Gastrointest Liver
Physiol 291: G689 –G699, 2006.
Guerra L, Fanelli T, Favia M, Riccardi SM, Busco G, Cardone
RA, Carrabino S, Weinman EJ, Reshkin SJ, Conese M,
Casavola V. Na⫹/H⫹ exchanger regulatory factor isoform 1 overexpression modulates cystic fibrosis transmembrane conductance
regulator (CFTR) expression and activity in human airway
16HBE14o- cells and rescues DeltaF508 CFTR functional expression in cystic fibrosis cells. J Biol Chem 280: 40925– 40933, 2005.
Guggino WB, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 7:
426 – 436, 2006.
Guner YS, Kiela PR, Xu H, Collins JF, Ghishan FK. Differential
regulation of renal sodium-phosphate transporter by glucocorticoids during rat ontogeny. Am J Physiol Cell Physiol 277: C884 –
C890, 1999.
Gupta N, Tarif SR, Seikaly M, Baum M. Role of glucocorticoids
in the maturation of the rat renal Na⫹/H⫹ antiporter (NHE3).
Kidney Int 60: 173–181, 2001.
Gutierrez-Ford C, Levay K, Gomes AV, Perera EM, Som T,
Kim YM, Benovic JL, Berkovitz GD, Slepak VZ. Characterization of tescalcin, a novel EF-hand protein with a single Ca2⫹binding site: metal-binding properties, localization in tissues and
cells, effect on calcineurin. Biochemistry 42: 14553–14565, 2003.
Haggie PM, Stanton BA, Verkman AS. Increased diffusional
mobility of CFTR at the plasma membrane after deletion of its
COOH terminal PDZ binding motif. J Biol Chem 279: 5494 –5500,
2004.
Hall RA, Premont RT, Chow CW, Blitzer JT, Pitcher JA,
Claing A, Stoffel RH, Barak LS, Shenolikar S, Weinman EJ,
Grinstein S, Lefkowitz RJ. The beta2-adrenergic receptor interacts with the Na⫹/H⫹-exchanger regulatory factor to control
Na⫹/H⫹ exchange. Nature 392: 626 – 630, 1998.
Hall RA, Spurney RF, Premont RT, Rahman N, Blitzer JT,
Pitcher JA, Lefkowitz RJ. G protein-coupled receptor kinase 6A
phosphorylates the Na(⫹)/H(⫹) exchanger regulatory factor via a
PDZ domain-mediated interaction. J Biol Chem 274: 24328 –24334,
1999.
Hamada K, Shimizu T, Matsui T, Tsukita S, Hakoshima T.
Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain. EMBO J 19: 4449 – 4462, 2000.
Han W, Kim KH, Jo MJ, Lee JH, Yang J, Doctor RB, Moe OW,
Lee J, Kim E, Lee MG. Shank2 associates with and regulates
Na⫹/H⫹ exchanger 3. J Biol Chem 281: 1461–1469, 2006.
Hanley RM, Shenolikar S, Pollack J, Steplock D, Weinman EJ.
Identification of calcium-calmodulin multifunctional protein kinase
II in rabbit kidney. Kidney Int 38: 63– 66, 1990.
Harris BZ, Lim WA. Mechanism and role of PDZ domains in
signaling complex assembly. J Cell Sci 114: 3219 –3231, 2001.
Hayashi H, Szaszi K, Coady-Osberg N, Furuya W, Bretscher
AP, Orlowski J, Grinstein S. Inhibition and redistribution of
NHE3, the apical Na⫹/H⫹ exchanger, by Clostridium difficile toxin
B. J Gen Physiol 123: 491–504, 2004.
Hayashi H, Szaszi K, Coady-Osberg N, Orlowski J, Kinsella
JL, Grinstein S. A slow pH-dependent conformational transition
underlies a novel mode of activation of the epithelial Na⫹/H⫹
exchanger-3 isoform. J Biol Chem 277: 11090 –11096, 2002.
He J, Lau AG, Yaffe MB, Hall RA. Phosphorylation and cell
cycle-dependent regulation of Na⫹/H⫹ exchanger regulatory factor-1 by Cdc2 kinase. J Biol Chem 276: 41559 – 41565, 2001.
He X, Tse CM, Donowitz M, Alper SL, Gabriel SE, Baum BJ.
Polarized distribution of key membrane transport proteins in the
rat submandibular gland. Pflügers Arch 433: 260 –268, 1997.
Hecht G, Hodges K, Gill RK, Kear F, Tyagi S, Malakooti J,
Ramaswamy K, Dudeja PK. Differential regulation of Na⫹/H⫹
exchange isoform activities by enteropathogenic E. coli in human
Physiol Rev • VOL
202.
203.
204.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
217.
219.
220.
863
intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol
287: G370 –G378, 2004.
Heiska L, Alfthan K, Gronholm M, Vilja P, Vaheri A, Carpen O.
Association of ezrin with intercellular adhesion molecule-1 and
-2 (ICAM-1 and ICAM-2). Regulation by phosphatidylinositol 4,
5-bisphosphate. J Biol Chem 273: 21893–21900, 1998.
Helmle-Kolb C, Di Sole F, Forgo J, Hilfiker H, Tse CM,
Casavola V, Donowitz M, Murer H. Regulation of the transfected
Na⫹/H⫹-exchanger NHE3 in MDCK cells by vasotocin. Pflügers
Arch 434: 123–131, 1997.
Hernando N, Deliot N, Gisler SM, Lederer E, Weinman EJ,
Biber J, Murer H. PDZ-domain interactions and apical expression
of type IIa Na/P(i) cotransporters. Proc Natl Acad Sci USA 99:
11957–11962, 2002.
Hernando N, Gisler SM, Pribanic S, Deliot N, Capuano P,
Wagner CA, Moe OW, Biber J, Murer H. NaPi-IIa and interacting
partners. J Physiol 567: 21–26, 2005.
Hernando N, Wagner CA, Gisler SM, Biber J, Murer H. PDZ
proteins and proximal ion transport. Curr Opin Nephrol Hypertens
13: 569 –574, 2004.
Hihnala S, Kujala M, Toppari J, Kere J, Holmberg C, Hoglund
P. Expression of SLC26A3, CFTR and NHE3 in the human male
reproductive tract: role in male subfertility caused by congenital
chloride diarrhoea. Mol Hum Reprod 12: 107–111, 2006.
Hisamitsu T, Pang T, Shigekawa M, Wakabayashi S. Dimeric
interaction between the cytoplasmic domains of the Na⫹/H⫹ exchanger NHE1 revealed by symmetrical intermolecular cross-linking and selective co-immunoprecipitation. Biochemistry 43: 11135–
11143, 2004.
Honegger KJ, Capuano P, Winter C, Bacic D, Stange G,
Wagner CA, Biber J, Murer H, Hernando N. Regulation of
sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange
protein directly activated by cAMP (EPAC). Proc Natl Acad Sci
USA 103: 803– 808, 2006.
Hoogerwerf WA, Tsao SC, Devuyst O, Levine SA, Yun CH, Yip
JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, Donowitz M.
NHE2 and NHE3 are human and rabbit intestinal brush-border
proteins. Am J Physiol Gastrointest Liver Physiol 270: G29 –G41,
1996.
Hryciw DH, Ekberg J, Ferguson C, Lee A, Wang D, Parton RG,
Pollock CA, Yun CC, Poronnik P. Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds. Interaction of Na⫹-H⫹
exchange regulatory factor-2 with ClC-5. J Biol Chem 281: 16068 –
16077, 2006.
Hu MC, Fan L, Crowder LA, Karim-Jimenez Z, Murer H, Moe
OW. Dopamine acutely stimulates Na⫹/H⫹ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation. J Biol Chem 276: 26906 –
26915, 2001.
Hu Z, Wang Y, Graham WV, Su L, Musch MW, Turner JR.
MAPKAPK-2 is a critical signaling intermediate in NHE3 activation
following Na⫹-glucose cotransport. J Biol Chem 81: 24247–24253,
2006.
Huang P, Steplock D, Weinman EJ, Hall RA, Ding Z, Li J,
Wang Y, Liu-Chen LY. kappa Opioid receptor interacts with
Na(⫹)/H(⫹)-exchanger regulatory factor-1/Ezrin-radixin-moesinbinding phosphoprotein-50 (NHERF-1/EBP50) to stimulate Na(⫹)/
H(⫹) exchange independent of G(i)/G(o) proteins. J Biol Chem
279: 25002–25009, 2004.
Hung AY, Sheng M. PDZ domains: structural modules for protein
complex assembly. J Biol Chem 277: 5699 –5702, 2002.
Hunte C, Screpanti E, Venturi M, Rimon A, Padan E, Michel
H. Structure of a Na⫹/H⫹ antiporter and insights into mechanism of
action and regulation by pH. Nature 435: 1197–1202, 2005.
Hwang JI, Heo K, Shin KJ, Kim E, Yun C, Ryu SH, Shin HS,
Suh PG. Regulation of phospholipase C-beta 3 activity by Na⫹/H⫹
exchanger regulatory factor 2. J Biol Chem 275: 16632–16637, 2000.
Ikeda T, Schmitt B, Pouyssegur J, Wakabayashi S, Shigekawa
M. Identification of cytoplasmic subdomains that control pH-sensing of the Na⫹/H⫹ exchanger (NHE1): pH-maintenance, ATP-sensitive, flexible loop domains. J Biochem 121: 295–303, 1997.
Ikuma M, Kashgarian M, Binder HJ, Rajendran VM. Differential regulation of NHE isoforms by sodium depletion in proximal
87 • JULY 2007 •
www.prv.org
864
221.
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
MARK DONOWITZ AND XUHANG LI
and distal segments of rat colon. Am J Physiol Gastrointest Liver
Physiol 276: G539 –G549, 1999.
Inoue H, Nakamura Y, Nagita M, Takai T, Masuda M, Nakamura N, Kanazawa H. Calcineurin homologous protein isoform 2
(CHP2), Na⫹/H⫹ exchangers-binding protein, is expressed in intestinal epithelium. Biol Pharm Bull 26: 148 –155, 2003.
Ishiki M, Klip A. Minireview: recent developments in the regulation of glucose transporter-4 traffic: new signals, locations, partners. Endocrinology 146: 5071–5078, 2005.
Jacob P, Rossmann H, Lamprecht G, Kretz A, Neff C, Lin-Wu
E, Gregor M, Groneberg DA, Kere J, Seidler U. Down-regulated
in adenoma mediates apical Cl⫺/HCO⫺
3 exchange in rabbit, rat,
human duodenum. Gastroenterology 122: 709 –724, 2002.
Janecki AJ, Janecki M, Akhter S, Donowitz M. Basic fibroblast
growth factor stimulates surface expression and activity of Na(⫹)/
H(⫹) exchanger NHE3 via mechanism involving phosphatidylinositol 3-kinase. J Biol Chem 275: 8133– 8142, 2000.
Janecki AJ, Montrose MH, Tse CM, de Medina FS, Zweibaum
A, Donowitz M. Development of an endogenous epithelial Na⫹/H⫹
exchanger (NHE3) in three clones of Caco-2 cells. Am J Physiol
Gastrointest Liver Physiol 277: G292–G305, 1999.
Janecki AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM,
Khurana S, Donowitz M. Subcellular redistribution is involved in
acute regulation of the brush border Na⫹/H⫹ exchanger isoform 3
in human colon adenocarcinoma cell line Caco-2. Protein kinase
C-mediated inhibition of the exchanger. J Biol Chem 273: 8790 –
8798, 1998.
Jiang Z, Asplin JR, Evan AP, Rajendran VM, Velazquez H,
Nottoli TP, Binder HJ, Aronson PS. Calcium oxalate urolithiasis
in mice lacking anion transporter Slc26a6. Nat Genet 38: 474 – 478,
2006.
Jin XH, Siragy HM, Guerrant RL, Carey RM. Compartmentalization of extracellular cGMP determines absorptive or secretory
responses in the rat jejunum. J Clin Invest 103: 167–174, 1999.
Jung Kang Y, Su Jeon E, Jin Lee H, Oh YS, Suh PG, Sup Jung
J, Donowitz M, Ho Kim J. NHERF2 increases platelet-derived
growth factor-induced proliferation through PI-3-kinase/Akt-,
ERK-, Src family kinase-dependent pathway. Cell Signal 16: 791–
800, 2004.
Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA,
Donowitz M, Hisaminato A, Fujiwara T, Ito Y, Cantley LC,
Yaffe MB. TAZ: a novel transcriptional co-activator regulated by
interactions with 14-3-3 and PDZ domain proteins. EMBO J 19:
6778 – 6791, 2000.
Kandasamy RA, Orlowski J. Genomic organization and glucocorticoid transcriptional activation of the rat Na⫹/H⫹ exchanger Nhe3
gene. J Biol Chem 271: 10551–10559, 1996.
Kandasamy RA, Yu FH, Harris R, Boucher A, Hanrahan JW,
Orlowski J. Plasma membrane Na⫹/H⫹ exchanger isoforms
(NHE-1, -2, -3) are differentially responsive to second messenger
agonists of the protein kinase A and C pathways. J Biol Chem 270:
29209 –29216, 1995.
Kandror KV, Pilch PF. Compartmentalization of protein traffic in
insulin-sensitive cells. Am J Physiol Endocrinol Metab 271: E1–
E14, 1996.
Kapus A, Grinstein S, Wasan S, Kandasamy R, Orlowski J.
Functional characterization of three isoforms of the Na⫹/H⫹ exchanger stably expressed in Chinese hamster ovary cells. ATP
dependence, osmotic sensitivity, role in cell proliferation. J Biol
Chem 269: 23544 –23552, 1994.
Karim ZG, Chambrey R, Chalumeau C, Defontaine N, Warnock
DG, Paillard M, Poggioli J. Regulation by PKC isoforms of
Na⫹/H⫹ exchanger in luminal membrane vesicles isolated from
cortical tubules. Am J Physiol Renal Physiol 277: F773–F778, 1999.
Kaunisto KM, Rajaniemi HJ. Expression and localization of the
Na⫹/H⫹ exchanger isoform NHE3 in the rat efferent ducts. J Androl
23: 237–241, 2002.
Kawagishi R, Tahara M, Sawada K, Morishige K, Sakata M,
Tasaka K, Murata Y. Na⫹/H⫹ exchanger-3 is involved in mouse
blastocyst formation. J Exp Zool A Comp Exp Biol 301: 767–775,
2004.
Keller KM, Wirth S, Baumann W, Sule D, Booth IW. Defective
jejunal brush border membrane sodium/proton exchange in assoPhysiol Rev • VOL
239.
240.
241.
242.
243.
244.
245.
246.
247.
248.
249.
250.
251.
252.
253.
254.
255.
256.
ciation with lethal familial protracted diarrhoea. Gut 31: 1156 –
1158, 1990.
Kennedy DJ, Leibach FH, Ganapathy V, Thwaites DT. Optimal
absorptive transport of the dipeptide glycylsarcosine is dependent
on functional Na⫹/H⫹ exchange activity. Pflügers Arch 445: 139 –
146, 2002.
Kennedy DJ, Raldua D, Thwaites DT. Dual modes of 5-(N-ethylN-isopropyl)amiloride modulation of apical dipeptide uptake in the
human small intestinal epithelial cell line Caco-2. Cell Mol Life Sci
62: 1621–1631, 2005.
Kenny AJ, Booth AG, George SG, Ingram J, Kershaw D, Wood
EJ, Young AR. Dipeptidyl peptidase IV, a kidney brush-border
serine peptidase. Biochem J 157: 169 –182, 1976.
Kerjaschki D, Farquhar MG. The pathogenic antigen of Heymann
nephritis is a membrane glycoprotein of the renal proximal tubule
brush border. Proc Natl Acad Sci USA 79: 5557–5561, 1982.
Khadeer MA, Tang Z, Tenenhouse HS, Eiden MV, Murer H,
Hernando N, Weinman EJ, Chellaiah MA, Gupta A. Na⫹-dependent phosphate transporters in the murine osteoclast: cellular
distribution and protein interactions. Am J Physiol Cell Physiol
284: C1633–C1644, 2003.
Khadilkar A, Iannuzzi P, Orlowski J. Identification of sites in
the second exomembrane loop and ninth transmembrane helix of
the mammalian Na⫹/H⫹ exchanger important for drug recognition
and cation translocation. J Biol Chem 276: 43792– 43800, 2001.
Khurana S, Kreydiyyeh S, Aronzon A, Hoogerwerf WA, Rhee
SG, Donowitz M, Cohen ME. Asymmetric signal transduction in
polarized ileal Na(⫹)-absorbing cells: carbachol activates brushborder but not basolateral-membrane PIP2-PLC and translocates
PLC-gamma 1 only to the brush border. Biochem J 313: 509 –518,
1996.
Khurana S, Nath SK, Levine SA, Bowser JM, Tse CM, Cohen
ME, Donowitz M. Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na⫹/H⫹ exchange. J Biol Chem 271: 9919 –9927, 1996.
Kiela PR, Guner YS, Xu H, Collins JF, Ghishan FK. Age- and
tissue-specific induction of NHE3 by glucocorticoids in the rat
small intestine. Am J Physiol Cell Physiol 278: C629 –C637, 2000.
Kim E, Sheng M. PDZ domain proteins of synapses. Nat Rev
Neurosci 5: 771–781, 2004.
Kim EJ, Jung YW, Kwon TH. Angiotensin II AT1 receptor blockade changes expression of renal sodium transporters in rats with
chronic renal failure. J Korean Med Sci 20: 248 –255, 2005.
Kim JH, Lee-Kwon W, Park JB, Ryu SH, Yun CH, Donowitz M.
Ca(2⫹)-dependent inhibition of Na⫹/H⫹ exchanger 3 (NHE3) requires an NHE3–E3KARP-alpha-actinin-4 complex for oligomerization and endocytosis. J Biol Chem 277: 23714 –23724, 2002.
Kim JY, Han W, Namkung W, Lee JH, Kim KH, Shin H, Kim E,
Lee MG. Inhibitory regulation of cystic fibrosis transmembrane
conductance regulator anion-transporting activities by Shank2.
J Biol Chem 279: 10389 –10396, 2004.
Kinsella JL, Heller P, Froehlich JP. Na⫹/H⫹ exchanger: proton
modifier site regulation of activity. Biochem Cell Biol 76: 743–749,
1998.
Kitano K, Yusa F, Hakoshima T. Structure of dimerized radixin
FERM domain suggests a novel masking motif in COOH terminal
residues 295–304. Acta Crystallograph Sect F Struct Biol Cryst
Commun 62: 340 –345, 2006.
Kiwull-Schone H, Wiemann M, Frede S, Bingmann D, Wirth
KJ, Heinelt U, Lang HJ, Kiwull P. A novel inhibitor of the
Na⫹/H⫹ exchanger type 3 activates the central respiratory CO2
response and lowers the apneic threshold. Am J Respir Crit Care
Med 164: 1303–1311, 2001.
Klanke CA, Su YR, Callen DF, Wang Z, Meneton P, Baird N,
Kandasamy RA, Orlowski J, Otterud BE, Leppert M. Molecular
cloning and physical and genetic mapping of a novel human
Na⫹/H⫹ exchanger (NHE5/SLC9A5) to chromosome 16q22 1.
Genomics 25: 615– 622, 1995.
Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl
PM. Insulin activates Na⫹/H⫹ exchanger 3: biphasic response and
glucocorticoid dependence. Am J Physiol Renal Physiol 283:
F532–F539, 2002.
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
257. Klisic J, Nief V, Reyes L, Ambuhl PM. Acute and chronic regulation of the renal Na/H exchanger NHE3 in rats with STZ-induced
diabetes mellitus. Nephron Physiol 102: p27–p35, 2005.
258. Klisic J, Zhang J, Nief V, Reyes L, Moe OW, Ambuhl PM.
Albumin regulates the Na⫹/H⫹ exchanger 3 in OKP cells. J Am Soc
Nephrol 14: 3008 –3016, 2003.
259. Knickelbein R, Aronson PS, Atherton W, Dobbins JW. Sodium
and chloride transport across rabbit ileal brush border. I. Evidence
for Na-H exchange. Am J Physiol Gastrointest Liver Physiol 245:
G504 –G510, 1983.
260. Knickelbein R, Aronson PS, Schron CM, Seifter J, Dobbins
JW. Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling. Am J Physiol Gastrointest Liver Physiol 249: G236 –G245,
1985.
261. Kocher O, Cheresh P, Lee SW. Identification and partial characterization of a novel membrane-associated protein (MAP17) upregulated in human carcinomas and modulating cell replication and
tumor growth. Am J Pathol 149: 493–500, 1996.
262. Kocher O, Pal R, Roberts M, Cirovic C, Gilchrist A. Targeted
disruption of the PDZK1 gene by homologous recombination. Mol
Cell Biol 23: 1175–1180, 2003.
263. Kocher O, Yesilaltay A, Cirovic C, Pal R, Rigotti A, Krieger M.
Targeted disruption of the PDZK1 gene in mice causes tissuespecific depletion of the high density lipoprotein receptor scavenger receptor class B type I and altered lipoprotein metabolism.
J Biol Chem 278: 52820 –52825, 2003.
264. Kocinsky HS, Girardi AC, Biemesderfer D, Nguyen T,
Mentone S, Orlowski J, Aronson PS. Use of phospho-specific
antibodies to determine the phosphorylation of endogenous
Na⫹/H⫹ exchanger NHE3 at PKA consensus sites. Am J Physiol
Renal Physiol 289: F249 –F258, 2005.
265. Koo BK, Jung YS, Shin J, Han I, Mortier E, Zimmermann P,
Whiteford JR, Couchman JR, Oh ES, Lee W. Structural basis of
syndecan-4 phosphorylation as a molecular switch to regulate signaling. J Mol Biol 355: 651– 663, 2006.
266. Kovbasnjuk O, Edidin M, Donowitz M. Role of lipid rafts in
Shiga toxin 1 interaction with the apical surface of Caco-2 cells.
J Cell Sci 114: 4025– 4031, 2001.
267. Krishnan S, Rajendran VM, Binder HJ. Apical NHE isoforms
differentially regulate butyrate-stimulated Na absorption in rat distal colon. Am J Physiol Cell Physiol 285: C1246 –C1254, 2003.
268. Kujala M, Tienari J, Lohi H, Elomaa O, Sariola H, Lehtonen E,
Kere J. SLC26A6 and SLC26A7 anion exchangers have a distinct
distribution in human kidney. Nephron Exp Nephrol 101: e50 –58,
2005.
269. Kurashima K, D’Souza S, Szaszi K, Ramjeesingh R, Orlowski
J, Grinstein S. The apical Na(⫹)/H(⫹) exchanger isoform NHE3
is regulated by the actin cytoskeleton. J Biol Chem 274: 29843–
29849, 1999.
270. Kurashima K, Szabo EZ, Lukacs G, Orlowski J, Grinstein S.
Endosomal recycling of the Na⫹/H⫹ exchanger NHE3 isoform is
regulated by the phosphatidylinositol 3-kinase pathway. J Biol
Chem 273: 20828 –20836, 1998.
271. Kurashima K, Yu FH, Cabado AG, Szabo EZ, Grinstein S,
Orlowski J. Identification of sites required for down-regulation of
Na⫹/H⫹ exchanger NHE3 activity by cAMP-dependent protein kinase. Phosphorylation-dependent and -independent mechanisms.
J Biol Chem 272: 28672–28679, 1997.
272. Kwon TH, Nielsen J, Kim YH, Knepper MA, Frokiaer J,
Nielsen S. Regulation of sodium transporters in the thick ascending limb of rat kidney: response to angiotensin II. Am J Physiol
Renal Physiol 285: F152–F165, 2003.
273. Laghmani K, Preisig PA, Moe OW, Yanagisawa M, Alpern RJ.
Endothelin-1/endothelin-B receptor-mediated increases in NHE3
activity in chronic metabolic acidosis. J Clin Invest 107: 1563–1569,
2001.
274. Laghmani K, Sakamoto A, Yanagisawa M, Preisig PA, Alpern
RJ. A consensus sequence in the endothelin-B receptor second
intracellular loop is required for NHE3 activation by endothelin-1.
Am J Physiol Renal Physiol 288: F732–F739, 2005.
275. Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun CC, Kalbacher
H, Gregor M, Seidler U. The down regulated in adenoma (dra)
Physiol Rev • VOL
276.
277.
278.
279.
280.
281.
282.
283.
284.
285.
286.
287.
288.
289.
290.
291.
292.
865
gene product binds to the second PDZ domain of the NHE3 kinase
A regulatory protein (E3KARP), potentially linking intestinal Cl⫺/
⫹
⫹
HCO⫺
exchange. Biochemistry 41: 12336 –
3 exchange to Na /H
12342, 2002.
Lamprecht G, Seidler UE. The emerging role of PDZ adapter
proteins for regulation of intestinal ion transport. Am J Physiol
Gastrointest Liver Physiol 291: G766 –G777, 2006.
Lamprecht G, Weinman EJ, Yun CH. The role of NHERF and
E3KARP in the cAMP-mediated inhibition of NHE3. J Biol Chem
273: 29972–29978, 1998.
Lang F, Henke G, Embark HM, Waldegger S, Palmada M,
Bohmer C, Vallon V. Regulation of channels by the serum and
glucocorticoid-inducible kinase: implications for transport, excitability and cell proliferation. Cell Physiol Biochem 13: 41–50, 2003.
Lardy H, Thomas M, Noordine ML, Bruneau A, Cherbuy C,
Vaugelade P, Philippe C, Colomb V, Duee PH. Changes induced
in colonocytes by extensive intestinal resection in rats. Dig Dis Sci
51: 326 –332, 2006.
Lau AG, Hall RA. Oligomerization of NHERF-1 and NHERF-2 PDZ
domains: differential regulation by association with receptor
carboxyl-termini and by phosphorylation. Biochemistry 40: 8572–
8580, 2001.
Lee J, Ha JH, Kim S, Oh Y, Kim SW. Caffeine decreases the
expression of Na⫹/K⫹-ATPase and the type 3 Na⫹/H⫹ exchanger in
rat kidney. Clin Exp Pharmacol Physiol 29: 559 –563, 2002.
Lee MG, Ahn W, Choi JY, Muallem S, Kim KH. A novel Na⫹dependent transporter and NHE3 mediate H⫹ efflux in the luminal
membrane of the pancreatic duct: regulation by cAMP. J Korean
Med Sci 15 Suppl: S29 –S30, 2000.
Lee MG, Schultheis PJ, Yan M, Shull GE, Bookstein C, Chang
E, Tse M, Donowitz M, Park K, Muallem S. Membrane-limited
expression and regulation of Na⫹-H⫹ exchanger isoforms by P2
receptors in the rat submandibular gland duct. J Physiol 513:
341–357, 1998.
Lee-Kwon W, Johns DC, Cha B, Cavet M, Park J, Tsichlis P,
Donowitz M. Constitutively active phosphatidylinositol 3-kinase
and AKT are sufficient to stimulate the epithelial Na⫹/H⫹ exchanger 3. J Biol Chem 276: 31296 –31304, 2001.
Lee-Kwon W, Kawano K, Choi JW, Kim JH, Donowitz M.
Lysophosphatidic acid stimulates brush border Na⫹/H⫹ exchanger
3 (NHE3) activity by increasing its exocytosis by an NHE3 kinase
A regulatory protein-dependent mechanism. J Biol Chem 278:
16494 –16501, 2003.
Lee-Kwon W, Kim JH, Choi JW, Kawano K, Cha B, Dartt DA,
Zoukhri D, Donowitz M. Ca2⫹-dependent inhibition of NHE3
requires PKC alpha which binds to E3KARP to decrease surface
NHE3 containing plasma membrane complexes. Am J Physiol Cell
Physiol 285: C1527–C1536, 2003.
Leheste JR, Rolinski B, Vorum H, Hilpert J, Nykjaer A,
Jacobsen C, Aucouturier P, Moskaug JO, Otto A, Christensen
EI, Willnow TE. Megalin knockout mice as an animal model of
low molecular weight proteinuria. Am J Pathol 155: 1361–1370,
1999.
Leong PK, Devillez A, Sandberg MB, Yang LE, Yip DK, Klein
JB, McDonough AA. Effects of ACE inhibition on proximal tubule
sodium transport. Am J Physiol Renal Physiol 290: F854 –F863,
2006.
Leong PK, Yang LE, Holstein-Rathlou NH, McDonough AA.
Angiotensin II clamp prevents the second step in renal apical NHE3
internalization during acute hypertension. Am J Physiol Renal
Physiol 283: F1142–F1150, 2002.
Leong PK, Yang LE, Lin HW, Holstein-Rathlou NH, McDonough AA. Acute hypotension induced by aortic clamp vs. PTH
provokes distinct proximal tubule Na⫹ transporter redistribution
patterns. Am J Physiol Regul Integr Comp Physiol 287: R878 –
R885, 2004.
Leung GP, Tse CM, Chew SB, Wong PY. Expression of multiple
Na⫹/H⫹ exchanger isoforms in cultured epithelial cells from rat
efferent duct and cauda epididymidis. Biol Reprod 64: 482– 490,
2001.
Levine SA, Montrose MH, Tse CM, Donowitz M. Kinetics and
regulation of three cloned mammalian Na⫹/H⫹ exchangers stably
87 • JULY 2007 •
www.prv.org
866
293.
294.
295.
296.
297.
298.
299.
300.
301.
302.
303.
304.
305.
306.
307.
308.
309.
310.
311.
MARK DONOWITZ AND XUHANG LI
expressed in a fibroblast cell line. J Biol Chem 268: 25527–25535,
1993.
Levine SA, Nath SK, Yun CH, Yip JW, Montrose M, Donowitz
M, Tse CM. Separate COOH terminal domains of the epithelial
specific brush border Na⫹/H⫹ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth
factors. J Biol Chem 270: 13716 –13725, 1995.
Li X, Ding J, Liu Y, Brix BJ, Fliegel L. Functional analysis of
acidic amino acids in the cytosolic tail of the Na⫹/H⫹ exchanger.
Biochemistry 43: 16477–16486, 2004.
Li X, Galli T, Leu S, Wade JB, Weinman EJ, Leung G, Cheong
A, Louvard D, Donowitz M. Na⫹-H⫹ exchanger 3 (NHE3) is
present in lipid rafts in the rabbit ileal brush border: a role for rafts
in trafficking and rapid stimulation of NHE3. J Physiol 537: 537–
552, 2001.
Li X, Leu S, Cheong A, Zhang H, Baibakov B, Shih C,
Birnbaum MJ, Donowitz M. Akt2, phosphatidylinositol 3-kinase,
PTEN are in lipid rafts of intestinal cells: role in absorption and
differentiation. Gastroenterology 126: 122–135, 2004.
Li X, Liu Y, Kay CM, Muller-Esterl W, Fliegel L. The Na⫹/H⫹
exchanger cytoplasmic tail: structure, function, interactions with
tescalcin. Biochemistry 42: 7448 –7456, 2003.
Li X, Zhang H, Cheong A, Leu S, Chen Y, Elowsky CG,
Donowitz M. Carbachol regulation of rabbit ileal brush border
Na⫹-H⫹ exchanger 3 (NHE3) occurs through changes in NHE3
trafficking and complex formation and is Src dependent. J Physiol
556: 791– 804, 2004.
Li XX, Xu J, Zheng S, Albrecht FE, Robillard JE, Eisner GM,
Jose PA. D(1) dopamine receptor regulation of NHE3 during development in spontaneously hypertensive rats. Am J Physiol Regul
Integr Comp Physiol 280: R1650 –R1656, 2001.
Li Y, Li J, Straight SW, Kershaw DB. PDZ domain-mediated
interaction of rabbit podocalyxin and Na(⫹)/H(⫹) exchange regulatory factor-2. Am J Physiol Renal Physiol 282: F1129 –F1139,
2002.
Licht C, Laghmani K, Yanagisawa M, Preisig PA, Alpern RJ.
An autocrine role for endothelin-1 in the regulation of proximal
tubule NHE3. Kidney Int 65: 1320 –1326, 2004.
Liedtke CM, Raghuram V, Yun CC, Wang X. Role of a PDZ1
domain of NHERF1 in the binding of airway epithelial RACK1 to
NHERF1. Am J Physiol Cell Physiol 286: C1037–C1044, 2004.
Liedtke CM, Yun CH, Kyle N, Wang D. Protein kinase C epsilondependent regulation of cystic fibrosis transmembrane regulator
involves binding to a receptor for activated C kinase (RACK1) and
RACK1 binding to Na⫹/H⫹ exchange regulatory factor. J Biol Chem
277: 22925–22933, 2002.
Lin X, Barber DL. A calcineurin homologous protein inhibits
GTPase-stimulated Na-H exchange. Proc Natl Acad Sci USA 93:
12631–12636, 1996.
Lin X, Voyno-Yasenetskaya TA, Hooley R, Lin CY, Orlowski J,
Barber DL. Galpha12 differentially regulates Na⫹-H⫹ exchanger
isoforms. J Biol Chem 271: 22604 –22610, 1996.
Lozupone F, Lugini L, Matarrese P, Luciani F, Federici C,
Iessi E, Margutti P, Stassi G, Malorni W, Fais S. Identification
and relevance of the CD95-binding domain in the N-terminal region
of ezrin. J Biol Chem 279: 9199 –9207, 2004.
Lucioni A, Womack C, Musch MW, Rocha FL, Bookstein C,
Chang EB. Metabolic acidosis in rats increases intestinal NHE2
and NHE3 expression and function. Am J Physiol Gastrointest
Liver Physiol 283: G51–G56, 2002.
Lundgren O, Peregrin AT, Persson K, Kordasti S, Uhnoo I,
Svensson L. Role of the enteric nervous system in the fluid and
electrolyte secretion of rotavirus diarrhea. Science 287: 491– 495,
2000.
Madara JL. Sodium-glucose cotransport and epithelial permeability. Gastroenterology 107: 319 –320, 1994.
Madara JL, Pappenheimer JR. Structural basis for physiological
regulation of paracellular pathways in intestinal epithelia. J Membr
Biol 100: 149 –164, 1987.
Madjdpour C, Bacic D, Kaissling B, Murer H, Biber J. Segmentspecific expression of sodium-phosphate cotransporters NaPi-IIa
and -IIc and interacting proteins in mouse renal proximal tubules.
Pflügers Arch 448: 402– 410, 2004.
Physiol Rev • VOL
312. Magro F, Fraga S, Soares-da-Silva P. Signaling of short- and
long-term regulation of intestinal epithelial type 1 Na⫹/H⫹ exchanger by interferon-gamma. Br J Pharmacol 145: 93–103, 2005.
313. Maher MM, Gontarek JD, Bess RS, Donowitz M, Yeo CJ. The
Na⫹/H⫹ exchange isoform NHE3 regulates basal canine ileal Na⫹
absorption in vivo. Gastroenterology 112: 174 –183, 1997.
314. Maher MM, Gontarek JD, Jimenez RE, Donowitz M, Yeo CJ.
Role of brush border Na⫹/H⫹ exchange in canine ileal absorption.
Dig Dis Sci 41: 651– 659, 1996.
315. Mahon MJ, Cole JA, Lederer ED, Segre GV. Na⫹/H⫹ exchangerregulatory factor 1 mediates inhibition of phosphate transport by
parathyroid hormone and second messengers by acting at multiple
sites in opossum kidney cells. Mol Endocrinol 17: 2355–2364, 2003.
316. Mahon MJ, Donowitz M, Yun CC, Segre GV. Na(⫹)/H(⫹) exchanger regulatory factor 2 directs parathyroid hormone 1 receptor
signalling. Nature 417: 858 – 861, 2002.
317. Mahon MJ, Segre GV. Stimulation by parathyroid hormone of a
NHERF-1-assembled complex consisting of the parathyroid hormone I receptor, phospholipase Cbeta, actin increases intracellular
calcium in opossum kidney cells. J Biol Chem 279: 23550 –23558,
2004.
318. Mailander J, Muller-Esterl W, Dedio J. Human homolog of
mouse tescalcin associates with Na(⫹)/H(⫹) exchanger type-1.
FEBS Lett 507: 331–335, 2001.
319. Markert T, Vaandrager AB, Gambaryan S, Pohler D, Hausler
C, Walter U, De Jonge HR, Jarchau T, Lohmann SM. Endogenous expression of type II cGMP-dependent protein kinase mRNA
and protein in rat intestine. Implications for cystic fibrosis transmembrane conductance regulator. J Clin Invest 96: 822– 830, 1995.
320. Matovcik LM, Haimowitz B, Goldenring JR, Czernik AJ,
Gorelick FS. Distribution of calcium/calmodulin-dependent protein kinase II in rat ileal enterocytes. Am J Physiol Cell Physiol 264:
C1029 –C1036, 1993.
320a.Matsushita M, Sano Y, Yokoyama S, Takai T, Inoue H, Mitsui
K, Todo K, Ohmori H, Kanazawa H. Loss of calcineurin, homologous protein 1 (CHP1) in chicken B lymphoma DT4O cells destabilizes Na⫹/H⫹ exchanger isoform 1 (NHE1) protein. Am J Physiol
Cell Physiol. In press.
321. McDonough AA, Biemesderfer D. Does membrane trafficking
play a role in regulating the sodium/hydrogen exchanger isoform 3
in the proximal tubule? Curr Opin Nephrol Hypertens 12: 533–541,
2003.
322. McDonough AA, Leong PK, Yang LE. Mechanisms of pressure
natriuresis: how blood pressure regulates renal sodium transport.
Ann NY Acad Sci 986: 669 – 677, 2003.
323. McSwine RL, Musch MW, Bookstein C, Xie Y, Rao M, Chang
EB. Regulation of apical membrane Na⫹/H⫹ exchangers NHE2 and
NHE3 in intestinal epithelial cell line C2/bbe. Am J Physiol Cell
Physiol 275: C693–C701, 1998.
324. Meder D, Shevchenko A, Simons K, Fullekrug J. Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical
domain during polarization of MDCK cells. J Cell Biol 168: 303–313,
2005.
325. Melvin JE, Park K, Richardson L, Schultheis PJ, Shull GE.
Mouse down-regulated in adenoma (DRA) is an intestinal Cl(-)/
HCO(3)(-) exchanger and is up-regulated in colon of mice lacking
the NHE3 Na(⫹)/H(⫹) exchanger. J Biol Chem 274: 22855–22861,
1999.
326. Mennone A, Biemesderfer D, Negoianu D, Yang CL, Abbiati T,
Schultheis PJ, Shull GE, Aronson PS, Boyer JL. Role of sodium/
hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes. Am J Physiol Gastrointest
Liver Physiol 280: G247–G254, 2001.
327. Misik AJ, Perreault K, Holmes CF, Fliegel L. Protein phosphatase regulation of Na⫹/H⫹ exchanger isoform I. Biochemistry 44:
5842–5852, 2005.
328. Miyazaki E, Sakaguchi M, Wakabayashi S, Shigekawa M,
Mihara K. NHE6 protein possesses a signal peptide destined for
endoplasmic reticulum membrane and localizes in secretory organelles of the cell. J Biol Chem 276: 49221– 49227, 2001.
329. Miyazaki H, Anzai N, Ekaratanawong S, Sakata T, Shin HJ,
Jutabha P, Hirata T, He X, Nonoguchi H, Tomita K, Kanai Y,
Endou H. Modulation of renal apical organic anion transporter 4
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
330.
331.
332.
333.
334.
335.
336.
337.
338.
339.
340.
341.
342.
343.
344.
345.
346.
function by two PDZ domain-containing proteins. J Am Soc Nephrol 16: 3498 –3506, 2005.
Moe OW. Acute regulation of proximal tubule apical membrane
Na/H exchanger NHE-3: role of phosphorylation, protein trafficking, regulatory factors. J Am Soc Nephrol 10: 2412–2425, 1999.
Moe OW, Amemiya M, Yamaji Y. Activation of protein kinase A
acutely inhibits and phosphorylates Na/H exchanger NHE-3. J Clin
Invest 96: 2187–2194, 1995.
Mohler PJ, Kreda SM, Boucher RC, Sudol M, Stutts MJ,
Milgram SL. Yes-associated protein 65 localizes p62(c-Yes) to the
apical compartment of airway epithelia by association with EBP50.
J Cell Biol 147: 879 – 890, 1999.
Morales FC, Takahashi Y, Kreimann EL, Georgescu MM.
Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 organizes
ERM proteins at the apical membrane of polarized epithelia. Proc
Natl Acad Sci USA 101: 17705–17710, 2004.
Mrkic B, Tse CM, Forgo J, Helmle-Kolb C, Donowitz M, Murer
H. Identification of PTH-responsive Na/H-exchanger isoforms in a
rabbit proximal tubule cell line (RKPC-2). Pflügers Arch 424: 377–
384, 1993.
Mukherjee S, Kallay L, Brett CL, Rao R. Mutational analysis of
the intramembranous H10 loop of yeast Nhx1 reveals a critical role
in ion homeostasis and vesicle trafficking. Biochem J 398: 97–105,
2006.
Muller T, Wijmenga C, Phillips AD, Janecke A, Houwen RH,
Fischer H, Ellemunter H, Fruhwirth M, Offner F, Hofer S,
Muller W, Booth IW, Heinz-Erian P. Congenital sodium diarrhea
is an autosomal recessive disorder of sodium/proton exchange but
unrelated to known candidate genes. Gastroenterology 119: 1506 –
1513, 2000.
Muller U, Brandsch M, Prasad PD, Fei YJ, Ganapathy V,
Leibach FH. Inhibition of the H⫹/peptide cotransporter in the
human intestinal cell line Caco-2 by cyclic AMP. Biochem Biophys
Res Commun 218: 461– 465, 1996.
Murtazina R, Kovbasnjuk O, Steplock D, Hogema B, Weinman
E, de Jonge H, Donowitz. M. Two-photon microscopic measurement of brush border (BB) NHE3 activity demonstrates that
NHERF1 is necessary for cAMP inhibition of NHE3 in mouse
proximal tubule but not in ileum. Gastroenterology 128: A367, 2005.
Murtazina R, Booth BJ, Bullis BL, Singh DN, Fliegel L. Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na⫹/H⫹
exchanger. Eur J Biochem 268: 4674 – 4685, 2001.
Murtazina R, Kovbasnjuk O, Donowitz M, Li X. Na⫹/H⫹ exchanger NHE3 activity and trafficking are lipid Raft-dependent.
J Biol Chem 281: 17845–17855, 2006.
Musch MW, Bookstein C, Xie Y, Sellin JH, Chang EB. SCFA
increase intestinal Na absorption by induction of NHE3 in rat colon
and human intestinal C2/bbe cells. Am J Physiol Gastrointest
Liver Physiol 280: G687–G693, 2001.
Nakamura N, Tanaka S, Teko Y, Mitsui K, Kanazawa H. Four
Na⫹/H⫹ exchanger isoforms are distributed to Golgi and post-Golgi
compartments and are involved in organelle pH regulation. J Biol
Chem 280: 1561–1572, 2005.
Nakamura T, Shibata N, Nishimoto-Shibata T, Feng D,
Ikemoto M, Motojima K, Iso ON, Tsukamoto K, Tsujimoto M,
Arai H. Regulation of SR-BI protein levels by phosphorylation of
its associated protein, PDZK1. Proc Natl Acad Sci USA 102: 13404 –
13409, 2005.
Naoe Y, Arita K, Hashimoto H, Kanazawa H, Sato M, Shimizu
T. Structural characterization of calcineurin B homologous protein
1. J Biol Chem 280: 32372–32378, 2005.
Narins SC, Park EH, Ramakrishnan R, Garcia FU, Diven JN,
Balin BJ, Hammond CJ, Sodam BR, Smith PR, Abedin MZ.
Functional characterization of Na(⫹)/H(⫹) exchangers in primary
cultures of prairie dog gallbladder. J Membr Biol 197: 123–134,
2004.
Nath SK, Hang CY, Levine SA, Yun CH, Montrose MH,
Donowitz M, Tse CM. Hyperosmolarity inhibits the Na⫹/H⫹ exchanger isoforms NHE2 and NHE3: an effect opposite to that on
NHE1. Am J Physiol Gastrointest Liver Physiol 270: G431–G441,
1996.
Physiol Rev • VOL
867
347. Nath SK, Kambadur R, Yun CH, Donowitz M, Tse CM. NHE2
contains subdomains in the COOH terminus for growth factor and
protein kinase regulation. Am J Physiol Cell Physiol 276: C873–
C882, 1999.
348. Nguyen R, Reczek D, Bretscher A. Hierarchy of merlin and ezrin
N- and COOH terminal domain interactions in homo- and heterotypic associations and their relationship to binding of scaffolding
proteins EBP50 and E3KARP. J Biol Chem 276: 7621–7629, 2001.
349. Nishiki T, Augustine GJ. Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2⫹-dependent neurotransmitter
release. J Neurosci 24: 8542– 8550, 2004.
350. Noonan WT, Woo AL, Nieman ML, Prasad V, Schultheis PJ,
Shull GE, Lorenz JN. Blood pressure maintenance in NHE3deficient mice with transgenic expression of NHE3 in small intestine. Am J Physiol Regul Integr Comp Physiol 288: R685–R691,
2005.
351. Noshiro R, Anzai N, Sakata T, Miyazaki H, Terada T, Shin HJ,
He X, Miura D, Inui K, Kanai Y, Endou H. The PDZ domain
protein PDZK1 interacts with human peptide transporter PEPT2
and enhances its transport activity. Kidney Int 70: 275–282, 2006.
352. Nourry C, Grant SG, Borg JP. PDZ domain proteins: plug and
play! Sci STKE 2003: RE7, 2003.
353. Numata M, Orlowski J. Molecular cloning and characterization of
a novel (Na⫹,K⫹)/H⫹ exchanger localized to the trans-Golgi network. J Biol Chem 276: 17387–17394, 2001.
354. Numata M, Petrecca K, Lake N, Orlowski J. Identification of a
mitochondrial Na⫹/H⫹ exchanger. J Biol Chem 273: 6951– 6959,
1998.
355. Obukhov AG, Nowycky MC. TRPC5 activation kinetics are modulated by the scaffolding protein ezrin/radixin/moesin-binding
phosphoprotein-50 (EBP50). J Cell Physiol 201: 227–235, 2004.
356. Oh YS, Jo NW, Choi JW, Kim HS, Seo SW, Kang KO, Hwang JI,
Heo K, Kim SH, Kim YH, Kim IH, Kim JH, Banno Y, Ryu SH,
Suh PG. NHERF2 specifically interacts with LPA2 receptor and
defines the specificity and efficiency of receptor-mediated phospholipase C-beta3 activation. Mol Cell Biol 24: 5069 –5079, 2004.
357. Orlowski J, Grinstein S. Diversity of the mammalian sodium/
proton exchanger SLC9 gene family. Pflügers Arch 447: 549 –565,
2004.
358. Orlowski J, Grinstein S. Na⫹/H⫹ exchangers of mammalian cells.
J Biol Chem 272: 22373–22376, 1997.
359. Orlowski J, Kandasamy RA, Shull GE. Molecular cloning of
putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, mRNA tissue expression of the
rat Na/H exchanger NHE-1 and two structurally related proteins.
J Biol Chem 267: 9331–9339, 1992.
360. Palmada M, Embark HM, Wyatt AW, Bohmer C, Lang F. Negative charge at the consensus sequence for the serum- and glucocorticoid-inducible kinase, SGK1, determines pH sensitivity of
the renal outer medullary K⫹ channel, ROMK1. Biochem Biophys
Res Commun 307: 967–972, 2003.
361. Palmada M, Poppendieck S, Embark HM, van de Graaf SF,
Boehmer C, Bindels RJ, Lang F. Requirement of PDZ domains
for the stimulation of the epithelial Ca2⫹ channel TRPV5 by the
NHE regulating factor NHERF2 and the serum and glucocorticoid
inducible kinase SGK1. Cell Physiol Biochem 15: 175–182, 2005.
362. Pang T, Hisamitsu T, Mori H, Shigekawa M, Wakabayashi S.
Role of calcineurin B homologous protein in pH regulation by the
Na⫹/H⫹ exchanger 1: tightly bound Ca2⫹ ions as important structural elements. Biochemistry 43: 3628 –3636, 2004.
363. Pang T, Su X, Wakabayashi S, Shigekawa M. Calcineurin homologous protein as an essential cofactor for Na⫹/H⫹ exchangers.
J Biol Chem 276: 17367–17372, 2001.
364. Pang T, Wakabayashi S, Shigekawa M. Expression of calcineurin B homologous protein 2 protects serum deprivation-induced cell death by serum-independent activation of Na⫹/H⫹ exchanger. J Biol Chem 277: 43771– 43777, 2002.
365. Park K, Olschowka JA, Richardson LA, Bookstein C, Chang
EB, Melvin JE. Expression of multiple Na⫹/H⫹ exchanger isoforms in rat parotid acinar and ductal cells. Am J Physiol Gastrointest Liver Physiol 276: G470 –G478, 1999.
87 • JULY 2007 •
www.prv.org
868
MARK DONOWITZ AND XUHANG LI
366. Park KS, Jeong MS, Kim JH, Jang SB. Overexpression, purification, characterization of PDZ domain proteins NHERF and
E3KARP in Escherichia coli. Protein Expr Purif 40: 197–202, 2005.
367. Pearson MA, Reczek D, Bretscher A, Karplus PA. Structure of
the ERM protein moesin reveals the FERM domain fold masked by
an extended actin binding tail domain. Cell 101: 259 –270, 2000.
368. Pedrosa R, Gomes P, Hopfer U, Jose PA, Soares-da-Silva P.
Gialpha3 protein-coupled dopamine D3 receptor-mediated inhibition of renal NHE3 activity in SHR proximal tubular cells is a
PLC-PKC-mediated event. Am J Physiol Renal Physiol 287: F1059 –
F1066, 2004.
369. Pedrosa R, Gomes P, Soares-da-Silva P. Distinct signalling cascades downstream to Gsalpha coupled dopamine D1-like NHE3
inhibition in rat and opossum renal epithelial cells. Cell Physiol
Biochem 14: 91–100, 2004.
370. Pedrosa R, Gomes P, Zeng C, Hopfer U, Jose PA, Soaresda-Silva P. Dopamine D3 receptor-mediated inhibition of Na⫹/H⫹
exchanger activity in normotensive and spontaneously hypertensive rat proximal tubular epithelial cells. Br J Pharmacol 142:
1343–1353, 2004.
371. Peng Y, Amemiya M, Yang X, Fan L, Moe OW, Yin H, Preisig
PA, Yanagisawa M, Alpern RJ. ET(B) receptor activation causes
exocytic insertion of NHE3 in OKP cells. Am J Physiol Renal
Physiol 280: F34 –F42, 2001.
372. Peng Y, Moe OW, Chu T, Preisig PA, Yanagisawa M, Alpern
RJ. ETB receptor activation leads to activation and phosphorylation of NHE3. Am J Physiol Cell Physiol 276: C938 –C945, 1999.
373. Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ. ClC-5
Cl⫺ channel disruption impairs endocytosis in a mouse model for
Dent’s disease. Nature 408: 369 –373, 2000.
374. Poggioli J, Karim Z, Paillard M. [Effect of angiotensin ii on
Na⫹/H⫹ exchangers of the renal tubule]. Nephrologie 19: 421– 425,
1998.
375. Praetorius J, Andreasen D, Jensen BL, Ainsworth MA, Friis
UG, Johansen T. NHE1, NHE2, NHE3 contribute to regulation of
intracellular pH in murine duodenal epithelial cells. Am J Physiol
Gastrointest Liver Physiol 278: G197–G206, 2000.
376. Pribanic S, Gisler SM, Bacic D, Madjdpour C, Hernando N,
Sorribas V, Gantenbein A, Biber J, Murer H. Interactions of
MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal
tubular cells. Am J Physiol Renal Physiol 285: F784 –F791, 2003.
377. Pushkin A, Abuladze N, Newman D, Muronets V, Sassani P,
Tatishchev S, Kurtz I. The COOH termini of NBC3 and the 56-kDa
H⫹-ATPase subunit are PDZ motifs involved in their interaction.
Am J Physiol Cell Physiol 284: C667–C673, 2003.
378. Pushkin A, Clark I, Kwon TH, Nielsen S, Kurtz I. Immunolocalization of NBC3 and NHE3 in the rat epididymis: colocalization
of NBC3 and the vacuolar H⫹-ATPase. J Androl 21: 708 –720, 2000.
379. Putney LK, Denker SP, Barber DL. The changing face of the
Na⫹/H⫹ exchanger, NHE1: structure, regulation, cellular actions.
Annu Rev Pharmacol Toxicol 42: 527–552, 2002.
380. Quinones H, McLeroy P, Hu MC, Price E, Mumby MC, Moe
OW. Protein phosphatase (PP2A) and the acute regulation of exchanger NHE3 by dopamine (DA): direct interaction with NHE3.
Am Soc Nephrol 8A: A41, 2001.
381. Raghuram V, Hormuth H, Foskett JK. A kinase-regulated mechanism controls CFTR channel gating by disrupting bivalent PDZ
domain interactions. Proc Natl Acad Sci USA 100: 9620 –9625, 2003.
382. Raghuram V, Mak DD, Foskett JK. Regulation of cystic fibrosis
transmembrane conductance regulator single-channel gating by
bivalent PDZ-domain-mediated interaction. Proc Natl Acad Sci
USA 98: 1300 –1305, 2001.
383. Rajendran VM, Binder HJ. Characterization of Na-H exchange in
apical membrane vesicles of rat colon. J Biol Chem 265: 8408 –
8414, 1990.
384. Rajendran VM, Geibel J, Binder HJ. Characterization of apical
membrane Cl-dependent Na/H exchange in crypt cells of rat distal
colon. Am J Physiol Gastrointest Liver Physiol 280: G400 –G405,
2001.
385. Rajendran VM, Geibel J, Binder HJ. Chloride-dependent Na-H
exchange. A novel mechanism of sodium transport in colonic
crypts. J Biol Chem 270: 11051–11054, 1995.
Physiol Rev • VOL
386. Ramakrishna BS, Venkataraman S, Srinivasan P, Dash P,
Young GP, Binder HJ. Amylase-resistant starch plus oral rehydration solution for cholera. N Engl J Med 342: 308 –313, 2000.
387. Reczek D, Berryman M, Bretscher A. Identification of EBP50: a
PDZ-containing phosphoprotein that associates with members of
the ezrin-radixin-moesin family. J Cell Biol 139: 169 –179, 1997.
388. Reinlib L, Mikkelsen R, Zahniser D, Dharmsathaphorn K,
Donowitz M. Carbachol-induced cytosolic free Ca2⫹ increases in
T84 colonic cells seen by microfluorimetry. Am J Physiol Gastrointest Liver Physiol 257: G950 –G960, 1989.
389. Repishti M, Hogan DL, Pratha V, Davydova L, Donowitz M,
Tse CM, Isenberg JI. Human duodenal mucosal brush border
Na(⫹)/H(⫹) exchangers NHE2 and NHE3 alter net bicarbonate
movement. Am J Physiol Gastrointest Liver Physiol 281: G159 –
G163, 2001.
390. Rocha F, Musch MW, Lishanskiy L, Bookstein C, Sugi K, Xie Y,
Chang EB. IFN-gamma downregulates expression of Na⫹/H⫹ exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe
cells. Am J Physiol Cell Physiol 280: C1224 –C1232, 2001.
391. Rood RP, Emmer E, Wesolek J, McCullen J, Husain Z, Cohen
ME, Braithwaite RS, Murer H, Sharp GW, Donowitz M. Regulation of the rabbit ileal brush-border Na⫹/H⫹ exchanger by an
ATP-requiring Ca2⫹/calmodulin-mediated process. J Clin Invest 82:
1091–1097, 1988.
392. Rossier BC. The epithelial sodium channel: activation by membrane-bound serine proteases. Proc Am Thorac Soc 1: 4 –9, 2004.
393. Rossmann H, Jacob P, Baisch S, Hassoun R, Meier J, Natour
D, Yahya K, Yun C, Biber J, Lackner KJ, Fiehn W, Gregor M,
Seidler U, Lamprecht G. The CFTR associated protein CAP70
interacts with the apical Cl⫺/HCO⫺
3 exchanger DRA in rabbit small
intestinal mucosa. Biochemistry 44: 4477– 4487, 2005.
394. Rossmann H, Sonnentag T, Heinzmann A, Seidler B, Bachmann O, Vieillard-Baron D, Gregor M, Seidler U. Differential
expression and regulation of Na⫹/H⫹ exchanger isoforms in rabbit
parietal and mucous cells. Am J Physiol Gastrointest Liver
Physiol 281: G447–G458, 2001.
395. Rutherford PA, Pizzonia JH, Biemesderfer D, Abu-Alfa A,
Reilly R, Aronson PS. Expression of Na⫹-H⫹ exchanger isoforms
NHE1 and NHE3 in kidney and blood cells of rabbit and rat. Exp
Nephrol 5: 490 – 497, 1997.
396. Sabolic I, Herak-Kramberger CM, Ljubojevic M, Biemesderfer D, Brown D. NHE3 and NHERF are targeted to the basolateral
membrane in proximal tubules of colchicine-treated rats. Kidney
Int 61: 1351–1364, 2002.
397. Saksena S, Gill RK, Syed IA, Tyagi S, Alrefai WA, Ramaswamy
K, Dudeja PK. Inhibition of apical Cl⫺/OH⫺ exchange activity in
Caco-2 cells by phorbol esters is mediated by PKCepsilon. Am J
Physiol Cell Physiol 283: C1492–C1500, 2002.
398. Saksena S, Gill RK, Tyagi S, Alrefai WA, Sarwar Z,
Ramaswamy K, Dudeja PK. Involvement of c-Src and protein
kinase C delta in the inhibition of Cl⫺/OH⫺ exchange activity in
Caco-2 cells by serotonin. J Biol Chem 280: 11859 –11868, 2005.
399. Sandu C, Artunc F, Palmada M, Rexhepaj R, Grahammer F,
Hussain A, Yun C, Alessi DR, Lang F. Impaired intestinal NHE3
activity in the PDK1 hypomorphic mouse. Am J Physiol Gastrointest Liver Physiol 291: G868 –G876, 2006.
400. Sangan P, Rajendran VM, Geibel JP, Binder HJ. Cloning and
expression of a chloride-dependent Na⫹-H⫹ exchanger. J Biol
Chem 277: 9668 –9675, 2002.
401. Sardet C, Franchi A, Pouyssegur J. Molecular cloning, primary
structure, expression of the human growth factor-activatable
Na⫹/H⫹ antiporter. Cell 56: 271–280, 1989.
402. Schmieder S, Nagai M, Orlando RA, Takeda T, Farquhar MG.
Podocalyxin activates RhoA and induces actin reorganization
through NHERF1 and Ezrin in MDCK cells. J Am Soc Nephrol 15:
2289 –2298, 2004.
403. Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani
M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T,
Giebisch G, Aronson PS, Lorenz JN, Shull GE. Renal and
intestinal absorptive defects in mice lacking the NHE3 Na⫹/H⫹
exchanger. Nat Genet 19: 282–285, 1998.
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
404. Schwark JR, Jansen HW, Lang HJ, Krick W, Burckhardt G,
Hropot M. S3226, a novel inhibitor of Na⫹/H⫹ exchanger subtype
3 in various cell types. Pflügers Arch 436: 797– 800, 1998.
405. Scott RO, Thelin WR, Milgram SL. A novel PDZ protein regulates
the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor. J Biol Chem 277: 22934 –22941, 2002.
406. Seidler U, Rossmann H, Jacob P, Bachmann O, Christiani S,
Lamprecht G, Gregor M. Expression and function of Na⫹HCO⫺
3
cotransporters in the gastrointestinal tract. Ann NY Acad Sci 915:
1–14, 2000.
407. Shenolikar S, Minkoff CM, Steplock DA, Evangelista C, Liu
M, Weinman EJ. N-terminal PDZ domain is required for NHERF
dimerization. FEBS Lett 489: 233–236, 2001.
408. Shenolikar S, Voltz JW, Cunningham R, Weinman EJ. Regulation of ion transport by the NHERF family of PDZ proteins. Physiology 19: 362–369, 2004.
409. Shenolikar S, Voltz JW, Minkoff CM, Wade JB, Weinman EJ.
Targeted disruption of the mouse NHERF-1 gene promotes internalization of proximal tubule sodium-phosphate cotransporter type
IIa and renal phosphate wasting. Proc Natl Acad Sci USA 99:
11470 –11475, 2002.
410. Shiue H, Musch MW, Wang Y, Chang EB, Turner JR. Akt2
phosphorylates ezrin to trigger NHE3 translocation and activation.
J Biol Chem 280: 1688 –1695, 2005.
411. Sidhu SS, Bader GD, Boone C. Functional genomics of intracellular peptide recognition domains with combinatorial biology
methods. Curr Opin Chem Biol 7: 97–102, 2003.
412. Silva NL, Haworth RS, Singh D, Fliegel L. The carboxyl-terminal region of the Na⫹/H⫹ exchanger interacts with mammalian heat
shock protein. Biochemistry 34: 10412–10420, 1995.
413. Silver DL, Wang N, Vogel S. Identification of small PDZK1-associated protein, DD96/MAP17, as a regulator of PDZK1 and plasma
high density lipoprotein levels. J Biol Chem 278: 28528 –28532,
2003.
414. Silviani V, Colombani V, Heyries L, Gerolami A, Cartouzou G,
Marteau C. Role of the NHE3 isoform of the Na⫹/H⫹ exchanger in
sodium absorption by the rabbit gallbladder. Pflügers Arch 432:
791–796, 1996.
415. Simpson JE, Walker NM, Schweinfest CW, Clarke LL. Downregulated in adenoma (DRA, SLC26a3) is the predominant ClHCO-3 exchanger in the lower villous epithelium of murine duodenum. Gastroenterology 130: 374, 2006.
416. Sitaraman SV, Wang L, Wong M, Bruewer M, Hobert M, Yun
CH, Merlin D, Madara JL. The adenosine 2b receptor is recruited
to the plasma membrane and associates with E3KARP and Ezrin
upon agonist stimulation. J Biol Chem 277: 33188 –33195, 2002.
417. Slepkov ER, Chow S, Lemieux MJ, Fliegel L. Proline residues in
transmembrane segment IV are critical for activity, expression and
targeting of the Na⫹/H⫹ exchanger isoform 1. Biochem J 379:
31–38, 2004.
418. Smith WJ, Nassar N, Bretscher A, Cerione RA, Karplus PA.
Structure of the active N-terminal domain of Ezrin. Conformational
and mobility changes identify keystone interactions. J Biol Chem
278: 4949 – 4956, 2003.
419. Soleimani M, Watts BA III, Singh G, Good DW. Effect of
long-term hyperosmolality on the Na⫹/H⫹ exchanger isoform
NHE-3 in LLC-PK1 cells. Kidney Int 53: 423– 431, 1998.
420. Somwar R, Sumitani S, Taha C, Sweeney G, Klip A. Temporal
activation of p70 S6 kinase and Akt1 by insulin: PI 3-kinase-dependent and -independent mechanisms. Am J Physiol Endocrinol
Metab 275: E618 –E625, 1998.
421. Steinbrecher KA, Mann EA, Giannella RA, Cohen MB. Increases in guanylin and uroguanylin in a mouse model of osmotic
diarrhea are guanylate cyclase C-independent. Gastroenterology
121: 1191–1202, 2001.
422. Su X, Pang T, Wakabayashi S, Shigekawa M. Evidence for
involvement of the putative first extracellular loop in differential
volume sensitivity of the Na⫹/H⫹ exchangers NHE1 and NHE2.
Biochemistry 42: 1086 –1094, 2003.
423. Suh PG, Hwang JI, Ryu SH, Donowitz M, Kim JH. The roles of
PDZ-containing proteins in PLC-beta-mediated signaling. Biochem
Biophys Res Commun 288: 1–7, 2001.
Physiol Rev • VOL
869
424. Sun AM, Liu Y, Centracchio J, Dworkin LD. Expression of
Na⫹/H⫹ exchanger isoforms in inner segment of inner medullary
collecting duct. J Membr Biol 164: 293–300, 1998.
425. Sun AM, Liu Y, Dworkin LD, Tse CM, Donowitz M, Yip KP.
Na⫹/H⫹ exchanger isoform 2 (NHE2) is expressed in the apical
membrane of the medullary thick ascending limb. J Membr Biol
160: 85–90, 1997.
426. Sun C, Mierke DF. Characterization of interactions of Na⫹/H⫹
exchanger regulatory factor-1 with the parathyroid hormone receptor and phospholipase C. J Pept Res 65: 411– 417, 2005.
427. Sun F, Hug MJ, Bradbury NA, Frizzell RA. Protein kinase A
associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin. J Biol Chem 275: 14360 –14366,
2000.
428. Sun F, Hug MJ, Lewarchik CM, Yun CH, Bradbury NA,
Frizzell RA. E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance
regulator in airway cells. J Biol Chem 275: 29539 –29546, 2000.
429. Sundaram U. Mechanism of intestinal absorption. Effect of
clonidine on rabbit ileal villus and crypt cells. J Clin Invest 95:
2187–2194, 1995.
430. Swiatecka-Urban A, Boyd C, Coutermarsh B, Karlson KH,
Barnaby R, Aschenbrenner L, Langford GM, Hasson T,
Stanton BA. Myosin VI regulates endocytosis of the cystic fibrosis
transmembrane conductance regulator. J Biol Chem 279: 38025–
38031, 2004.
431. Szaszi K, Grinstein S, Orlowski J, Kapus A. Regulation of the
epithelial Na(⫹)/H(⫹) exchanger isoform by the cytoskeleton. Cell
Physiol Biochem 10: 265–272, 2000.
432. Szaszi K, Kurashima K, Kaibuchi K, Grinstein S, Orlowski J.
Role of the cytoskeleton in mediating cAMP-dependent protein
kinase inhibition of the epithelial Na⫹/H⫹ exchanger NHE3. J Biol
Chem 276: 40761– 40768, 2001.
433. Szaszi K, Kurashima K, Kapus A, Paulsen A, Kaibuchi K,
Grinstein S, Orlowski J. RhoA and rho kinase regulate the epithelial Na⫹/H⫹ exchanger NHE3. Role of myosin light chain phosphorylation. J Biol Chem 275: 28599 –28606, 2000.
434. Szaszi K, Paulsen A, Szabo EZ, Numata M, Grinstein S,
Orlowski J. Clathrin-mediated endocytosis and recycling of the
neuron-specific Na⫹/H⫹ exchanger NHE5 isoform. Regulation by
phosphatidylinositol 3⬘-kinase and the actin cytoskeleton. J Biol
Chem 277: 42623– 42632, 2002.
435. Tai YH, Decker RA, Marnane WG, Charney AN, Donowitz M.
Effects of methylprednisolone on electrolyte transport by in vitro
rat ileum. Am J Physiol Gastrointest Liver Physiol 240: G365–
G370, 1981.
436. Takeda T. Podocyte cytoskeleton is connected to the integral
membrane protein podocalyxin through Na⫹/H⫹-exchanger regulatory factor 2 and ezrin. Clin Exp Nephrol 7: 260 –269, 2003.
437. Tang Y, Tang J, Chen Z, Trost C, Flockerzi V, Li M, Ramesh V,
Zhu MX. Association of mammalian trp4 and phospholipase C
isozymes with a PDZ domain-containing protein, NHERF. J Biol
Chem 275: 37559 –37564, 2000.
438. Tattersall A, Meredith D, Furla P, Shen MR, Ellory C, Wilkins
R. Molecular and functional identification of the Na(⫹)/H(⫹) exchange isoforms NHE1 and NHE3 in isolated bovine articular chondrocytes. Cell Physiol Biochem 13: 215–222, 2003.
439. Taylor L, Guerina VJ, Donowitz M, Cohen M, Sharp GW.
Calcium and calmodulin-dependent protein phosphorylation in rabbit ileum. FEBS Lett 131: 322–324, 1981.
440. Terawaki S, Maesaki R, Hakoshima T. Structural basis for
NHERF recognition by ERM proteins. Structure 14: 777–789, 2006.
441. Terawaki S, Maesaki R, Okada K, Hakoshima T. Crystallographic characterization of the radixin FERM domain bound to the
COOH terminal region of the human Na⫹/H⫹-exchanger regulatory
factor (NHERF). Acta Crystallogr D Biol Crystallogr 59: 177–179,
2003.
442. Thelin WR, Hodson CA, Milgram SL. Beyond the brush border:
NHERF4 blazes new NHERF turf. J Physiol 567: 13–19, 2005.
443. Thevenet L, Albrecht KH, Malki S, Berta P, Boizet-Bonhoure
B, Poulat F. NHERF2/SIP-1 interacts with mouse SRY via a different mechanism than human SRY. J Biol Chem 280: 38625–38630,
2005.
87 • JULY 2007 •
www.prv.org
870
MARK DONOWITZ AND XUHANG LI
444. Thomas GM, Rumbaugh GR, Harrar DB, Huganir RL. Ribosomal S6 kinase 2 interacts with and phosphorylates PDZ domaincontaining proteins and regulates AMPA receptor transmission.
Proc Natl Acad Sci USA 102: 15006 –15011, 2005.
445. Thomson RB, Wang T, Thomson BR, Tarrats L, Girardi A,
Mentone S, Soleimani M, Kocher O, Aronson PS. Role of
PDZK1 in membrane expression of renal brush border ion exchangers. Proc Natl Acad Sci USA 102: 13331–13336, 2005.
446. Thwaites DT, Ford D, Glanville M, Simmons NL. H(⫹)/soluteinduced intracellular acidification leads to selective activation of
apical Na(⫹)/H(⫹) exchange in human intestinal epithelial cells.
J Clin Invest 104: 629 – 635, 1999.
447. Thwaites DT, Kennedy DJ, Raldua D, Anderson CM, Mendoza
ME, Bladen CL, Simmons NL. H/dipeptide absorption across the
human intestinal epithelium is controlled indirectly via a functional
Na/H exchanger. Gastroenterology 122: 1322–1333, 2002.
448. Tse CM, Brant SR, Walker MS, Pouyssegur J, Donowitz M.
Cloning and sequencing of a rabbit cDNA encoding an intestinal
and kidney-specific Na⫹/H⫹ exchanger isoform (NHE-3). J Biol
Chem 267: 9340 –9346, 1992.
449. Tse CM, Levine SA, Yun CH, Brant SR, Pouyssegur J, Montrose MH, Donowitz M. Functional characteristics of a cloned
epithelial Na⫹/H⫹ exchanger (NHE3): resistance to amiloride and
inhibition by protein kinase C. Proc Natl Acad Sci USA 90: 9110 –
9114, 1993.
450. Tse CM, Levine SA, Yun CH, Montrose MH, Little PJ, Pouyssegur J, Donowitz M. Cloning and expression of a rabbit cDNA
encoding a serum-activated ethylisopropylamiloride-resistant epithelial Na⫹/H⫹ exchanger isoform (NHE-2). J Biol Chem 268:
11917–11924, 1993.
451. Tse CM, Ma AI, Yang VW, Watson AJ, Levine S, Montrose MH,
Potter J, Sardet C, Pouyssegur J, Donowitz M. Molecular
cloning and expression of a cDNA encoding the rabbit ileal villus
cell basolateral membrane Na⫹/H⫹ exchanger. EMBO J 10: 1957–
1967, 1991.
452. Tse M, Levine S, Yun C, Brant S, Counillon LT, Pouyssegur J,
Donowitz M. Structure/function studies of the epithelial isoforms
of the mammalian Na⫹/H⫹ exchanger gene family. J Membr Biol
135: 93–108, 1993.
453. Tsuganezawa H, Preisig PA, Alpern RJ. Dominant negative
c-Src inhibits angiotensin II induced activation of NHE3 in OKP
cells. Kidney Int 54: 394 –398, 1998.
454. Tsukita S, Oishi K, Sato N, Sagara J, Kawai A, Tsukita S. ERM
family members as molecular linkers between the cell surface
glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 126:
391– 401, 1994.
455. Tsunoda S, Zuker CS. The organization of INAD-signaling complexes by a multivalent PDZ domain protein in Drosophila photoreceptor cells ensures sensitivity and speed of signaling. Cell Calcium 26: 165–171, 1999.
456. Turban S, Beutler KT, Morris RG, Masilamani S, Fenton RA,
Knepper MA, Packer RK. Long-term regulation of proximal tubule acid-base transporter abundance by angiotensin II. Kidney Int
41: 1143–1150, 2006.
457. Turban S, Wang XY, Knepper MA. Regulation of NHE3, NKCC2,
NCC abundance in kidney during aldosterone escape phenomenon:
role of NO. Am J Physiol Renal Physiol 285: F843–F851, 2003.
458. Turner JR, Black ED. NHE3-dependent cytoplasmic alkalinization is triggered by Na(⫹)-glucose cotransport in intestinal epithelia. Am J Physiol Cell Physiol 281: C1533–C1541, 2001.
459. Turner JR, Black ED, Ward J, Tse CM, Uchwat FA, Alli HA,
Donowitz M, Madara JL, Angle JM. Transepithelial resistance
can be regulated by the intestinal brush-border Na(⫹)/H(⫹) exchanger NHE3. Am J Physiol Cell Physiol 279: C1918 –C1924, 2000.
460. Turner JR, Madara JL. Physiological regulation of intestinal
epithelial tight junctions as a consequence of Na(⫹)-coupled nutrient transport. Gastroenterology 109: 1391–1396, 1995.
461. Ueyama A, Yaworsky KL, Wang Q, Ebina Y, Klip A. GLUT-4myc
ectopic expression in L6 myoblasts generates a GLUT-4-specific
pool conferring insulin sensitivity. Am J Physiol Endocrinol Metab
277: E572–E578, 1999.
462. Vaandrager AB, Smolenski A, Tilly BC, Houtsmuller AB,
Ehlert EM, Bot AG, Edixhoven M, Boomaars WE, Lohmann
Physiol Rev • VOL
463.
464.
465.
466.
467.
468.
469.
470.
471.
472.
473.
474.
475.
476.
477.
478.
479.
480.
SM, de Jonge HR. Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl⫺ channel activation. Proc Natl Acad Sci USA 95:
1466 –1471, 1998.
Valiente M, Andres-Pons A, Gomar B, Torres J, Gil A, Tapparel C, Antonarakis SE, Pulido R. Binding of PTEN to specific
PDZ domains contributes to PTEN protein stability and phosphorylation by microtubule-associated serine/threonine kinases. J Biol
Chem 280: 28936 –28943, 2005.
Van de Graaf SF, Hoenderop JG, van der Kemp AW, Gisler
SM, Bindels RJ. Interaction of the epithelial Ca(2⫹) channels
TRPV5 and TRPV6 with the intestine- and kidney-enriched PDZ
protein NHERF4. Pflügers Arch 452: 407– 417, 2006.
Venema K, Belver A, Marin-Manzano MC, Rodriguez-Rosales
MP, Donaire JP. A novel intracellular K⫹/H⫹ antiporter related to
Na⫹/H⫹ antiporters is important for K⫹ ion homeostasis in plants.
J Biol Chem 278: 22453–22459, 2003.
Vidyasagar S, Barmeyer C, Geibel J, Binder HJ, Rajendran
VM. Role of short-chain fatty acids in colonic HCO(3) secretion.
Am J Physiol Gastrointest Liver Physiol 288: G1217–G1226, 2005.
Vidyasagar S, Rajendran VM, Binder HJ. Three distinct mechanisms of HCO⫺
3 secretion in rat distal colon. Am J Physiol Cell
Physiol 287: C612–C621, 2004.
Wade JB, Liu J, Coleman RA, Cunningham R, Steplock DA,
Lee-Kwon W, Pallone TL, Shenolikar S, Weinman EJ. Localization and interaction of NHERF isoforms in the renal proximal
tubule of the mouse. Am J Physiol Cell Physiol 285: C1494 –C1503,
2003.
Wade JB, Welling PA, Donowitz M, Shenolikar S, Weinman
EJ. Differential renal distribution of NHERF isoforms and their
colocalization with NHE3, ezrin, ROMK. Am J Physiol Cell Physiol
280: C192–C198, 2001.
Wakabayashi S, Bertrand B, Ikeda T, Pouyssegur J, Shigekawa
M. Mutation of calmodulin-binding site renders the Na⫹/H⫹ exchanger (NHE1) highly H⫹-sensitive and Ca2⫹ regulation-defective.
J Biol Chem 269: 13710 –13715, 1994.
Wakabayashi S, Hisamitsu T, Pang T, Shigekawa M. Kinetic
dissection of two distinct proton binding sites in Na⫹/H⫹ exchangers by measurement of reverse mode reaction. J Biol Chem 278:
43580 – 43585, 2003.
Wakabayashi S, Hisamitsu T, Pang T, Shigekawa M. Mutations
of Arg440 and Gly455/Gly456 oppositely change pH sensing of
Na⫹/H⫹ exchanger 1. J Biol Chem 278: 11828 –11835, 2003.
Wakabayashi S, Ikeda T, Iwamoto T, Pouyssegur J, Shigekawa
M. Calmodulin-binding autoinhibitory domain controls “pH-sensing” in the Na⫹/H⫹ exchanger NHE1 through sequence-specific
interaction. Biochemistry 36: 12854 –12861, 1997.
Wakabayashi S, Ikeda T, Noel J, Schmitt B, Orlowski J,
Pouyssegur J, Shigekawa M. Cytoplasmic domain of the ubiquitous Na⫹/H⫹ exchanger NHE1 can confer Ca2⫹ responsiveness to
the apical isoform NHE3. J Biol Chem 270: 26460 –26465, 1995.
Wakabayashi S, Pang T, Su X, Shigekawa M. A novel topology
model of the human Na(⫹)/H(⫹) exchanger isoform 1. J Biol Chem
275: 7942–7949, 2000.
Wakabayashi S, Shigekawa M, Pouyssegur J. Molecular physiology of vertebrate Na⫹/H⫹ exchangers. Physiol Rev 77: 51–74,
1997.
Wang D, Sun H, Lang F, Yun CC. Activation of NHE3 by dexamethasone requires phosphorylation of NHE3 at Ser663 by SGK1.
Am J Physiol Cell Physiol 289: C802–C810, 2005.
Wang P, Wang JJ, Xiao Y, Murray JW, Novikoff PM, Angeletti
RH, Orr GA, Lan D, Silver DL, Wolkoff AW. Interaction with
PDZK1 is required for expression of organic anion transporting
protein 1A1 on the hepatocyte surface. J Biol Chem 280: 30143–
30149, 2005.
Wang S, Raab RW, Schatz PJ, Guggino WB, Li M. Peptide
binding consensus of the NHE-RF-PDZ1 domain matches the
COOH terminal sequence of cystic fibrosis transmembrane conductance regulator (CFTR). FEBS Lett 427: 103–108, 1998.
Wang S, Yue H, Derin RB, Guggino WB, Li M. Accessory protein
facilitated CFTR-CFTR interaction, a molecular mechanism to potentiate the chloride channel activity. Cell 103: 169 –179, 2000.
87 • JULY 2007 •
www.prv.org
NHE3 BINDING PARTNERS AND COMPLEXES
481. Wang Y, Cai H, Cebotaru L, Hryciw DH, Weinman EJ, Donowitz M, Guggino SE, Guggino WB. ClC-5: role in endocytosis in
the proximal tubule. Am J Physiol Renal Physiol 289: F850 –F862,
2005.
482. Wang Z, Petrovic S, Mann E, Soleimani M. Identification of an
apical Cl⫺/HCO⫺
3 exchanger in the small intestine. Am J Physiol
Gastrointest Liver Physiol 282: G573–G579, 2002.
483. Wang Z, Wang T, Petrovic S, Tuo B, Riederer B, Barone S,
Lorenz JN, Seidler U, Aronson PS, Soleimani M. Renal and
intestinal transport defects in Slc26a6-null mice. Am J Physiol Cell
Physiol 288: C957–C965, 2005.
484. Ward RE, Schweizer L, Lamb RS, Fehon RG. The protein 4.1,
ezrin, radixin, moesin (FERM) domain of Drosophila Coracle, a
cytoplasmic component of the septate junction, provides functions
essential for embryonic development and imaginal cell proliferation. Genetics 159: 219 –228, 2001.
485. Watanabe C, Kato Y, Ito S, Kubo Y, Sai Y, Tsuji A. Na⫹/H⫹
exchanger 3 affects transport property of H⫹/oligopeptide transporter 1. Drug Metab Pharmacokinet 20: 443– 451, 2005.
486. Watson RT, Pessin JE. Intracellular organization of insulin signaling and GLUT4 translocation. Recent Prog Horm Res 56: 175–
193, 2001.
487. Watson RT, Shigematsu S, Chiang SH, Mora S, Kanzaki M,
Macara IG, Saltiel AR, Pessin JE. Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and
GLUT4 translocation. J Cell Biol 154: 829 – 840, 2001.
488. Watts BA III, George T, Good DW. The basolateral NHE1
Na⫹/H⫹ exchanger regulates transepithelial HCO⫺
absorption
3
through actin cytoskeleton remodeling in renal thick ascending
limb. J Biol Chem 280: 11439 –11447, 2005.
489. Watts BA III, Good DW. Hyposmolality stimulates apical membrane Na(⫹)/H(⫹) exchange and HCO(3)(⫺) absorption in renal
thick ascending limb. J Clin Invest 104: 1593–1602, 1999.
490. Weinman E, Hanley R, Morell G, Yuan N, Steplock D, Bui G,
Shenolikar S. Regulation of the renal Na⫹-H⫹ exchanger by calcium calmodulin-dependent multifunctional protein kinase II.
Miner Electrolyte Metab 18: 35–39, 1992.
491. Weinman EJ, Boddeti A, Cunningham R, Akom M, Wang F,
Wang Y, Liu J, Steplock D, Shenolikar S, Wade JB. NHERF-1 is
required for renal adaptation to a low-phosphate diet. Am J Physiol
Renal Physiol 285: F1225–F1232, 2003.
492. Weinman EJ, Cunningham R, Wade JB, Shenolikar S. The role
of NHERF-1 in the regulation of renal proximal tubule sodiumhydrogen exchanger 3 and sodium-dependent phosphate cotransporter 2a. J Physiol 567: 27–32, 2005.
493. Weinman EJ, Dubinsky W, Shenolikar S. Regulation of the renal
Na⫹-H⫹ exchanger by protein phosphorylation. Kidney Int 36:
519 –525, 1989.
494. Weinman EJ, Minkoff C, Shenolikar S. Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by
PKA. Am J Physiol Renal Physiol 279: F393–F399, 2000.
495. Weinman EJ, Mohanlal V, Stoycheff N, Wang F, Steplock D,
Shenolikar S, Cunningham R. Longitudinal study of urinary excretion of phosphate, calcium, uric acid in mutant NHERF-1 null
mice. Am J Physiol Renal Physiol 290: F838 –F843, 2006.
496. Weinman EJ, Steplock D, Bui G, Yuan N, Shenolikar S. Regulation of renal Na⫹-H⫹ exchanger by cAMP-dependent protein
kinase. Am J Physiol Renal Fluid Electrolyte Physiol 258: F1254 –
F1258, 1990.
497. Weinman EJ, Steplock D, Donowitz M, Shenolikar S. NHERF
associations with sodium-hydrogen exchanger isoform 3 (NHE3)
and ezrin are essential for cAMP-mediated phosphorylation and
inhibition of NHE3. Biochemistry 39: 6123– 6129, 2000.
498. Weinman EJ, Steplock D, Shenolikar S. Acute regulation of
NHE3 by protein kinase A requires a multiprotein signal complex.
Kidney Int 60: 450 – 454, 2001.
499. Weinman EJ, Steplock D, Shenolikar S. cAMP-mediated inhibition of the renal brush border membrane Na⫹-H⫹ exchanger requires a dissociable phosphoprotein cofactor. J Clin Invest 92:
1781–1786, 1993.
500. Weinman EJ, Steplock D, Shenolikar S. NHERF-1 uniquely
transduces the cAMP signals that inhibit sodium-hydrogen exPhysiol Rev • VOL
501.
502.
503.
504.
505.
506.
507.
508.
509.
510.
511.
512.
513.
514.
515.
516.
517.
518.
519.
871
change in mouse renal apical membranes. FEBS Lett 536: 141–144,
2003.
Weinman EJ, Steplock D, Tate K, Hall RA, Spurney RF, Shenolikar S. Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF). J Clin Invest 101: 2199 –2206, 1998.
Weinman EJ, Steplock D, Wade JB, Shenolikar S. Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition
of Na⫹/H⫹ exchange in OK cells. Am J Physiol Renal Physiol 281:
F374 –F380, 2001.
Weinman EJ, Steplock D, Wang Y, Shenolikar S. Characterization of a protein cofactor that mediates protein kinase A regulation
of the renal brush border membrane Na⫹-H⫹ exchanger. J Clin
Invest 95: 2143–2149, 1995.
Weinman EJ, Wang Y, Wang F, Greer C, Steplock D, Shenolikar S. A COOH terminal PDZ motif in NHE3 binds NHERF-1 and
enhances cAMP inhibition of sodium-hydrogen exchange. Biochemistry 42: 12662–12668, 2003.
Wells KM, Rao R. The yeast Na⫹/H⫹ exchanger Nhx1 is an
N-linked glycoprotein. Topological implications. J Biol Chem 276:
3401–3407, 2001.
Wiederkehr MR, Di Sole F, Collazo R, Quinones H, Fan L,
Murer H, Helmle-Kolb C, Moe OW. Characterization of acute
inhibition of Na/H exchanger NHE-3 by dopamine in opossum
kidney cells. Kidney Int 59: 197–209, 2001.
Wiederkehr MR, Zhao H, Moe OW. Acute regulation of Na/H
exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation. Am J Physiol Cell Physiol 276: C1205–C1217, 1999.
Wiemann M, Kiwull-Schone H, Frede S, Bingmann D, Kiwull
P. Brainstem NHE-3 expression and control of breathing. Adv Exp
Med Biol 551: 39 – 44, 2004.
Wilson JM, Laurent P, Tufts BL, Benos DJ, Donowitz M, Vogl
AW, Randall DJ. NaCl uptake by the branchial epithelium in
freshwater teleost fish: an immunological approach to ion-transport protein localization. J Exp Biol 203: 2279 –2296, 2000.
Woo AL, Gildea LA, Tack LM, Miller ML, Spicer Z, Millhorn
DE, Finkelman FD, Hassett DJ, Shull GE. In vivo evidence for
interferon-gamma-mediated homeostatic mechanisms in small intestine of the NHE3 Na⫹/H⫹ exchanger knockout model of congenital diarrhea. J Biol Chem 277: 49036 – 49046, 2002.
Woo AL, Noonan WT, Schultheis PJ, Neumann JC, Manning
PA, Lorenz JN, Shull GE. Renal function in NHE3-deficient mice
with transgenic rescue of small intestinal absorptive defect. Am J
Physiol Renal Physiol 284: F1190 –F1198, 2003.
Wormmeester L, Sanchez de Medina F, Kokke F, Tse CM,
Khurana S, Bowser J, Cohen ME, Donowitz M. Quantitative
contribution of NHE2 and NHE3 to rabbit ileal brush-border
Na⫹/H⫹ exchange. Am J Physiol Cell Physiol 274: C1261–C1272,
1998.
Xu H, Chen R, Ghishan FK. Subcloning, localization, expression
of the rat intestinal sodium-hydrogen exchanger isoform 8. Am J
Physiol Gastrointest Liver Physiol 289: G36 –G41, 2005.
Xu J, Li XX, Albrecht FE, Hopfer U, Carey RM, Jose PA.
Dopamine(1) receptor, G(salpha), Na(⫹)-H(⫹) exchanger interactions in the kidney in hypertension. Hypertension 36: 395–399,
2000.
Xu L, Dixit MP, Nullmeyer KD, Xu H, Kiela PR, Lynch RM,
Ghishan FK. Regulation of Na⫹/H⫹ exchanger-NHE3 by angiotensin-II in OKP cells. Biochim Biophys Acta 1758: 519 –526, 2006.
Xue J, Douglas RM, Zhou D, Lim JY, Boron WF, Haddad GG.
Expression of Na⫹/H⫹ and HCO⫺
3 -dependent transporters in
Na⫹/H⫹ exchanger isoform 1 null mutant mouse brain. Neuroscience 122: 37– 46, 2003.
Yamamoto Y, Taniguchi K. Distribution of pH regulators in the
rat laryngeal nerve: the spatial relationship between Na⫹/HCO⫺
3
cotransporters and Na⫹/H⫹ exchanger type 3. Neurosci Lett 368:
127–129, 2004.
Yammani RR, Seetharam S, Seetharam B. Cubilin and megalin
expression and their interaction in the rat intestine: effect of thyroidectomy. Am J Physiol Endocrinol Metab 281: E900 –E907,
2001.
Yang L, Leong PK, Chen JO, Patel N, Hamm-Alvarez SF,
McDonough AA. Acute hypertension provokes internalization of
87 • JULY 2007 •
www.prv.org
872
520.
521.
522.
523.
524.
525.
526.
527.
528.
529.
530.
531.
532.
533.
MARK DONOWITZ AND XUHANG LI
proximal tubule NHE3 without inhibition of transport activity.
Am J Physiol Renal Physiol 282: F730 –F740, 2002.
Yang LE, Leong PK, Ye S, Campese VM, McDonough AA.
Responses of proximal tubule sodium transporters to acute injuryinduced hypertension. Am J Physiol Renal Physiol 284: F313–F322,
2003.
Yang LE, Maunsbach AB, Leong PK, McDonough AA. Differential traffic of proximal tubule Na⫹ transporters during hypertension
or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes. Am J
Physiol Renal Physiol 287: F896 –F906, 2004.
Yang LE, Maunsbach AB, Leong PK, McDonough AA. Redistribution of myosin VI from top to base of proximal tubule microvilli
during acute hypertension. J Am Soc Nephrol 16: 2890 –2896, 2005.
Yang X, Amemiya M, Peng Y, Moe OW, Preisig PA, Alpern RJ.
Acid incubation causes exocytic insertion of NHE3 in OKP cells.
Am J Physiol Cell Physiol 279: C410 –C419, 2000.
Yeo CJ, Barry K, Gontarek JD, Donowitz M. Na⫹/H⫹ exchange
mediates meal-stimulated ileal absorption. Surgery 116: 388 –394,
1994.
Yip JW, Ko WH, Viberti G, Huganir RL, Donowitz M, Tse CM.
Regulation of the epithelial brush border Na⫹/H⫹ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor
and phorbol esters is not through changes in phosphorylation of
the exchanger. J Biol Chem 272: 18473–18480, 1997.
Yip KP, Tse CM, McDonough AA, Marsh DJ. Redistribution of
Na⫹/H⫹ exchanger isoform NHE3 in proximal tubules induced by
acute and chronic hypertension. Am J Physiol Renal Physiol 275:
F565–F575, 1998.
Yonemura S, Hirao M, Doi Y, Takahashi N, Kondo T, Tsukita
S, Tsukita S. Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, ICAM-2. J Cell Biol 140: 885– 895, 1998.
Yun CC. Concerted roles of SGK1 and the Na⫹/H⫹ exchanger
regulatory factor 2 (NHERF2) in regulation of NHE3. Cell Physiol
Biochem 13: 29 – 40, 2003.
Yun CC, Chen Y, Lang F. Glucocorticoid activation of Na(⫹)/
H(⫹) exchanger isoform 3 revisited. The roles of SGK1 and
NHERF2. J Biol Chem 277: 7676 –7683, 2002.
Yun CC, Palmada M, Embark HM, Fedorenko O, Feng Y,
Henke G, Setiawan I, Boehmer C, Weinman EJ, Sandrasagra
S, Korbmacher C, Cohen P, Pearce D, Lang F. The serum and
glucocorticoid-inducible kinase SGK1 and the Na⫹/H⫹ exchange
regulating factor NHERF2 synergize to stimulate the renal outer
medullary K⫹ channel ROMK1. J Am Soc Nephrol 13: 2823–2830,
2002.
Yun CH, Gurubhagavatula S, Levine SA, Montgomery JL,
Brant SR, Cohen ME, Cragoe EJ Jr, Pouyssegur J, Tse CM,
Donowitz M. Glucocorticoid stimulation of ileal Na⫹ absorptive
cell brush border Na⫹/H⫹ exchange and association with an increase in message for NHE-3, an epithelial Na⫹/H⫹ exchanger
isoform. J Biol Chem 268: 206 –211, 1993.
Yun CH, Lamprecht G, Forster DV, Sidor A. NHE3 kinase A
regulatory protein E3KARP binds the epithelial brush border
Na⫹/H⫹ exchanger NHE3 and the cytoskeletal protein ezrin. J Biol
Chem 273: 25856 –25863, 1998.
Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman
EJ, Donowitz M. cAMP-mediated inhibition of the epithelial brush
border Na⫹/H⫹ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci USA 94: 3010 –3015, 1997.
Physiol Rev • VOL
534. Yun CH, Tse CM, Donowitz M. Chimeric Na⫹/H⫹ exchangers: an
epithelial membrane-bound N-terminal domain requires an epithelial cytoplasmic COOH terminal domain for regulation by protein
kinases. Proc Natl Acad Sci USA 92: 10723–10727, 1995.
535. Zachos N, Li X, Hodson C, Cha B, Chen Y, Milgram S,
Donowitz M. Brush border (BB) PDZ proteins and IKEPP (intestinal and Kidney Epithelial PDZ Domain Protein) differentially
regulate NHE3 in response to elevated Ca2⫹ inhibition with PDZK1
and stimulation with IKEPP (Abstract). Gastroenterology 128: A32,
2005.
536. Zachos N, Li X, Kovbasnjuk O, Sarker R, Hogema B, de Jonge
H, Donowitz M. Elevated intracellular calcium [Ca2⫹]i inhibits
NHE3 activity by a PDZK1 (NHERF3) dependent process in which
NHE3 and PDZK1 bind basally and dissociate with elevated [Ca2⫹]I
(Abstract). Gastroenterology 130: A51, 2006.
537. Zachos NC, Tse M, Donowitz M. Molecular physiology of intestinal Na⫹/H⫹ exchange. Annu Rev Physiol 67: 411– 443, 2005.
538. Zhang M, Wang W. Organization of signaling complexes by PDZdomain scaffold proteins. Acc Chem Res 36: 530 –538, 2003.
539. Zhang Y, Magyar CE, Norian JM, Holstein-Rathlou NH,
Mircheff AK, McDonough AA. Reversible effects of acute hypertension on proximal tubule sodium transporters. Am J Physiol Cell
Physiol 274: C1090 –C1100, 1998.
540. Zhang Y, Mircheff AK, Hensley CB, Magyar CE, Warnock DG,
Chambrey R, Yip KP, Marsh DJ, Holstein-Rathlou NH,
McDonough AA. Rapid redistribution and inhibition of renal sodium transporters during acute pressure natriuresis. Am J Physiol
Renal Fluid Electrolyte Physiol 270: F1004 –F1014, 1996.
541. Zhang Y, Norian JM, Magyar CE, Holstein-Rathlou NH,
Mircheff AK, McDonough AA. In vivo PTH provokes apical
NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition. Am J
Physiol Renal Physiol 276: F711–F719, 1999.
542. Zhao H, Shiue H, Palkon S, Wang Y, Cullinan P, Burkhardt JK,
Musch MW, Chang EB, Turner JR. Ezrin regulates NHE3 translocation and activation after Na⫹-glucose cotransport. Proc Natl
Acad Sci USA 101: 9485–9490, 2004.
543. Zhao H, Wiederkehr MR, Fan L, Collazo RL, Crowder LA, Moe
OW. Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of
protein kinase a and NHE-3 phosphoserines 552 and 605. J Biol
Chem 274: 3978 –3987, 1999.
544. Zhou M, Sevilla L, Vallega G, Chen P, Palacin M, Zorzano A,
Pilch PF, Kandror KV. Insulin-dependent protein trafficking in
skeletal muscle cells. Am J Physiol Endocrinol Metab 275: E187–
E196, 1998.
545. Zizak M, Bartonicek D, Cha B, Murtazina R, Kim JH, LeeKwon W, Gorelick F, Tse M, Donowitz M. Calcium/calmodulin
dependent protein kinase II constitutively binds and regulates the
ileal BB Na/H exchanger NHE3. Gastroenterology 124: T1034, 2003.
546. Zizak M, Cavet ME, Bayle D, Tse CM, Hallen S, Sachs G,
Donowitz M. Na(⫹)/H(⫹) exchanger NHE3 has 11 membrane
spanning domains and a cleaved signal peptide: topology analysis
using in vitro transcription/translation. Biochemistry 39: 8102–
8112, 2000.
547. Zizak M, Lamprecht G, Steplock D, Tariq N, Shenolikar S,
Donowitz M, Yun CH, Weinman EJ. cAMP-induced phosphorylation and inhibition of Na(⫹)/H(⫹) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE
regulatory factor. J Biol Chem 274: 24753–24758, 1999.
87 • JULY 2007 •
www.prv.org