From the Oral and Pharyngeal Cancer Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892
Receptors coupled to heterotrimeric GTP-binding
proteins (G proteins) are integral membrane proteins involved in the
transmission of signals from the extracellular environment to the
cytoplasm. The best known family of G protein-coupled receptors
(GPCRs),1 currently
comprising more than 1000 members, exhibits a common structural motif
consisting of seven membrane-spanning regions (1) (Fig.
ins;1873f1}1). A diverse array of external stimuli including neurotransmitters, hormones, phospholipids, photons, odorants, certain taste ligands, and growth factors can activate specific members of this receptor family and promote interaction between the receptor and the G protein on the intracellular side of the
membrane. This causes the exchange of GDP for GTP bound to the G
protein
INTRODUCTION
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Introduction
References
subunit and apparently the dissociation of the
heterodimers. In turn, GTP-bound G protein
subunits or
complexes initiate intracellular signaling responses by acting on
effector molecules such as adenylate cyclases or phospholipases or
directly regulating ion channel or kinase function (Fig. 1, and see
below). Sixteen distinct mammalian G protein
subunits have been
molecularly cloned and are divided into four families based upon
sequence similarity:
s,
i,
q, and
12. Similarly, eleven G protein
subunits and five G protein
subunits have been identified.
Thus, GPCRs are likely to represent the most diverse signal
transduction systems in eukaryotic cells. The biochemical and
biological consequences of such diversity in subunit composition and
coupling specificity for each receptor have just begun to be
elucidated. In this review, we will briefly describe the role of G
proteins and their coupled receptors in normal growth control and
tumorigenesis and then focus on current efforts to elucidate the
signaling pathways connecting this class of cell surface receptors to
nuclear events regulating gene expression.
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Fig. 1.
Diversity of the G protein-coupled receptor
signal transduction system. See text for details. DAG,
diacylglycerol; IP3, inositol
trisphosphate.
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Proliferative Signaling through G Protein-coupled Receptors |
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Proliferative signaling has generally been associated with
polypeptide growth factor receptors that possess an intrinsic protein tyrosine kinase activity (2). A variety of oncogenes have been found to
code for mutated forms of these receptors (3) and their ligands (4) or
for molecules that participate in their growth-promoting pathways (5).
On the other hand, GPCRs have been traditionally linked to
tissue-specific, fully differentiated cell functions (1). However,
GPCRs are also expressed in proliferating cells, and they have been
implicated in embryogenesis, tissue regeneration, and growth
stimulation (reviewed in Ref. 6). In this regard, many ligands acting
via GPCRs, including thrombin, bombesin, bradykinin, substance P,
endothelin, serotonin, acetylcholine, gastrin, prostaglandin F2, and
lysophosphatidic acid, are known to elicit a mitogenic response in a
variety of cell types (reviewed in Refs. 6 and 7), and recent gene
knock-out studies indicate that certain GPCRs are essential for cell
growth under physiological conditions (8). Furthermore, accumulating
evidence indicates that GPCRs and their signaling molecules can harbor
oncogenic potential. For example, the mas oncogene, which
encodes a putative GPCR, was initially cloned using standard
transfection assays by virtue of its ability to induce tumors in mice
(9). Subsequently, serotonin 1C (10), muscarinic m1, m3, and m5 (11),
and adrenergic
1 (12) receptors were shown effectively to transform
contact-inhibited cultures of rodent fibroblasts when persistently
activated. Together these studies demonstrated that GPCRs can behave as
agonist-dependent oncogenes and prompted several groups to
explore the transforming potential of G protein
subunits. In recent
studies, constitutively active mutants of G
i,
G
q, G
0, G
12, and
G
13 were shown to behave as transforming genes in a
variety of cell types (reviewed in Ref. 13).
The recent discovery of activating mutations in GPCRs and G proteins in
several disease states, including cancer, further supports a role for
GPCRs in normal and aberrant growth control. For example, mutationally
activated Gs results in hyperplasia of endocrine cells
and has been found in human thyroid and pituitary tumors (reviewed in
Ref. 13) and in the McCune-Albright syndrome, a disease in which
multiple endocrine glands exhibit autonomous hyperproliferation (14).
Interestingly, activated G
s contributes significantly to
hyperplasia only in tissues where cAMP stimulates proliferation, thus
acting as an oncogene referred to as the gsp oncogene (15).
Activating mutations have also been identified for G
i2,
referred to as the gip2 oncogene, in a subset of ovarian sex
cord stromal tumors and adrenal cortical tumors (16). On the other
hand, G
12, referred as the gep oncogene (17,
18), was isolated as a transforming gene from a soft tissue
sarcoma-derived cell (19), although its role in tumorigenesis remains
unclear. Naturally occurring activated mutations in members of the
G
q family have not yet been described.
At the receptor level, the identification of constitutively active thyroid-stimulating hormone receptor mutations in 30% of thyroid adenomas (20) provided a direct link between this class of receptors and human cancer. Similarly, mutationally activated luteinizing hormone receptors have been identified in a form of familial male precocious puberty, which results from hyperplastic growth of Leydig cells (21). Perhaps more frequently than activating mutations, paracrine and autocrine stimulation of multiple GPCRs for neuropeptides and prostaglandins has been implicated in a number of human neoplasias, including small cell lung carcinoma (22), colon adenomas and carcinomas (23), and gastric hyperplasia and cancer (24). Sequences encoding functional GPCRs have also been found in the genome of transforming DNA viruses, including herpesvirus saimiri (25) and Kaposi's sarcoma-associated herpesvirus (26). Currently available evidence suggests that, at least for Kaposi's sarcoma-associated herpesvirus, these viral GPCRs are sufficient to subvert normal growth control.
The mechanism(s) whereby GPCRs regulate cell proliferation remain poorly understood. Although inhibition of adenylyl cyclase has been observed in cells responding to growth-promoting agents acting on Gi-coupled receptors, there is no formal proof that induction of DNA synthesis results from decreasing intracellular levels of cAMP. Conversely, several lines of investigation have implicated phosphatidylinositol bisphosphate (PIP2) hydrolysis as a critical component of mitogenesis (6). However, recent studies using mutant tyrosine kinase receptors suggested that PIP2 hydrolysis is neither necessary nor sufficient for mitogenesis (27, 28). Furthermore, a number of GPCR agonists induce the PIP2 turnover pathway but fail to stimulate growth when added alone to quiescent cells (29). Although the interpretation of this body of information can be hampered by the fact that each study has been performed in a different cell line, collectively it indicates that additional effector pathways might participate in the proliferative response to GPCR stimulation.
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Role of MAP Kinase in Proliferative Pathways |
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Critical molecules participating in the transduction of proliferative signals have just begun to be identified. One such example is the family of extracellular signal-regulated kinases (ERKs) or MAP kinases, whose enzymatic activity increases in response to mitogenic stimulation. These kinases play a central role in mitogenic signaling, as impeding their function prevents cell proliferation in response to a number of growth-stimulating agents (30). Furthermore, aberrant functioning of proteins known to be upstream of MAPK can induce cells to acquire the transformed phenotype, and constitutive activation of the MAPK pathway is itself sufficient for tumorigenesis (31, 32). Thus, MAPKs appear to be a critical component of growth-promoting pathways. The stimulation of tyrosine kinase receptors provokes the activation of MAPKs in a multistep process. For example, essential molecules linking epidermal growth factor receptors to MAP kinase include the adaptor protein GRB2/SEM-5, a guanine nucleotide exchange protein such as SOS, the small GTP-binding protein p21ras, and a cascade of protein kinases defined sequentially as MAP kinase kinase kinase, represented by c-Raf-1, and MAP kinase kinase such as MEK1 and MEK2 (reviewed in Ref. 32). MEKs ultimately phosphorylate p44mapk and p42mapk, also known as ERK1 and ERK2, respectively, on both threonine and tyrosine residues, thereby increasing their enzymatic activity. In turn, MAP kinases phosphorylate and regulate the activity of key enzymes and nuclear proteins, which ultimately regulate the expression of genes essential for proliferation (reviewed in Ref. 33). Because of the proposed central role of MAPK in proliferative pathways, many laboratories have recently addressed the nature of those molecules connecting GPCRs to MAP kinases.
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Signaling from G Protein-coupled Receptors to MAP Kinase Involves
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As an approach to explore the mechanism of MAPK activation
by GPCRs, several laboratories have used the transient coexpression of
an epitope-tagged form of MAPK together with GPCRs in readily transfectable cell lines, such as COS-7 cells. In this cellular setting, it was observed that MAPK was potently activated upon ligand
addition by either Gq-coupled or Gi-coupled
receptors, respectively, in a pertussis toxin-insensitive and
-sensitive fashion (34-36). However, under identical experimental
conditions, activated forms of Gi2, G
q,
Gs, or G12 were not able to induce MAPK
activation (35).
The failure of activated G subunits to mimic receptor stimulation of
MAPK activity and the accumulating evidence supporting an active role
for the G
dimers in signal transmission (37) prompted exploration
of the role of
complexes in signaling to the MAPK pathway. This
led to the observation that membrane-bound forms of
heterodimers
can directly elicit signaling pathways leading to MAPK activation (35)
and prompted the search for molecules acting downstream of G
in
this biochemical route. In a variety of experimental conditions, it was
shown that MAPK activation by
subunits required neither PLC-
nor PKC activation but was blocked by dominant interfering mutants of
the GTP-binding protein Ras (34, 35) and that
subunits can
induce the accumulation of Ras in the GTP-bound, active form (34).
Taken together, these findings indicated that signaling from GPCRs to
MAPK involves
subunits of heterotrimeric G proteins acting on a
Ras-dependent pathway and provided strong evidence that the
GPCR signaling pathway converges at the level of Ras with that emerging
from receptors of the tyrosine kinase class.
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The Pathway Linking GPCRs and G![]() ![]() |
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The inhibitory effect of genistein on lysophosphatidic
acid-induced MAPK activation provided the first indirect indication that tyrosine kinases might mediate the activation of MAPK by GPCRs
(38). Furthermore, several groups observed that activation of GPCRs in
a variety of cellular systems leads to the rapid phosphorylation of the
adaptor protein Shc on tyrosine residues and the consequent formation
of Shc-GRB2 complexes (39, 40). Searching for candidate tyrosine
kinases, Luttrell et al. (41) have recently obtained evidence that Src, or a Src-like kinase, links to activation of
the Ras-MAPK pathway through phosphorylation of Shc and the recruitment
of GRB2 and SOS. That report was soon followed by several studies
describing the implication of other non-receptor tyrosine kinases
linking GPCRs to MAPK. These include Src-like kinases such as Fyn, Lyn,
and Yes and the more distantly related Syk (42, 43) and a novel
Ca2+ and PKC-dependent protein tyrosine kinase,
Pyk2 (44-46). The latter is closely related to focal adhesion kinase,
which is involved in the formation of focal complexes containing Src,
paxillin, dynamin, and Grb2 after integrin binding. Focal adhesion
kinase can also be activated by GPCRs (47, 48) and may possibly be involved in GPCR signaling to MAPK. Tyrosine kinases of the receptor class have also been implicated in GPCR signaling; both PDGF and epidermal growth factor receptors were recently shown to become phosphorylated in response to GPCR agonists (49, 50) and to play a role
in MAPK activation by GPCRs by recruiting signaling complexes
containing Shc and GRB2. In short, it is becoming increasingly clear
that a number of non-receptor tyrosine kinases and tyrosine kinase
receptors can link GPCRs to the Ras-MAPK pathway. However, the relative
contribution of each of these kinases in GPCR signaling to MAPK is
still unclear and under current investigation.
Additional potential links between G and the Ras-MAPK pathway
have been recently identified. They include the protein tyrosine phosphatase SH-PTP1 (51) and Ras-GRF, a distinct Ras guanine nucleotide
exchange factor expressed in neuronal cells, which can be activated in
response to GPCR stimulation or upon coexpression of G
(52). In
addition, several groups observed that wortmannin, a
phosphatidylinositol 3-kinase (PI3K) inhibitor, can diminish MAPK
activation by GPCRs (see Ref. 53), and a novel PI3K isotype, termed
PI3K
, that is activated by G
complexes (54) was found to play
a critical role in linking Gi-coupled receptors and G
to the MAPK signaling pathway (55). In this case, PI3K
was found to
act downstream from G
and upstream of Src-like kinases, thus
suggesting a potential mechanism whereby heterotrimeric G proteins can
regulate non-receptor tyrosine kinases.
Ras-independent activation of MAPK by GPCRs has also been reported (56,
57), although it was defined as such primarily based on the failure to
observe accumulation of Ras in the GTP-bound form in response to GPCR
stimulation. However, as dominant interfering mutants of Ras can
diminish MAPK activation, even in systems where GTP-bound Ras was not
readily demonstrable (56), it is still possible that undetected amounts
of Ras in the GTP-bound form might be sufficient to cooperate with
other pathways to induce MAPK activation. Alternatively, in certain
cellular backgrounds, GPCRs might be able to utilize pathways bypassing
the requirement for Ras activation. One such potential Ras-independent
pathway might help explain the activation of MAPK by constitutively
active Gi2, the gip2 oncogene, which can be
observed in only a limited number of cell types (58). Another putative
Ras-independent pathway might involve PKC, as direct activation of PKC
by phorbol esters can induce MAPK in a Ras-dependent or
Ras-independent fashion (59, 60). Consequently, in cells where PKC can
directly activate signaling pathways leading to MAPK activation, it is
expected that MAPK activation by Gq-coupled receptors would
not strictly require Ras. In this line, Gq-coupled receptor
activation of MAPK has been shown to be PKC-dependent (60),
fully PKC-independent (61), or partially PKC-dependent
(62).
We can conclude that multiple molecules may mediate MAPK activation by
GPCRs and G. The expression of some of these molecules follows a
restricted tissue distribution (44, 52, 54), which might help explain
the seemingly conflicting results obtained by different groups
analyzing the relative contribution of each pathway in different cell
lines and tissue culture systems. The nature of the biochemical routes
utilized to communicate GPCRs to the MAPK pathway would then be
expected to depend heavily on the repertoire of signaling molecules
available in each particular tissue and cell type.
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G Protein-coupled Receptors Activate the Jun Kinase (JNK) Pathway by a Novel Biochemical Route |
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The studies described above strongly suggest that both GPCRs and tyrosine kinase receptors can activate Ras, thereby initiating a cascade of events leading to MAPK activation and transcriptional regulation. However, activation of GPCRs was found to induce a clearly distinct pattern of expression of immediate early genes, including those of the jun and fos family (64). In particular, activation of GPCRs but not tyrosine kinase receptors for PDGF led, in NIH 3T3 cells, to a remarkable expression of c-jun (64). This response did not correlate with MAPK activation (64), thus suggesting that GPCRs control a distinct biochemical route regulating gene expression. Furthermore, recent work demonstrated that a novel family of enzymes closely related to MAPK, named Jun kinases (JNKs) (65) or stress-activated protein kinases (SAPKs) (66), selectively phosphorylates and regulates the activity of the c-Jun protein. Based on those findings, the ability to signal to JNK by cell surface receptors was further investigated. Interestingly, in NIH 3T3 cells, GPCRs but not PDGF receptors were found potently to activate JNK (64), thus establishing that the GPCR signaling pathways diverge at the level of JNK from those utilized by tyrosine kinase receptors.
Although it was initially thought that JNKs were located downstream
from Ras, this hypothesis was in conflict with the lack of activation
of JNK by PDGF or by other agonists acting on receptors that are known
to couple to the Ras pathway (64, 66). Soon, it was found that the
Ras-related small GTP-binding proteins Rac1 and Cdc42 initiate an
independent kinase cascade regulating JNK activity (67) and that Rac
and Cdc42 are an integral part of the signaling route linking many cell
surface receptors, including GPCRs, to JNK (68). More recent work has
identified many components of this pathway and has shown that JNK is
potently activated by several naturally occurring human oncogenes
(reviewed in Ref. 69). Further examination of the G protein subunits
linking GPCRs to JNK provided evidence that free dimers (68)
and, in some cellular systems, G
12 (70) transfer signals
from this class of receptors to JNK.
The pathway(s) connecting GPCRs to other, recently discovered members
of the MAPK superfamily, such as ERK6, ERK5, and SAPK4, have not yet
been defined. However, GPCRs have recently been shown to activate a
novel pathway that involves the transcriptional regulation of the serum
response factor by the small GTP-binding protein Rho (71), and a recent
study suggests that both G12 and G might connect
GPCRs to Rho and to serum response factor (72). Those molecules linking
GPCRs and heterotrimeric G proteins to Rho remain undefined.
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Conclusion |
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The emerging picture from recent reports is that in mammalian
cells, subunits of heterotrimeric G proteins communicate GPCRs
with the MAPK and JNK pathways acting, respectively, on a Ras and
Rac1/Cdc42-dependent biochemical route. These findings together strongly suggest that
complexes provide a molecular bridge between heterotrimeric G proteins and small GTP-binding proteins. This connection is strikingly similar to the pathway linking
G protein-coupled pheromone receptors to MAPK-related enzymes in the
budding yeast Saccharomyces cerevisiae. In yeast, the G
protein
subunit can initiate activity of a MAPK cascade by binding
an exchange factor for the small GTP-binding protein Cdc42, and then
this GTP-binding protein physically interacts with the most upstream
kinase, Ste20, causing its activation (73). An additional scaffolding
protein, Ste5, binds yeast
and several components of this MAPK
cascade. In mammalian cells a number of sequentially acting molecules
are required instead to connect GPCRs and G
to Ras, including
tyrosine kinases, lipid kinases, adapter molecules, PKC, and certain
Ras guanine nucleotide exchange factors. However, it is still possible
that heterotrimeric G proteins might directly regulate the activity of
yet to be identified guanine nucleotide exchange factors for
Rho-related GTPases, similar to those shown in yeast. In this
line, no mammalian homologue for Ste5 has been described so far.
Surprisingly, however, a very recent report suggests that a
PDZ-containing protein acts as a scaffold, linking several signaling
molecules to G
q in the visual system of the fruit fly
(63). Thus, it is conceivable that still unidentified scaffolding
proteins might also participate in the mammalian pathway connecting
heterotrimeric G proteins to MAPK cascades.
We can conclude that the molecular complexity of the signaling pathways connecting GPCRs to the nucleus has just begun to be appreciated. These pathways involve an unsuspected number of biochemical routes, including those connecting heterotrimeric G proteins to small GTP-binding proteins of the Ras and Rho family, their regulated kinases, and their nuclear targets (Fig. 2). Further work in this area is expected to help identify the nature of all contributing molecules, as well as to elucidate fully their functional relationships. Emerging areas of interest also include exploring how all these signaling events that are initiated upon agonist binding to GPCRs, including second messenger generating systems, cytoskeletal changes, and physical interaction of heterotrimeric G protein subunits with molecules regulating kinase cascades, are integrated in space and time to elicit biologically relevant responses, including normal and aberrant cell growth.
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FOOTNOTES |
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* This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. This is the third article of three in the "Signaling by Heterotrimeric G Proteins Minireview Series."
To whom correspondence should be addressed: Cell Growth Control
Section, Oral and Pharyngeal Cancer Branch, NIDR, National Institutes
of Health, Bldg. 30, Rm. 212, 9000 Rockville Pike, Bethesda, MD
20892-4330. Tel.: 301-496-6259; Fax: 301-402-0823.
1 The abbreviations used are: GPCR, G protein-coupled receptor; PIP2, phosphatidylinositol bisphosphate; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAP or ERK kinase; PLC, phospholipase C; PKC, protein kinase C; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; JNK, Jun kinase; SAPK, stress-activated protein kinase.
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REFERENCES |
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