Physiol Rev 95: 377– 404, 2015
doi:10.1152/physrev.00015.2014
THE EVOLVING IMPACT OF G PROTEIN-COUPLED
RECEPTOR KINASES IN CARDIAC HEALTH
AND DISEASE
Priscila Y. Sato, J. Kurt Chuprun, Mathew Schwartz, and Walter J. Koch
Center for Translational Medicine and Department of Pharmacology, Temple University School of Medicine,
Philadelphia, Pennsylvania; and Advanced Institutes of Convergence Technology, Suwon, South Korea
Sato PY, Chuprun JK, Schwartz M, Koch WJ. The Evolving Impact of G ProteinCoupled Receptor Kinases in Cardiac Health and Disease. Physiol Rev 95: 377– 404,
2015; doi:10.1152/physrev.00015.2014.—G protein-coupled receptors (GPCRs)
are important regulators of various cellular functions via activation of intracellular
signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and
subsequent ␤-arrestin-mediated mechanisms including phosphorylation, internalization, and either
receptor degradation or resensitization. The seven-member GRK family varies in their structural
composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus
our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated
signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been
highly implicated in various etiologies of cardiovascular disease including hypertension and heart
failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in
cardiac homeostasis and disease progression, and herein we review the current data on GRKs
related to cardiac disease and discuss their potential in the development of novel therapeutic
strategies in cardiac diseases including heart failure.
L
I.
II.
III.
IV.
V.
VI.
INTRODUCTION
G PROTEIN-COUPLED RECEPTOR...
G PROTEIN-COUPLED RECEPTOR...
G PROTEIN-COUPLED RECEPTOR...
OTHER GRKs AND THE...
CONCLUSIONS
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I. INTRODUCTION
The body maintains its “physiological homeostasis” by
constantly sending signals to organs, which receive and
translate circulating stimuli into appropriate cellular responses. The ability of cells to communicate with each other
is paramount for the maintenance of this homeostasis. This
communication is achieved through molecules, also known
as agonists, which bind to receptors activating downstream
signal transduction pathways.
One of the major agonist-receptor interactions is that of G
protein-coupled receptors (GPCRs) (155). Although there
is not a solid consensus on classification, this superfamily of
receptors may be considered to encompass protein members from six different classes: class A (rhodopsin-like),
class B (secretin-receptor like), class C (metabotropic-glutamate/pheromone), class D (fungal mating pheromone),
class E (cAMP), and frizzled-smoothened (8, 38, 77, 78,
130). Although the exact size of this superfamily is un-
known, human genome sequence analysis has predicted
that nearly 800 human genes are GPCRs (25). Commonly
known GPCRs include: ␤-adrenergic receptors (␤-ARs),
␣-adrenergic receptors (␣-ARs), rhodopsin, endothelin receptors, adenosine receptors, and others (77). These receptors are integral membrane proteins characterized by seven
membrane-spanning domains, which upon agonist binding
change their conformation triggering heterotrimeric G proteins to bind to it in the intracellular space and subsequently
activating downstream signal transduction pathways (45,
205, 206, 214) (see FIGURE 1). Agonist binding to GPCRs
promote conformational changes (206) which allow for the
dissociation of heterotrimeric G proteins into ␣ and ␤␥
subunits, stimulating activation of downstream proteins
such as protein kinase A, Src, MEK5, phosphatidylinositol
3-kinase (PI3K), GPCR kinase (GRK), etc. While activation
of signaling pathways by these receptors is extremely important, so is deactivation. When the balance of activation
and deactivation is impaired, imperative cellular mechanisms become faulty, and if not corrected in a timely manner, this signaling can become irreversibly detrimental to
the body, leading to diseased states.
Deactivation of GPCR-elicited signaling is mainly accomplished by GRKs, and this is termed desensitization. Termination of signaling via GPCRs is done through a two-step
mechanism: first, the GRK phosphorylates the active receptor (and only this form of the receptor), leading to confor-
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SATO ET AL.
Resting (GDP-bound) state
G-protein
coupled receptor
Agonist-binding and
Gα-Gβγ association
GDP Gα Gβγ
Agonist
Receptor
recycling
GDP Gα
RGS
P
P
P
P
Receptor
endocytosis
GDP Gα Gβγ
Gβγ
P
RGS binding and
GTP hydrolysis
Nucleotide
exchange
GRK
Proteosomal
degradation
Effector
Effector
GTP Gα
GRK
β-arrestin
P P
P
Gβγ
Gα-Gβγ dissociation,
GRK2 and β-arrestin
association
GTP
GTP
P
GTP Gα Gβγ
GDP
GT
Signaling
FIGURE 1. G protein-coupled receptor activation and reactivation. At rest, G␣␤␥ is bound to GDP and both
receptor and G␣␤␥ are not associated. Upon agonist binding, G␣␤␥ associates with the receptor followed by
GDP-GTP nucleotide exchange. G␣ and G␤␥ dissociate from the receptor with its respective effector proteins
leading to downstream signaling. At the active receptor, GRKs bind and phosphorylate the receptor leading to
receptor conformational changes and subsequent ␤-arrestin binding. ␤-Arrestin-bound receptors are then
primed for clathrin-mediated endocytosis and sorted for proteosomal degradation or receptor recycling.
Concurrently, RGS binds to G␣ leading to GTP hydrolysis and promoting the receptor to its initial resting state.
mational changes that promote binding of ␤-arrestin; and
second, phosphorylated and ␤-arrestin bound GPCRs are
no longer able to bind to and activate G proteins (132, 228,
274). FIGURE 1 depicts a schematic of GPCR activation,
signaling, and desensitization. Briefly, agonist binding to
the receptor causes nucleotide exchange, G␣-G␤␥ dissociation, and downstream signaling through effector activation.
The G␣-G␤␥ dissociation mobilizes GRKs to phosphorylate the activated receptor, a process which may vary depending on the GRK isoforms (see sect. IIC). Serine (Ser)
and threonine (Thr) phosphorylation by GRKs at the
COOH terminus of the receptor promotes ␤-arrestin mobilization and binding to the receptor. This may further lead
to clathrin-induced endocytosis and reactivation or degradation of the receptor. Although receptor endocytosis reactivation/recycling is an extremely important and complex
mechanism involving many other proteins, it is beyond the
scope of this review and better detailed by Moore et al.
(163). Lastly, activation of GTPases causes G protein trimer
reformation and repriming of the receptor for another
round of stimulation. In this review, we focus on the mul-
378
tifaceted functions of GRKs including their evolving nonGPCR or noncanonical activity that appears to be critical
for cardiovascular homeostasis and disease.
The first description of phosphorylation-mediated deactivation of GPCRs was provided in the 1970s where the
photoreceptor rhodopsin, a prototypical GPCR, was rapidly deactivated by phosphorylation by “opsin or rhodopsin kinase” (also known as GRK1) (28, 133, 270).
Later, the same deactivation mechanism was proposed in
many other GPCRs (reviewed in Ref. 35) including ␤2ARs (21). Several studies have demonstrated that agonist-specific desensitization of the ␤2-AR was associated
with receptor phosphorylation (19, 229, 243). GRK2
was then identified (initially named the ␤-AR kinase,
␤-ARK1) and shown to be able to phosphorylate ␤-ARs
as well as rhodopsin in a light-dependent manner (19,
21). It was only when GRK2 was identified and characterized to possess properties similar to GRK1 that the
hypothesis was put forth of a possible family of new
proteins: the GRK family (16, 19).
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G PROTEIN-COUPLED RECEPTOR KINASES
II. G PROTEIN-COUPLED RECEPTOR KINASES
Visual subfamily
1
A. GRK Family Members
The GRK family is composed of seven members. This family of kinases is quite small considering that they are responsible for deactivating potentially more than 800 GPCR gene
products as only a small percent of these receptors are not
regulated by phosphorylation (78). Although identification
of phosphorylation-induced desensitization of GPCRs by
rhodopsin kinase (GRK1) (270) and GRK2 (or ␤-ARK1)
(18, 21) opened the doors to this novel deactivation mechanism, it was only after cloning of GRK2 in 1989 that the
idea of a new eukaryotic serine/threonine (Ser/Thr) protein
kinase family fully emerged. Promptly thereafter, new
members of this family were identified and included: GRK3
(20), GRK4 (6), GRK5 (134, 192), GRK6 (17), and GRK7
(101, 269). Based on sequence similarity and gene structure
analysis, vertebrate GRKs are further divided into three
subcategories: visual or rhodopsin-kinases subfamily (comprising of GRK1 and GRK7), ␤-AR kinases subfamily
(comprising of GRK2 and GRK3), and the GRK4 subfamily
(comprising of GRK4, GRK5, and GRK6) (see FIGURE 2).
The GRK family of proteins possesses shared structural and
functional hallmarks. These proteins are mainly composed
of three regions: a short 25-residue proximal NH2-terminal
region unique to the GRK family of kinases, a regulator of
G protein signaling (RGS) homology domain (RH), which
is interrupted by a Ser/Thr kinase domain (KD). This
⬃500 –520 amino acid residue KD belongs to the AGC
kinase family and is shared by all GRKs (reviewed in Refs.
95, 112). The entire NH2 terminus is highly conserved
among GRKs though the NH2-terminal region is unique to
the GRK family of kinases and implicated in GPCR binding
(reviewed in Refs. 95, 112). The COOH terminus is the
most diverse region among GRK subfamilies. GRK1 and
GRK7 have short COOH-terminal prenylation sequences
(101, 113). GRK2 and GRK3 contain pleckstrin homology
(PH) domains implicated in binding anionic phospholipids
and G␤␥ subunits (34, 60, 128, 187). GRK2 has been recently implicated to localize to mitochondria (43, 83).
GRK2 does not have a classical mitochondrial localization
sequence, so it is not clear what is the required minimal
sequence necessary for this novel localization. GRK4 and
GRK6 have palmitoylation sites (194, 241) as well as lipidbinding capabilities (118). GRK5 membrane targeting relies
on a phospholipid binding domain (residues 552–562) (196).
Alternative splicing has been identified for GRK4 (4 splice
variants) (194, 216) and GRK6 (4 splice variants) (75, 161,
193, 257); however, not all splice variants appear to be expressed across species. More recently, a nuclear localization
sequence (NLS) has been identified within GRK5 (amino acids
388 –395) (120), which happens to be a common feature in all
GRK4 subfamily members (119). In addition, a nuclear export
signal (NES, amino acids 259 –265) has also been identified
42
184
514
532
563
K219
GRK1
1
40
531
513 553
185
K220
GRK7
βARK subfamily
1
36
184
513 553
661 689
513 553
661 688
K220
GRK2
1
36
184
K220
GRK3
GRK4 subfamily
1
38
181
508 525
578
K216K217
GRK4
1
38
180
259 265 388 395
507 525
590
K215
GRK5
1
38
180
507 525
576
K215K216
GRK6
N-terminal region
PH domain
RH domain
Nuclear localization signal
C-terminus
Nuclear export signal
Kinase domain
FIGURE 2. GRK topology and subfamily distribution. All GRKs are
comprised of a NH2-terminal region (green), followed by an RH domain
(pink) interrupted by a kinase domain (yellow) and a COOH terminus
(orange). Visual or rhodopsin-kinases subfamily comprises of GRK1
(also known as rhodopsin kinase) and GRK7, with the unique capability
of prenylation in the COOH terminus. The ␤ARK subfamily comprises of
GRK2 and GRK3, having a characteristic PH domain. The GRK4 subfamily is comprised of GRK4, GRK5, and GRK6. GRK5 has a unique
nuclear localization signal (in blue) and nuclear export signal (in dark
green). Numbers in black correspond to amino acids at start or end of
each region. GRK4 and GRK6 residue numbers correspond to GRK4␣
and GRK6A variants, respectively. Numbers in red correspond to residues responsible for catalytic activity.
(119). FIGURE 2 depicts the tri-domain schematic of all members of the GRK family that include the above-described features. Catalytic activity is dependent on specific lysine residues
and shown for each isoform.
B. GRK Tissue Localization
Most GRKs are ubiquitously expressed; however, there is
selective tissue distribution. GRK1 and GRK7, members of
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SATO ET AL.
the rhodopsin-kinase subfamily, are generally restricted to
the retina where they primarily target rhodopsin and cone
opsin, respectively. Within the retina, these two isoforms
are particularly restricted to rod and cone photoreceptors,
although both are also present in pinealocytes (41, 231,
283, 284). GRK4 is highly expressed in the testes (194),
although it has also been shown to be expressed in proximal
tubule cells in kidneys (73, 258), in the brain (217), and in
the uterus myometrium (29). In particular, two GRK4 variants (GRK4␣ and GRK4␥) have been reported to signal
through D1 and D3 dopamine receptors within the kidney
(73, 258). Essentially all the remaining isoforms are ubiquitously expressed with varying degrees of expression depending on type of tissue. GRK2 primarily targets several
GPCRs including ␤-ARs and angiotensin II type 1 receptors, and it is expressed in most tissues. GRK3 targets olfactory receptors, thrombin receptors, kappa-opioid CRF1
(57, 219), and ␣1- and ␣2–ARs (12, 246). It is found in
most tissues, but most prominently in the olfactory epithelium. GRK5 can phosphorylate ␤-ARs, adenosine receptors, and M2 muscarinic receptors, and it is expressed in
most tissues (14).
TABLE 1 shows tissue distribution of various GRK isoforms
(reviewed in Ref. 112). Pertinent to this review is the expression of cardiac GRKs. In the human heart there is evidence for expression of GRK2, GRK3, GRK5, and GRK6
(67). The most prominent cardiac GRK isoforms are 2 and
5, although 3 and 6 have also been reported at low levels (1,
67, 162). Moreover, it is important to note that a particular
GRK does not have to be the most prominently expressed
GRK in a tissue to be crucial for a specific GPCR signaling
regulation as its cellular localization and activity are key
measures determining GPCR-GRK interactions. More recently, GRK3 and GRK4 have been described to be present
in the thyroid nodules and to be upregulated in hyperfunc-
Table 1. GRK isoform tissue distribution
GRK
Tissue Localization
Reference Nos.
1
2
Retina⬎⬎pineal
Leukocytes⬎front cortex of
brain⬎⬎heart⬎lung⬎kidney,
thyroid nodules
Brain⬎spleen⬎heart, lung, kidney,
thyroid nodules
Testis⬎⬎⬎⬎brain, thyroid nodules
Heart, placenta, lung⬎skeletal
muscle⬎brain, liver,
pancreas⬎kidney, thyroid
nodules
Brain, skeletal
muscle⬎pancreas⬎heart, lung,
kidney, placenta⬎liver, thyroid
nodules
Retina, particularly cones
146, 171
16, 65, 135,
262
3
4
5
6
7
380
20, 58, 135,
209, 262
6, 262
134, 262
17, 98, 262
101, 269
tioning thyroids, which may also indirectly impact cardiac
function (262).
C. Canonical Subcellular Targeting of GRK
Isoforms
Agonist-mediated activation of GPCRs is obviously critical
for cellular responses; however, sustained GPCR stimulation is deleterious in part because of receptor desensitization and eventually downregulation. If novo or de novo
activation of receptors does not occur, this downregulation
of receptors may not only lead to loss of responsiveness to
agonists but also cellular response disarray. It is the primary
job of GRKs to reverse the harmful effects of chronic GPCR
stimulation. To accomplish this goal, GRKs are canonically
thought to be present mainly in the cellular cytoplasm or
close to the plasma membrane. Certain GRK isoforms have
specific sequence trademarks that allow them to preferentially target specific subcellular compartments. In general,
membrane targeting of all GRKs towards activated GPCRs
involves residues and domains within the COOH terminus
of these kinases (see TABLE 2).
GRK1 and GRK7 are localized directly to the plasma membrane after GPCR activation as both isoforms possess a
short COOH-terminal prenylation sequence (101, 113),
and the attached lipid molecules act as lipid anchors facilitating attachment to cell membranes. This cellular localization is advantageous for visual GRKs as they perform their
function of “probing” for active rhodopsins or cone opsins.
As such, visual GRKs perform a two-dimensional search
instead of a three-dimensional diffusion, which is essential
for their ultra-rapid signal termination. This means that
because GRK1 and GRK7 attachment to the membrane is
facilitated by lipid anchoring, when GPCRs become active,
these specific kinases have the advantage of searching for
active receptors in the x- and y-position only since they are
already closely associated with the membrane. On the contrary, all other GRKs do not have this facilitated mode of
attachment, having to perform an x, y, and z search for
activated receptors.
The ␤-ARK family of kinases (GRK2 and GRK3) are membrane targeted from the cytosol through binding to the
membrane-anchored G␤␥ subunits of G proteins following
their dissociation after GPCR activation (34, 187). Indeed,
GRK2 and GRK3 were originally found to be localized in
the cytoplasm and then recruited to the membrane as a
response to agonist-occupied receptors (19, 21, 242). This
binding occurs within the COOH terminus and includes the
PH domain (34, 189). Because G␤␥ subunits are generated
by activated receptors, free G␤␥ recruits these GRK isoforms to the locale of active GPCRs, thereby greatly enhancing GPCR phosphorylation only when needed ensuring the fidelity of the desensitization process of only agonistoccupied receptors. Nevertheless, it is important to note
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Table 2. GRK subcellular targeting at baseline conditions
GRK
Tissue Localization
Reference Nos.
1
2
Cytoplasm very close to plasma membrane (rod outer segments)
Cytoplasm (free or associated with actin, actinin,
microtubules)⬎plasma membrane ⬎⬎⬎mitochondria
Cytoplasm⬎plasma membrane
Splice variants below:
␣: Cytoplasm⬎ plasma membrane ⬎⬎⬎nucleus
␤: Cytoplasm⬎ plasma membrane ⬎⬎⬎nucleus
␥: Cytoplasm⬎ plasma membrane ⬎⬎⬎nucleus
␦: Cytoplasm ⬎ plasma membrane ⬎⬎⬎nucleus
Cytoplasm ⬎⬎⬎nucleus
Mainly in the cytoplasm, splice variants below:
A: Cytoplasm and close to plasma membrane⬎⬎⬎nucleus
B: Cytoplasm and close to plasma membrane⬎⬎⬎nucleus
C: Cytoplasm and close to plasma membrane⬎⬎⬎nucleus
D: Exclusively in the nucleus
Cytoplasm very close to plasma membrane (cone outer segments)
39, 224
19, 21, 43, 83, 168, 242, 281
3
4
5
6
7
that the fidelity of the desensitization process is aided by
the preference of GRKs for the agonist-occupied conformation of the receptor as a substrate, as agonist occupancy can
increase GPCR substrate affinity manyfold. Thus, like
GRK1 and GRK7, the ␤-ARK kinases attach to the membrane via a prenyl group; however, these kinases “pirate”
the lipid anchors from G␤␥ and do not have them directly
attached to their C-tail.
The G␤␥ binding domain within the COOH-terminal PH
domain of GRK2 (FIGURE 2) was originally determined biochemically (128). Following the structural information of
GRK2 at the atomic level, the key residues for G␤␥ binding
were confirmed (143). Importantly, peptides from this region can block the membrane localization of GRK2 and
prevent ␤-AR and GPCR desensitization (128). The shortest effective GRK2-G␤␥ blocking peptide is a 28-mer peptide. However, most studies over the last 20 years have used
the 194-amino acid residue peptide of the entire COOH
terminus of GRK2, ␤-ARKct (128, 129), which has been
shown to be effective both in vitro and in vivo at inhibiting
GRK2 activity (discussed in detail below).
Lastly, subcellular targeting of the GRK4 subfamily involves protein hallmarks and modifications also present in
the COOH termini. GRK4 and GRK6 carry both palmitoylation sites as well as lipid-binding positively charged
elements which promote a biased membrane localization
(147, 194, 240, 241). Though lacking the palmitoylation
site, the COOH terminus of GRK5 is capable of binding
phospholipids, thus distinctly promoting plasma membrane
targeting (251). These features likely enhance lipid bilayer
affinity and kinase activity. Interestingly, GRK6A splice
variant contains elements that both promote and inhibit
membrane localization. While it contains the features men-
19, 21, 242, 281
119
119
119
119
119,
281
119,
119,
119,
119,
41
120, 281
257
257
257
257
tioned above, it also contains acidic residues that inhibit
membrane localization (118, 257). A nonpalmitoylated
form of GRK6A is detected in the cytoplasm and nucleus,
although nuclear function still remains unclear (118). Surprisingly, GRK6B and GRK6C, which do not contain the
palmitoylation sites, are still capable of localizing to the
plasma membrane in cultured cells (257). Interestingly,
membrane localization is rescued when the string of acidic
residues located in the COOH terminus of GRK6A is
mutated to neutral or basic residues in nonpalmitoylated
GRK6A. This suggests that palmitoylation is not the dominant mechanism in which GRK4 subfamily of proteins
achieve their preferential membrane localization. In addition, a fourth isoform for GRK6 (GRK6D) has been proposed to lack the catalytic domain and exclusively localize
to the nucleus, although its function is yet to be determined
(257).
In vitro, different GRK isoforms may phosphorylate different GPCRs, and differential subcellular targeting and tissue
expression levels might contribute to explaining the functional specificity observed in vivo (see TABLE 1). For instance, GRK2 is capable of phosphorylating rhodopsin in a
light-dependent manner (18), yet GRK1 is the isoform involved in rhodopsin deactivation in vivo as GRK2 does not
perform this function in GRK1 knockout mice (39). GRK2
and GRK3 are both expressed in the brain and HEK cells. In
HEK cells, both isoforms behave similarly, yet in the brain,
GRK3 is more membrane-associated that GRK2 (4, 33).
Although enormous effort has been expended to understand GRK receptor specificity, the mechanism for receptor
preference still remains unclear. Receptor phosphorylation
“bar coding” has been suggested as one of the ways specificity might be determined (166), yet this concept may not
entirely account for GRK selectivity. In this study, phos-
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SATO ET AL.
phorylation patterns of the receptor by GRKs lead to diverse ␤-arrestin-related activities. Yet, it did not explain
why a specific GRK was more prone to phosphorylate a
receptor over another. GRK receptor specificity has been
analyzed in hybrid transgenic mouse hearts. For example,
GRK2, GRK3, and GRK5 overexpressing mice were bred
with cardiac-specific transgenic mice overexpressing constitutively activated ␣1B-ARs. The results revealed that different GRKs had varying effects on this receptor. While GRK3
desensitized ␣1B-ARs very well, GRK2 had no effect, and
the effects of GRK5 were mixed (68).
In another study using just GRK2 and GRK5 cardiac overexpressing transgenic mice, it was found that both of these
cardiac GRKs desensitize ␤-ARs quite well, while GRK2
could also attenuate signaling through angiotensin II receptors, and GRK5 had no effect on these receptors (212).
GRK5 and GRK2 also differently effected cardiac adenosine responses, respectively desensitizing or having no effect
(212). Clearly, GRKs have receptor selectivity in vivo; however, the mechanism of receptor preference still remains
unknown. Different expression levels of various GRK isoforms in a cell-specific manner may be one of the ways in
which GRKs accomplish different tasks in various tissues,
emphasizing the importance of spatial-temporal expression. Nevertheless, relative levels of expression in a particular tissue do not necessarily explain receptor-kinase specificity. Yet, how bar coding, cell-specific expression, or
structural characteristics play a role in receptor specificity
still requires further investigation.
D. Mechanism of Action of GRKs
The molecular basis in which GRKs first recognize active
receptors still remains a matter of speculation. The most
plausible hypothesis is termed “fly casting,” in which the
disordered NH2 terminus of GRKs forms an initial lowaffinity interaction with the active receptor (55, 226). In the
case of GRK1 and GRK7 which have a prenylation sequence thought to involve lipid anchoring to membranes,
this would mean that the search is in two dimensions, increasing its chance of finding active-receptors and fulfilling
its ultra-fast response requirement. The other subfamilies
are thought to involve a three-dimensional search, although
proximity to the membranes would also permit for these
kinases to be readily available to deactivate the receptor in
a timely manner.
Crystal structures of specific GRKs have proven essential in
understanding possible mechanisms of action and activation processes. These concepts have been well summarized
and recently reviewed in References 95 and 104. The bestdescribed function of GRKs is the ability to phosphorylate
active GPCRs. With the exception of GRK4␣, which has
been shown to be constitutively active and capable of phosphorylating unstimulated GPCRs (160, 203), the activity of
382
GRKs appear to depend on the functional state of the target. In fact, docking to the active-receptor seems to directly
activate GRKs (42, 172). Phosphorylation of the active receptor can in itself decrease G protein coupling (274), but
most importantly, it allows for ␤-arrestin to bind and block
the cytoplasmic surface of receptors, thus preventing interaction with effector enzymes that are necessary to initiate
downstream signaling (132).
A recent structure of GRK6 in a “closed conformation”
revealed an ordered NH2 terminus engaged in extensive
interactions with both the small lobe and the C-tail of the
kinase domain (26). These interactions are physically impossible when GRK6 is in a more open conformation (144).
More interesting is the fact that the contact points of these
residues in the NH2 terminus and the kinase domain are
highly conserved in all GRKs and that site-directed mutagenesis revealed them to be essential for phosphorylation
of active GPCRs and soluble substrates (26, 103, 105, 174,
238). In particular, mutations in hydrophobic residues in
the NH2-terminal helix of the kinase abolished receptor but
not peptide phosphorylation, suggesting the necessity of
these residues for GPCR-GRK interactions (26). Although
it seems evident how GRKs achieve an active conformation,
one can only infer the mechanism in which an active receptor would stabilize and potentiate GRK activity. Recently, a
structure of ␤-arrestin complexed to a GPCR phosphopeptide provided particular evidence of a receptor interacting
interface to ␤-arrestin (227). Structural understanding of
GRK complexed to the receptor would greatly aid in unveiling precisely how this interaction occurs. Although
no such structural evidence currently exists, it is known that
after a signaling event, the whole process of desensitization
starts by phosphorylation of the activated receptor by
GRKs. In particular, GRKs phosphorylate at one or more
Ser/Thr residues located on the intracellular domains of the
receptor, particularly sites located in the COOH terminus
and in the intracellular loop between TM5 and TM6 of the
receptor (127).
Docking of GRKs to active receptors has been proposed to
require the NH2 terminus of GRKs (26, 103, 173) as well as
other regions outside the kinase domain (10, 64). In addition, in order for GRKs to phosphorylate active GPCRs
efficiently, they require the presence of a cytoplasmic pocket
(not present in the receptor’s inactive state) as well as the
presence of negatively charged lipids (46, 204, 236). The
size and physical properties of this cytoplasmic pocket in
GPCRs are expected to be highly conserved while the sequence and size of the cytoplasmic loops of the receptors
may greatly differ. Thus the conservation of this complimentary cytoplasmic pocket within various GPCRs potentially allows GRKs to phosphorylate a broad array of
GPCR substrates. The rationale behind this hypothesis lies
in the fact that this pocket in the activated receptor would fit
in conformation and charge to that of GRKs in their active
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conformation. Once docked, the kinase would adopt a
closed conformation and lead to phosphorylation of substrates in close proximity (104).
The RH domain within the NH2 terminus (FIGURE 2) of
GRKs has also been implicated in GPCR phosphorylation.
Canonical RGS proteins are GTPase activating proteins
(GAP), attenuating G protein-mediated signaling by promoting GTPase activity of GTP-G␣ (reviewed in Ref. 102).
GRK2 and GRK3 can bind to G␣q through its RH domain
(36). However, RH domains of GRKs have low or no GAP
activity, and their ability to halt signaling is primarily due to
directly binding and sequestering activated G␣q (36) or direct hindrance of GPCRs (63, 64). In addition, GRK-RH
domain-mediated signaling can also occur independently of
the receptor-G␣q process. GRK2 is capable of scavenging
G␣q/11 inhibiting insulin-induced glucose transport (254).
Likewise, it can inhibit endothelin-1-mediated glucose transport by interfering with G␣q/11(242). Recently, GRK2 has
been shown to specifically regulate insulin receptor signaling in the heart by phosphorylating IRS-1 and blocking
glucose transporter membrane translocation (50), which
demonstrates a physiological role for GRK2 acting towards
a non-GPCR substrate.
E. Regulation of GRKs
Canonically, there is the necessity for GRKs to be regulated
at the moment it phosphorylates activated receptors. Phosphorylated active receptors allow for the binding of ␤-arrestins, which triggers the formation of clathrin-coated pits
and progression through endocytic pathways (see FIGURE
1). These internalized compartments may contain receptors, GRK2, ␤-arrestin, as well as other proteins characteristic of endocytic vesicles (reviewed in Ref. 163). Once internalized, these vesicles are sorted for degradation (desensitization) or recycling (resensitization). The presence of
GRKs in these internalized active-receptor vesicles may
physically prevent the kinase from performing its function
on other activated GPCRs. Additionally, prior to vesicle
formation and internalization, the binding of ␤-arrestin to
the active-receptor-GRK complex leads to further recruitment of other proteins. One of these recruited proteins is
Src, which has been reported to phosphorylate GRK2, diminishing its activity (177). Recently, it has been demonstrated that ␤-arrestin association to the phosphorylated
receptor occurs in a biphasic mode. First, there is a “loose”
hanging interaction between the C-tail of the receptor and
the NH2-terminal domain of arrestin. Analogous to a fishing line, the receptor samples for potential interactions at
high rate. Second, there is an insertion of the finger loop into
the receptor core leading to longitudinal arrangement of
arrestin, also known as fully engaged “tight” interaction
(228). This initial “fishing” of the receptor is only possible
after the receptor has been phosphorylated by GRKs. Subsequent signaling, also known as GPCR biased signaling,
occurs after ␤-arrestin is recruited to the phosphorylated
receptor, a topic which is further expanded in section IIF.
Although circulating agonists, such as catecholamines, can
induce activation of GRKs mediated by GPCRs, multiple
molecular mechanisms have been proposed to regulate
GRKs in the cellular cytoplasm. While some regulatory
mechanisms may be common to all GRKs, the fine tuning of
kinase activity may be different for each GRK subtype.
GRK1 has been shown to be capable of regulating its activity by autophosphorylation at Ser21, Ser488, and Thr489
(31, 173). In addition, while having no effect on GRK2, the
calcium-binding protein recoverin is able to inhibit GRK1.
As calcium levels change in response to changes in light,
recoverin regulates GRK1 (40, 280). In HEK cells, PKA
directly binds to and phosphorylates GRK2 at Ser685, enhancing G␤␥ binding and promoting membrane translocation (54). PKC has been shown to modulate GRK5 and
GRK2. PKC is able to phosphorylate GRK5 and inhibit its
activity severalfold due to both decreased affinity and activity for the active receptor (195). PKC-mediated GRK5
phosphorylation most likely occurs at Ser572, and either
Ser566 or Ser568, although PKC can also phosphorylate
autoregulatory sites Ser484 and/or Thr485 (195). On the
other hand, PKC phosphorylation enhances GRK2 activity two- to threefold, possibly by increasing the ability of
GRK2 to bind membranes and by disinhibiting tonic calmodulin regulation (48, 131, 276). GRK5 has also been
shown to be modulated by calmodulin through a calmodulin-binding domain on GRK5 (197).
GRK2 is a short-lived protein (half-life of ⬃1 h in HEK,
COS-7, Jurkat, and C6 glioma cells) that is polyubiquitinated and actively degraded by the proteasome proteolytic
pathway with increasing degradation in response to activation of ␤2-ARs (177, 180). Interestingly, in HL60 cells, the
half-life of GRK2 was estimated to be ⬃20 h (148). With
such a relative short half-life, transcriptional regulation
might be an important factor for GRK regulation, although
little is known about molecular mechanisms directly involved in modulating GRK mRNA levels. Initially, the
GRK2 promoter was considered to be a general “housekeeping” gene (181). Studies of the GRK2 promoter region
revealed several notable features, which included no evidence for TATA box, absence of CCAAT boxes in the proximal region upstream of the predicted start site, and the first
300 bases immediately upstream of the ATG are extremely
GC rich (87%) (181). In addition, there are predicted secondary structures in the 5’ region of the mRNA, which may
serve as posttranscriptional regulators of expression (181).
Nevertheless, evidence does exist on potential modulators
of GRK2 mRNA content. In the heart, circulating catecholamines and ␤-AR activity correlate with GRK2
mRNA expression (111). Mitogenic T-cell stimulation by
exposing mononuclear leukocytes to PHA led to increased
GRK2 mRNA levels, while no change was observed in
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SATO ET AL.
mRNA expression of GRK5 and GRK6 (59). More intriguing are the analysis of the human GRK2 gene promoter in
aortic smooth muscle cells, where agents that led to physiological vasoconstriction or hypertrophy increased GRK2
promoter activity, and on the contrary, proinflammatory
cytokines such as interleukin-1␤, tumor necrosis factor-␣,
or interferon-␥ decreased the activity of the GRK2 promoter (202).
As mentioned above, active GPCR-GRK complex recruits
␤-arrestins. ␤-Arrestins can also interact with the cytosolic
tyrosine kinase, c-Src, recruiting it to the complex. c-Src has
been shown to phosphorylate GRK2 on tyrosine residues,
marking it for degradation (177). In addition, in vitro studies have shown that c-Src modulates ␣1B-AR activity
through GRK2 and GRK3 (5) even though in vivo experiments show that GRK2 has no effect towards ␣1B-ARs
(68). Moreover, GRK2 can also be phosphorylated by p38
(141) and ERK1 (188) mitogen-activated protein (MAP)
kinases at Ser670. This phosphorylation results in diminished activity (less phosphorylation of the active receptor)
(188) and increased degradation (71). The phosphorylation
of Ser670 of GRK2 by MAP kinases has recently been
shown to cause the localization of this GRK2 to mitochondria (43), which will be discussed in more detail below.
Specifically relevant to cardiovascular regulation, mechanical stretch has also been implicated in activating GRKs. In
neonatal rat ventricular myocytes, mechanical stretch led to
activation of GRK2 through angiotensin II type I receptor,
G␣q, and PKC (153). Inflatable balloon stretch in explanted
hearts activated GRK5 and GRK6 but not GRK2 (201). In
addition, as detailed above, GRK2 and GRK3 contain
within their COOH terminus a PH domain that is involved
in G␤␥ binding. Free G␤␥ binds to these GRKs within this
region at multiple sites (34) with high affinity and are required for GPCR phosphorylation (189). Moreover, a regulatory G␤␥-binding site has also been described at the
NH2 terminus of GRK2 (70). Phospholipids have also been
reported to modulate the activity of GRKs (34) through the
PH domain of GRK2 and GRK3 or through amino acids
547–560 of GRK5 (34, 169, 185, 196). Another regulation
at the PH domain level of GRK2 and GRK3 is the ability to
bind to phosphatidylinositol 4,5-bisphosphate (PIP2) and
other acidic phospholipids. PIP2 binds to the NH2 terminus
of the PH domain and is able to increase by threefold GRK2
phosphorylation of membrane receptors (60, 61).
Recently, another mode of GRK2-specific regulation has
been described as this kinase is capable of being S-nitrosylated, and the dynamic modification at Cys340 when nitric
oxide (NO) bioavailability is high causes inhibition of
GRK2 activity towards GPCRs (271). GRK2 has been
found to interact with endothelial NO synthase (eNOS),
and these two molecules reciprocally inhibit one another
with eNOS-NO bioavailability altering the in vivo activity
384
of GRK2 (106). Importantly, in studies using a GRK2
knock-in mouse where Cys340 was mutated to a Ser and
incapable of being S-nitrosylated, GRK2 activity was
higher. This led to increased myocyte cell death after ischemic injury, and these mice were resistant to NO-mediated
cardioprotection (106). More recently, studies in heterozygous GRK2 knockout (KO) mice revealed that these mice
are more resistant to vascular remodeling, mechanical alterations, and hypertension induced by angiotensin II, partially through the preservation of NO bioavailability since
these mice have less GRK2 to inhibit eNOS (9). Therefore,
the unique interaction between GRK2 and the eNOS-NO
system is a powerful and dynamic mechanism for regulating
this GRK isoform, especially in the cardiovascular system.
Of particular importance, GRKs have also been implicated
in binding and sometimes phosphorylating other nonGPCR substrates. The first description of a non-GPCR substrate for GRKs was tubulin (37, 96, 186), and both GRK2
and GRK5 are capable of phosphorylating tubulin. Interestingly, phosphorylation of tubulin by GRK2 is enhanced
in the presence of G␤␥ and phospholipids (both of which
promote GRK localization to the membrane) as well as in
the presence of active GPCRs. It is known that active
GPCRs can enhance GRK phosphorylation of peptides up
to 100-fold (42, 172). Thus one may infer that any potential
substrate in close proximity to GRKs might become phosphorylated. This led to the suggestion that phosphorylation
of non-GPCR substrates might be related to opportunity
and location, being at the right place at the right time. In
support of this hypothesis is the “high gain” phosphorylation in the retina (22, 23), phosphorylation of IRS-1 by
GRK2 in response to insulin (50) and GRK2 phosphorylation of p38 (183).
On the other hand, GRKs have been hypothesized to possess functions beyond its kinase capabilities. GRKs have
been proposed to be modulators of cellular functions in a
phosphorylation-independent manner due to their ability to
interact with a variety of signaling and trafficking proteins,
such as actin (79), actinin (80), tubulin (37), G␣ (36), G␤␥
(187), clathrin (225), PI3K (164), GIT (190), caveolin (36,
88), lipids, etc. GRKs have been placed in the center of
regulatory processes involving cell migration (179), metabolism (50, 52, 254), and apoptosis (43). Further investigation is needed to address these GRK-mediated intermolecular interactions and how they link to various cellular processes. Overall, these noncanonical activities of GRKs may
play key roles in physiological signaling including in the
heart as detailed below.
F. GRKs Directing GPCR-Biased Signaling
GPCR signaling is rapidly attenuated by GRK phosphorylation of receptors and subsequent binding of the protein
␤-arrestin (191, 237). ␤-Arrestin binding to the activated
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G PROTEIN-COUPLED RECEPTOR KINASES
receptor acts as a clathrin adaptor ultimately triggering the
receptor for internalization (91). However, prior to receptor downregulation, activated GPCRs could be “functionally selective” or “biased” towards G protein or ␤-arrestinmediated signaling. It has been shown that the ␤2-AR
cytoplasmic end helices VI and VII adopt two major conformational states depending on its ligand. Receptor agonists primarily affect G protein specific active state of helix
VI, whereas ␤-arrestin biased ligands predominantly affect
helix VII (140).
In addition, the “pattern” of which a receptor is phosphorylated may dictate a “bar code” for ␤-arrestin function.
For example, GRK2 phosphorylation of the ␤2-AR results
in waning of signaling and/or receptor internalization,
whereas GRK6-phosphorylation of ␤2-ARs induces recruitment of ␤-arrestin that leads to extracellular signalregulated kinase 1/2 (ERK1/2) signaling (166). Distinct
GRK2 and GRK6 phosphorylation sites have been identified on the ␤2-AR (166). Moreover, work in other receptors
has also shown the requirement of GRK2 (and GRK3)mediated receptor phosphorylation for GPCR internalization, and GRK5 and GRK6-mediated receptor phosphorylation for ␤-arrestin-mediated ERK1/2 signaling (121, 123,
126, 207, 222, 285). In support of this GRK-mediated
GPCR-biased signaling are the studies using carvedilol, a
␤-AR antagonist that has been shown to be a weak ␤-arrestin-biased agonist that can activate ERK after an apparent
action of GRK6 (277).
Interestingly, in myocytes, ␤-arrestin “biased” signaling has
also been shown to involve AT1Rs, in particular stretchmediated activation of AT1Rs signals through ␤-arrestin
“biased” pathways (201). ␤-Arrestin has also been implicated in mediating transactivation of the epidermal growth
factor receptor (EGFR) downstream of ␤1-AR signaling
(167, 252). More intriguing is the fact that this ␤-arrestindependent transactivation of EGFR is cardioprotective, independent of G protein activation, and requires the actions
GRK5 and GRK6 (167). Interestingly, this novel pathway
shows a trend towards increase in activity when GRK2 is
absent, which supports the overall hypotheses that biased,
␤-arrestin-dependent signaling is indeed directed by the actions of specific GRKs notion of GRK dependence of ␤-arrestin function (167). In fact, it appears that biased GPCR
signaling is determined by which GRK phosphorylates a
given receptor (166). Thus GRK phosphorylation of GPCRs is a significant cellular event as it cannot only desensitize the active receptor but also dictate downstream signaling, functionally selecting for G protein or enabling ␤-arrestin-mediated pathways.
G. Mouse Models of Altered GRK Levels
Undoubtedly mouse models have proven to be an essential tool in understanding in vivo GRK functions. Various
mouse models have aided in understanding these multifaceted kinases and their regulation. The first GRK
mouse model was presented in Science 1995 by Koch et
al. (129). Here, cardiac-specific overexpression of GRK2
in vivo led to reduced functional coupling of ␤-ARs,
dampened adenylyl cyclase activity, and attenuated isoproterenol-stimulated myocardial contractility. In the
same paper, the authors showed that expression of the
␤-ARKct, the peptide inhibitor of GRK2, led to enhanced
contractility in the presence or absence of isoproterenol
(129). Many other mouse models followed this initial
report as listed in TABLE 3. Of note, it is important to
highlight that the only GRK global KO that is embryonically lethal is that of GRK2 (117). This is important to
note as particularly significant since GRK2 is the predominant isoform expressed during embryogenesis
(220). In fact, GRK2 transcripts are detected at 7.5 days
post coitum in multipotent cells in early mesoderm and
neural crest (220). Interestingly, in mice, while global
GRK2 knockout (KO) is embryonically lethal, cardiacspecific ablation of GRK2 is not (158). In fact, normal
heart structure, cardiac function, and gene expression
were observed in cardiac-specific GRK2 KOs when
Nkx2.5-Cre was used. These mice did exhibit enhanced
inotropic sensitivity to isoproterenol, impaired lusitropic
tachyphylaxis, and aggravated catecholamine toxicity
during chronic ␤-AR stimulation (158). This differs significantly from the situation when GRK2 is eliminated
from cardiac myocytes after birth as discussed below
(199). Of note, it is important to mention recent studies
crossing heterozygous GRK5 and GRK6 knockout mice
previously described (84, 85) to generate double GRK5
and GRK6 knockout mice which are embryonically lethal (32).
Among genetic in vivo models of GRKs, involving manipulation of GRKs, GRK2 is the best studied isoform, with
many mouse models revealing the different structural elements of this kinase (see TABLE 3). The majority of GRKbased genetically engineered mouse models have been targeted to cardiovascular tissues and have paved the way that
proves that GRKs, especially GRK2 and GRK5, play critical roles in normal and pathophysiological cardiac regulation and function. The remaining portion of this review will
focus on discussions concerning key biologically, physiologically, and translationally significant aspects of cardiovascular GRK function that have been largely gained from
mouse models. Importantly, these mouse studies have also
been largely complemented by human studies where applicable as well as studies in larger animal species. The understanding of GRK function and regulation provided by these
mouse models and other studies provides evidence that
GRKs and their targeted inhibition represent a potential
new and powerful drug class that may yield new treatments
for cardiovascular disorders.
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SATO ET AL.
Table 3. Mouse models of GRKs
GRK
Isoform
Chromosome
in Humans
1
13
2
3
11
22
Chromosome
in Mouse
8
19
5
4
4
5
5
10
19
6
5
7
3
13
Year of
Publication
Genotype
Phenotype
1999
GRK1 knockout
Loss of light-dependent phosphorylation of
rhodopsin. Single photon responses
became larger and long-lasting.
39
2010
GRK1 overexpression
Photoreceptor damage when exposed to
bright light
272
1996
GRK2 knockout
Embryonic lethality-hypoplasia of
ventricular myocardium. Heterozygous
mice are supersensitive to ␤-AR
stimulation.
117
2006
GRK2-flox knockout
(cardiac)
Viable mouse, enhanced ionotropy to
isoproterenol though develop
accelerated catecholamine toxicity to
chronic treatment; lusitropic
tachyphylaxis.
158
1995
GRK2 overexpression
(cardiac)
Decreased ␤-AR signaling, attenuated
ventricular contractility in the presence
of isoproterenol.
129
2002
GRK2 overexpression
(vascular smooth
muscle restricted)
␤-AR signaling attenuation, vasodilation in
response to isoproterenol was
attenuated, resting mean blood arterial
blood pressure elevation; hypertrophy.
69
1995
␤ARKct (495–689)
(cardiac)
Enhanced contractility with or without
isoproterenol stimulation
129
2003
␤ARKnt (50–145)
(cardiac)
Cardiac hypertrophy, enhanced ␤-AR
density, and signaling.
125
2013
GRK2-C340S
Loss of nitrosylation-mediated
cardioprotection postischemic injury.
106
1997
GRK3 knockout
Supersensitivity to olfactory stimuli.
182
1998
GRK3 overexpression
(cardiac)
Normal ␤-AR biochemical signaling and
hemodynamic function. Decreased
MAP kinase activity in response to
thrombin.
110
2008
GRK3ct (cardiac)
Hypertension, ␣1-receptor
hyper-responsiveness.
261
GRK4 knockout
Not characterized
⫹
2002
GRK4␥-A142V
Hypertension, altered dopamine signaling
73, 268
1996
GRK5 overexpression
(cardiac)
␤-Adrenergic receptor desensitization,
unchanged ANG II receptor stimulation.
212
1999
GRK5 knockout
Exon 6 and 7 flanked by LoxP. Muscarinic
supersensitivity and impaired receptor
desensitization.
85
2011
GRK5 knockout
Exon 3 partially deleted and replaced with
␤gal. Diminished LPS-induced neutrophil
infiltration in the lung with no
differences in liver and spleen.
Decreased cytokine/chemokine levels.
176
2012
GRK5-flox knockout
(cardiac)
Cardioprotection posttransaortic
constriction.
85, 89
2008
GRK5-Q41 (cardiac)
No protective effects against increased
LV mass and chamber size induced by
isoproterenol.
137
2008
GRK5-L41 (cardiac)
GRK5-L41 decreases ␤1-AR signaling by
enhancing desensitization.
137
2008
GRK5 overexpression
⌬NLS (cardiac)
Normal pathology after transaortic
constriction.
157
2013
GRK5 and GRK6
double knockout
Embryonically lethal.
32
2003
GRK6 knockout
Impaired desensitization of postsynaptic
D2-like dopamine receptors and
dopaminergic supersensitivity to
psychostimulants. Alterations in
immune response and inflammation.
76, 84,
124
*
*GRK7 is present in cones; therefore, there is no GRK7 equivalent in mouse as these animals are color blind.
⫹
Knockout animal deposited at Jackson Laboratories by Dr. Lefkowitz, yet there is no published work related
to this mouse model.
386
Reference
Nos.
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G PROTEIN-COUPLED RECEPTOR KINASES
III. G PROTEIN-COUPLED RECEPTOR
KINASE 2 AND THE CARDIOVASCULAR
SYSTEM
A. GRK2 and Heart Failure
Heart failure is the end point of many cardiac diseases
which include but are not restricted to myocardial infarction, hypertension, coronary artery disease, cardiomyopathies, congenital heart diseases, and others. To prevent
heart failure, it is vital to understand the molecular events
that interfere with ␤-AR signaling homeostasis. One of the
hallmarks of heart failure is increased activation of the sympathetic nervous system, leading to an increase in circulating catecholamines (reviewed in Ref. 151). In the heart, that
translates into increased activation of the ␤-ARs. This augmentation in stimulus is thought to be initially a compensatory mechanism, to reestablish cardiac performance, increasing heart rate and contractility. Unfortunately, if this
increase in signal transduction remains unabated, it can
lead to the deleterious effects seen in heart failure. Data
have shown that these pathological events are triggered by
enhanced GRK2 activity in both the heart and the sympathetic nervous system (151, 213).
The myocardium uses positive chronotropic and ionotropic
effects mediated by ␤1-ARs and ␤2-ARs through Gs coupling to accomplish the demand elicited by increased agonists. In the nonfailing heart, 75% of ␤-ARs are ␤1, with
distribution patterns fairly similar among all cardiac chambers (reviewed in Ref. 14). Interestingly, chronic stimulation of ␤-ARs has been reported to produce opposing outcomes; stimulation of ␤1 and ␤2 would lead to deleterious
and cardioprotective effects, respectively (86). Although
initially thought to be only present in adipose tissue, functional ␤3-ARs are also upregulated in the failing myocardium (44, 62). Thus upregulation of active ␤-ARs increases
the necessity for GRKs to turn off this increase in ␤-AR
signal transduction. Indeed, chronic catecholamine exposure in vivo can lead to upregulated GRK2 (111). Continued activation of ␤1-ARs and ␤2-ARs on myocytes following cardiac stress/injury leads to their desensitization via
enhanced GRK2 activity and chronically this leads to receptor downregulation. Importantly, a hallmark characteristic
of the failing heart is the loss of ␤-AR-mediated inotropic
reserve manifested by both a loss (⬃50%) of ␤-AR receptor
density but also overdesensitization of remaining receptors,
attributed to increased GRK activity (151, 213).
Importantly, human studies have provided compelling evidence that during heart failure there is both increased catecholamine circulation as well as increased myocardial levels
of GRKs (reviewed in Ref. 178). While in an aging human
there are no changes in GRK activities (136), alterations in
cardiac GRKs are evident in the development of human
heart failure (136, 184, 253). GRK activity was reported to
be elevated in end-stage heart failure patients (136). Increased myocardial and lymphocyte levels of GRK2 have
been reported in human heart failure (100, 108). Myocardial biopsy samples and peripheral blood lymphocytes from
23 human transplant patients also revealed an increase in
both GRK2 and GRK5, although there was no correlation
between myocardial and lymphocyte levels. The authors on
this study found that in myocardial tissues, the ␤1/␤2 ratio
was 57/42%, which differs from normal 80/20% ratio, and
they also found significant increases in both GRK5 and
GRK2 (2). In peripheral blood lymphocytes, the authors
observed a substantial increase in GRK2 levels and a ␤1/␤2
ratio of 3/96% (2). Human patients with ischemic and dilated cardiomyopathies have elevated GRK2 mRNA levels
(3-fold) as well as increased activity (2-fold) (253). GRK2
levels were elevated in all chambers in human hearts of left
ventricular overload disorders as well as in dilated cardiomyopathies (67). More recently, mRNA and protein levels
of GRKs in left ventricular myocardium were analyzed
from 31 patients with advanced heart failure undergoing
transplantation. Compared with control nonfailing left
ventricular samples, advanced heart failure patients possessed augmented levels of GRK2 and GRK5, but not
GRK3 (3).
Animal models recapitulating human heart failure have
yielded substantial and important mechanisms involving
GRKs and how the increase in levels can correlate to the
unfavorable prognostic cardiac pathology. These models
have been extensively discussed in References 14, 95, and
178. Of note are the studies in rats subjected to surgical
coronary artery occlusion. Hypertrophic failing hearts
showed increased GRK2 protein and activity while no major change was observed in hypertrophic nonfailing hearts
(250). Another study in hypertrophic cardiomyopathic
mouse hearts showed increased GRK2 protein expression
and activity compared with nontransgenic littermate controls (NLC) (81). In rats subjected to left coronary artery
ligation, myocardial mRNA and protein levels of GRK2
and GRK5 were elevated in failing hearts (259). In spontaneously hypertensive heart failure rats, GRK2 increased at
the intercalated disk in 6-, 12-, and 24-mo-old animals,
while no change was observed in total protein expression
(281). In mice, pressure overload following transverse aortic constriction also led to an increase in cytosolic and membrane GRK (47).
The most abundant circulating catecholamines are epinephrine, norepinephrine, and dopamine. Circulating epinephrine and norepinephrine primarily derive from two major
sources in the body. Sympathetic nerve endings are capable
of releasing their own stored norepinephrine directly onto
the cardiac muscle. Alternatively, upon acetylcholine stimulation of nicotinic cholinergic receptors at the adrenal medulla, chromaffin cells release previously synthesized and
stored epinephrine (mainly) and norepinephrine (reviewed
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SATO ET AL.
in Ref. 151). This is pertinent to heart failure as sympathetic
nervous system hyperactivity chronically stimulates ␤-ARs
leading to increased GRK levels and worsens prognosis.
Adrenal levels of GRK2 have been shown to be upregulated
in chronic heart failure, leading to augmented catecholamine release due to downregulation of ␣2-ARs in chromaffin cells (149). Moreover, in rats, modulation of adrenal
catecholamine release has been manipulated by overexpression of GRK2 or expression of ␤-ARKct (152). More important are the studies in mice specifically harboring deleted
GRK2 in chromaffin cells. At 4 wk postmyocardial infarction, adrenal-targeted GRK2 KO animals showed decreased circulating catecholamines and consequently reduced sympathetic activity. This was associated with enhanced cardiac function and improved ␤-adrenergic reserve
compared with control animals (150). Overall, the link between adrenal GRK2 and ␣2-AR-mediated negative feedback on catecholamine release appears important and to
have great implication in adverse cardiac remodeling and
progression of heart failure.
Due to the strong correlation between GRK2 levels and
heart failure, GRK2 inhibition has been proposed as a potential treatment for cardiac injury. Studies in a mouse
model expressing the ␤-ARKct as a peptide inhibitor of
GRK2 has added significant evidence supporting this notion. Cardiac-targeted ␤-ARKct transgenic expression in
mice led to a phenotype of significantly enhanced cardiac
function and increased sensitivity to acute ␤-AR agonist
exposure (129). Reciprocally, cardiac-targeted transgenic
mice with enhanced levels of GRK2, at levels found in failing human myocardium, leads to a phenotype of significantly aberrant ␤-AR signaling and a loss of inotropic reserve (129). These mice with cardiac ␤-ARKct expression
have also been used to rescue several models of heart failure
including dilated cardiomyopathy (210) and hypertrophic
cardiomyopathy (82, 97). The Muscle Lim Protein (MLP)
KO mouse, which is a model of dilated cardiomyopathy,
was the first heart failure mouse model rescued by ␤-ARKct
expression as GRK2 inhibition in myocytes from birth resulted in a MLP KO mouse that never developed pathology
(210). Calsequestrin (CSQ) is a sarcoplasmic reticulum calcium-binding protein. Mice overexpressing this protein
have a severe hypertrophic cardiomyopathy phenotype
with shortened survival (97). Surprisingly, mice expressing
the ␤-ARKct and overexpressing CSQ have significantly
enhanced survival that was synergistically potentiated by
␤-AR blocker treatment (86). This was the first example
that GRK2 inhibition could be complementary and even
more beneficial than the ␤-blockade, which is a standard of
care for human heart failure.
The inhibition of GRK2 by ␤-ARKct expression is anticipated to be mixed as it will also block other G␤␥-dependent
signaling processes so to ultimately prove that GRK2 inhibition is the therapeutic target of ␤-ARKct in heart failure.
388
Studies have been carried out using the GRK2 KO mice. As
stated, global homozygous GRK2 mice are lethal (117).
However, heterozygous KO mice are viable and present
with enhanced cardiac function essentially identical to
␤-ARKct transgenic mice (211). Interestingly, these KO
mice with 50% less GRK2 expressed in their hearts could be
further enhanced functionally with cardiac ␤-ARKct expression (211). GRK2 has also been genetically ablated only
in cardiomyocytes at or after birth, and these animals are
protected from heart failure development after myocardial
infarction showing that GRK2 activity is indeed pathological in the heart following cardiac injury (199). In fact,
induction of cardiomyocyte GRK2 gene ablation after postmyocardial infarction in mice significantly improved global
cardiac function and reversed adverse ventricular remodeling (199). Overall, these studies in mice show that the primary target of ␤-ARKct expression is GRK2 inhibition,
although there appears to be slight intricate mechanisms
that are unique to ␤-ARKct expression versus GRK2 KO.
One of these is the regulation of the L-type calcium channel
that is enhanced with loss of both GRK2 and ␤-ARKct
expression (200, 263). ␤-ARKct expression disinhibits the
L-type current through G␤␥ binding (263), while the loss of
GRK2 enhances L-type currents independent of G␤␥ (200).
Other mechanistic differences may exist, but these models
clearly show that limiting GRK2 activity in the heart after
injury prevents and even reverses heart failure.
Studies beyond the mouse have solidified GRK2 inhibition
via the ␤-ARKct as a viable therapeutic strategy for heart
failure (reviewed in Ref. 260). First, adenoviral-mediated
intracoronary-mediated myocardial delivery of the ␤ARKct to rabbit models of cardiac dysfunction and heart
failure has led to improved cardiac function (221, 248,
273). In fact, when ␤-ARKct was expressed before a myocardial infarction, the prevention of ␤-AR desensitization
and downregulation was shown to prevent heart failure
development (273). Recently, preclinical studies using
adeno-associated virus-serotype-6 (AAV6) to express
␤-ARKct have shown reversal of heart failure in both rats
(208) and pigs (198). These data have been submitted to
the United States Food and Drug Administration, and a
phase I clinical trial utilizing ␤-ARKct gene therapy for
GRK2 inhibition is currently underway with a prospective start date in 2016.
Additional evidence in support of the potential that GRK2
inhibition has in treating heart failure patients comes from
recent studies revealing that paroxetine, a drug that targets
serotonin reuptake centrally and that is currently used to
treat depression and central nervous system-related disorders, is capable of inhibiting GRK2 as an off-target (249).
Paroxetine has been shown to increase myocyte contractility through GRK2 inhibition, and further studies will investigate whether it can improve cardiac function in heart failure models (249). Interestingly, paroxetine is known to
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G PROTEIN-COUPLED RECEPTOR KINASES
have developmental cardiovascular side effects if given to
pregnant mothers (154), and this is consistent with GRK2
KO mice (117). Although paroxetine may not be an ideal
candidate for heart failure therapy, currently it is a prototype molecule that can lead medicinal chemists to develop
the first selective small molecule pharmacological GRK2
inhibitor. Of note, another compound gallein, which is not
a candidate for a viable human drug, has been shown recently to reverse the CSQ transgenic mouse model of heart
failure (122). Gallein actually targets G␤␥ binding and so is
analogous to ␤-ARKct expression. Although other targets
of G␤␥ binding were found, the primary mechanisms for
heart failure reversal with gallein was G␤␥-GRK2 inhibition (122). Overall, evidence strongly supports the fact that
inhibition of GRK2 in treating heart failure patients can be
beneficial though ␤-ARKct gene therapy or emerging small
molecules, and this will probably become a clinical reality in
the near future.
B. GRK2 and Acute Cardiac Injury
In humans, neurohormonal activation such as adrenergic
overdrive has been observed in patients acutely after cardiac
stress/injury including after a myocardial infarction (230).
In addition, increased levels of GRK2 have also been reported in other human studies of disease states (reviewed in
Refs. 14, 27). In animal models of cardiac injury, GRK2
upregulation is one of the first cardiac molecular changes
seen (159, 273). Of note is the observation that increased
levels of GRK2 correlates with clinical severity and left
ventricular stroke volume (3). In a 2-yr follow up study in
humans with acute coronary syndrome, GRK2 levels increased during ST-segment elevation in myocardial infarction and correlated with decreased ejection fraction and
end-systolic volume (218). In mice, upregulation of GRK2
was observed as early as 7 days after transverse aortic restriction (47), and increased levels of GRK2 appear to precede the prognosis of overt heart failure (7, 109).
Therefore, it is clear that GRK2 upregulation in myocardium occurs acutely after myocardial injury with significant
involvement in chronic progression of disease. Importantly,
studies have recently shown that altered GRK2 levels and
activity also have profound and significant effects in the
heart following acute cardiac injury (30, 72). First, when
cardiac-targeted GRK2 overexpressing transgenic mice are
subjected to ischemia/reperfusion (I/R) injury, they present
with significantly increased injury (i.e., cardiac infarction
size), and this was due to increased myocyte cell death due
to apoptosis in the ischemic region (30). Interestingly,
␤-ARKct transgenic mice displayed significant cardioprotection after I/R injury (30). This protection against acute
ischemic injury due to GRK2 inhibition with ␤-ARKct was
confirmed in cardiac-specific GRK2 KO mice as both mice
with loss of cardiomyocyte GRK2 at birth or induced loss of
GRK2 in adulthood had significant cardioprotection after
I/R injury (72). The mechanisms responsible for the prodeath activity of GRK2 and cardioprotection of lower
GRK2 activity and expression are not entirely clear, although in the transgenic study, inhibition of eNOS-NO
system via GRK2 was observed while ␤-ARKct increased
cardiac NO levels after I/R and improved survival signaling
(30). This is especially interesting due to the recent discovery of a dynamic interrelationship between GRK2 and
eNOS in the myocyte (106). In the cardioprotected GRK2
KO mice, an inhibition of cytochrome c release from mitochondria was observed as well as decreased myocyte apoptosis due to lower caspase-2 and -9 activities (72). Thus it
appears that acute GRK2 inhibition is a target for novel
therapeutic intervention as ischemic cardioprotection without the necessity for preconditioning of the myocardium.
Paroxetine, as a GRK2 inhibitor, although not ideal for the
chronic treatment of heart failure, may be a candidate for
acute single-dose administration around the time of reperfusion of postmyocardial infarction patients and is being
considered for such preclinical testing due to potential cardioprotective properties.
The cause of increased apoptosis after acute ischemia with
enhanced GRK2 activity is not entirely understood, but the
above studies in GRK2 KO mice are consistent with mitochondrial-dependent cell death pathways being involved.
This is especially interesting since studies by our lab (43)
and others (83, 168) have shown GRK2 to be localized to
mitochondria, which is a novel area of cellular localization
for this GRK. This is of particular significance to I/R injury
as the mitochondrion is the major source of energy to cells.
However, there is still no consensus in functional role for
mitochondrial GRK2. Of note, mitochondria from mice
overexpressing GRK2 possess premature opening of the
membrane permeability transition pore compared with
nontransgenic littermate mice (43). Studies in HEK cells
have argued that GRK2 overexpression is involved in increasing ATP content and I/R in quadriceps femoris muscle
of Cre-deleted GRK2 mice leads to decreased ATP content
compared with floxed control mice (83). On the other hand,
our lab has shown that GRK2 localization to the mitochondria is detrimental to the cardiac outcome post-I/R. GRK2
has been shown to interact with Hsp90 (148) and to be
phosphorylated by ERK at Ser670 (188). In cardiac cells,
translocation of GRK2 to the mitochondria is mediated by
ERK phosphorylation of GRK2 and Hsp90 binding (43).
With the use of geldanamycin or N-acetylcysteine (Hsp90
inhibitor and antioxidant, respectively), post-I/R injury
translocation of GRK2 to the mitochondria was attenuated
(43). Also, the use of COOH-terminal mutations of GRK2
where Ser670 was replaced with alanine revealed a loss of
mitochondrial localization following oxidative stress and,
importantly, less apoptosis indicating that GRK2 targeting to the mitochondrial is a prerequisite for its pro-death
activities (43). FIGURE 3A shows the proposed mechanism
for GRK2 translocation to the mitochondria. FIGURE 3B
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SATO ET AL.
A
B
Ischemia/Reperfusion
(Oxidative stress)
GPCR
ROS
GRK2
ROS
NAC
ROS
BARKCT
P
ERK
GRK2
Hsp90
Geldamycin
Mitochondria
FIGURE 3. Mitochondrial GRK2 translocation in oxidative stress. A: cellular stressful events (such as
ischemia/reperfusion) lead to the generation of reactive oxygen species (ROS) and subsequent activation of
extracellular signal-regulated kinases (ERK), which in turn phosphorylate G protein-coupled receptor kinase 2
(GRK2) at residue 670. Phosphorylated GRK2 then interacts with heat shock protein 90 (Hsp90) mediating
translocation to the mitochondria. ␤ARKct is a peptide inhibitor of GRK2. Treatment with the antioxidant
N-acetylcysteine inhibits ROS generation. Geldamycin (GA) is an inhibitor of Hsp90. B: immunogold electron
microscopy of cardiac tissue from nontransgenic littermate control (NLC) on top and GRK2-overexpressing
(GRK2Tg) animals on bottom. Gold particle labeling shows GRK2 present at baseline in mitochondria of both
genotypes though overexpression of GRK2 seems to also translate to increase amount of GRK2 in the
mitochondria. Original experiment first described in Chen et al. (43). Scale bars are 1 ␮m. It is clear that basally
GRK2 is already residing within the mitochondria (B), independent of mechanisms shown in A. Therefore,
GRK2 may play a role in normal mitochondrial homeostasis. It is not yet clear what this can be, but data
suggest mitochondrial GRK2 may regulate ATP levels (83).
shows immunogold electron micrographs from cardiac
tissue of GRK2-overexpressing mice (GRK2Tg; bottom)
and a nontransgenic littermate control mouse (NLC;
top). This clearly shows the localization of GRK2 to the
mitochondria and that overexpression of GRK2 leads to
increased mitochondria GRK2 even at baseline. The functional impact of this localization is not yet clear but under
active investigation.
C. GRK2 and Hypertension
Increase in blood pressure can be mediated through increase
in catecholamines (norepinephrine or epinephrine), which
act primarily on ␣-ARs (␣1 and ␣2), potentially altering
peripheral resistance. Other hypertensive neurohormones
such as angiotensin II and endothelin-1 can also induce
chronic GPCR activation that can lead to increased peripheral resistance. Alterations in peripheral resistance can ulti-
390
mately lead to an attenuation of vasodilation, subsequently
increasing cardiac afterload. Hypertension dramatically increases the risk for ischemic heart disease, which can in turn
lead to stroke as well as cardiac and renal failure. Although
the kidney and heart are at the core of blood pressure control, the contractile state of the vasculature is of major importance, due to the fact that blood flow is inversely proportional to the radius of the vessel to the fourth power
(reviewed in Ref. 99). Interestingly, GRK2 levels are increased in young patients with hypertension compared with
both normotensively young and old patients (92). In agreement are the studies in hypertensive patients showing increased peripheral blood lymphocyte GRK2 levels (116).
Overall, increased GRK2 levels are correlated with increased blood pressure (94). In hypertensive rat models,
there is increased GRK2 in both lymphocytes and vascular
smooth muscle (93). Importantly, vascular-targeted increase of GRK2 via transgenesis is sufficient to increase
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G PROTEIN-COUPLED RECEPTOR KINASES
mean arterial blood pressure by ⬃20% compared with
control NLC mice (69). The increased levels of GRK2 in
vascular smooth muscle were also accompanied by thickening of the vessels and cardiac hypertrophy (69). This
was attributed to the enhanced desensitization of GPCRs
that relax blood vessels such as ␤2-ARs (69). Of course,
the mechanism for how GRK2 may induce hypertension
is not fully understood. Data with ␤-blockers would suggest it may not be entirely due to uncoupling of ␤-ARs,
and it is important to keep in mind that the noncanonical
actions of GRK2 such as MAP kinase regulation may be
in play here.
GRK2-dependent changes in blood pressure and vascular
homeostasis may also occur within endothelial cells where
GRK2 is expressed. Consistent with results in cardiomyocytes (30, 106), lowering GRK2 levels in vascular endothelial cells increases NO bioavailability, which can lead to
enhanced vasorelaxation, and this can protect against hypertension in mice (9, 245). Beneficial effects of lowering
GRK2 in endothelial cells also include improved insulin
signaling as recently shown in spontaneously hypertensive
rats (279) and in a mouse model of type 2 diabetes (244).
These data coupled with vascular smooth muscle targeting
support GRK2 inhibition, not only in heart failure, but for
hypertension as well. However, it must be noted that GRK2
has also been shown to be important in regulating endothelial inflammation and oxidative stress (51, 239). For example, lower GRK2 exclusively in murine endothelial cells was
recently shown to be associated with increased reactive oxygen species (51). This is intriguing since lower GRK2
in endothelial cells in mice with global loss of GRK2
(heterozygous GRK2 KO mice) are protective against oxidative stress (9). Thus more research is needed to reconcile
these findings.
Race is also a factor that contributes to the development
of hypertension (138, 223). GRK2 levels were measured
in a cohort of 133 African-American patients. Quantification of GRK2 levels in lymphocytes of hypertensive
patients (systolic pressure above 130 mmHg) showed a
2-fold increase in protein expression and a greater than
40% increase in GRK2 activity (53). In addition, the
same study found a direct correlation of GRK2 (but not
GRK5) between plasma norepinephrine levels and systolic blood pressure in black adults. Overall, elevated
levels of GRK2 in lymphocytes and vascular smooth muscle have been implicated in both human hypertension and
animal models of the disease. This increase in GRK2
levels and activity seem to be very pertinent especially
when hypertension takes the overt turn to heart failure
(99). Many have suggested using levels of GRK2 in lymphocytes as a biomarker in heart failure at least to monitor response to treatment (108). It may also be a risk
predictor of heart failure development.
IV. G PROTEIN-COUPLED RECEPTOR
KINASE 5 AND CARDIAC DISEASE
A. GRK5 and Its Role in Pathological
Cardiac Hypertrophy and Heart Failure
In addition to GRK2, GRK5 is found at high levels in the
heart (2, 67). In fact, of all the tissues expressing GRK5 in
the body, its highest levels are found in myocardium (134;
also see TABLE 1). Interestingly, in myocardial tissue of
transplanted hearts, GRK5 was shown to be the most commonly expressed GRK (2). Recently, many studies in human subjects have suggested GRK5 as a key player in the
development of cardiac hypertrophy and heart failure.
GRK5 levels were increased by 68% in the left ventricle of
hearts with dilated cardiomyopathy and by 48% in volumeoverload patients (67). In the failing myocardium, GRK5
protein was upregulated, and an increase in GRK5 protein
and mRNA was observed in patients treated with ␤-agonists compared with those under ␤-blocker treatment. In
this study, ␤-blockade led to GRK5 mRNA levels that were
comparable to nonfailing hearts, although protein content
only showed a decreased trend compared with failing hearts
(3). Finally, in human studies comprised of ischemic, dilated, and nonischemic/nondilated cardiomyopathies, expression levels of GRK5 inversely correlated with left ventricular end-systolic and diastolic diameters (162). On the
basis of these human studies, it has been proposed that
GRK5 is a potential therapeutic target for the treatment of
heart failure (reviewed in Ref. 107).
Like GRK2, the canonical function of GRK5 is to terminate
GPCR signaling. Indeed, cardiac-targeted GRK5 overexpressing transgenic mice have severely attenuated ␤-AR signaling and myocardial in vivo responsiveness, demonstrating that, like GRK2, this kinase can act as a ␤-AR kinase in
vivo (212). Cardiac-targeted overexpression of GRK2 or
GRK5 in mice has been shown to result in different regulation of GPCR signaling. For instance, adenosine receptors
are targeted by the overexpression of GRK5 but not GRK2,
while angiotensin II receptors are targeted by the overexpression of GRK2 and not GRK5 (212). Of importance to
cardiac function, recent studies have revealed functions for
GRK5 beyond its canonical role of regulating GPCRs.
GRK5 can bind to calmodulin with an affinity 40 times
higher than GRK2 (215). The binding to calmodulin prevents membrane-mediated signaling, although phosphorylation of soluble substrates is unchanged (49, 79). This
binding of calmodulin has been shown to be essential for
the non-GPCR activity of GRK5 in the heart as this causes
the nuclear accumulation of GRK5 (90). GRK5 has also
been shown to bind to DNA and to have an active nuclear
localization signal (NLS) corresponding to amino acids
388 –395 in the catalytic domain (see FIGURE 2). Altering
the NLS to keep GRK5 out of the nucleus does not appear
to significantly affect GPCR kinase activity (120). More
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SATO ET AL.
recently, GRK5 has been shown to possess a nuclear export
signal (NES) corresponding to amino acids 259 –265 (see
FIGURE 2) (119). Of note, the NLS within the catalytic domain appears to be a common feature of GRK4 subfamily
members (119). Following cardiac hypertrophic stimulation, which causes calcium-calmodulin activation, GRK5
translocates to the nucleus where it has noncanonical activity as a class II histone deactylase (HDAC) kinase and induces gene transcription through the derepression of MEF2
(157, 282). This has been shown in transgenic GRK5 overexpressing mice to lead to exaggerated cardiac hypertrophy
and accelerated heart failure (157). Importantly, transgenic
mice overexpressing a GRK5 with its NLS mutated have
normal responses to hypertrophic stimulation showing that
it is this novel activity of GRK5 responsible for the pathology (157, 282). To confirm the importance of GRK5 to
cardiac pathology when there is abnormal hypertrophic
stress, GRK5 KO mice (both global and cardiac-specific)
have significantly less hypertrophy and improved function
after ventricular pressure overload (89). These data demonstrate the first known pathophysiological role for a noncanonical activity of a GRK, and a schematic of this mechanism of GRK5 in pathological cardiac hypertrophy is
shown in FIGURE 4A. Additionally, studies in myocytes of
spontaneously hypertensive heart failure rats show GRK5
protein localization to the nucleus (FIGURE 4B), further supporting the notion of GRK5 redistribution in cardiac diseased states.
Genome-wide RNA interference in Drosophila identified
G protein-coupled receptor kinase (Gprk)2, which in
other organisms is homologous to GRK5, to be a conserved regulator of NF␬⟩ (255). Further studies have also
confirmed GRK5 as a mediator of NF␬⟩ signaling in
mammals, with potential implications in cardiac hypertrophy and other cardiovascular pathologies, although
the mechanism of action is still not fully understood
(114, 170, 232). NF␬⟩ is a highly regulated dimeric transcription factor present as heterodimers (p50:p65) or homodimers (p50:p50 or p65:p65) (87). Extracellular stimulus induces the activation of I␬⟩ kinase (IKK)-mediated
phosphorylation and degradation of NF␬⟩ repressor,
I␬⟩␣, causing NF␬⟩ to be free to shuttle to the nucleus
(13, 66). However, this recent view has been challenged
by the findings that NF␬⟩:I␬⟩␣ complexes are able to
continuously shuttle between cytoplasm and nucleus by
nuclear transporters (24, 87). With that in mind, studies
in HEK and BAEC cells overexpressing GRK5 showed
nuclear accumulation of I␬⟩␣, which was linked to inhibition of NF␬⟩ signaling. The mechanism suggested in
this study was due to a physical interaction between
GRK5-RH domain (residues 50 –176) and I␬⟩␣ NH2 terminus rather than phosphorylative events (232). In addition, in support of this mechanism, intracardiac injection
of a NH2-terminal peptide of GRK5 (residues 1–176 containing the RH domain) reduced left ventricular hyper-
392
trophy by inhibiting NF␬⟩-mediated hypertrophic gene
expression (233). A schematic view of this mechanism is
shown in FIGURE 5A. Nevertheless, in neonatal myocytes
exposed to the hypertrophic agonist, phorbol 12-myristate 13-acetate (PMA), there were increased levels of
GRK5, p50, and p65. Expression of dominant negative
I␬⟩␣, usage of N-acetylcysteine, or silencing of p65
showed decreased levels of GRK5. Activation of NF␬⟩
led to binding of p50 and p65 to the GRK5 promoter and
subsequent increase in GRK5 expression after hypertrophic stimuli (115). Conversely to the mechanism proposed in FIGURE 5A, overexpression of GRK5 increased
levels of p50 and p65, and silencing of GRK5 led to
decreased levels of p50 and p65 and loss of NF␬⟩ DNA
binding activity (114). A schematic of the latter mechanism is shown in FIGURE 5B.
Moreover, in other studies, GRK5 has also been implicated
in mediating NF␬⟩ activation in macrophages, and these
cells are important in injured myocardium (175, 176).
Overexpression of GRK5 in Raw 264.7 macrophages enhanced TNF-␣-mediated NF␬⟩ activity, and knockdown of
GRK5 led to diminished NF␬⟩ activity, in agreement with
the mechanism in FIGURE 5B. Moreover, in macrophages,
GRK2 and GRK5 interacted with I␬⟩␣ NH2 terminus (in
agreement with FIGURE 5A), and GRK5 (but not GRK2)
phosphorylated I␬⟩␣ at the same residues as I␬⟩␣ kinase
does (Ser32 and Ser36) (175). Due to the role of NF␬⟩
signaling in inflammation, studies have also looked at how
GRK5 levels modulate inflammation. In a sepsis model,
GRK5 KO mice showed less inflammation than its control,
with diminished expression of NF␬⟩-driven genes and decreased phosphorylation of I␬⟩␣ (170, 176). In agreement,
lipopolysaccharide (LPS)-induced I␬⟩␣ phosphorylation
was significantly reduced in macrophages from GRK5 KO
mice. LPS-induced p65 translocation to the nucleus and
NF␬⟩ DNA binding was significantly diminished in the
GRK5 KO mice (176).
B. GRK5 Polymorphism and Its Role in
Cardiac Disease
Genetic polymorphisms have been suggested to be involved
in inter-individual differences in response to ␤-AR activation and its outcome to disease. GRK5 nonsynonymous
polymorphisms have been reported in humans while there is
none in GRK2 (137). In particular, many studies have
looked into GRK5 residue 41 glutamine to leucine polymorphism due to its high prevalence in the population from
African genetic background. GRK5-L41 has been reported
to represent a gain-of-function polymorphism by enhancing
␤2-ARs uncoupling during persistent agonist exposure
(266). This polymorphism has been suggested to present a
greater risk for loss of bronchodilatory function during
␤-AR agonist stimulation in asthma patients (266). Studies
in transgenic mice bearing cardiac GRK5-L41 showed an
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G PROTEIN-COUPLED RECEPTOR KINASES
A
Epinephrine/Angiotensin II
Endothelin-1
GPCR
GPCR
GRK5
Gq
Gq GRK5
Calmodulin
GRK5
Calmodulin
Exit
nucleus
GRK5
14-3-3
P
P
HDAC5
IP3 receptor
Calmodulin
HDAC5
Hypertrophic genes
MEF2
DNA
Nucleus
B
a
b
c
d
e
f
g
h
enhanced uncoupling of isoproterenol-stimulated ␤-adrenergic signaling compared with GRK5-Q41 or nontransgenic littermate mice (137). Interestingly, GRK5-L41 and
GRK5-Q41 show similar nuclear localization and ability to
stimulate HDAC nuclear export (282) (see FIGURE 4). In
this study, diminished ␤-AR-signaling was viewed to be
beneficial as it was interpreted as mimicking ␤-blockade. In
fact, African-American GRK5-L41 heart failure patients
showed prolonged survival or prolonged time for cardiac
transplantation (137). In addition, in a cohort of 2,673
FIGURE 4. Agonist-mediated GRK5
translocation to the nucleus. A: catecholamine or angiotensin II-mediated receptor
activation leads to calmodulin activation
through Gq. Calmodulin binds to the NH2
terminus of GRK5 dislodging from the
plasma membrane and promoting nuclear
localization. Nuclear GRK5 interaction to
calmodulin is then stabilized by IP3-regulated calcium release. Nuclear GRK5 can
act as an HDAC5 kinase, de-repressing
MEF2 and inducing hypertrophic gene
transcription. On the other hand, endothelin-1 receptor activation leads to its desensitization via GRK5 which cannot interact
with calmodulin thus preventing nuclear
translocation. B: cardiomyocytes from control rats (left column) and spontaneously hypertensive heart failure rats (SHHF) (right
column). Myocytes are shown at different
ages which correspond to: 2 (first row), 6
(second row), 12 (third row), and 24 mo old
(fourth row). Confocal images labeling for
GRK5 reveal a redistribution of GRK5 to the
nucleus in SHHF rats. Scale bar equals 50
␮m. [Image from studies of Yi et al. (281),
with permission from John Wiley and Sons,
Inc.]
acute coronary syndrome patients, improved outcome was
observed in African-American patients harboring GRK5L41 (56). Moreover, analysis of human patients carrying
GRK5-L41 was found to be protective in treated hypertensive patients with coronary artery disease, although no influence in blood pressure response to antihypertensives was
seen (142). Yet, another study, looking at patients with left
ventricular apical ballooning syndrome showed an increased prevalence of GRK5-L41 polymorphism (235), although no link to prognosis was drawn in this study. Over-
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SATO ET AL.
A
B
GPCR
GPCR
GRK5
GRK5
GRK5
IκBα
GRK5
NFκB
IκBα
NFκB
GRK5
GRK5
NFκB
NFκB
NFκB
NFκB
IκBα
IκBα
NFκB
IκBα
NFκB
NFκB
IκBα
NFκB-driven genes
and GRK5
NFκB-driven genes
DNA
DNA
FIGURE 5. Potential mechanisms of interaction involving GRK5 and NF␬⟩ pathway. A: NF␬⟩-I␬⟩␣ complex
can shuttle between cytoplasm and nucleus. Alternatively, I␬⟩␣ can be phosphorylated by I␬⟩␣-kinase leading
to its degradation. Free NF␬⟩ can then enter the nucleus and activate transcription. GRK5 RH domain can bind
to I␬⟩␣ COOH terminus. Upregulation of GRK5 leads to the increase of nuclear I␬⟩␣, which can in turn inhibit
NF␬⟩ and NF␬⟩-driven transcription. B: alternatively, upregulation of GRK5 has been proposed to upregulate
NF␬⟩ proteins (p50 and p65). This upregulation in NF␬⟩ would lead to its translocation to the nucleus and
activation of gene transcription. In addition, NF␬⟩ can also activate the transcription of GRK5.
all, it is important to point out that while nuclear events
may be an important factor observed in these polymorphisms, canonical GPCR phosphorylation and desensitization as well as noncanonical induction of ␤-arrestin-biased
signaling due to altered GRK5 activity may be a contributing factor to what has been observed with this GRK5 polymorphism.
More recently, intronic short tandem repeat (STR) (CAn)
polymorphisms of GRK5 have been linked to type 2 diabetes mellitus (T2DM) (278). A cohort of 1,164 patients was
classified in four groups (normal fasting glucose, impaired
fasting glucose, impaired glucose tolerance, and T2DM),
and five intronic CAn alleles in GRK5 were identified. CA16
was identified as the only GRK5 STR to be associated with
abnormal blood glucose (278), yet the mechanism in which
this would alter transcription is still unknown. Overall,
GRK5 polymorphisms represent a potential mechanism in
which genetic variability could associate with cardiac disease susceptibility and risk factor. Nevertheless, further investigation needs to address mechanism of action, polymorphisms that are silent versus nonsilent, and which cardiac
conditions GRK5 polymorphisms would mostly influence,
as there are also reports of GRK5 polymorphisms not associating to certain diseases (74, 247).
394
V. OTHER GRKs AND THE
CARDIOVASCULAR SYSTEM
A. GRK3 and the Heart
GRK3 has been shown to be expressed in the human heart
although to a much lesser extent than GRK2 and GRK5 (2,
67, 162). Clinical studies have shown that decreased heart
performance inversely correlates with levels of GRK3
(162). Other studies, however, have reported no change in
GRK3 levels in heart failure patients (3). Nevertheless, discrepancies such as these may be explained, at least in part,
by varying heart failure etiologies, such as the augmented
levels of GRK3 in volume overload patients and unchanged
levels of GRK3 in dilated cardiomyopathy hearts (67).
Studies in mice have been particularly helpful in unveiling
the potential functional role of cardiac GRK3 (see TABLE 3).
GRK3 KO mice developed normally, albeit displaying impairment in odorant-induced desensitization of cAMP responses (182). Cardiac-restricted expression of GRK3
peptide inhibitor (GRK3ct, analogous to the ␤-ARKct of
GRK2) in mice led to hypertension derived from increased
cardiac output, hyperkinetic myocardium, and increased
␣1-AR responsiveness (261). No alterations in cardiac mass
or left ventricular dimensions were observed in GRK3ct
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G PROTEIN-COUPLED RECEPTOR KINASES
mice at baseline. Nevertheless, after chronic ventricular
pressure-overload, preserved cardiac function was observed
only in GRK3ct transgenic mice compared with NLC control mice (264). This preservation in function after pressure
overload was observed even though there was similar induction of hypertrophic gene expression as seen in control
mice (264). Based on this, during development of heart
failure, it can be inferred that GRK3 may play a role in
receptor responsiveness without modulating hypertrophic
responses (as in the case of GRK5). In addition, mice overexpressing GRK3 in the heart have no hypertrophy (68) and
possessed normal ␤-AR-signaling both biochemically and
functionally, although altered thrombin-mediated signaling
was observed (110). In hybrid mice studies, GRK3 overexpression has been highly implicated in diminishing ␣1-adrenergic receptor signaling (68). While studies in mice have
provided clues as to potential cardiac responses driven by
GRK3, further studies still need to elucidate the role of
GRK3 in human heart failure.
B. GRK4 and GRK6 in Hypertension
To a much lesser extent, GRK4 and GRK6 have also been
implicated in cardiac dysfunction. Since the levels of GRK4
and GRK6 in the human heart are rather low (67), it has
been hypothesized that most of the cardiac impact by these
two isoforms is through an indirect mechanism, possibly
through altered blood pressure. GRK4 is the main GRK
isoform in the testes, although it is also expressed in the
kidneys and brain, and at minor levels in other organs (see
TABLE 1) (194, 256). GRK4 has been reported to possess
four alternative splice variants (194). Furthermore, GRK4
single nucleotide polymorphisms have been previously reported and include R65L, A142V, and A486V. GRK4A486V has been associated with hypertension in Italian
patients (15), in Chinese patients (267), and in an Australian cohort of patients (234). Although in these three studies
no association between hypertension and GRK4-R65L or
GRK4-A142V variant was seen, increasing copies of
GRK4-R65L and GRK4-A142V were associated with reduced atenolol-induced diastolic blood pressure lowering
(256). In fact, mice harboring the GRK4-A142V variant
had increased arterial blood pressure compared with control animals (73). GRK4-A142V mice have defective coupling between D1 dopamine receptors and GRK4 in the
renal proximal tubule; thus the kidneys are unable to properly excrete sodium, leading to hypertension (73). In addition to the above-mentioned human studies, no single site
GRK4 polymorphism showed significant association with
hypertension in a population from Ghana (275). Yet, in
another study, GRK4-A486V has also been implicated with
hypertension, as has R65L, but only when in conjunction
with other polymorphisms (156). To clarify discrepancies,
meta-analysis of all five human studies was recently conducted and subgroup analysis by ethnicity revealed that
GRK4-A486V inversely correlated with hypertension
among East Asians, while it positively correlates among
Europeans. In addition, in Europeans GRK4-R65L also
correlated with hypertension (139). Overall, GRK4 polymorphisms may have an impact in the heart by promoting
hypertensive phenotypes; however, inconsistent results in
human studies have yet to provide solid evidence in this
matter.
GRK6 has four alternative splice variants (75, 161, 193,
257), and its association with hypertension has been studied
to a much lesser extent. GRK6 mRNA has been reported to
be barely detectable in human hearts (67). Due to its close
relationship with GRK5 and due to its minor expression in
the heart, efforts have attempted to determine whether hypertension-mediated heart failure induces changes in GRK6
expression. In a rat model of spontaneous hypertensive
heart failure (SHHF), GRK6 expression was increased in
12- and 24-mo-old animals compared with age-matched
controls with an increased cytoplasmic-to-membrane ratio
as seen by confocal imaging (281). Increased localization to
the membrane and kinase activity are proposed to occur
because of palmitoylation of GRK6 (241). GRK6-A contains three cysteines in the COOH terminus known to serve
as substrates for palmitoylation. GRK6-B (slice variant-B)
lacks those palmitoylation sites but has a site for protein
kinase phosphorylation, while GRK6-C has none of the
these two motifs, and GRK6-D lacks catalytic activity (161,
193). While GRK6 A-C differ in their COOH terminus,
which is involved in membrane targeting and receptor interaction, in COS-6 cells GRK6-D is exclusively localized to
the nucleus (257). Importantly, three of the GRK6 variants
identified in mouse are likely to exist in humans and rats
(257). The link between GRK6 levels, GRK6 variants, and
hypertension-mediated heart failure still needs further investigation.
Immune response and inflammation are important cellular
mechanisms that may also impact the heart. Important to
note are the studies reporting the involvement of GRK6 in
lymphocyte chemotaxis and inflammation (76, 124). Lymphocytes from GRK6 KO mice possess impaired chemotactic ability to respond to CXCL12, which is more pronounced in T cells than B cells (76). This effect is specific to
GRK6 as other GRK isoforms were not able to rescue the
impaired migratory behavior. Polymorphonuclear neutrophils (PMN) are involved in defense against infection, and
leukotriene (LT) B4 is one of the most potent chemoattractants of PMN. Studies in GRK6 KO mice revealed increased
PMN response towards LTB4 and stromal cell-derived factor 1 (SDF-1) (124). Loss of GRK6 in mice reduces and
enhances chemotaxis in lymphocytes and PMN, respectively, suggesting a cell-specific role of GRK6. Acute inflammation response was also increased in GRK6 KO mice
(124), and GRK6 deficiency in mice has been implicated
with autoimmune disease related to impaired apoptotic cell
clearance (165). Nevertheless, GRK6-mediated immune re-
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SATO ET AL.
sponse and inflammation impact on the heart still remain to
be fully elucidated.
VI. CONCLUSIONS
GPCR signaling is a dynamic process that can quickly adapt
to meet cellular demands. This signaling highly depends on
proteins that regulate its activity. Two protein families that
are intricately linked in the regulation of the activated receptors are the GRKs and ␤-arrestins. GRKs have a wide
array of physiological impact, and alterations in GRKs have
been linked to many noncardiac pathologies such as bipolar
disease (11), Alzheimer’s disease (168), rheumatoid arthritis (145), multiple sclerosis (265), Parkinson’s disease (33),
and others. This review focused on GRKs, and in particular,
the role of cardiac GRKs in maintaining myocardial function in diseased and nondiseased conditions. GRK2 and
GRK5 are the main cardiac isoforms. While both isoforms
have been shown to mediate canonical GPCR receptor signaling, unique non-receptor-dependent regulatory roles
have been recently uncovered. For example, GRK2 has been
reported to be involved in mitochondrial homeostasis and
GRK5 in nuclear transduction signaling. The noncanonical
localization of these GRKs opens new possibilities and exciting potential, as it provides a new avenue in which GRKs
could mechanistically exert function in the myocardium.
␤-Blockers are one of the most widely used drugs in the
treatment of hypertension and congestive heart failure. Like
␤-blockers, the GRKs are also involved in terminating ␤-AR
signaling. Changes in GRK levels or inactivation of GRKs
may reduce or enhance ␤-AR-mediated signaling, subsequently altering downstream signaling pathways. Human
studies suggest that GRK2 and GRK5 are increased in expression and activity in various cardiac diseases. Inhibition
of GRK2, as seen by the expression of the ␤-ARKct as well
as the targeted KO of GRK2 in myocytes, has been shown
to improve cardiac function in several animal models of
cardiomyopathy. It is well established that alterations in
GRK levels lead to changes in signaling pathways, which
may regulate apoptosis, inflammation, hypertrophy, and/or
transcription. While canonical signaling of GRKs in the
heart exacerbates progression to heart failure, novel, noncanonical molecular mechanisms of action for GRK2 and
GRK5 in the mitochondria and nucleus, respectively, hold
promise for future drug development to limit the pathology
evoked by upregulation of these kinases after cardiac stress/
injury. With the elucidation of novel GPCR biased signaling
mediated via ␤-arrestins have led to more importance for
GRK-mediated phosphorylation as GRKs enable this
type of signaling. In fact, which GRK can phosphorylate
a given receptor appears to control ␤-arrestin independent signaling.
In addition, new evidence on the role of nonabundantly
expressed cardiac GRK isoforms (GRK3, GRK4, and
396
GRK6) is intriguing. These isoforms, though expressed at
low levels at baseline, may be important and relevant in
diseased states. Finally, it is worth emphasizing that the
GRKs are expressed at different levels in various tissues,
and the challenge remains to understand the role that GRKs
play in various cardiac conditions. The overall correlation
between GRK levels to different cardiac disease etiologies
still remains to be determined, and further investigation will
no doubt continue to uncover important translational aspects that GRKs present in the cardiovascular system.
ACKNOWLEDGMENTS
Present address of M. Schwartz: Advanced Institutes of
Convergence Technology, Suwon, South Korea.
Address for reprint requests and other correspondence:
W. J. Koch, W. W. Smith Chair in Cardiovascular Medicine, Professor and Chair, Dept. of Pharmacology, Director, Center for Translational Medicine, Temple University
School of Medicine, 3500 N. Broad St., 940 MERB, Philadelphia, PA 19140 (e-mail: Walter.koch@temple.edu).
GRANTS
W. J. Koch is the W. W. Smith Chair in Cardiovascular
Medicine and is funded by National Heart, Lung, and
Blood Institute Grants R37 HL061690, R01 HL085503,
P01 HL091799, P01 HL75443 (Project 2), and P01
HL108806 (Project 3). P. Y. Sato is supported by a postdoctoral fellowship from the Great Rivers Affiliate of the
American Heart Association.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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