Current Medicinal Chemistry, 2000, 7, 911-943
911
Signal Transduction Pathways of G Protein-coupled Receptors
and Their Cross-Talk with Receptor Tyrosine Kinases: Lessons
from Bradykinin Signaling
Claus Liebmann*a and Frank-D. Böhmerb
Institute of Biochemistry and Biophysics, Biological and Pharmaceutical Faculty (a) and
Research Group `Molecular Cell Biology`, Medical Faculty (b), Friedrich-SchillerUniversity Jena, D-07743 Jena, Germany
Abstract: G protein-coupled receptors (GPCRs) represent a major class of
drug targets. Recent investigation of GPCR signaling has revealed
interesting novel features of their signal transduction pathways which may be
of great relevance to drug application and the development of novel drugs. Firstly, a single class
of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G
proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive
initiation of different signaling chains. Secondly, the different signaling pathways emanating
from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal
modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic
signals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogen
activated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases
(RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly
understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs)
which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs.
Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases,
including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules
such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are
likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes
previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may
be of much interest for modulating GPCR-mediated biological responses.
1. Introduction
G Protein-coupled receptors (GPCRs) constitute a
superfamily of transmembrane proteins that transduce
extracellular signals to the intracellular level. More than
1000 of GPCRs are known up to now - tendency
increasing. Their agonists differ in size and structure
ranging from large glycoproteins to simple molecules
such as amines or nucleosides. Agonist binding to a
GPCR leads to activation of a heterotrimeric G protein,
which in turn is linked to either activating or inhibiting
second messenger pathways. The resulting change in
second messenger concentration then leads to further
downstream effector events, frequently activation of
protein kinases.
In many cases a single receptor can activate
different G proteins and thereby induce dual or multiple
*Address correspondence to this author at the Institute of Biochemistry
and Biophysics, Friedrich-Schiller-University Jena, Philosophenweg 12,
D-07743 Jena, Germany; Tel.: +49-3641-949357; Fax : +49-3641949352; E-mail: bglicl@rz.uni-jena.de
0929-8673/00 $19.00+.00
signaling routes [1]. In other cases, multiple receptors
can converge on a single G protein which has the
capability of integrating different signals [1]. Stimulation
of a particular GPCR may result not only in activation of a
single signaling pathway but also to subsequent
interactions with those activated by the other GPCRs.
These interactions between different receptor-coupled
signal transduction pathways are termed cross-talk. In
that way, synergistic interactions may be produced
which result in an amplification of a coincident signal
within the same cell playing a role in "fine-tuning" of
multiple receptor-signaling pathways [2]. On the other
hand, the signal transduction of one receptor may be
also negatively regulated by that of another receptor,
by feedback effects or by initiation of a parallel inhibitory
pathway.
Meanwhile it has become clear that GPCRs are not
only involved in the regulation of metabolic or excitatory
cellular responses but are also implicated in cell
proliferation [3] (Fig. 1 ). Many ligands of GPCRs which
are known as classical hormones or neurotransmitters,
© 2000 Bentham Science Publishers B.V.
912 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
Liebmann and Böhmer
Fig. (1). Schematic representation of GPCR-induced transmembrane signaling.
such as vasopressin, angiotensin II, endothelin,
substance P, bradykinin, acetylcholin or serotonin,
were found to elicit a mitogenic response in various
cells [3,4]. Thus, for example, the nonapeptide
bradykinin
(Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)
which regulates blood pressure and smoooth muscle
tone has been found to stimulate growth in small cell
lung cancer (SCLC) cells [5]. There is increasing
evidence indicating that GPCRs can function as
agonist-dependent oncoproteins [6].
What are the mechanisms by which a GPCR can
modulate cellular growth? One answer is cross-talk:
Beside the interactions between different GPCR
signaling pathways, stimulation of GPCRs may namely
also result in activation of a pathway which receptor
tyrosine kinases (RTK) employ for stimulation of cell
growth, the so-called "MAP kinase (MAPK) pathway"
[2,4,6,7]. Growth factor RTKs frequently stimulate
MAPK via recruitment of a complex of Shc, Grb2 and
Sos proteins to the cell membrane, leading to
subsequent activation of the small GTP-binding protein
Ras, the two protein kinases Raf and MEK and finally of
MAPK [8]. Once activated MAPK translocates to the
nucleus where transcription factors are phosphorylated
and subsequently activated [8]. GPCRs that mediate
growth stimulatory effects activate key effector
molecules of the MAPK pathway, including the RTK,
Ras, Raf, or MAPK. Although MAPK activation may not
be required for all GPCR-mediated growth responses,
MAPK could be a convergence point of growthpromoting signals arriving from both RTKs and different
GPCRs. Bradykinin, for example again, was also
reported to stimulate MAPK activation, e.g. in
fibroblasts [9], SCLC cells [10], or ventricular myocytes
[11].
In this review, we discuss principle signaling routes
linking GPCRs to the MAPK pathway and, vice versa,
leading to modulation of GPCR signalling pathways by
RTK-induced tyrosine phosphorylation. As an example
we will discuss the recent progress in the evaluation of
cross-talk mechanisms between the bradykinin B2
receptor (B2R) and the EGF receptor (EGFR).
The majority of the modern pharmaca is targeted to
GPCRs [12]. Uncovering of the complexity of GPCR
signaling, of cross-talk mechanisms and of interaction
with RTKs has interesting implications for drug
development. Previously known GPCR effectors may
Signal Transduction Pathways of G Protein-coupled Receptors
have unexpected effects via affecting complex
signaling networks. This may lead to unwanted side
effects but also to novel applications of known drugs.
Compounds affecting recently discovered GPCR signal
transduction pathways as RTK transactivation may
present paradigms for novel principles to modulate
GPCR signaling.
2. Basic Principles
Transduction
2.1.
GPCR
Subtypes
of
Subfamilies
GPCR
and
Signal
Receptor
All GPCRs consist of an extracellular N-terminal
segment, seven transmembrane helices (TM1-TM7),
which form the TM core, three extracellular loops, and
three loops and the C-terminal segment exposed to
the cytoplasm. A fourth cytoplasmic loop may be
formed when the C-terminal segment is palmitoylated at
a Cys-residue. The seven TMs are arranged into a
membrane-spanning boundle in the counterclockwise
direction from TM1 to TM7 as viewed from the
extracellular surface [13]. The N-terminal segment is
the site of glycosylation and ligand binding, the Cterminal
segment
allows
palmitoylation
and
phosphorylation as prerequisites for desensitization
and internalization. The intracellular loops transmit the
signal from the receptor to G protein. The TM core does
not form a pocket or tunnel structure as conceivable
but is tightly packed by hydrogen bonds and salt
bridges [13]. There are several excellent reviews
focussing on GPCR structure, conformation, and
activation [13,14-16]. On the basis of ligand binding
and receptor activation several GPCR subfamilies have
been classified [14,16]:
1.
GPCRs with ligand binding to the core
exclusively
(photons,
biogenic
amines,
nucleosides,
lysophosphatidic
acid,
eicosanoids)
2.
GPCRs with ligand binding partially in both TM
core and exoloops (short peptides)
3.
GPCRs with ligand binding to the exoloops and
the N-terminal segment (large polypeptides, e.g.
glucagon)
4.
GPCRs whose ligands bind to the N-terminus
exclusively (glycoproteins, e.g. LH, TSH)
5.
GPCRs whose ligands bind to a long N-terminal
segment that subsequently interacts with the
membrane-associated receptor domaine (small
neurotransmitters) and
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
6.
913
GPCRs with protease ligands that act through
cleavage of the N-terminal segment which in turn
activates the receptor (e.g. thrombin)
Within these subfamilies, the receptors for the
various endogenous ligands may occur as receptor
subtypes which are distiguished by their different
pharmacological agonist and antagonist profiles,
differences
in
their
(cDNA-deduced)
primary
sequences, and in many cases in different signal
transduction mechanisms [17]. For example, molecular
exploration has revealed the existence of 9 subtypes
of the adrenergic receptor (α 1A, α 1B, α 1D, α 2A, α 2B,
α 2C, β1, β2, β3), or 5 subtypes of the muscarinic receptor
(M1-M5), or 7 subtypes of the opioid receptor (µ1, µ2,
δ1, δ2, κ 1, κ 2, κ 3).
For bradykinin, three receptor subtypes with distinct
pharmacological profiles have been cloned [18-20] and
characterized. The bradykinin B2 receptor (B2R) is
constitutively expressed in various cells and mediates
the physiological and pathophysiological effects of
bradykinin such as vasodilation, bronchoconstriction,
inflammatory reactions or pain sensations. Expression
of the B1 receptor is only induced under
pathophysiological conditions such as injury (for review
see [21]). In addition, there are presumably tissuespecific splice variants of the B2R [22]. Bradykinin and
kallidin ([Lys0]BK) are equipotent natural agonist of the
B2R but do not act at the B1R. The principal B1R
agonist is [des-Arg9]BK. For both subtypes specific
antagonists have been developed such as Hoe 140 (DArg[Hyp 3,Thi 5, D-Tic7, Oic8]BK) for the B2R and
[Leu 8,des-Arg9]BK for the B1R. Recently, a third type
of kinin receptor was described in chicken and termed
ornithokinin receptor [20] where bradykinin is inactive
but the B2R antagonist Hoe 140 acts as full agonist.
The existence of receptor subtypes represent the
first level of specificity in GPCR signal transduction: a
single agonist may activate distinct signaling pathways
via receptor subtypes which are encoded by different
genes and display distinct coupling specificities.
2.2.
Posttranslational
GPCRs:
Glycosylation,
Phosphorylation
Modifications
Palmitoylation
of
and
The N-terminal segments of most GPCRs are
glycosylated on asparagyl residues. Glycosylation may
be important for the functional expression and cell
surface localization of receptors [23] and may
contribute to the ligand binding [24], depending on the
cell type.
Most GPCRs contain one or two conserved cysteine
residues in their C-terminal cytoplasmic domain that
appear to be generally palmitoylated. Palmitoylation is
914 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
Liebmann and Böhmer
unique among lipid modification of proteins: it is
reversible and adjustable [25]. There are several lines
of evidence indicating that palmitoylation is important
for intracellular trafficking of GPCRs and their cell
surface expression but not for ligand binding or G
protein activation [26,27].
Phosphorylation of GPCRs on seryl or threonyl
residues represents a mechanism for the rapid control
of receptor function and regulates the association of a
GPCR with other proteins as well as their subcellular
localization. This type of covalent modification
terminates or attenuates receptor signaling via
desensitization [28]. Receptor desensitization by
phosphorylation is mediated either by G proteincoupled receptor kinases (GRKs) or by second
messenger
kinases
[28,29].
Heterologous
desensitization of a particular GPCR is a feed back
regulation by second messenger kinases such as
protein kinase A (PKA) or protein kinase C (PKC) which
may be activated via a signaling pathway of the target
receptor or via a separate GPCR. Phosphorylation
leads to changes in the receptor conformation and,
subsequently, results in an impaired interaction with G
proteins.
Ligand-induced or homologous desensitization
requires GRKs and their functional cofactors, the
arrestins. Both the GRK family and the arrestin family
include at least six members [29]. In a first step the
agonist-occupied receptors are phosphorylated by a
GRK. Then, an arrestin binds to the phosphorylated
GPCR and disrupt the interaction between receptors
and G proteins. Desensitization represents the first
step of internalization and down-regulation [28]. The
cytosolically localized GRKs may be translocated to the
membrane and thereby activated by the agonistTable 1.
occupied receptor but also by other factors such as
PKC or calmodulin [30]. Therefore, homologous or
heterologous receptor phosphorylation on Ser- or Thrresidues might be an important mechanism in cross-talk
regulation.
Tyrosine phosphorylation of GPCRs
discussed in a later chapter of this article.
2.3. Heterotrimeric
Mediators
G
Proteins
will
as
be
Signal
The heterotrimeric guanine-nucleotide-binding
regulatory proteins (G proteins) are composed of a 3652 kDa α-subunit, a 35-36 kDa β-subunit, and an 8-10
kDa γ -subunit. The β-and γ -subunits are assembled
into βγ-complexes that act as functional units. G
proteins are usually classified into four major
subfamilies that share some common features: Gs, G i,
Gq/11 and G12 (Table 1). Multiple isoformes of each
subunit have been identified up to date including 20
different α-, 6 β- and 12 γ -subunits. Interestingly, not all
the possible combinations of β- and γ - subunits can be
formed. G proteins underlie a cycle of activation and
deactivation that transmits the signal from the receptor
to the effectors (reviewed in [31, 34]). Receptor
activation triggers the exchange of GDP (bound in the
inactive state) for GTP on the α-subunit of a G protein
coupled to the receptor. This results in dissociation of
the complex into receptor, GTP-liganded Gα - and Gβγ units. The free receptor has a reduced affinity for its
agonist which is released. Gα -GTP and/or Gβγ can
interact with their target effectors. The slow intrinsic
GTPase activity of Gα terminates the α-induced
effector association. Gα - GDP re-associates the free βγcomplexes thereby also terminating βγ-mediated
Classification of G Proteins. Data are Summarized from [1,31-34]
Class
Subtyp
Toxin
Effectors
Receptors
Gs
αs, αolf
CTX
AC stimulation; Ca2+-channels
β1/β2-AR; V2-R; D 1-R; A2-R; odorantR
Gi
αi1-3, αo,
PTX
AC inhibition;
α2-AR; M2/M4-R; SSTR;
αt1-2; αgust
PTX
regulation of K+ - and Ca2+
µ-/δ-OR; TR; D2-R;
αz
-
-channels; cGMP-PDE
A1-R; LPA-R
αq, α11,
-
PLCβ activation
AT II-R; ET-R; B2R;
α14-16
-
α12,13
-
Gq
G12
M 1/M3-R; V1-R; P2Y-R
Na+ /K+ -exchange; Bruton`s
tyrosine Kinase/ras GAP
TxA2-R; LPA-R
Abbreviations used are explained in the text. Additional abbreviations: α/β-AR= adrenergic receptors; V1/2 -R= vasopressin receptors; D1/2 -R= dopamine
receptors; A1/2 -R=adenosine receptors; M1-4-R= muscarinic receptors; SSTR= somatostatin receptors; µ/δ-OR= opioid receptors; TR= taste receptor; LPA-R=
lysophosphatidic acid receptor; AT II-R= angiotensin II receptor; ET-R= endothelin receptors; B2R= bradykinin receptor; P2Y = purinergic receptor; TxA2-R=
thromboxane A2 receptor
Signal Transduction Pathways of G Protein-coupled Receptors
signaling. The reconstituted heterotrimeric G protein
interacts again with the receptor and can begin the
cycle anew.
Several of the α-subunits are substrates for
covalent modifications by either cholera toxin (CTX) or
pertussis toxin (PTX). CTX catalyzes the NAD+ dependent ADP-ribosylation of a conserved arginine
residue of Gα s and Gα t proteins, resulting in an
inhibition of GTPase activity and, therefore, constitutive
activation of Gα . PTX induces ADP-ribosylation of most
Gα -subunits of the Gi/o-family (except of Gα z), which
results in inhibition of receptor-G protein-coupling.
These toxins are important tools for identifying and
discriminating G protein-mediated responses.
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
915
The functional properties of G proteins are
influenced by the covalent attachment of three types of
lipids (for review see [35,36]). All α-subunits (with
exception of transducin) are reversibly palmitoylated by
labile thioester bonds to N-terminal cysteine residues.
In addition, α-subunits of the Gi/o family are
myristoylated at conserved N-terminal glycine residues.
This lipid modification is formed by a stable amide
linkage and irreversible. The γ -subunits of all G proteins
are prenylated by a stabile thioether bond between
prenyl groups and cysteine residues. The precise
function of these lipid modifications is not yet known
but they appear to facilitate the targeting of G proteins
to the membrane and the localization of G proteins to
specific membrane subdomains, e.g. caveolae [36].
Fig. (2). Adenylate cyclase isoform II (A) and phospholipase Cγ (B) as typical examples for convergence and integration of
signals at the level of single key molecules within signaling networks. Abbreviations are explained in the text.
916 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
The α-subunits of G proteins have different contact
regions to receptors, βγ-subunits, and effectors. There
is accumulating evidence suggesting that a region of
the C-terminal segment of Gα is important for the
coupling selectivity in receptor-G protein-interaction
[37]. For example, in elegant molecular and
biochemical studies has been shown that in α i/osubunits the C-terminal residues -4 (cysteine), -3
(glycine), and -1 (phenylalanine/tyrosine) and in α q/11subunits the C-terminal residues -3 (asparagine) and -5
(glutamate) play key roles in determining the receptor
selectivity [38,39]. The first 25 amino acids in the Nterminal part of α-subunits are essential for βγ-binding
whereas the effector binding region partially overlaps
the putative βγ-binding region. Therefore, the αsubunit cannot simultaneously bind effector and βγ
[31]. In addition to the α-subunit, the C-terminal region
of Gβ and the C-terminal part of Gγ may be involved in
receptor coupling and specificity [40].
2.4. The Main Effector Systems of GPCR
Signaling:
Isoforms,
Multiple
Second
Messengers,
and
Second
Messengerderived Mediators
2.4.1. Adenylate cyclase (AC)
Classically, adenylate cyclase responds to GPCRinduced stimulatory or inhibitory regulation, mediated
either by Gsα or Giα , respectively. Meanwhile at least 9
mammalian AC isoforms have been cloned and
functionally characterized (for review see [41-43]). All
isozymes can be stimulated by Gα s and,
experimentally, with the plant diterpene forskolin
(except AC-IX) but they differ in their response to other
regulatory molecules. Based on their sequence
homology, the ACs have been divided into several
subfamilies with similar patterns of regulation.
Adenylate cyclase type I (AC-I) can be stimulated by
Ca2+/calmodulin and inhibited by Giα - and βγ-subunits.
AC-II may be also activated by Ca2+/calmodulin but is
not inhibited by βγ-complexes. AC-VIII defines ist own
subfamily although its regulatory properties correspond
to those of AC-III. Type V and VI AC constitute a
subfamily that is only activated by Gsα and may be
inhibited by Giα as well as submicromolar
concentrations of Ca2+ but is insensitive to calmodulin
and is not affected by βγ-complexes. The subfamily of
AC-II, AC-IV, and AC-VII is insensitive towards Ca2+, can
be stimulated by activated PKC and is sensitive to βγcomplexes which are capable of enhancing the
stimulatory effect of Gsα (Fig. 2 A ). The regulation of
AC-IX is still unclear. In addition, cyclic nucleotide
phosphodiesterases (PDEs) that play a crucial role in
cAMP signal termination, are protein products of a
multigene family displaying arround 30 isoforms [43].
The intracellular target of cAMP is protein kinase A
(PKA) which consists of two cAMP-binding regulatory
subunits (R) and two catalytic subunits (C). R exists in
Liebmann and Böhmer
two forms, R-I and R-II. The PKA-RI complex is
cytosolically localized and its immobilization needs the
interaction with additional regulatory proteins, the Akinase-anchoring proteins (AKAPs). The PKA-RII
complex is particulate-associated. Thus, multiple
isoforms of AC, PDE and PKA combined with spatial
and temporal control provide an immense cross-talk
potential of the cAMP system.
2.4.2.
Phospholipases,
Lipidkinases,
and
Phospholipid-derived
Second
Messenger
Systems
Constitute Their own
Signaling
Network
The membrane lipid phosphatidylinositol 4,5bisphosphate (PIP2) represents the substrate for
phospholipase C (PLC). The hydrolysis of PIP2 results
in the simultaneous production of two second
messengers, inositol 1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG), which mediate intracellular Ca2+release and the activation of PKC, respectively [44]. To
date 3 subfamilies of PLC are known including 4 PLCβ
isozymes, 2 PLCγ isozymes, and 4 PLCδ isoforms (for
review see [45]). The PLCβ isozymes are activated by
α-subunits of the Gq/11 family but may be also
stimulated by βγ-complexes of Gq/11, Gi or Gz proteins.
The biological importance of PLCδ isozymes is not yet
clear. It is assumed that activation of PLCδ might be an
event secondary to receptor-mediated activation of
other PLCs or Ca2+-channels [45]. The two PLCγ
isozymes are structurally distinct from the other PLCs
because they contain two SH2 domains and one SH3
domain (SH = Src homology; see next chapter). Thus,
PLCγ is activated by RTKs of growth factors via binding
to their autophosphorylated tyrosine residues and the
subsequent phosphorylation of PLCγ at the tyrosines
771, 783, and 1254. The PLCγ isozymes may be also
activated by nonreceptor phosphotyrosine kinases
(PTKs) such as Src or Pyk2 (see also chapter 3). In
various cells, these PTKs can be activated by GPCRs.
In addition, PLCγ isozymes may be activated directly by
several lipid-derived second messengers of GPCRs in
the absence of tyrosine phosphorylation. Thus,
GPCRs coupled to PLD or PI3-kinases may stimulate
PLCγ through generation of their second messengers
phosphatidic acid (PA) or PIP3, respectively [45]. Like
AC-II, PLCγ represents a typical point of signal
integration and may play a key role in cross-talk (Fig.
2 B ).
In addition to PLCs, PIP2 serves also as a substrate
for the receptor-regulated class I phosphoinositide 3kinases (PI3Ks) which phosphorylate PIP2 to
PtdIns(3,4,5)P3 (PIP3) [46,47]. One subtype of PI3K,
the p110 PI3Kγ , has been shown to be directly
activated by βγ-complexes from Gi proteins [48]. In
addition to PIP3 production, this enzyme has the
capacity to activate the MAPK pathway [49]. Two other
Signal Transduction Pathways of G Protein-coupled Receptors
isoforms, the PI3Ks α and β are dimeric molecules
consisting of the p110 catalytic subunit and a p85
adaptor protein that contains two SH2 domains and
links the p110 subunit to RTK signaling pathways.
Recently it was reported that also the PI3Kβ can be
alternatively activated by βγ-complexes from G proteins
[50]. The PI3K-produced second messenger PIP3 is
rapidly degradated to PtdIns (3,4)P2, both molecules
act as second messengers and can directly activate
several PKC isoforms and/or the serine/threonine
protein kinases PDK and PKB/Akt. Activated PKB/Akt
provides a survival signal that protects cells from
apoptosis [51].
PIP2 is not only substrate for PLCs and PI3Ks but
also cofactor for the phospholipase D (PLD) which
represents another GPCR-regulated effector enzyme
[52]. PLD hydrolyzes phosphatidylcholine (PC) to
phosphatidic acid (PA) and choline in a cell-specific
response to various stimuli including thrombin,
vasopressin, endothelin, angiotensin II or bradykinin.
At least 3 mammalian PLD isoforms have been
identified up to now (for review see [52]). Downstream
of GPCRs, PLD regulation occurs by activation via PKC
or by activation via small G proteins such as ARF (ADPribosylation factor) or Rho (a monomeric G protein).
PLD could contribute to the intracellular signaling by
several mechanism. Firstly, PA has been implicated as a
lipid second messenger in the regulation of protein
kinases [54] activation of PLCγ [45], and other
signaling molecules, including the protein-tyrosine
phosphatase SHP-1 [55]. Secondly, in most cells PA is
rapidly converted to DAG through the action of
phosphadidate phosphohydrolase. Thus, PLD-derived
DAG is implicated in the late phase activation of PKC
[56]. Thirdly, PA is converted to lysophosphatidic acid
(LPA) by a specific PLA2. LPA is known as an
intercellular signaling molecule that is rapidly released
and affects cells by acting on ist own GPCR. LPA may
be involved in the regulation of a variety of cellular
responses, e.g. cell proliferation, smooth muscle
contraction, platelet aggregation, or neurotransmitter
release [57]. Interestingly, the LPA receptor is capable
of coupling to both G i and Gq/11 proteins thus activating
dual pathways [57]. Therefore, LPA generated from PA
is a typical example that a second messenger of a given
GPCR signalling pathway may produce a mediator
which acts through another GPCR in an autocrine or
paracrine manner.
Another phospholipase that is stimulated by
numerous agonists of GPCRs but also by growth
factors in a variety of cells is the cytosolic 85 kDa
phospholipase A2 (PLA2) (for review see [58-60]).
Stimulation of PLA 2 leads to the release of arachidonic
acid (AA) and, subsequently, to the production of
eicosanoids [61]. Activation of PLA2 by GPCRs
requires increase in cytosolic Ca2+ for the association of
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
917
PLA 2 to the membrane phospholipid substrate and
requires also phosphorylation that is obviously
catalyzed by activated MAPK (ERK2). PKC and PKA
can phosphorylate PLA2 in vitro but there is no
evidence for a direct phosphorylation in vivo [60].
Nevertheless, PKC activation can lead indirectly to
PLA 2 activation by triggering the MAPK pathway (see
chapter 4). Activation of PLA2 represents the direct
pathway of AA release. Alternatively, AA may be
released by a DAG-lipase from DAG which may be
produced either via PLCβ, PLCγ , or the PLD pathway.
AA from the various sources may be converted via the
cyclooxygenase pathway , for example, to PGE2 that
can bind to and activate 4 different prostanoid receptor
subtypes (EP1-4). EP2 and EP4 couple to Gs and
stimulate adenylate cyclase activity. EP 1 couples to
Gq/11 and activates PLCβ. EP 3 is able to regulate three
different sets of G proteins including Gq/11, Gi/o, and Gs.
This is another example for a signaling mechanism
where second messengers can produce mediator
molecules acting via GPCRs anew and utilize the same
set of effectors as their first messengers.
We have to go back once more to the Gq/11/PLC βpathway which is chiefly producing the multiple second
messengers IP3 and DAG. IP3 binds to a tetrameric IP3
receptor in the endoplasmatic reticulum (ER) and
triggers the release of Ca2+ from the ER resulting in an
intracellular increase in cytosolic Ca2+ from
approximately100 nM to approximately 1 µM. In terms
of signalling, Ca 2+ has to bind to trigger proteins such
as calmodulin that realize the messenger function of
Ca2+ (for review see [62,63]).
The intracellular target for DAG is protein kinase C
(PKC) that belongs to the serine/threonine protein
kinases. PKC is thought to be essentially involved in
the
regulation
of
cellular
proliferation
and
differentiation. The PKC superfamily consists of several
isoforms. Based on their domain structure and their
regulation they have to be divided into at least 3
subfamilies: "convential" cPKCs (α, βI, βII, and γ ), which
are sensitive to Ca2+, DAG, and phorbol esters; "novel"
nPKCs (δ, ε, η, and θ), which are activated by DAG and
phorbol esters but are independent of Ca2+; and
"atypical" aPKCs (ζ, λ, and µ), which are insensitive to
Ca2+, DAG and phorbol esters and may be regulated by
phospholipids, such as PA (for review see [56, 64-66].
Recently, the PKC superfamily was supplemented by
the newly discovered PKC kinases (PRKs) consisting
of at least 3 members (PRK1-3) [66]. Like the aPKCs,
they are insensitive to Ca2+, DAG and phorbol esters.
Phorbol esters are potent tumor promoters and can
substitute for DAG in activating cPKCs and nPKCs.
They are not metabolized like DAG, evoke a prolonged
activation of PKC and are useful tools in studying PKCregulated pathways in vivo.
918 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
Liebmann and Böhmer
Table 2. Typical Bradykinin Signalling Pathways : Multiple Coupling and Cell Specificity
Cell/Tissue
B2R Effector
Mechanism
Ref.
A431 cells, guinea pig ileum, others
PLCβ (stimulation)
via Gq/11
[71,72]
fibroblasts, rat myometrial cells
PLA2 (stimulation)
partially via Gi
[73.74]
tracheal cells
PLD (activation)
unknown
[75]
A431 cells
AC (stimulation)
via Gs
[76]
airway smooth muscle cells
AC (stimulation)
via Gq/11, PKC, MAPK, PLA2, PGE2
[77]
PC-12 cells
AC (stimulation)
via Gq/11, Ca2+/calmodulin
[78]
Rat uterus, GPI
AC (inhibiton)
via Gi
[79, 80]
NG 108-15 cells
Ca2+-activated K+ channels (activation)
via Gq/11, IP3, Ca2+
[81]
NG 108-15 cells
N-type Ca2+ channels (inhibition)
via G13, Rac, p38 MAPK
[82]
Thus, all signaling routes that generate DAG may
result in the activation of distinct PKC isoforms. In
addition, the PKC isoforms ζ, ε, and δ are known as
targets for PIP3, the second messenger of PI3Ks
[67,68].
Finally, it should be noted that not only membrane
enzymes but also K+ and Ca2+ channels may be
effectors for GPCRs (for review see [69]). Recently,
also cross-talk at the level of Ca2+ channels was
reported resulting from PKC-mediated phosphorylation
that antagonizes βγ-induced inhibition of Ca2+
channels [70]. This cross-talk mechanism provides a
first example that not only effector enzymes but also
ion channels have the potential for the integration of
multiple signaling inputs.
2.4.3.
Bradykinin
B2
Signal Transduction
Receptor-mediated
In dependency on the cell or tissue investigated the
pleiotropic hormone bradykinin has been shown to be
capable of activating different G proteins and multiple
signaling pathways (Table 2). In the majority of cases,
however, and in most cells bradykinin stimulates PLCβ
via a Gq/11 protein.
2.5.Complexity of GPCR Signaling:
Coupling, Switching, and Cross-talk
Multiple
It is well known that many individual receptors are
capable of interacting with different G proteins [1,83].
For example, the human thyrotropin (TSH) receptor or
also the bradykinin B2 receptor (Table 1) can interact
with members from each of the four major subfamilies of
α-subunits. The specificity of interaction may be
determined by the agonist concentration or by different
kinetics. Thus, low concentrations of TSH lead to
stimulation of AC whereas higher concentrations
induce activation of PLCβ [84]. The activation of
multiple coupling GPCRs, consequently results in
multifunctional signaling. Interactions between the
particular pathways of a single GPCR with multiple
signal transduction within the same cell could be
classified as homologous cross-talk. Such interactions
may occur at different levels of signal transduction.
Recently, the group of Lefkowitz [85] has
demonstrated that the β-AR can "switch" from G s to G i.
The "switch on" of Gi requires the proceeding
activation of Gs and the stimulation of the cAMP
pathway. Activated PKA phosphorylates the β-AR
leading to receptor coupling to Gi. Gβγ -complexes
released from Gi then activate MAPK in a Src- and Rasdependent manner [85]. Thus, this "switch on"
mechanism induces cross-talk at the receptor level.
Another example representing a "switch off"
mechanism comes from our own work about BK
signaling in A431 cells [76]. In this cell line, BK induces
a rapid G q/11-mediated activation of PLCβ resulting in
stimulation of PKC translocation. In a dual pathway BK
slowly activates G s followed by an increase in cAMP
production and PKA activation that leads to an inhibiton
of PKC translocation (Fig. 3 ).
In addition, numerous interactions between
signaling pathways of different GPCRs have been
described (for review see [2]). This type of interaction,
therefore, could be classified as "heterologous crosstalk". Synergistic cross-talk between Gi- and Gqcoupled
receptors,
e.g.
adenosine
A1- or
neuropeptide Y (NPY)-receptors and α 1-adrenergic
receptors often result in augmentation of physiological
responses such as smooth muscle contractions [2].
Such synergistic interactions seem to play an important
role in the "fine tuning" of multiple receptor signaling
pathways [2].
Signal Transduction Pathways of G Protein-coupled Receptors
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
919
Fig. (3). Dual signaling pathway of the bradykinin B2 receptor (B2R) in A431 cells: a rapid Gq/11 -mediated activation of the
PLCβ/PKC pathway is negatively modulated by a slowly induced but independent and Gs -mediated activation of the cAMP/PKA
pathway [76].
On the other hand, a single G protein can be
activated by multiple receptors (Table 1) thus acting as
convergence point [1,83]. The specificity of receptor
coupling to a mutual effector system may be
determined by the composition of heterotrimeric G
proteins. For example, using the antisense
oligonucleotide technique Kleuss et al. [86, 87]
showed in excellent studies that in GH3 cells inhibition
of Ca2+- channels by somatostain receptors is mediated
by Go2β1γ 3 whereas inhibition by M4 muscarinic
receptors is mediated by G o1β1γ 4. Another mechanism
for selective regulation of GPCR signaling pathways
may be provided by the newly discovered RGS
(regulators of G protein signaling) proteins [88, 89].
These RGS proteins function obviously as GAPS
(GTPase-activating proteins) for heterotrimeric G
proteins and and enhance the speed of GTP hydrolysis
thereby controling the kinetics of G protein signaling.
Members of the RGS family have been shown to
display selectivity for different Gα -subunits such as
RGS2 for α q or RGS1/3 for α i [89]. Thus, the cellspecific expression of different GRS proteins may alow
the independent control of multiple signaling pathways
of GPCRs.
3. Receptor Tyrosine Kinase (RTK)
Signaling and MAP Kinase Pathways
3.1. Ligand-dependent Activation of RTKs
Protein tyrosine kinases (PTKs) have central
functions for the regulation of cell proliferation and
other cell functions. Many polypeptidic mitogenic
factors ("growth factors") elicit their cellular responses
via receptors of the transmembrane receptor tyrosine
kinase (RTK) family [90-92]. Well known examples are
the receptors for epidermal growth factor [93,94], the
platelet derived growth factors (PDGFs) [95,96] or the
fibroblast growth factors (FGFs) [97-99]. The biological
effects of RTK stimulation include effects, largely
positive, on cell proliferation ("growth"), cell
differentiation, locomotion and cell survival (Fig. 4 ). In
contrast, receptors for cytokines, a class of growth- and
920 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
differentiation-modulating
polypeptides
for
hematopoietic and immune cells, are devoid of intrinsic
enzymatic activity and need to recruit cytoplasmic PTKs
for signaling [100]. While many aspects of signaling
from RTKs and cytokine receptors have similarities, the
latter class of receptors shall not be discussed further in
this review. RTKs consist of an extracellular ligand
binding domain, a single transmembrane domain and
an intracellular domain which harbors the conserved
tyrosine kinase subdomain and variable regulatory
sequences [101]. The hitherto known molecules in this
receptor family fall into 13 classes, assigned largely on
the basis of homology within their intracellular tyrosine
kinase domain [102]. More than 100 genes for RTKs
may exist in the human genome [101]. The functional
unit of an RTK consists of at least two receptor
molecules or higher oligomers [103-105]. Receptor
dimers or oligomers can be homomers or heteromers of
related [106,107], possibly also of unrelated RTKs . In
the absence of ligand, receptor dimer (oligomer)
formation apparantly occurs spontaneously with low
efficiency [108]. Solely overexpression of a given RTK
may trigger sufficient dimer formation leading to
signaling in the absence of a ligand [107]. This has
Liebmann and Böhmer
frequently been observed in high level overexpression
experiments [109,110] and in cancer cells
overexpressing an RTK. The receptors in the insulin
receptor family [111,112] present an exception since
they consist of disulfid-bridged dimers in the absence
of the ligand. Ligand bindig initiates signaling by
triggering dimer/oligomer formation or, in case of the
insulin receptor family, by triggering interaction of a
preformed receptor dimer. Various molecular means
are employed by RTK ligands to accomplish receptor
dimer stabilization [105]. Monomeric ligands as EGF
drive dimerization by interacting with receptor subunits
in a 1:1 stoichiometry [113]. Dimerization may be
consequence of conformational changes in the ligandoccupied receptors or may result from a bivalent
interaction of both ligands with both receptor subunits
in the dimer [114]. Other ligands are dimeric and
interact with two receptor subunits at the time, as in
case of PDGFs [104].
As a consequence of receptor dimerization the two
subunits trans-phosphorylate each other on tyrosine
residues. Two types of phosphorylations can be
distinguished: Phosphorylations in the "activation
Fig. (4). Multiple signaling pathways activated by RTKs and negatively controlled by PTPs in absence and presence of an RTK
ligand. The mechanisms as well as abbreviations are explained in the text.
Signal Transduction Pathways of G Protein-coupled Receptors
loop" in the kinase subdomain which lead to elevation
of kinase activity and phosphorylations in other parts of
the intracellular domain which create binding sites for
signaling molecules. Crystal structures of the insulin
receptor catalytic domain [115] and the FGF-receptor 1
catalytic domain [116] have revealed the mechanism of
activation of these kinases [117]. The activation loop, a
flexible structure identified in many protein kinases, in
its unphosphorylated state can occupy partially (FGF
receptor -1) or completely (insulin receptor) the active
site of the kinase. Phosphorylation leads to
displacement of the activation loop and in turn provides
access for substrates to the kinase active site. A
phosphorylation site corresponding to tyrosine 1162 in
the insulin receptor kinase is conserved in RTKs
suggesting that this mechanism of activation is a
common principle. However, for example in case of the
PDGFβ-receptor substantial residual kinase activity can
be detected in a receptor variant with the 857 tyrosine
(corresponds to 1162 in insulin receptor) mutated
[118] or selectively blocked by a kinase inhibitor [119].
Thus, stringency of regulation by activation loop
phosphorylation varies among the different RTKs.
Multiple
tyrosine
residues
can
further
be
phosphorylated in the RTK cytoplasmic domains, both
C- or N-terminally of the catalytic subdomain or, in RTKs
with a split kinase domain as PDGF receptors, also in
the "kinase insert" [95, 96]. These sites, in their
phosphorylated form, present recognition and binding
sites for signaling molecules posessing different types
of phosphotyrosine binding domains as SH2-domains
[120-122], or PTB domains [123, 124]. SH2 ("srchomology 2"-domains) are protein modules of about
100 amino acids which are found in numerous proteins
and recognize phosphotyrosine in the context of up to
6 C-terminal amino acids. In contrast, PTB
("phosphotyrosine binding") domains recognize
phosphotyrosine and 1-6 N-terminally situated amino
acids. Another type of protein domains involved in
signal transduction are SH3 ("src-homology 3")
domains. They consist of about 60 amino acids and
interact with other proteins in a phosphorylationindependent
manner
recognizing
proline-rich
sequences. These domains and a still increasing
number of recently discovered protein-protein
interaction domains are apparently required to achieve
efficiency and specificity in highly ordered signal
transduction complexes. Multiple interactions of the
RTK cytoplasmic domains with signaling molecules
form the basis for initiation of multiple intracellular
signaling events (Fig. 4 ). Several signaling chains may
be necessary to elicit together a particular biological
response. On the other hand, they form the basis for
different types of biological responses which can be
mediated via the same receptor as stimulation of cell
growth, cell locomotion, cell differentiation and
prevention of apoptosis.
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
3.2. Specificity and Redundancy
Downstream Signaling
in
921
RTK
Downstream signaling molecules which bind to
trans-phosphorylated RTKs fall in two classes:
enzymes and adaptor molecules. An example for the
former is the phospholipase Cγ [125], which binds via
tandem SH2-domains to different activated RTKs, is in
turn phosphorylated and itself activated. An example
for the latter is the p85 subunit of PI3kinase α, which
docks to several growth factor receptors and permits in
turn binding and thus membrane recruitment of the PI3
kinase catalytic subunit p110α [126, 127]. Adaptor
molecules can also recruit further adaptors, which
recruit other singaling molecules and so forth [122]. An
example is the family of Shc adaptor molecules, which
are phosphorylated subsequent to RTK binding and in
their phosphorylated form are able to bind the adaptor
molecule Grb2 [128]. Thus, RTKs present scaffolding
molecules for the assembly of multiple signaling
proteins which can interact in a chain of signal
transduction events [129].
Many RTKs have been shown to interact with the
same set of signaling proteins. For example, Shc and
Grb2 have been demonstrated to bind to multiple RTKs
including the EGFR [130], the EGFR-related receptor
HER2/erbB2 [131] and the hepatocyte growth factor
(HGF) receptor Met [132]. On the other hand, there are
also pronounced specificities. Thus, PLCγ cannot bind
to the insulin receptor [111] or Grb2 binds only with low
affinity directly to the PDGFβ-receptor [133]. Certain
RTKs have even quite specific interaction partners.
The insulin receptor family RTKs accomplish most of
their signaling activity via recruitment of relatively
specific docking proteins, the insulin receptor
substrates (IRS), which in their phosphorylated form
then interact with further signaling molecules [111].
Other examples are the FGF receptor 1 which elicits
much of its signaling via the FRS2 adaptor [134, 135]
and the Met/HGF receptor RTK which exhibits a
particularly strong interaction with the docking protein
Gab1 [136].
3.3. RTK Interaction
Tyrosine Kinases
with
Further
Protein
Another level of complexity of RTK signaling has
emerged when it was found that RTKs utilize also
cytoplasmic PTKs for signal transmission [137].
Cytoplasmic PTKs fall into 9 classes [102, 138].
Members of the Src-family of PTKs, comprising for
example the intensely investigated PTK pp60c-src
(designated as "Src" throughout this review), its
oncogenic counterpart v-Src and the PTKs Yes (pp62cyes), Fyn (p59fyn), Lyn (p56lyn) and Lck (p56lck) harbor
one SH2-domain and one SH3 domain in addition to
922 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
the catalytic domain. Negative regulation of these
kinases occurs by phosphorylation on tyrosine
residues in the C-terminus, leading to interaction with
the intramolecular SH2-domain. Positive regulation is
then possible by dephosphorylation of this
phosphotyrosine by protein-tyrosine phosphatases
(PTPs, see chapter 3.4). Src, Fyn and Yes may have
apparently very similar functions. Several assays used
for detecting involvement of Src in cellular functions
cannot differentiate between these kinases. Another
family of cytoplasmic PTKs is named after its prototype
focal adhesion kinase (Fak), a 125kDa enzyme which
cooperates with Src in integrin signaling and regulation
of the assembly of cell-matrix adhesion complexes. A
relative of Fak is Pyk2, a PTK which is activated by Ca2+
and likely to be involved in downstream signaling of
some GPCRs. Finally, the family of JAK ("janus
kinases") should be mentioned here, which includes
also Tyk2 as a member. These PTKs function
downstream of cytokine receptors but may also interact
with some RTKs. A paradigm for interaction of an RTK
with cytoplasmic PTKs present the PDGF-receptors
which have the capacity to recruit Src family kinases,
including p60 c-src and activate them in turn [139-141].
Src family kinase activation has been suggested to be
essential for mitogenic signaling of the PDGFβ-receptor
[142]. Similar observations have been made with the
EGFR, although RTK- Src kinase complexes are less
readily demonstrable in this case [142-145]. Src-family
kinases do appear, however, to not only simply mediate
a signal but, to also phosphorylate the RTKs and
thereby modulate signaling. Src phosphorylation sites
have been mapped on the EGFR, the PDGFβ-receptor
and the IGF-1 receptor [146-151]. Mutation of a main
Src-phosphorylation site on the PDGFβ-receptor leads
to a reduced mitogenic signaling and a more
pronounced stimulation of chemotaxis by PDGF via the
mutant receptor [150]. For the EGFR and the IGF-1
receptor, increased RTK activity subsequent to
phosphorylation by Src has been shown [146, 151].
Other cytoplasmic tyrosine kinases which have been
shown to interact with RTKs are for example FER, a
member of the fes/fps family of nontransmembrane
receptor tyrosine kinases [152, 153] and possibly
members of the JAK/Tyk family [154].
3.4. Inactivation Mechanisms for RTKs
Tyrosine phosphorylation needs to be tightly
regulated. Loss of this tight regulation may lead to
aberrant cell behaviour as uncontrolled proliferation.
For many tyrosine kinases including RTKs oncogenic
variants exist which have constitutive activity as a
common property [155]. In case of RTKs, tight
regulation is on the one hand provided by the
necessity of ligand stimulation for activation. On the
other hand, several negative regulation mechanisms
Liebmann and Böhmer
exist which allow termination of signaling, and, evenly
important, silencing of the RTK basal signaling activity
in the absence of a ligand. Ligand-independent
activation of RTKs [107] (discussed below) by various
and quite diverse cell treatments including stimulation
of GPCRs clearly shows that RTKs may have a signaling
activity in the absence of ligand which is normally
silenced. There are paradigms for negative regulation
of RTKs at all levels of RTK activation, including
interference with ligand binding [156], with receptor
dimerization
[157]
and
with
the
tyrosine
phosphorylations executed by the receptor kinase
[158]. Also, receptor internalization [159-162] and
phosphorylation of the receptor cytoplasmic domains
by heterologous serin-threonine kinases are means of
RTK inactivation, which are utilized by the cell. The
EGFR is subject to phosphorylation by PKC at Thr 654
[163], leading to attenuation of receptor signaling.
Also, phosphorylations at Thr 669, Ser 1002, 1046 and
1047 [164-167] may modulate EGF RTK negatively. A
modulation
of
signaling
by
PKC-dependent
phosphorylation has also been shown for other RTKs
[168] and may be an important general regulation
principle.
Another means or negative RTK control are the
actions of protein-tyrosine phosphatases (PTPs) (Fig.
4 ). Since this type of interactions may provide an
important link for cross-talk with the GPCR family of
receptors (see chapter 4.4), we will describe it in some
detail.
Ligand-activated
RTKs
are
rapidly
dephosphorylated by cellular PTPs, shown and
investigated in some detail for example for the EGFR,
PDGFβ-receptor or the insulin receptor [169-173].
Dephosphorylation parameters for not ligand-activated
receptors are difficult to evaluate, however, treatment
of cells with PTP inhibitors as vanadate [174],
phenylarsineoxide [175, 176], hydrogen peroxide
[177] or diamide [178, 179] leads to phosphorylation of
many RTKs. Thus, "basal" activity of RTKs is probably
quite significant and under negative control of PTPs.
Over the last 10 years PTPs have emerged as a large
family of quite diverse molecules [180-182]. More than
100 PTP species may exist [183] and hitherto almost
50 different individual PTPs have been shown to be
expressed in a single cell type [184]. PTPs can broadly
be classified in cytoplasmic and transmembrane, or
"receptor-like" enzymes [185]. The former posess one
PTP catalytic domain and various types of proteinprotein interaction or membrane targeting domains.
The tramsmembrane PTPs have frequently two
catalytic domains and variable extracellular domains.
Depending on the PTP subclass, these may recognize
cell surface proteins, matrix components or soluble
ligands. For example, the PTPs RPTPµ and k have
been shown to interact homophilically [186-188].
RPTPβ has a carboanhydrase-like motif in the
extracellular domain which can interact with contactin, a
Signal Transduction Pathways of G Protein-coupled Receptors
neuronal cell surface protein [189]. Also, RPTPβ has
been shown to interact with the matrix protein tenascin,
cellular adhesion molecules (CAMs) and pleiotrophin, a
heparin-binding neurite-promoting factor (reviewed in
[190]). Hitherto, it is unclear whether these interactions
can affect intracellular PTP activity. From crystal
structure data for RPTPα and from experiments with a
chimeric molecule consisting of the EGFR extracellular
domain and the RPTP CD45 intracellular domain it has
been proposed that for certain RPTPs dimerization may
lead to inactivation of the first (membrane-proximal)
catalytic domain, which harbors most of PTP activity
[191-193]. Among the cytoplasmic PTPs much
attention has been devoted to SHP-1 (HCP, SH-PTP1,
PTP1C) and SHP-2 (syp, SH-PTP2), the only known
mammalian PTPs which posess SH2-domains [194,
195]. Their tandem SH2-domains target these PTPs to
cellular phosphotyrosine-containing proteins including
RTKs. Both PTPs exist in a relatively inactive "closed"
conformation, with the N-terminal SH2-domain blocking
the active site. Occupation of the SH2-domain leads to
activation of PTP activity [196, 197]. SHP-2 is
ubiquitously expressed and mediates positive signals
for many RTKs and also cytokine receptors by a
hitherto unknown mechanism involving the PTP
catalytic activity. For some receptors, however, SHP-2
may also negatively control signaling steps (reviewed in
[198]). SHP-1 negatively regulates various types of
receptors
in
hematopoietic
cells,
including
immunoreceptors [199, 200], cytokine receptors [201],
and the RTKs Kit/SCF-receptor [202] and CSF-1
receptor [203]. Apart from hematopoietic cells, SHP-1
is also expressed in epithelial cells [204]. It can
associate with the EGFR and negatively regulate
signaling of this receptor at least in cells with relatively
high EGFR levels [55, 205, 206]. While SHP-1 has
been assigned to various RTKs as a likely or possible
negative regulator, in most cases the important PTPs
for a given RTK signaling regulation are unknown. It is
quite possible that several PTPs are involved in
different aspects of signal regulation. The EGFR may
serve as an example of this. In transient coexpression
systems multiple PTPs have the capacity to
dephosphorylate the receptor [109]. Interaction of the
receptor has been described with the cytoplasmic
PTPs SHP-1 and SHP-2 (via SH2-domains) [205],
PTP1B and T-cell PTP (with so-called "substrate
trapping mutants" of the PTPs, forming stable enzymesubstrate complexes) [207, 208] and PTPs of the PTPPEST-family [209]. Also, transmembrane PTPs are
likely to modulate EGFR signaling. Inducible
overexpression of the transmembrane PTP RPTPσ in
A431 cells leads to reduced EGFR phosphorylation
and a reduced capacity of the cells to form colonies in
soft agar. Partial suppression of endogenous RPTPσ
by inducible expression of an RPTPσ antisenseconstruct leads to elevated receptor phosphorylation
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
923
and soft agar colony forming capacity [210]. Thus,
RPTPσ appears to control aspects of EGFR signaling in
A431 cells. Understanding regulation of PTP activity is
only in its beginning. This concerns effects of known or
putative ligands for RPTPs, mentioned above as well as
possible effects of intracellular effectors. Activity of a
number of PTPs has been shown to be modulated by
Ser/Thr phosphorylation. For example,
phosphorylation of SHP-1 by PKC leads to inhbition [211],
PKC-phosphorylation of the transmembrane PTP
RPTPα to elevation [212] of PTP activity. Various PTPs
are also phosphorylated on tyrosine residues, which
has likewise been suggested do modulate activity
[213]. Several cytosolic and transmembrane PTPs
undergo partial proteolytic cleavages which modify
cellular localization and may also affect activity towards
cellular substrates [214]. Finally, lipid second
messengers can modulate PTP activity as shown for
phosphatidic acid and the PTP SHP-1 [55]. In
conclusion, PTPs and regulation of their activity
provide important but hitherto poorly understood
pathways to modulate RTK signaling activity in a
positive and negative manner.
3.5. RTK-mediated MAP-Kinase Activation
An important signaling pathway which links RTKs to
cell proliferation and possibly other biological
endpoints is the so-called "MAP-kinase cascade". This
term refers to a signal transmission chain from the
membrane to the nucleus, which is conserved from
yeast to mammals and consists of a small G-protein
followed by three consecutively activated protein
kinases (for recent reviews see [215-217]). In
multicellular organisms RTKs have aquired the capacity
to activate one variant of this chain, whose last
enzymes in this case are the closely related Ser/Thrspecific; proline-directed "extracelluar signal regulated
kinases" (ERKs) 1, and 2, also designated "mitogenactivated protein kinases" (MAPK) p44 and p42,
respectively. In the literature frequently the term "MAP
kinase" is used as a synonym for ERK1/2, as we do in
the other chapters of this article. The main pathway for
mitogenic signaling from RTKs like the EGFR or the
PDGFβ−receptor involves the following
steps:
Subsequent
to
RTK
activation
and
transphosphorylation occurs binding of a complex
consisting of the adaptor molecule Grb2 and the
guanine-nucleotide exchange factor (GEF) Sos for the
small G-protein Ras. Ras, a 21kDa protein is the
prototype for numerous small G-proteins in the cell (for
review see [218-222]). They all have in common that
they are active, i.e. capable to interact with "effector
proteins" in the GTP-loaded state. They have a small
intrinsic GTPase activity, which is greatly enhanced by
specific GTPase-activating proteins (GAPs). GTPloading of inactive, GDP-loaded small G-proteins is
924 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
accomplished with the help of GEFs as the
aforementioned Sos. Binding of the Grb2-Sos comples
to the receptor is mediated by the Grb2 SH2-domain
and can be either directly to the tyrosinephosphorylated RTK as in case of EGFR or indirectly via
the phosphorylated adaptor Shc as in case of the
PDGF receptor. Membrane-recruited Sos now enables
a GDP-GTP-exchange on Ras. All this occurs at the
inner surface of the plasma membrane, where Ras is
bound via a farnesyl-anchor (Fig. 5 ). GTP-loaded Ras
has a high affinity for the first kinase in the MAPKcascade, the product of the cellular protooncogene craf, Raf-1 and recruits it to the membrane. Membrane
recruitment of Raf-1 leads in an incompletely
understood manner to activation of Raf-1. Probably
additional proteins [223] and membrane lipids [224] as
well as phosphorylation [225] contribute to activation of
Ras-bound Raf-1. Activated Raf-1 phosphorylates its
donwstream kinases MKK1or 2 (MAP-kinase kinase;
also termed MEK for MAP/ERK-kinase) on two Ser
residues, leading to activation of MKK. MKKs are
kinases with "dual specificity", i.e. they can
phosphorylate Ser/Thr and Tyr residues. Dual
phosphorylation on Thr and Tyr in the activation loop of
Liebmann and Böhmer
MAP kinases ERK2/1 confers ERK activation.
Activated ERKs can phosphorylate multiple cellular
target proteins, including transcription factors
(subsequent to nuclear translocation of Erks), the p90
ribosomal S6 kinase (Rsk90), microtubule-associated
proteins (MAPs) and phospholipase A2. These
phosphorylations link activation of the MAP kinase
pathway to cell growth and transformation. It should be
noted that several members in the pathway upstream
from Erks are transforming when overexpressed in a
constitutively active form, including Ras, Raf and MEK,
emphasizing that this is a main pathway for mitgogenic
signaling.
Biological responses initiated via the MAPK pathway
seem to critically depend on the duration of MAPK
activation. For example in PC12 cells, sustained MAPK
stimulation via the nerve growth factor (NGF) receptor
TrkA leads to a differentiation response, while a short
wave of MAPK activation via the EGFR mediates a
mitogenic response. In other cells a sustained
activation of MAPK may be necessary for eliciting a
mitogenic response [226]. An intersting feature of the
MAPK pathway is negative feedback regulation. Erks
Fig. (5). MAP kinase pathways are routed by scaffold proteins and negatively controlled by multiple phosphatases.
Signal Transduction Pathways of G Protein-coupled Receptors
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
925
can phosphorylate and thereby negatively regulate
upstream molecules in the chain, notably Sos. Also, for
example the EGFR can be phosphorylated on Thr 669
by Erk2.
presents another level of regulation and possibilities for
cross-talk with signaling pathways emanating from
GPCR.
There are multiple variants of the MAPK signaling
chain, starting with the existence of different isoforms
of Ras, Raf, MKK, and ERK [217]. Parallel pathways on
the basis of distinct members of the MAPK-family with
corresponding upstream components and distinct
downstream targets and biological effects exist. In
yeast probably 6 members of the MAPK family exist
[227]. They do not couple to RTKs (those do not exist
in yeast) but to nutrient sensors or the pheromone
receptor, a GPCR. In animal cells there are likewise
multiple members of the MAPK-family. Other members,
which are relatively well investigated apart from
ERK1/2, are the NH2-terminal Jun-kinase/stress
activated protein kinase (JNK/SAPK) and p38. Many
RTKs can activate ERKs, some growth factor receptors
have also been shown to activate JNK. JNK and p38
are both activated by various stress factors. In addition
to ERK1/2 further MAPK-family members may be
important for RTK signaling. For example the mitogenic
signal of the EGFR has recently been shown to require
activation of ERK5 (also known as "big molecular
weight kinase" BMK) [228].
3.6 Ligand-independent Activation of RTKs
Specificity within a given MAP kinase cascade is on
the one hand provided by specificities of the upstream
and downstream elements for each other. On the other
hand, scaffolding of different components of the chain
by specific scaffold proteins seems to be an additional
mean of providing specificity and efficient coupling of
the signal transducing components [229, 230]. For
example, MP1 and JIP1 are recently identified proteins
which bind members of the ERK or JNK cascade,
respectively, and may provide a scaffold for routing the
signal within the cascade (Fig. 5 ).
MAPK signaling is subject to negative regulation by
phosphatases (reviewed in [231]). One class of
phosphatases responsible for inactivation of MAP
kinases are the dual-specificity MAP kinase
phosphatases (MKPs). Members of this family, for
example CL100/MKP1 are products of "immeadiate
early genes" which are rapidly activated upon growth
factor stimulation. Multiple MKPs have pronounced
selectivities for certain MAP kinase members and thus
for feedback inhibition of the respective cascade [232,
233]. Also, PTPs can inactivate MAP kinases by
dephosphorylating solely the phosphotyrosine in the
MAPK activation loop [234]. Finally, the regulatory
phosphothreonine on MAP kinase familiy members can
be subject to dephosphorylation by Ser/Thr specific
phosphatases [231]. In conclusion, inactivation of the
MAP kinase cascades by phosphatases, either dualspecificity or tyrosine-specific or Ser/Thr-specific,
Various quite diverse agents and cell treatments
have been observed to induce activation of RTKs in
the absence of ligand. These include cell treatments
with "adverse agents", for example UV [235] or X-ray
irradiation [236]. Also, activation of different GPCRs has
been shown to lead to RTK activation. This pathway
has been designated "transactivation" and is discussed
in detail below (chapter 4.4). Finally, integrin and cellcell adhesion molecule activation have been shown to
result in RTK activation [237].
From the current knowledge of RTK activation and
signaling control pathways different mechanisms could
be envisaged for ligand-independent RTK activation:
ligand-independent RTK dimer/oligomer formation,
heterologous phosphorylations and inactivation of
"silencing" mechanisms.
Some evidence has been obtained for the last
possibility in case of UV-mediated RTK activation. PTPs
which keep RTKs silent and inactivate them after ligand
stimulation perform catalysis with particiation of a
reactive cysteine in their active center and are therefore
very susceptible to oxidation. UV treatment of cells
could recently been shown to inactivate membrane
bound PTPs for the EGFR and the PDGF receptor,
most likely via an oxidative mechanism [110]. It seems
quite likely that various adverse agents can lead to
generation of reactive oxygen species
and
subsequently to PTP inactivation. These inactivation
mechanisms may in fact be partially reversible and play
even a role in ligand-activated signal transduction
[238]. Hydrogen peroxide generation subsequent to
RTK activation has been shown for the PDGF receptor
to be essential for eliciting a mitogenic signal [239].
EGFR activation is accompanied by hydrogen peroxide
generation [240] and a reversible inactivation of the
PTP PTP1B [241]. PTP activity, however, is likely to be
subject to regulation by further mechanisms, as
phosphorylation or proteolysis, outlined above. This
should in turn also modulate activity of PTP-regulated
RTKs.
An interesting question is to what extent
heterologous tyrosine kinases may be capable of RTK
activation. RTK phosphorylation by Src-family kinases is
possible and may be sufficient to generate signal
molecule docking sites and subsequent signal
generation [137]. This would in principle be possible
without participation of the RTK intrinsic tyrosine kinase
activity. For the case of GPCR transactivation, however,
926 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
Liebmann and Böhmer
participation of RTK intrinsic activity has been
demonstrated (see chapter 4.4). It is an interesting
possibility that Src-family kinases or other heterologous
tyrosine kinases could also execute such RTK
phosphorylations that would lead to RTK activation.
Indeed, phosphorylation sites for Src on the EGFR are
partially within the kinase domain and may regulate
kinase activation [146, 148]. Also, PDGFβ-receptor was
found to become phosphorylated on many sites
including Tyr 857, the site presumably involved in
kinase activation, by Src kinase in vitro [150]. Thus, Srcfamily kinases and possibly other tyrosine kinases may
be capable of direct RTK activation although this
possibility clearly requires further investigation.
4. Signaling Pathways from GPCRs
MAP Kinase
to
Like RTKs, many GPCRs can induce mitogenic
responses or contribute to neoplastic growth of human
tumors (reviewed in [242]. Depending on the cell type,
mitogenic responses can be mediated by Gi/o, Gq/11, Gs,
or G 12 proteins. [4,6,79]. For example, constitutively
active mutants of G protein α-subunits have been
identified in various tumor cells, and GTPase deficient
forms of G s, Gi/o, and G12 have been demonstrated to
induce cellular growth after expression in several cell
lines [243].
The pathways coupling GPCRs to nuclear
responses are of high complexity and may differ from
cell type to cell type. However, stimulation of various
GPCRs was found to induce activation of key effector
molecules of RTK signaling, including Ras, Raf, and
MAPK. These observations led to the assumption that
MAPK might be a point of convergence for proliferative
signals emanating from both different G proteins and
RTKs (Fig. 6 ).
It is thought that both the α-subunits and βγcomplexes of heterotrimeric G proteins are capable of
mediating activation of MAPK. Obviously, in PTXsensitive pathways MAPK is activated through βγcomplexes from Gi/o proteins whereas in PTXinsensitive pathways the activation of MAPK is
mediated via α q/11-subunits and PKC [4,7,8]. Gsmediated regulatory effects on MAPK activity may be
either stimulatory or inhibitory. The role of G12/13
proteins in MAPK activation is not yet clear. Very
recently, the binding of Gα 12 to Bruton`s tyrosine
kinase (Btk) and the stimulation of Btk and of Gap1m
which is a RasGTPase-activating protein (rasGAP) was
reported [33].
These findings indicate, for the first time, the
possibility of a direct link between heterotrimeric and
monomeric G proteins.
Intensive research led to the identification of various
protein kinases that could make the link between G
proteins and the MAPK cascade. Recently, a novel
principal pathway of MAPK activation has been
discovered termed transactivation [243, 244].
Fig. ( 6 ) . MAP kinase activation via RTKs and by various GPCRs may involve Ras and different G protein subtypes. A
schematic overview.
Signal Transduction Pathways of G Protein-coupled Receptors
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
927
Fig. (7). Principle mechanisms of MAPK activation via G i/o -coupled receptors. The different and possible biochemical routes
are explained in the text.
Transactivation stands
for
ligand-independent
activation of RTKs triggered by GPCRs [107]. Despite
the efforts made in the last years the early mechanisms
of MAPK activation by agonists of GPCRs remain poorly
understood. However, in the following we will attempt
to describe the most likely biochemical routes
connecting GPCRs to MAPK.
4.1.
Principle
Mechanisms
of
Activation by G i/o-coupled Receptors
MAPK
There are several lines of evidence suggesting that
MAPK activation by G i-coupled receptors involves the
βγ-complexes and is Ras-dependent. Thus, activation
of MAPK via Gi is attenuated by coexpression of the αsubunits of transducin which acts to sequester Gβγ
[245]. This was supported by similar results obtained in
Rat-1 cells with βARKct, a carboxy-terminal fragment of
the β-adrenergic receptor kinase that also acts as βγsequestrant and significantly inhibited activation of
both Ras and MAPK via the Gi-coupled LPA receptor
[246]. Further, overexpression of Gβγ subunits results
in activation of MAPK [245, 247] whereas a
constitutively active mutant of Giα did not increase
MAPK activity in COS-7 cells [248]. It was
demonstrated that MAPK activation by βγ-subunits
required neither PLCβ nor PKC (reviewed in [4]) but
was blocked by dominant negative Ras [245]. In
addition, βγ-stimulated MAPK activation is inhibited by
tyrosine kinase inhibitors such as genistein suggesting
the involvement of a tyrosine kinase in this pathway
[247]. Taken together, all experimental data led
assume that Ras or an effector molecule upstream of
Ras should be the target for the Giβγ -mediated
mitogenic signaling. The question remained how the
βγ-complexes might regulate Ras. First answer came
from the laboratory of Lefkowitz. This group
demonstrated that βγ-subunits stimulated tyrosine
phosphorylation and thereby activation of the adaptor
protein p52 Shc thus inducing the formation of ShcGrb2 complexes [249, 250]. Interstingly, Gβγ stimulated phosphorylation of Shc has been also found
to be inhibited by Wortmannin, a specific inhibitor of
PI3K suggesting an additional involvement of PI3K
upstream of Shc [249]. Then Luttrell et al. [251]
provided evidence that the non-receptor tyrosine
kinase Src mediates the βγ-induced tyrosine
phosphorylation of Shc. Later, several studies
described the involvement of more Src-like kinases
such as Fyn, Lyn, and Yes [252] or also, recently, of an
unknown non-Src cytosolic tyrosine kinase that
induces the interaction of a p100 kDa protein with Grb2
[253].
Another candidate that has been implicated in ShcGrb2-Sos complex formation is the FAK-related non-
928 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
receptor tyrosine kinase PYK2. This enzyme is
predominantly expressed in neuronal cells and may link
both Gi and Gq with Grb2-Sos in a Ca2+- and Srcdependent pathway as was shown for LPA- and BKreceptors in PC-12 cells [254].
On the other hand, PI3Kγ has been shown to play a
major role in Gβγ -mediated activation of MAPK. Under
endogenous conditions βγ-sensitive PI3K activity has
been described in neutrophils and platelets [255, 256],
and PI3Kγ has been shown to activate MAPK when
expressend in COS-7 cells in response to Gi-derived
βγ-complexes [49]. PI3Kγ can be activated due to
direct interaction with βγ-complexes [48] or due to a
constitutive association with the p101 βγ-sensitive
protein [257]. PI3Kγ was found to act upstream of Srclike kinases suggesting another putative link to the
Shc-Grb2-Sos complex [49]. Furthermore, we [258]
and others [50, 259] have recently shown that the
p85/p110 PI3Kβ may play a role downstream of Gi- or
Gq-coupled receptor signalling, too. The putative
signaling pathways connecting Gi/o-coupled receptors
to MAPK are summarized in Fig. (7 ).
Very recently, once more the group of Lefkowitz
provided evidence which probably opened a new
dimension in our understanding of mitogenic signal
transduction (reviewed in [260]). Firstly, stimulation of
β-adrenergic receptor expressed in HEK 293 cells
resulted in a Gi-coupled, βγ-mediated and Rasdependent activation of MAPK. However, the
interaction of β-AR with Gi required a prior receptor
coupling to Gs subsequently leading to cAMP
production and activation of PKA. PKA-induced
receptor phosphorylation was found to be a
prerequisite for switching the receptor from Gs to Gi
thereby activating another signaling pathway. [261].
Furthermore, in HEK 293 cells additionally expressing
dominant negative mutants of β-arrestin or dynamin
which are known to block receptor endocytosis the βAR-mediated activation of MAPK was prevented [262].
The inhibitors of receptor internalization were found to
specifically block the interaction between Ras-bound
Raf and the cytosolic MEK. Thus, GRKs and arrestin
mediating the uncoupling and internalization of GPCRs
may play also an essential role in the GPCR-induced
MAPK activation [260, 262].
4.2. Principle Mechanisms by which Gq/11coupled Receptors May Activate MAPK
Receptors coupled to PTX-insensitive G proteins of
the Gq/11 family are thougth to mediate MAPK activation
via their α-subunits. Their signaling is mostly insensitive
to βγ-sequestrants (reviewed in [4,7]). Gq/11-coupled
receptors may activate MAPK by either Ras-
Liebmann and Böhmer
independent or also Ras-dependent pathways [4,7].
The Ras-independent pathway shows, e.g. in COS
cells or CHO cells expressing Gq-coupled receptors no
sensitivity to a dominant negative mutant of Ras and
may involve activation of PKC and Raf. Gq-mediated
signalling in CHO cells has been demonstrated to be
inhibited by down-regulation of PKC by chronic
exposure to phorbol esters and by dominant negative
Raf [247]. Phorbol esters as direct activators of PKC
have been reported to activate MAPK in various cells.
In addition, expression of constitutively active mutants
of PKC showed that at least in vitro the PKC isoforms
α,β,δ, ε, and η have the potential to activate MAPK at
the level of Raf-1. The aPKC ζ cannot increase Raf-1
activity and stimulates MEK by an independent
mechanism [263]. Recently, much interest has been
focussed at a possible role of the PKC isoforms α and
especially ε as activators of Raf-1 in vivo. For example, a
dominant negative mutant of PKC ε inhibited both
proliferation of NIH 3T3 cells and Raf activation in COS
cells whereas active PKC ε overcame the inhibitory
effects of dominant negative Ras in NIH 3T3 cells [264].
Furthermore, constitutively active mutants of PKCα
as well as PKC ε overcame the inhibitory effects of
dominant negative mutants of the other PKC isotype
[264]. In addition, PKC ε can be co-precipitated with
Raf-1 from Sf-9 insect cells and PKC ε transformed NIH
3T3 cells [265]. PKC ε was demonstrated to be
activated by both phosphatidylinositol (PI)- and
phosphatidylcholin (PC)-derived DAG [266], and PChydrolysis was shown to induce Raf-1 activation [267].
Moreover, PKC ε may be also activated via the PI3K
pathway thus representing a point of convergence for
several lipid-derived second messengers. Taken
together, there is increasing evidence that Raf-1
activation by PKC ε may play a critical role in mitogenic
signalling of Gq/11-coupled receptors.
On the other hand, agonists of Gq/11-coupled
receptors such as bombesin, bradykinin, or
vasopressin as well as phorbol esters have been also
described to stimulate Src family kinases transiently
thereby activating MAPK in a Ras-dependent manner
[268]. According to a hypothesis proposed by Della
Rocca et al. [269] activation of Src kinases by Gq/11coupled receptors might be the final event of a cascade
including the increase in cytosolic Ca2+ in response to
IP3 and the Ca2+-calmodulin mediated activation of
Pyk2 or of a related tyrosine kinase which
phosphorylates Src. Indeed, stimulation of Gq-coupled
BK receptors in PC-12 cells was demonstrated to
activate PyK2 and subsequently Src and MAPK [254].
Src may phosphorylate either a RTK or Shc leading to
the formation of a Shc-Grb2-Sos complex. The most
likely biochemical routes connecting Gq/11-coupled
receptors to The MAPK pathway are summarized in Fig.
(8 ).
Signal Transduction Pathways of G Protein-coupled Receptors
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
929
Fig. (8). Principle mechanisms of MAPK activation in response to of G q/11 -coupled receptor stimulation. The putative links of
Gq/11 to MAPK are explained in the text.
4.3.
Gs-coupled
Receptors
and
MAPK:
Differential
and
Controversial
Effects
of
cAMP and βγ-Subunits
The role of Gs in the regulation of cell growth and
MAPK activity appears to be extremely cell-type
specific. In various cells such as smooth muscle cells
[270], adipocytes [271], or fibroblasts [272], cAMP has
been shown to inhibit the MAPK cascade. In these
cells, cAMP activates PKA which in turn
phosphorylates Raf-1 thereby decreasing the affinity of
Raf for Ras as well as Raf kinase catalytic activity [273].
In PC-12 cells, in contrast, cAMP stimulates the MAPK
pathway and induces neuronal differentiation [274]. In
COS-7 cells, Faure et al. [248] reported that the
expression of a constitutively active mutant of Gs,
treatment with forskolin or dibutyryl cAMP, or
stimulation of transiently expressed, Gs-coupled LH
receptor resulted in activation of MAPK. Contradictory
results have been presented by Crespo et al. [275]
suggesting dual effects of β-adrenergic receptors on
MAPK activity in COS-7 cells. In this study, stimulation
of β-AR simultaneously led to βγ-dependent activation
and cAMP-mediated inhibition of MAPK. The balance
between these two mechanisms was postulated do
determine the outcome of the signal to MAPK [275].
However, in view of the present knowledge it may be
assumed that not βγ-complexes from Gs but those
released from G i after switching of β-AR from Gs to Gi
[261] should mediate the activation of MAPK.
Very recently, a new family of cAMP-binding
proteins was discovered that exhibit properties of a
guanine necleotide exchange factor (GEF). These
cAMP-GEFs are capable of activating monomeric G
proteins in a cAMP-dependent and PKA-independent
manner suggesting a direct coupling of cAMPmediated signaling to Ras superfamily signaling [276].
4.4. A New Role for RTKs in GPCR Signaling:
Different Pathways Lead to Transactivation
RTKs are not only receptors for specific peptidic
growth factors but also essentially involved in the
mitogenic signalling of GPCRs where they may act as
scaffold proteins, signal mediaters, and signal
integrators. First evidence for ligand-independent
tyrosine phosphorylation of RTKs by a GPCR came in
1995 from reports demonstrating the tyrosine
phosphorylation of PDGFR by angiotensin II [277] in rat
smooth muscle cells or of EGFR by bradykinin in human
keratinocytes [278]. Then, in two excellent papers,
Daub et al. [243, 244] described the transactivation of
EGFR in diverse cell types such as Rat-1 cells, HaCat
keratinocytes, mouse astrocytes as well as in COS-7
cells (Fig. 9 A ). In Rat-1 cells it was shown that mitogens
930 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
such as endothelin, LPA and thrombin mediate both
MAPK activation and DNA synthesis via activation of
EGFR [243]. In COS-7 cells transiently expressing Gior G q-coupled receptors was demonstrated that both
types of GPCRs stimulated Shc phosphorylation as well
as MAPK activity via EGFR transactivation [244]. Since
inhibition of PI3K did not affect EGFR tyrosine
phosphorylation but abolished MAPK activation PI3K
was supposed to act downstream of EGFR. Similar
results were obtained with the Src-inhibitor PP-1
leading to the assumption that also Src should be
involved in the signaling pathway downstream of
EGFR. Independently, Luttrell et al. from the
Lefkowitz`group provided evidence that at least Gicoupled receptors may induce EGFR tyrosine
phosphorylation via Src-family tyrosine kinases [279].
They proposed, in contrast, that a PI3K-dependent
step might lie upstream of Src kinase activation. Src
kinase, indeed, possesses a domain with high affinity
to PIP3 the second messenger product of PI3K [280].
In contrast again, recently published data suggest that
in HeLa cells the intrinsic EGFR tyrosine kinase
contributes to the LPA-stimulated MAPK pathway and
c-Src is probably not involved [281]. However, this
finding in HeLa cells does not exclude that members of
the Src family may be involved in other cell types.
Furthermore, the same GPCR may induce
transactivation of different RTKs depending on the cell
type. This has been recently demonstrated for LPA
which transactivates EGFR in COS-7 cells but is also
capable of using the PDGFR in L cells that lack EGFR
[282].
In neuronal cells EGFR transactivation appears to be
dependent on Ca2+. In PC-12 cells, for example,
bradykinin-induced tyrosine phosphorylation of EGFR
upstream of Shc and MAPK was reported [283]. This
effect of BK was absent in PC-12 cells pretreated with
EGTA. In other cells, however, EGFR transactivation
was found to be independent of Ca2+ [284]. In addition
to the differences in Ca2+-sensitivity or the role of Src,
the high degree of cell specificity within the
mechanisms involved in EGFR transactivation by
GPCRs is also reflected by different modes of action of
PKC (Fig. 9 ). Although activation of PKC has been
widely shown to play a key role in MAPK activation by
Gq-coupled receptors, in COS-7 cells the Gq-mediated
transactivation of EGFR was postulated without any
attempt to modify PKC activity in these cells [244]. This
is surprising all the more because in 1995 Coutant et al.
[278] already showed that in HaCaT human
keratinocytes
bradykinin
induces
tyrosine
phosphorylation of EGFR via a PKC-dependent
pathway. Similar results were reported for human 293
cells stably transfected with m1 muscarinic receptors. In
these cells carbachol-induced EGFR transactivation
Liebmann and Böhmer
was potently inhibited by a PKC inhibitor and the
phorbol ester PMA could mimick the carbachol effect
suggesting the PKC-dependency of EGFR tyrosine
phoshorylation [284] (Fig. 9 B ). A completely different
mechanism of EGFR transactivation was found in GN4
rat liver epithelial cells where angiotensin II (A II) is
capable of activating MAPK via two pathways. Under
normal conditions, A II stimulates MAPK via a PKCdependent, Ras-independent pathway. In PKCdepleted cells, the A II receptor can switch and
activates MAPK via an equipotent Ras-dependent
pathway including EGFR tyrosine phosphorylation.
Thus, the latent transactivation route represents an
alternative pathway to MAPK that is masked by PKC
and uncovered when the PKC pathway breaks down
[285] (Fig. 9 C ).
Moreover, in COS-7 cells transiently co-transfected
with the human bradykinin B2 receptor and MAPK we
found a pathway of MAPK activation that includes the
independent and equipotent stimulation of both PKC
and EGFR [329]. Activation of MAPK in response to BK
is prevented by inhibitors of PKC as well as EGFR.
Inhibitors of PI3K or Src failed to affect MAPK activation
by BK. PKC translocation studies and coexpression of
inactive and constitutively active mutants of different
PKC isoforms provided evidence for a critical role of the
PKC isozymes α and ε in BK signalling towards MAPK.
BK induced tyrosine phosphorylation of the EGFR that
was independent of PKC. Since blockade of the EGFR
did also not influence BK-stimulated increase in
phosphatidylinositol phosphate formation, PKC should
act neither upstream nor downstream of EGFR but in a
permanent dual signalling pathway. MAPK activation by
BK requires signals from both pathways which
represent a two-key system for regulating an enzyme
which is critically involved in the control of cell growth
(Fig. 9 D ).
As described above, a common theme in the
literature on RTK transactivation by GPCR is the
possible involvement of Src-family kinases. They may
induce signal transduction by either phosphorylating
RTKs on docking sites for signaling molecules or via
direct activation of intrinsic RTK activity. As outlined in
chapter 3, it is not entirely clear, how Src-family kinases
can possibly accomplish RTK activation. One would
have to assume that they execute phosphorylations of
the RTK molecules which are functionally equivalent to
trans-phosphorylations in RTK dimers. Such a model
would be in agreement with some of the available data
on Src-phosphorylation sites in different RTKs but this
mechanism has not been fully established. Further,
while tyrosine phosphorylation in the RTK "activation
loop" has been established as activation mechanism for
some RTKs as FGF receptor, insulin receptor or
Signal Transduction Pathways of G Protein-coupled Receptors
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
931
Fig. (9). Different modes of EGFR transactivation. (A) Principle mechanisms of RTK (EGFR, PDGFR) transactivation without
consideration of PKC. Several authors implicate PI3Kγ either upstream or downstream of RTK [244, 279]. (B) In human 293 cells
PKC was found to be involved in M1AchR-mediated EGFR transactivation. It is not yet clear whether PKC induces activation of
a cytosolic phosphotyrosine kinase (PTK) or inactivation of a phosphotyrosine phosphatase (PTP) subsequently leading to
enhanced tyrosine phosphorylation of EGFR. In that model, activated EGFR was demonstrated to open a K+ channel in the
membrane [284]. (C) Latent dual pathway of EGFR transactivation by angiotensin II. In GN4 cells AII activates MAPK
dominantly via the PKC/Raf pathway . When the PKC pathway is cancelled MAPK is alternatively activated via EGFR
transactivation [285]. (D) Activation of MAPK by BK via a permanent dual pathway. Both activation of PKC and EGFR
transactivation are necessary for the stimulation of MAPK activity by BK.
Met/HGF receptor, respective evidence is still missing
for other RTKs. In fact, it is likely that for other RTKs, in
particular EGFR regulation by activation loop
phosphorylations is less important. Thus, the possible
role of heterologous Src-kinase phosphorylations is
even more questionable.
In contrast, it should be considered that a main
pathway for RTK activation may be instead the
interference with negative regulatory pathway
mechanisms (see chapter 3.4) in particular with the
activity of RTK-silencing PTPs. Their activity could well
be affected by GPCR signaling events, as changed
932 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
Ca2+-levels, lipid second messengers or various
protein kinases including Src-family kinases. This kind
of mechanism is not easy to demonstrate. EGF
receptor dephosphorylation is a very rapid process
[286-288] with t1/2 <<2min. It can be detected in intact
cells as a decay of phosphotyrosine on the EGFR after
quenching the kinase with specific cell-permeable
blockers, subsequent to ligand stimulation [288]. A
moderate attenuation of the dephoshporylation rate, in
particular if it would affect only the not ligand-stimulated
receptor, may be impossible to monitor with this
technique. Use of general PTP inhibitors, on the other
hand, will lead to rapid hyperphosphorylation of many
cellular proteins which will likewise make conclusions as
to the possible involvement of PTP in transactivation
difficult. We believe, however, that the involvement of
PTPs in GPCR-mediated RTK activation requires much
attention and possibly the development of novel tools
to monitor RTK-directed PTP activity.
4.5. Expression Models versus Endogenous
Conditions: Varying Mechanisms of MAPK
Activation by GPCRs in Tumor Cell Lines
The majority of models describing mitogenic
pathways from a GPCR to MAPK are based upon
experimental data obtained by coexpression of
epitope-tagged MAPK together with GPCRs and often
Liebmann and Böhmer
additional proteins of interest in COS-7 cells or other
transfectable cell lines. However, the procedure of
transfection might and the overexpression of certain
proteins will result in artificial and abnormal conditions
within the cell compared with the native cell. Therefore,
it should be considered that in general in
overexpression models only signaling mechanisms can
be detected which represent putative pathways which
may not necessarily reflect the real situation under
endogenous conditions. In natural cells or tumor cell
lines expressing individual amounts of signal
transducing molecules and different isoforms of
effector molecules, for instance, completely different
links between GPCRs and the MAPK cascade may be
observed compared with the signalling pathways after
stimulation of the same GPCR in an expression system.
In order to demonstrate such conflicting findings in an
expression model and in tumor cell lines two examples
from our own work may be mentioned. Thus, in COS-7
cells transiently transfected with the human B2R
bradykinin activated MAPK by a dual pathway and
requires the independent signalling via both PKC and
EGFR transactivation as described above. In contrast,
when we studied MAPK activation in response to BK in
the human colon carcinoma cell line SW-480 we
detected a hitherto unknown biochemical route to
MAPK [258]. Both BK-induced stimulation of DNA
synthesis and activation of MAPK were abolished by
two different inhibitors of PI3K, wortmannin and LY
Fig. (10). Bradykinin receptor signaling in the human colon carcinoma cell line SW-480. Here BK-induced activation of MAPK
was found to be mediated via a pathway involving the consecutive stimulation of G q/11 protein, PI3Kβ, and PKCε [258].
Signal Transduction Pathways of G Protein-coupled Receptors
294002, as well as by two different inhibitors of PKC,
bisindolylmaleimide and Ro 31-8220. Furthermore,
stimulation of SW-480 cells by BK led to both increased
formation of PIP3 and translocation of PKC ε that was
inhibited by wortmannin, too. Using subtype-specific
antibodies, only the p110 and p85 subunits of PI3Kβ
but not p110 α or p110 γ were detectable in SW-480
cells. Finally, p110 β co-immunoprecipitated with PKC ε
indicating a physical association between the two
proteins. These results suggest a novel pathway
involving the consecutive activation of Gq/11, PI3Kβ,
PKCε, and MAPK (Fig. 1 0 ).
Another cross-talk mechanism between B2R and
the MAPK pathway we observed in A 431 cells [Graneß
A.; Hanke S.; Boehmer F.-D.; Liebmann C.; in press]. In
this cell line BK induced a decrease in both basal and
EGF-stimulated tyrosine phosphorylation of EGFR by
stimulating the activity of a
phosphotyrosine
phosphatase. This effect can be demonstrated by
several experimental approaches including the
respective Western blots of EGFR as well as direct
measurement of PTPase activity using the [32P]
tyrosine-phosphorylated PTPase substrate raytide.
Moreover, BK was previously shown to also induce a
PKC-mediated phosphorylation of EGFR at Thr-654
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
933
resulting in EGFR desensitization [289]. It may be
concluded that BK affects the EGFR via both a
decrease of tyrosine phosphorylation by enhanced
PTPase activity and a decrease of EGFR sensitivity
towards EGF by PKC. Although the EGFR is
transinactivated by BK in A 431 cells, a BK-induced and
PKC-mediated activation of MAPK was observed (Fig.
1 1 ). These results provide evidence that not in all
cases EGFR transactivation is necessary for GPCRinduced MAPK activation.
Additionally, a single agonist may also induce
activation of MAPK by different signalling pathways
which can vary between different cell types stimulated.
For example, in GN4 cells the angiotensin II (AII)
receptor was reported to stimulate MAPK via a
dominant PKC pathway and a latent EGFR
transactivation pathway as already discussed [285]. In
rat cardiac myocytes, AII was found to activate MAPK via
PLC β, PKC, and Raf-1 independently of EGFR [290].
In contrast, in rat vascular smooth muscle cells a
pathway from AII receptor to MAPK was detected
involving the sequential activation of Gq/11, PI3K, PKC
ζ, and an association of PKCζ with Ras suggesting a
PKC/Ras dependent pathway that can by-pass the
EGFR [291]. Further, in rabbit renal proximal tubular
Fig. (11). Bradykinin B2 receptor signaling in the human epidermoid carcinoma cell line A431. BK attenuates EGFR by both
stimulation of PTP and of PKC. Activation of PTP results in a decreased tyrosine phosphorylation of EGFR and activated PKC
induces threonine phosphorylation of EGFR that leads to its desensitization towards EGF. Nevertheless, BK is capable of
stimulating MAPK activity via another PKC-dependent pathway. This is an example for a simultaneously occuring negative
modulation of EGFR (transinactivation) and positive regulation of MAPK activity by a GPCR in a cell-specific manner.
934 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
epithelial cells, AII activates MAPK via the AT2 receptor
subtype and a signaling route involving stimulation of Gi
and, subsequently, PLA2, the release of arachidonic
acid which in turn activates Shc and Ras [292]. In N1E115 neuroblastoma cells AT2 receptors mediate
inhibition of serum- or EGF-induced MAPK activation,
possibly via activation of a PTP (293).
These few examples may illustrate by which
immense complexity and cell specificity the cross talk
between GPCRs ant RTKs can occur.
5. RTK-induced Tyrosine Phosphorylation of GPCR Signaling Elements
Although relatively much is known about the
interaction of GPCRs with RTKs and the MAPK
cascade, the modulation of GPCR-coupled signal
transduction by RTK-induced tyrosine phosphorylation
is considerably less investigated. Meanwhile, however,
there is mounting evidence that cross-talk includes not
only the foreward regulation of the MAPK pathway by
GPCRs but also the regulation of GPCR signaling by
RTKs. Recently, at least three levels of GPCR signal
transduction were implicated to be favoured targets of
tyrosine phosphorylation by RTKs.
5.1. Tyrosine Phosphorylation of GPCRs
G protein-coupled receptors lack intrinsic tyrosine
kinase activity. Nevertheless, several agonists of GPCR
such as angiotensin II [294] or gastrin [295] have been
demonstrated
to
stimulate
phosphatidylinositol
metabolism by a signalling mechanism that involves
PLC γ and Src instead of PLCβ. In vascular smooth
muscle cells, for example, the AT1 receptor was
demonstrated to induce a transient tyrosine
phosphorylation of PLCγ 1 and a parallel IP3 formation
in a manner similar to that observed in response to
growth factors [294]. In addition, electroporation of antiSrc antibodies into these cells eliminated both tyrosine
phosphorylation of PLCγ and AII-induced stimulation of
IP3 production suggesting that activation of PLCγ
occurs downstream from activation of c-Src tyrosine
kinase [296]. Very recently, the activation of PLCγ by
AII was found to be accompanied by binding of PLCγ to
the AT1 receptor in dependency on AII stimulation as
well as tyrosine phosphorylation. A prerequisite of
PLC γ 1 binding appears to be phosphorylation of
tyrosine 319 in a YIPP motif in the C-terminal
intracellular domain of the AT1 receptor. This
phosphorylated tyrosine residue can be recognized by
a SH2-domain of PLCγ 1 [297]. It might be speculated
that the receptor serves as a scaffold for PLCγ 1
allowing its phosphorylation by Scr tyrosine kinase.
Liebmann and Böhmer
Whereas the AT1 receptor preferentially couples to
Gq/11 proteins, in colonic epithelial cells also the Gi
protein-coupled gastrin receptor was found to stimulate
PLC γ 1 via Src [295]. It may be assumed, therefore, that
both Gq/11- and Gi-coupled receptors have the ability to
act without G proteins by forming signal transduction
complexes which are thought to be typical for RTKs.
On the other hand, with respect to ligand-induced
tyrosine phosphorylation of the B2 bradykinin receptor
contradictory results have been reported. In human
fibroblasts the B2R was shown to be phosphorylated at
serine and threonine residues in response to BK and
neither phosphoamino acid analysis nor Western
blotting with anti-phosphotyrosine antibodies revealed
tyrosine phoyphorylation of the B2R [298]. In contrast,
in endothelial cells the BK-induced IP3 formation was
reported to be dependent on a transient tyrosine
phosphorylation of PLCγ 1 [299, 300]. In vascular
endothelial cells, tyrosine phoyphorylation of PLCγ 1
could be correlated with its binding to the C-terminal
intracellular domain of the B2R similar to the findings at
the AT 1 receptor [300]. Unfortunately, there is no clear
evidence for a ligand-induced tyrosine phosphorylation
of the B2R in this work.
It is worth to speculate, however, that tyrosine
phosphorylation of GPCRs might occur not only ligandinduced (homologous) but also as a cross-talk event
induced by activated RTKs (heterologous). In that way,
a RTK could use tyrosine-phosphorylated GPCRs as
scaffold proteins for ist own signal transduction
machinery.
5.2. Tyrosine phosphorylation of G proteins
Like other signalling proteins, also G proteins may
be phosphorylated and thereby modulated by different
protein kinases. Thus, phosphorylation of Giα by PKA
appears to impair the dissociation of Gi into α- and βγsubunits [301]. In contrast, the α-subunits of Gz as well
as G 12/13 are phosphorylated by various isoforms of
PKC resulting in a prevention of their association with
βγ-subunits [302-304]. In addition, recombinant forms
of Gsα were shown to be targets for PKC in vitro
suggesting a putative role of Gsα within the cross-talk
between receptors which activate PKCs and those
which stimulate adenylate cyclase via Gs [305].
First evidence concerning tyrosine phosphorylation
of G proteins was provided in 1992 by Hausdorff et al.
[306]. They demonstrated in vitro the tyrosine
phosphorylation of Gsα as well as other Gα subtypes
(Giα , Goα ) by activated Src. As a functional
consequence, phosphorylation of Gsα was found to
increase receptor-induced GDP/GTP exchange and
GTPase activity. Three years later, Moyers et al. [307]
identified the in vitro phosphorylation sites of Gsα
Signal Transduction Pathways of G Protein-coupled Receptors
mediated by Src. In an excellent work two sites of
phosphorylation were mapped, Tyr-37 located near the
N-terminus and Tyr-377 located at the site of receptor
binding in the C-terminus. Tyrosine phosphorylation of
Gsα , at these sites, therefore, could change its ability to
interact with both βγ-subunits and the receptor [307].
Almost at the same time the tyrosine phosphorylation
of recombinant Gsα by isolated EGF receptor tyrosine
kinase was reported [308]. Tyrosine-phosphorylated
Gsα showed enhanced GTP binding and GTPase
activity and an increased degree of adenylate cyclase
stimulation after reconstitution with S49 cycmembranes [308]. These findings suggested that also
a RTK has the ability to phosphorylate and thereby to
activate Gsα . In 1996, too, using several experimental
approaches we provided the first evidence for tyrosine
phosphorylation of Gsα in A431 cells in vivo [309].
Treatment of A431 cells with EGF abolished both
bradykinin- and isoprenaline-induced binding of the
stabile GTP analogon [35S]GTPγ S to Gsα and
decreased the BK- and guanyl nucleotide-induced
cAMP accumulation and adenylate cyclase stimulation.
In contrast, the BK-induced and Gq/11-mediated
formation of inositol phosphates was not affected by
EGF. Thus, tyrosine phosphorylation of Gsα by EGF
results in an activation of adenylate cyclase by EGF, on
the one hand, and a simultaneously occuring loss of
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
935
the susceptibility of Gsα to GPCRs (Fig. 1 2 ). These
findings emphasized a differential recruitment of Gsα
by GPCRs and EGFR as a novel cross-talk mechanism.
Moreover, also Gq/11 appears to be tyrosine
phosphorylated. Recently, stimulation of GPCRs
coupled to G q/11 was shown to induce phosphorylation
on Tyr356 and a PTK is required before G protein
activation. It was further demonstrated that this tyrosine
phosphorylation is apparently essential for the
activation of Gq/11 by agonist stimulation [310]. These
results demonstrate that tyrosine phosphorylation of
The Gα q/11 subunit by PTKs contributes to GPCRmediated activation of Gq/11.
Taken together, it may be assumed that depending
on the type of G protein and the type of cell both
GPCR-induced ( probably mediated by Src, Pyk2 or
others) and/or RTK-induced tyrosine phosphorylation
of Gα -subunits might represent a mechanism to
regulate the activation of G proteins.
5.3. Tyrosine phosphorylation of PKCs
Tyrosine phosphorylation of PKC isoforms such as
δ, ε, η, and ζ in response to Src family tyrosine kinases
in vitro has been reported [311] but the biological
Fig. (12). EGFR-induced tyrosine phosphorylation of G sα in A431 cells is accompanied by a loss of the susceptibility of α s to
activation via GPCRs and its ability to stimulate adenylate cyclase in response to activated GPCRs [309].
936 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
significance remains unclear. In transiently transfected
COS-7 cells overexpressing various PKC isoforms the
PKCs α, β1,γ , δ, ε, and ζ were found to be tyrosine
phosphorylated and catalytically activated in response
to H2O2 that is known to induce oxidative stress [312].
However, the tyrosine kinases that phosphorylate PKC
isoforms under these conditions are not yet known.
Among the PKC isoforms which are tyrosine
phosphorylated in vitro some interest has been
focussed to PKCδ. This isoform has been shown to be
phosphorylated on tyrosine in Ras-transformed cells
[313] and in response to various stimuli such as
phorbol esters, carbachol, or substance P [314]. In
addition, PKCδ is tyrosine phosphorylated after
stimulation of the EGFR signalling pathway involving
the activation of a member of Src kinase family and
leading to inhibition of PKCδ activity [311]. In v-Srctransformed fibroblasts the formation of a Src-PKCδ
complex in which PKCδ
becomes
tyrosine
phosphorylated and down-regulated was proposed as
a possible molecular mechanism [315]. As PKCδ
phosphorylation sites Tyr 52 and Tyr187 in the N-terminal
part have been identified [316, 317]. However, the
functional
consequences
of
PKCδ
tyrosine
phosphorylation remain controversial and involve both
activation and inhibition of catalytic activity.
Nevertheless, these findings implicate the
possibility that PKC may be regulated by different
signalling pathway in response to stimulation of GPCRs
as well as RTKs. One is the classical DAG-dependent
pathway through activation of PLCβ/γ or PLD. Another
signalling route may occur DAG-independent but
involving activation of PI3K. A third pathway might be
the tyrosine phosphorylation of PKCs in response to
RTKs stimulated directly by growth factors or
transactivated via GPCRs.
Nevertheless, these findings implicate the
possibility that PKC may be regulated by different
signaling pathways in response to stimulation of
GPCRs as well as RTKs. One is the classical DAGdependent pathway through activation of PLCβ/γ or
PLD. Another signaling route may occur DAGindependent but involving activation of PI3K. A thrid
pathway might be the tyrosine phosphorylation of
PKCs in response to RTKs either stimulated directly by
growth factors or transactivated via GPCRs.
6. Implications for Drug Development
Activity of GPCRs can be selectively modulated by
receptor-subtype specific agonists or antagonists.
Numerous drugs have been developed on the basis of
this principle. Progress in understanding of GPCR
signal transduction pathways creates new perspectives
Liebmann and Böhmer
on GPCR directed drug development. Cross-talk
beween different GPCRs and the discovery of multiple
pathways initiated by a single class of GPCR predict that
GPCR agonists/antagonists will have side effects
depending on the given coupling of the GPCR to
signaling pathways, even if they target a given receptor
subtype with absolute specificity. An example comes
from the bradykinin story. B2R antagonists have a
therapeutic potential as novel analgetic and antiinflammatory agents. Structural modifications of BK led
to the discovery of the peptidic B2R antagonist Hoe
140 which is highly potent and specific in all cells and
tissues tested so far. When we screened various tumor
cell lines for mitogenic effects of BK we used Hoe 140
as control. Very surprisingly, we found that in certain
tumor cells the antagonist Hoe 140 induced stronger
mitogenic effects than BK (C. Liebmann, unpublished
results). On the other hand, understanding of GPCR
post-receptor signaling mechanisms opens up the
possibility to interfere with downstream signaling steps.
This concept has been put forward as "signal
transduction therapy" initially mainly focussed on RTK
signaling [318], where development of efficient ligand
antagonists hitherto failed. Inhibitors of enzymes
mediating important steps of GPCR signaling would
block receptor effects. Alternatively, inhibition of
negatively regulating steps could be used to augment
signaling of a given GPCR. Interstingly, the recent
findings on GPCR cross-talk with RTKs has brought
into play specific tyrosine kinase inhibitors as a novel
class of effectors for GPCR signaling [319-321]. These
drugs have actually been instrumental for clarifying the
involvement of RTKs in aspects of GPCR signaling,
notably mitogenic signaling of GPCR. Such
compounds are for example specific inhibitors for the
EGFR of the anilino quinazoline family as AG1478 or
PD153035 (Table 3), specific inhibitors of the PDGF
receptors as the quinoxaline AG1296 and the
phenylaminopyrimidine CGP53716 [322] or the Srcfamily inhibitor PP1 [323]. The latter has, however,
recently been found to block the PDGFβ-receptor as
well [287]. Another class of kinase inhibitors with
increasing relevance for GPCR signaling are blockers of
enzymes in the MAPK signaling cascades, including
the inhibitor of MEK1/2 PD98059 [288] or the specific
p38-blocker SB203580 [324, 325]. One can anticipate
that the current rapid development in the field of kinase
inhibitors will certainly be of great value for the
pharmacological modulation of GPCR signaling.
Several kinase blockers have recently been put
forward to clinical trials, maily aiming at tumor therapy by
targeting RTK pathways [326]. Since, however, as
outlined above, pathways from GPCRs and tumorrelevant RTKs converge, overlap and cross-talk, one
could anticipate that GPCR-related side effects may
become observed.
Signal Transduction Pathways of G Protein-coupled Receptors
Table 3.
Current Medicinal Chemistry, 2000, Vol. 7, No. 9
Protein Kinase Inhibitors: Agents Which Block also
Mitogenesis
Selected references: [143, 286-288, 319, 322, 323]
GPCR-Mediated
MAPK
Activation
937
and
Compound
Inhibited enzyme
Concentration for near complete inhibition in
intact cells by maintaining selectivity
Remarks
AG1478 (AG1517)
PD153053
EGFR
10-300 nM
may inhibit other HER-family kinases
as well
AG1295/6
PDGFαR, PDGFβR
5-10 µM
PP1/AGL1872
Src-family kinases, PDGFβR
1-3 µM
PD98059
MEK
10 µM
As we have tried to illustrate, GPCR signaling is a
pleiotropic response which, as a rule, involves multiple
signaling chains. To what extent these pathways are
activated strongly depends on the cell type concerned.
Thus, interference with GPCR signaling is likely to
require a "cocktail" of signaling effectors which is
tailored for a given target tissue.
Frequently, pharmacological modulation of GPCR
signaling aims at stimulation or augmentation of
receptor function. Compounds affecting enzymes
which regulate signal transduction negatively would be
excellent tools in this respects. However, effector
discovery for such enzymes is only in its beginnings.
This concerns inhibitors of PTPs, of MAPK inactivating
dual-specificity phosphatases, of inositolpolyphosphate phosphatases, of phosphatidylinositolphosphate phosphatases and others. An exception present
inhibitors of phosphodiesterases, where a number of
compounds are in clinical trials and one drug has
recently been introduced to the market [327]. This
example illustrates the immense possibilities for drug
development aiming at interference with negatively
regulating signaling events.
One could assume that specificity for signal
transduction therapy will be the more difficult to attain
the further distal to the GPCR the targeted signaling
step is positioned. As outlined above, it becomes,
however, increasingly clear that the pathways used by a
given GPCR, the extent of cross-talk and feedback
reactions, are to a very high degree cell-specific. Thus,
interference with a given enzyme is likely to have very
different effects in different cell types and beneficial
effects may be obtained in a particular tissue whithout
negatively affecting others. It is difficult to predict to
what extent inhibition of a certain signaling step will
abolish signaling or rather shift signaling to another
pathway. Also, partial inhibition of a pathway which
contains negative feeback elements may affect
feeback inhibition to a greater extent than positive
signal generation and thus, lead to even augmented
signaling as net result. Further, most studies on
also antagonist of aryl hydrocarbon
receptor, inhibits cyclooxygenases
signaling mechanisms have been performed in
permanent cell lines or overexpression systems and
some if not many proposed mechanisms may reveal to
be not physiologically relevant. In conclusion,
predictions on selective in vivo effects of certain
effectors are currently hardly possible. Generation of
transgenic mice with inactivated genes for certain
signal-mediating molecules has revealed, however,
that the effects of even complete abolishment of
certain signaling steps in all tissues may be much more
selective than previously anticipated. An example is the
recent inactivation of the gene for p85α, an adaptor
molecule required for activation of the α and β- isoforms
of PI3 kinase [328]. This knockout led not to lethality,
as might have been anticipated but to a specific
impairment of B-cell immunity. One has further to
consider, that application of a signal transduction
enzyme inhibitor in vivo is unlikely to lead to complete
ablation of enzyme activity, rather more attenuation in
some tissues and less attenuation in others,
depending on pharmacokinetic parameters and
expression levels. This leaves further possibilities for
specific effects.
In conclusion, development of further signal
transduction effectors for GPCRs seems highly
warranted although the only partially understood
complexity of signaling makes predictions of in vivo
effects and disease indications difficult. It can,
however, be anticipated that many reasonably target
selective compounds will eventually find applications.
Note Added in Proof
A novel mechanism for EGFR transactivation by
GPCRs has been shown recently: GPCR-dependent
activation of a metalloproteinase leads to processing of
pro-heparin-binding (HB)-EGF and EGFR activation by
released HB-EGF. This process is suppressed by the
metalloproteinase inhibitor batimastat.
[Prenzel, N.; Zwick, E.; Daub, H.; Laserer, M.; Abraham,
R.; Wallasch, C., Ullrich, A. Nature, 1 9 9 9 , 402, 884].
938 Current Medicinal Chemistry, 2000, Vol. 7, No. 9
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