Regulation of Mitogen-Activated Protein Kinase Signaling ... · G protein-GPCR signal-transducing...

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The family of receptors that transmit signals throughthe activation of heterotrimeric GTP-binding proteins(G proteins) constitutes the largest group of cell sur-face proteins involved in signal transduction. Thesereceptors participate in a broad range of important bi-ological functions and are implicated in a number ofdisease states. More than half of all drugs currentlyavailable influence G protein-coupled receptors(GPCRs). These receptors affect the generation ofsmall molecules that act as intracellular mediators orsecond messengers, and can regulate a highly inter-connected network of biochemical routes controllingthe activity of several members of the mitogen-activat-ed protein kinase (MAPK) superfamily. They includeextracellular signal-regulated kinase 1 (ERK1) andERK2 (or p44MAPK and p42MAPK), c-Jun NH2-terminal ki-nases (JNKs), ERK5 (or BMK), and p38 MAPKs, in-cluding p38α (or CSBP-1), p38β, p38γ (or SAPK3 orERK6), and p38δ (or SAPK4). This review will focus onthe molecular mechanisms by which GPCRs signal tothe nucleus through this intricate network of secondmessenger-generating systems and MAPK signalingpathways, thereby affecting the expression of geneswhose products influence many biological processes,including normal and aberrant cell growth.

The G Protein-Coupled Receptor Signaling SystemWith more than 1000 members, the family of receptors that trans-mit signals through the activation of heterotrimeric GTP-bindingproteins (G proteins) represents the largest group of cell surfacereceptors encoded by the mammalian genome (>1% of humangenes). (A Glossary of abbreviations and description of proteinsmentioned in this review is available online.) Similarly, the com-pletion of the Caenorhabditus elegans genome project revealedthat more than 5% of C. elegans genes encode G protein-coupledreceptors (GPCRs) (~1100 GPCRs) (1). These receptors mediatebiological responses elicited by a remarkably diverse array ofstimuli, including growth factors, vasoactive polypeptides,chemoattractants, neurotransmitters, hormones, phospholipids,photons, odorants, and taste ligands, and are estimated to be thetarget for nearly 60% of all drugs currently available (2). Thesereceptors are also frequently referred to as heptahelical or serpen-tine receptors, because the best-known family of GPCRs containsa conserved structural motif consisting of seven α-helical mem-brane-spanning regions (3, 4) (Fig. 1). Activation of these recep-tors causes a profound change in the transmembrane α helices,which affects the conformation of intracellular loops and uncov-

ers previously masked G protein binding sites (5-7). The GPCR-G protein interaction in turn promotes the release of guanosinediphosphate (GDP) bound to the G protein α subunit and its ex-change for guanosine triphosphate (GTP), and causes a confor-mational change in three flexible “switch regions” of the Gα sub-unit, thus activating Gα and causing the exposure of effectorin-teraction sites in the βγ heterodimers (8-14). Activated G proteinsubunits then initiate intracellular signaling responses by actingon a variety of effector molecules.

Heterotrimeric G Proteins and Second Messenger-Generating SystemsAbout 20 mammalian G protein α subunits have been identified,which can be divided into four families based on their primarysequence similarity: Gs, Gi, Gq, and G12 (15). These G protein αsubunits regulate the activity of several second messenger-gener-ating systems. For example, the Gq family controls the activity ofphosphatidylinositolspecific phospholipases, such as phospholi-pase C-β (PLC-β), which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (16). IP3 and DAGin turn lead to an increase in the intracellular concentrations offree calcium [Ca2+]i and the activation of a number of protein ki-nases, including protein kinase C (PKC) (17-20) (Fig. 1). Themembers of the Gs family activate adenylyl cyclases, whereas Gifamily members can inhibit a subset of these enzymes, therebycontrolling the intracellular concentrations of adenosine 3´,5´-monophosphate (cAMP). Indeed, nine distinct adenylyl cyclaseshave been cloned so far, and each appears to be distinctly regulat-ed by Gs and Gi, as well as by βγ subunits, [Ca2+]i, and PKCs (21-23). Thus, the impact on the intracellular concentrations ofcAMP by agonists acting on GPCRs will be highly dependent onwhich adenylyl cyclases are expressed in each cell type. Gα sub-units of the Gi family, which includes Gαi1, Gαi2, Gαi3, Gαo,transducin (Gαt), and gustducin (Gαgust), also activate a variety ofphospholipases and phosphodiesterases, and promote the openingof several ion channels (12). The nature of the signaling path-ways controlled by the G12 family has just begun to be elucidat-ed. Members of this enigmatic Gα family, which includes Gα12and Gα13, provide links between GPCRs and the activation of thesmall GTP-binding protein Rho (24-28).

In addition to the many Gα subunits, 12 G protein γ subunitsand 6 G protein β subunits have been cloned. Gβγ dimers, when re-leased from the heterotrimeric complex upon Gα activation, canthemselves regulate the activity of many signaling molecules, in-cluding ion channels, phosphatidylinositol 3-kinases (PI3Ks),phospholipases, adenylyl cyclases, and receptor kinases (13). Thepossibility exists that distinct pools of Gβγ subunits may play dif-ferent roles in signal transmission (12). Taken together, this emerg-ing body of information shows the tremendous complexity of theG protein-GPCR signal-transducing system, whose biochemicaland biological consequences have just begun to be appreciated.

Regulation of Mitogen-Activated Protein Kinase Signaling Networks by G Protein-Coupled Receptors

J. Silvio Gutkind(Published 11 July 2000)

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The author is at the Oral and Pharyngeal Cancer Branch, NationalInstitute of Dental Research, National Institutes of Health, 30 Con-vent Drive, Building 30, Room 212, Bethesda, MD 20892-4330, USA.E-mail: [email protected]


G Protein-Coupled Receptors and Cell Growth ControlGPCRs are involved in many important biological functions,such as photo- and chemoreception, neurotransmission, regula-tion of secretion from endocrine and exocrine glands, exocyto-sis, chemotaxis, blood pressure control, platelet function, aswell as embryogenesis, angiogenesis, tissue regeneration, andcontrol of normal and aberrant cell growth. The ability to trans-duce proliferative signals has been frequently associated withthe activation of polypeptide growth factor receptors that havean intrinsic protein tyrosine kinase activity (29). However, theavailability of bacterial toxins, such as pertussis toxin that in-hibits the function of G protein α subunits of the Gi family(30), helped to establish that GPCRs mediate proliferative re-sponses to many mitogens, including thrombin (31, 32),lysophosphatidic acid (LPA) (33), and many other substancesthat are normally found in serum (34). In addition, pharmaco-logical and biochemical evidence indicates that a large numberof mitogens, including bombesin, vasopressin, bradykinin, sub-stance K, acetylcholine receptor agonists, angiotensin II, andmany others (35) stimulate cell proliferation by acting on re-ceptors coupled to G proteins that are insensitive to bacterialtoxins. If persistently activated, some of these GPCRs con-tribute to malignant transformation (36-39), and ultimately tohuman cancer (40-48). Furthermore, recent data suggest thatcertain DNA viruses also encode functional GPCRs, includinghuman cytomegalovirus (HCMV) (49, 50), Herpesvirus saimiri(HVS) (51), and the Kaposi’s sarcoma-associated herpesvirus(KSHV) (52). The latter is likely to represent a constitutivelyactive chemokine receptor, which harbors transforming poten-tial (52) and might actively participate in the angiogenic pro-cess that characterizes this typically AIDS-associated malig-nancy (53).

The ability of GPCRs to affect cell growth prompted sev-eral groups to examine whether their immediate downstreamtargets, the G proteins, also have transforming potential ifmutationally activated. GTPase-deficient mutants of Gαi,Gαq, Gαo, Gα12, and Gα13 were found to display oncogenicproperties when expressed in several cellular systems; andnaturally occurring activated mutants of certain G proteinswere also identified in various disease states, including can-cer (54). This led to the designation of certain activated Gαmutants as oncogenes, including Gαs, Gαi2, and Gα12, re-ferred to as the gsp (55), gip2 (56), and gep oncogenes (57,58), respectively.

The nature of the intracellular signaling pathways mediatingthe proliferative effects of G proteins and their coupled recep-tors is still poorly understood. Whereas conventional secondmessenger-generating systems, such as adenylyl cyclases, ionchannels, and phospholipases, were the focus of the early re-search efforts addressing this issue (35, 59), an emerging bodyof information indicates that additional effector pathways par-ticipate in proliferative signaling by GPCRs (48). In particular,GPCRs have been shown to activate members of the extracellu-lar signal-regulated kinases (ERKs) or mitogen-activated pro-tein kinases (MAPKs) family, which are key components of in-tracellular signaling pathways that control cell proliferation (60,61). The elucidation of the molecular mechanisms wherebyGPCRs activate MAPKs is believed to be central to understand-ing how these receptors regulate cell growth and has recentlybecome one of the most exciting areas of investigation in the Gprotein field.

The Classical MAPK PathwayThe initial search for candidate molecules mediating the mito-genic effects of growth factors such as epidermal growth factor(EGF) and platelet-derived growth factor (PDGF), which stimu-late receptor tyrosine kinases, led to the identification of a tyro-sine-phosphorylated protein of 42 kD (62, 63), which exhibitedsimilar electrophoretic characteristics to those of a phosphotyro-sine-containing molecule detected upon activation of PKC withphorbol esters (64) or in virally transformed cells (65). Soon af-ter, this molecule was identified as p42MAPK and was shown torequire a dual phosphorylation of both threonine and tyrosine forits full activation (66, 67). Molecular cloning of this moleculeand a highly related protein, p44MAPK, revealed that they belongto a family of serine-threonine kinases related to the kinasesFus3 (a yeast MAPK involved in the pheromone response) andKss1 (kinase suppressor of SST2, a yeast MAPK involved in fil-amentous growth) from the yeast Saccharomyces cerevisiae (68,69), which act as the final step in a kinase cascade that partici-pates in the pheromone-induced mating response (70). In mam-malian cells, p44MAPK and p42MAPK, also known as ERK1 (extra-cellular signal-regulated kinase 1) and ERK2, respectively, re-ferred to herein as MAPKs, are believed to be central compo-nents of proliferative pathways. Their enzymatic activity increas-es in response to mitogenic stimulation, impeding their functionprevents cell proliferation in response to many growth factors(71), and constitutive activation of molecules acting upstream ofMAPKs is sufficient for tumorigenesis (72, 73).

How growth factor receptors of the tyrosine kinase class acti-vate MAPKs appears now to be relatively well understood. Forexample, binding of EGF to its cognate receptors leads to the ty-rosine phosphorylation of several substrates including the EGFreceptor (EGFR) itself. Such phosphorylation sites serve as dock-ing sites for the binding of adapter proteins that contain structuralmotifs involved in protein-protein interaction. These adapter pro-teins include Grb2 and SEM5 (73), which possess a Src homolo-gy 2 (SH2) domain and two SH3 domains, and Shc (an adapterprotein in the Ras pathway), which possesses a phosphotyrosinebinding (PTB) domain, a SH2 domain, and a SH3 domain (74).Both PTB and SH2 domains bind phosphotyrosine-containingpolypeptides (75, 76). Shc is also a substrate for the EGFR, andupon tyrosine phosphorylation binds to the SH2 domain of Grb2(77). Thus, activation of EGFRs results in the association of Grb2to the EGFR, which also results in the recruitment of SOS, whichbinds to the SH3 domains of Grb2 (78) (Fig. 1). SOS stimulatesthe exchange of GDP bound to Ras for GTP (78) and initiates aprotein kinase cascade that includes, sequentially, a MAPK ki-nase kinase (MAPKKK), which phosphorylates and activates aMAPK kinase (MAPKK), which in turn phosphorylates and acti-vates a MAPK. MAPKs then phosphorylate and regulate the ac-tivity of key enzymes and nuclear proteins, which can ultimatelyregulate the expression of genes essential for cell proliferation(60). A-Raf, B-Raf, and Raf-1 are examples of MAPKKKs;MEK1 and MEK2 are examples of MAPKKs.

Signaling by G Protein-Coupled Receptors to theMAPK PathwayThe activation of MAPKs in response to agonists acting on GPCRsis well documented. These agonists include a variety of GPCR lig-ands of very diverse structure, including polypeptides, such asbombesin, endothelin-1, somatostatin, interleukin-8, luteinizinghormone-releasing hormone (LHRH), thyrotropin-releasing hor-

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mone (TRH), formyl-methionyl-leucyl-phenylalanine (fMLP) pep-tide, C5a peptide, oxytocin, and angiotensin II; lipid mediators,such as thromboxane A2, prostaglandin F2α, platelet-activatingfactor (PAF), sphingosine-1-phosphate, and LPA; and bioactiveamines, such as noradrenaline and serotonin. These agonists effec-tively activate MAPKs upon stimulation of appropriate cell types(79, 80). However, the nature of the biochemical routes linkingGPCRs to MAPK remains the subject of intense investigation.

Both Gi- and Gq-coupled receptors can potently stimulateMAPK activation. However, expression of activated mutants ofGq and Gi in most cases does not result in the activation ofMAPKs (81-83). Furthermore, although an activated form ofGαi2, the gip2 oncogene, induces MAPK activation when ex-pressed in rodent fibroblasts (84), this stimulation is pertussistoxin-insensitive, suggesting an indirect mechanism of activationfor MAPK, which likely results from the acquisition of additionalchanges in these cells during the process of cellular transforma-tion (84). Why the expression of activated G protein α subunitsoften fails to mimic receptor-mediated stimulation of MAPK isstill unclear, but these observations and findings of an importantfunction for free βγ subunits in signal transmission (13) prompted

several laboratories to ex-plore whether βγ het-erodimers participate in sig-naling to the MAPK path-way. Indeed, overexpressionof βγ subunits was found tobe sufficient to stimulateMAPKs (81, 82). Further-more, stimulation of MAPKactivity by coexpressed βγdimers did not require PKCactivation, but involved theactivation of Ras (82, 85).Taken together, these find-ings suggested that signalingfrom GPCRs to MAPK in-volves βγ heterodimers act-ing on a Ras-dependent path-way, thus providing the firstindication that βγ subunitslink G proteins to small GT-Pases of the Ras superfamily.Furthermore, these findingsestablished that the signaling

pathway linking GPCRs toMAPK converges with that used by

receptor tyrosine kinases at the level ofRas (Fig. 1).A role for nonreceptor tyrosine kinases.

The observation that genistein, a relatively non-specific tyrosine kinase inhibitor, diminishes the

activation of MAPK by LPA provided the first indica-tion that tyrosine kinases have a role in the activation of

MAPKs by GPCRs (86). Soon after, it was shown that stim-ulation of GPCRs could promote the rapid phosphorylation ofShc on tyrosine residues and the consequent formation of Shc-Grb2 complexes (87, 88). Several nonreceptor tyrosine kinaseshave been proposed to initiate this response. For example, Srcor Src-like kinases have been found to mediate the phosphory-lation of Shc by GPCRs and βγ subunits (89), and inhibitors ofSrc-like kinases diminish the activation of MAPK by Gq- andGi-coupled receptors (90). However, how Src is activated in re-sponse to GPCR agonists is not fully understood. Interestingly,studies on the mechanism by which β-adrenergic receptorsstimulate MAPK may provide some clues (91). In this case,binding of β-adrenergic agonists to their cognate G protein-linked receptors results in the rapid phosphorylation of the ago-nist-occupied receptor by the G protein-coupled receptor kinase(GRK) and the consequent recruitment of a protein known asarrestin to the GRK-phosphorylated receptor. In turn, arrestinfunctions as an adapter protein, binding both Src and the ago-nist-occupied receptor, thereby promoting the recruitment of theSrc kinase to the membrane and its consequent activation (91).The subsequent internalization of β-adrenergic receptor-ar-restin-Src complexes and their accumulation into clathrin-coat-ed vesicles appear to be required for the activation of MAPK(91). The process of receptor internalization (and signaling toMAPK) involves the phosphorylation of dynamin I, a GTPasethat participates in the formation of clathrin-coated vesicles, bySrc (92). However, receptor endocytosis may not be necessaryfor the stimulation of MAPK by other GPCRs (93, 94). Pertur-

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Fig. 1. Multiple pathwayslinking G protein-coupled re-ceptors to the MAPKs. Biochemi-cal routes stimulating Ras, as wellas novel pathways acting on Rap1,are depicted (see text for details). Ar-rows, positive stimulation; blockedlines, inhibition.


bation of the clathrin-mediated endocytic pathway may affectthe ability to stimulate MAPK even when provoked by recep-tors that do not undergo endocytosis (95). Furthermore, domi-nant negative mutants of dynamin I may prevent the activationof MAPKs by additional mechanisms that are independent ofits ability to inhibit the formation of endocytic vesicle andGPCR internalization (96, 97). Thus, components of the endo-cytic machinery may play multiple roles in signal transmission.Their relative contribution to the activation of MAPKs byGPCRs and other cell surface receptors in each cellular systemwill certainly be the focus of further exploration.

Signaling from GPCRs to MAPK may not require the activa-tion of Src and the phosphorylation of Shc and dynamin I incertain cell lines (98, 99). For example, in some cellular sys-tems the tyrosine phosphorylation of another dynamin isoform,dynamin II, by a yet to be identified kinase may play a key rolein signaling from LPA receptors to MAPK (100). Furthermore,in lymphoid cells deletion of Csk (COOH-terminal Src familykinase) or the Src-like tyrosine kinase Lyn prevents the activa-tion of MAPKs by Gq- but not Gi-coupled receptors; however,in cells lacking the Bruton’s tyrosine kinase (Btk), only the Gi-dependent response is affected (101, 102). In contrast, in lym-phoid cells lacking the nonreceptor tyrosine kinase Syk, bothGq- and Gi-dependent responses were inhibited (101, 102). AsBtk and Syk exhibit a very restricted tissue distribution, theseobservations suggest that GPCRs might use highly specializedpathways in certain cell types. Indeed, whereas activation ofSyk by GPCRs has been reported in some cells (103), this ki-nase or the highly related kinase Zap70 are activated in mastcells and T cells, respectively, after stimulation of multimericcell surface receptors but not in response to GPCRs (104, 105).

Another nonreceptor tyrosine kinase implicated in the acti-vation of MAPK by GPCRs is Pyk2, a Ca2+- and PKC-depen-dent kinase, which can mediate the activation of MAPK by bothGq- and Gi-coupled receptors when [Ca2+]i is increased as aconsequence of PLC activation by βγ subunits or by Gαq (106-108). This appears to be particularly important in certain fre-quently studied neuronal-like cell lines, such as PC12 cells(106, 107, 109). Pyk2 is closely related to FAK (focal adhesionkinase), which is part of the focal adhesion complex. In thiscase, integrin engagement leads to the recruitment of a multi-meric intracellular complex that includes FAK and Src, andleads to the phosphorylation of FAK in multiple sites, therebycreating docking sites for many adapter proteins, includingGrb2, paxillin, p130CAS, and Src (110, 111). FAK was alsoshown to be activated by Gq- and Gi-coupled receptors (112-114), and thus may represent an additional candidate to mediatein GPCR signaling to MAPK (109).

A role for receptor tyrosine kinases. Certain growth factorreceptor tyrosine kinases have been shown also to participate inGPCR signaling. For example, PDGF receptors (PDGFR) andEGF receptors (EGFR) become tyrosine-phosphorylated uponGPCR stimulation (115-118) and participate in MAPK activa-tion by GPCRs. In this case, the phosphorylation of tyrosine ki-nase receptors would be expected to provide docking sites forthe recruitment of signaling complexes leading to Ras activa-tion. How these growth factor receptors are activated in re-sponse to GPCR stimulation is still unclear. One such possibili-ty involves the activation of Src and the phosphorylation ofEGFR by this nonreceptor tyrosine kinase (119). Another inter-esting possibility is that activation of GPCRs may lead to the

proteolytic cleavage of latent agonist for the receptor tyrosinekinase. For example, transforming growth factor-α (TGF-α),amphiregulin, heparin-binding-epidermal growth factor (HB-EGF), and others are produced as inactive, membrane-spanningprohormones that are processed and released through regulatedproteolysis (120) by metalloproteases, most likely of theADAM (a disintegrin-like and metalloprotease domain-contain-ing protein) family (121) (Fig. 1). The activation of such pro-hormones might stimulate receptor tyrosine kinases in an au-tocrine manner and also act on receptor tyrosine kinases ex-pressed by neighboring cells. Such a mechanism of “inside-out”communication between GPCRs has been recently demonstrat-ed (122). In this case, EGFR transactivation upon GPCR stimu-lation was shown to involve the cleavage of pro HB-EGF by ametalloproteinase activity that is rapidly induced upon GPCR-ligand interaction. Activation of MAPK itself can result in theshedding of HB-EGF (123). Thus, whether the activation ofEGFR is the cause or the consequence of MAPK stimulation byGPCRs warrants further investigation.

We conclude that GPCRs make extensive use of nonreceptorand receptor tyrosine kinases that, upon autophosphorylation orphosphorylation of membrane-bound substrates, create dockingsites where proteins containing phosphotyrosine-binding do-mains (PTB and SH2 domains) can assemble in multimeric-sig-naling complexes leading to the recruitment of SOS and theconsequent activation of Ras. It is not, therefore, surprising thatthe nature of the endogenous tyrosine kinase(s) used in this bio-chemical route is frequently dictated by their availability or theavailability of yet to be identified accessory molecules in eachcellular setting. For example, detailed analysis of some of thecandidate tyrosine kinases (Src, Pyk2, and EGFR) revealedtheir distinct contribution in signaling to MAPK in a set ofcommonly used cell lines (109). In this case, EGFR and Pyk2were found to be critical for MAPK activation by Gq- and Gi-coupled receptors in Rat 1 and PC12 cells, respectively, where-as both played partial roles in HEK 293 cells; and Src activationwas important in all cases (109). These data illustrate how therelative contribution of tyrosine kinases and their downstreamtargets can be strictly tissue and cell type specific. This conceptemerges from what, at first glance, would appear to represent acollection of seemingly conflicting reports (124).

Signaling through Ras-GRF. Although the complexity of tyro-sine kinase pathways activated by GPCRs appears to be perplex-ing, in-depth analysis of signaling molecules used by GPCRs tostimulate MAPK in a variety of cellular systems is now providingthe first glimpse of the existence of additional biochemical routesthat might cooperate with, or even act instead of, those describedabove to connect GPCRs to MAPKs. Regarding the pathwaylinking GPCRs and Gβγ to Ras and MAPKs, additionalmolecules include the protein tyrosine phosphatase SH-PTP1(125), Ras-guanine-nucleotide releasing factor (Ras-GRF) (126),kinase suppressor of Ras-1 (KSR-1) (127), and PI3Kγ (128).Ras-GRF is a distinct Ras-guanine-nucleotide exchange factorexpressed in neuronal cells, and its activity is enhanced in re-sponse to GPCR stimulation, or upon coexpression of Gβγ, by amechanism involving calcium and the direct phosphorylation ofRas-GRF by a yet to be identified kinase (126). A Ras-GRF-re-lated protein, Ras-GRF2, is also expressed in nonneuronal-de-rived tissues (129) and might represent a good candidate to medi-ate the GPCR-initiated signaling in nonneuronal cells.

Signaling through PI 3-kinase. The early observation that

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wortmannin, a PI3K inhibitor, diminishes MAPK activation byGPCRs (130) provided the first indication of a role for this lipidkinase in GPCR signaling to MAPK. A likely candidate to sig-nal from GPCR to MAPK was the PI3K isoform PI3Kγ (131),which does not bind the p85 PI3K noncatalytic subunit and isnot stimulated by tyrosine phosphorylation, but is activated up-on physical interaction with Gβγ complexes (131). PI3Kγ wasfound to act downstream from Gβγ and upstream of Src-like ki-nases and Shc, Grb2, SOS, and Ras, suggesting a potentialmechanism by which G proteins can regulate nonreceptor tyro-sine kinases and, in turn, control the MAPK pathway (128). In-terestingly, recent evidence suggests that the ability to stimulateMAPK by PI3Kγ can be dissociated from its lipid-kinase activi-ty (132), which is consistent with the observation that PI3Kαcannot replace PI3Kγ function in the MAPK pathway, althoughPI3Kα does increase endogenous levels of phosphatidylinositoltrisphosphate (PIP3) (128). The ability to act as a protein kinaseor as a scaffold for signaling molecules may explain the uniqueproperties of PI3Kγ (133). Because this PI3K isoform exhibits arestricted tissue distribution, additional PI3K isoforms may par-ticipate in signaling through GPCRs, and accumulating evi-dence suggests that PI3Kβ can also be stimulated by GPCRs(134). The molecular dissection of the contribution of PI3Ksand their enzymatic products in signaling MAPK activation byGPCRs warrants further investigation.

Signaling through phospholipase C. The fact that Gq-cou-pled receptors can potently stimulate PLC-β suggests that theconsequent stimulation of PKC may contribute to MAPK acti-vation by this class of GPCRs. However, this is not a straight-forward issue, as the mechanism of activation of MAPK byPKC is still unclear. PKCs can phosphorylate Raf (135, 136),but this alone does not result in an increased ability of Raf tophosphorylate MEK (MAPK or ERK kinase) (137). On the oth-er hand, although DAG can stimulate the Ras exchange factor,Ras-GRP, expressed in lymphocytes (138, 139), PKC activationdoes not enhance the accumulation of Ras-GTP in the vast ma-jority of cell types (140). Nonetheless, PKC stimulation acti-vates MAPK in a Ras-dependent manner in several cellular set-tings (141-143). Furthermore, recently available data suggestthat PKCs stimulate Ras and MAPKs by a mechanism distinctfrom those known to mediate Ras activation by receptor tyro-sine kinases (144). Evidence for an increasing number ofmolecules that aid in the fine tuning of the Ras-Raf signalingsystem comes from the facts that (i) Ras alone is not sufficientto activate Raf fully in vitro (137), and (ii) Ras does not fullyactivate Raf when both are expressed in Sf 9 cells (145). Thus,PKCs might act directly on Raf to facilitate full activation ofRaf upon binding to Ras (143), as well as acting on themolecules involved in fine tuning the Ras-Raf interaction.

Gq-coupled receptors can activate MAPK in a PKC-dependent(142, 146), fully PKC-independent (147, 148), or partially PKC-dependent (149) manner. The requirement for PKC for MAPKactivation might be highly dependent on the level of receptor ex-pression and on the diversity of PKC isoforms and signalingmolecules expressed in each cell type. Another possible explana-tion for this discrepancy lies in the observation that PLC-β, adownstream target for Gαq, can also exert a potent GTPase-acti-vating protein (GAP) activity on G proteins of the Gq family(150). At low receptor density or high PLC-β expression, GPCRstimulation would activate PLC-β through Gαq; the PLC-β wouldenhance the rate of GTP hydrolysis by Gαq, which would limit

the availibility of the βγ complex because of the reassociation ofGDP-bound Gαq with βγ complexes and allow only the PKC-de-pendent pathway to proceed. Thus, the PLC-β to PKC pathwaywould be active, but the βγ to Ras pathway would have insuffi-cient time to occur under those conditions, resulting in only thePKC-dependent pathway of MAPK activation.

Signaling through Rap. Another mechanism by which Gprotein α subunits may also activate MAPK involves the regula-tion of the Ras-like GTPase Rap1. This molecule, originallyidentified as a biological antagonist of Ras (151), has receivedmuch attention, because it can block MAPK activation by com-peting with Ras for binding to Raf-1 and A-Raf, or it can stimu-late MAPK through the activation of B-Raf (152, 153) (Fig. 1).A guanine-nucleotide exchange factor for Rap1, named Epac,has been identified and shown to be activated by cAMP in aPKA (cAMP-dependent protein kinase)-independent manner(154). Gαi, Gαz, and Gαo can bind directly to Rap1 GAPs (155-157). In the case of Gαo, its GDP-bound inactive form bindsRap1GAP, thus preventing its activity and enhancing the accu-mulation of Rap1-GTP, which can stimulate MAPK in certainneuronal-derived cells (155). Gαi, on the other hand, binds anNH2-terminal extended form of Rap1GAP, termed Rap1GAPII,resulting in its enhanced activity and the consequent decreasedlevels of Rap1-GTP (156). In the latter case, this effect of Gαiwas shown to contribute to the Gβγ-dependent activation ofMAPK by decreasing the levels of Rap1-GTP that was acting asa Ras competitor. The activated form of Gαz also bindsRap1GAP, and forms stable molecular complexes with Rap1(157), suggesting that Rap1 can participate in the regulation ofMAPK by Gαz. These findings exemplify the complexity of theregulatory pathways used by GPCRs; these pathways act onmolecules whose final function, inhibitory or stimulatory, onMAPK may be highly dependent on the cell type or tissue un-der investigation (Fig. 1).

Signaling by G Protein-Coupled Receptors to OtherMAPK-Related PathwaysActivation of MAPKs frequently results in their rapid transloca-tion to the nucleus, where they phosphorylate and regulate thefunctional activity of various transcription factors (158). BecauseGPCRs and tyrosine kinases converge at the level of Ras to acti-vate MAPKs, activation of both classes of cell surface receptorswas expected to elicit a similar response by nuclear transcriptionfactors. However, this was found not to be the case (159).

Signaling through the JNK pathway. Expression of c-jun (atranscription factor proto-oncogene) in NIH 3T3 cells was in-duced only by GPCR stimulation but not by receptor tyrosinekinases, and this response did not correlate with MAPK activa-tion (159), suggesting that GPCRs control a distinct biochemi-cal route regulating gene expression. A family of enzymesstructurally related to MAPKs, termed c-Jun NH2-terminal ki-nases (JNKs) (160) or stress-activated protein kinases (SAPKs)(161), selectively phosphorylate c-Jun, thereby enhancing itstranscriptional activity. Consistent with this finding and the ear-lier observation that c-Jun enhances c-jun expression (162), itwas observed that GPCRs but not tyrosine kinase receptors po-tently activate JNK in murine fibroblasts (159).

These findings indicated that the GPCR signaling routesmight diverge at the level of JNK from those used by tyrosine ki-nase receptors. An unexpected prediction from these studies wasthat a distinct set of upstream signaling molecules might regulate

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JNK and MAPK. Two findings prompted several labs to examinewhether the Rho family of GTPases participates in GPCR signal-ing to the JNK pathway: (i) signaling from GPCRs to Jun wasMAPK independent, and (ii) Rac and Cdc42, two small GTPasesof the Rho family, could control the activity of PAK1, a serine-threonine kinase, in a manner similar to that of Ras acting on Raf(163). Indeed, Rac1 and Cdc42 were found to initiate an indepen-dent kinase cascade regulating JNK activity (164, 165). Othercomponents of this pathway include several MAPKKKs: MAPKor ERK kinase kinases (MEKK1, MEKK2, MEKK3, MEKK4,and MEKK5), ASK1 (apoptosis-stimulated kinase 1), MLK3(mixed-lineage kinase 3), TPL2 (tumor progression locus 2, alsoknown as Cot), TAK (TGF-β-activated kinase), MUK (MAPKupstream kinase), GCK (germinal center kinase), and PAK,which can all contribute to activation of JNKs, apparentlythrough two MAPKKKs, MKK4 (also called Sek or JNKK1) andMKK7 (166-169). So far, however, there is limited informationregarding the precise architecture of the signaling pathways inwhich each of these kinases act. Substrates for JNK include tran-scription factors of the c-Jun family, as well as other transcriptionfactors, including Elk-1 and Elk-2, Sap-1 (serum response factoraccessory protein-1), NFAT4 (nuclear factor of activated T cells4), and ATF2 (activating transcription factor 2) (169).

How GPCRs are linked to JNK is still unclear. Detailed ex-amination of the pathway linking GPCRs to JNK provided evi-dence that free βγ dimers (170) and Gα12 or Gα13 (171-174)convey signals from this class of receptors to JNK. Furthermore,in all cases JNK activation was blocked in cells expressing dom-inant negative mutants of Rac1 or Cdc42 or both, suggesting arole for these GTPases in the activation of JNK by GPCRs. Reg-ulation of GEFs may link βγ and Gα12 or Gα13 to Rac1 orCdc42. Cumulative work of several laboratories, however, sug-gests that a number of molecules might play this role: (i) Ras-GRF1, which upon βγ binding, can act as a Ras GEF and can in-duce guanine-nucleotide exchange on Rac1 through its Dbl ho-mology domain (DH domain) (175); (ii) Ras-GRF2, which ishighly related to Ras-GRF1 and can transmit calcium-initiatedsignals to Ras and Rac1 (176); (iii) Dbl, a GEF for Cdc42 andRho that binds βγ subunits, although that does not result in ademonstrable increase in its GEF activity (177); and (iv) Tiam1,a Rac-specific GEF that is phosphorylated on threonine residuesin a PKC-dependent manner in cells stimulated with LPA,bombesin, or bradykinin. Efforts to knock out the genes forthese GEFs should provide valuable information regarding theirrelative contribution in signaling to small GTPases by GPCRs.

Recent studies suggest that βγ subunits activate JNK prefer-entially through MKK4 by stimulating Rho and Cdc42, and to alesser extent through MKK7 acting downstream from Rac1(178), whereas Gα12 uses Src-like kinases to stimulate JNK(179). In line with the latter observations, it has been recentlyshown that Pyk2 and FAK can also participate in signaling toJNK through the recruitment of an adapter protein Crk (180,181) or paxillin (182), both of which are expected to activateGEFs for small GTPases of the Rho family (183, 184). Al-though a preliminary model of how GPCRs can stimulate smallGTPases of the Rho family to activate JNK is depicted in Fig.2, this model is likely to be redefined in the foreseeable future,when more biological and biochemical data become available.

Signaling through the p38 and ERK5 pathways. Much less isknown about the molecular mechanisms connecting GPCRs toeach member of the p38 family of kinases related to MAPKs and

to ERK5. In particular, the family of p38 kinases has grownrapidly over the past few years. To date, four p38 kinases havebeen described and named p38α (or CSBP-1), p38β, p38γ (alsoknown as SAPK3 or ERK6), and p38δ (or SAPK4), some ofwhich also have splice variants (169). This family of proteins ex-hibits a common Thr-Gly-Tyr phosphorylation motif and shows apattern of activation by stress insults and cell surface receptorsvery reminiscent of that displayed by JNKs (185). Although theupstream activators for these kinases are not yet well defined, po-tential p38 MAPKKKs include ASK1 and TAK1 (169). MKK6seems to be a very general stimulator for p38s, whereas MKK3and MKK4 preferentially phosphorylate p38α (186). The specifictranscription factors that can be regulated by p38s include cAMPresponsive element binding protein (CREB), ATF1 (activatingtranscription factor 1), ATF2, Max, CHOP (C/EBP homologousprotein), and MEF2C (169). In addition, these MAPKs can alsotrigger the activation of other serine-threonine kinases such asMnk1 (MAPK-interacting kinase 1) and Mnk2 and MAPKAPKs(MAPK-activated protein kinases) (169).

Although the activation of p38 by GPCRs has been docu-mented in primary cells, immortalized cell lines, perfused or-gans, and even after in vivo manipulations (186-196), there isvery limited knowledge on the mechanism of activation of thisparticular family of MAPKs. The p38α enzyme is activated byboth Gαq and Gβγ (197), and a role for the tyrosine kinase Btkin Gq-mediated stimulation has been suggested from observa-tions in lymphocytic cells (198). In addition, it has been shownthat Gα12 and Gα13 can activate the putative MAPKKK, ASK1,although the activation of p38s was not examined (199). Elec-trophysiological studies have implicated p38s downstream fromGα13 (200). Gq-coupled receptors can activate not only p38αand p38β, but also the most distantly related p38γ and p38δ en-zymes (201). We can conclude that how GPCRs activate p38MAPKs is still far from being fully understood, thus warrantingfurther investigation.

A MAPK distantly related to p42MAPK and p44MAPK has re-cently been characterized and termed BMK1 (big mitogen-acti-vated protein kinase 1) or ERK5. It contains a Thr-Glu-Tyr mo-tif in its activation loop, similar to that of MAPKs (202). Thiskinase is larger than any other known MAPK (~80 kD) and isselectively activated by MEK5 (203). ERK5 can be stimulatedby oxidative stress, and may also play a role in early gene ex-pression triggered by serum by directly phosphorylating thetranscription factors MEF2C (203) or c-Myc (204). Interesting-ly, the search for the mechanism whereby GPCRs regulate c-junexpression revealed a key role for transcription factors of theMEF2 family in the transcriptional response from the c-jun pro-moter (205). Furthermore, it has been recently observed thatGPCRs can potently activate ERK5, and that this MAPK linksGPCRs to the transcriptional activation of MEF2A andMEF2C, and to c-jun expression (201). Current work suggeststhat GPCRs activate ERK5 through Gαq and Gα13 by a still un-known pathway (206).

Scaffolding Proteins Provide Signal Specificity: ALesson from YeastGiven the increasing complexity of the biochemical routes regu-lating MAPKs, it is difficult to envision how the selectivity andspecificity in signal transduction can be achieved. However, therecent identification of molecules capable of binding compo-nents of MAPK cascades suggests a potential role for scaffold-

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ing proteins as the physical basis for the specificity of signaltransmission in mammalian cells. This is highly reminiscent ofthat in the yeast MAPK cascades, where specificity is achievedby the assembling of hierarchical protein modules. For example,in Saccharomyces cerevisiae, mating factors (α or a) activate Gprotein-linked pheromone receptors which, in turn, induce thedissociation of a G protein into α (GPA1) and βγ (Ste4 andSte18) subunits (70). Free βγ dimers then activate a serine-threo-nine kinase, Ste20, a process that involves a Cdc42 GEF, Cdc24(207), and Cdc42 (208), thereby stimulating the activity of a lin-ear cascade of kinases including, sequentially, Ste11 (a yeastMAPKKK) and Ste7 (a yeast MAPKK), which phosphorylateand activate the yeast MAPK homologs Fus3 and Kss1. Theconnection of Gβγ to small GTPases represents a biologicallyrelevant example of a pathway extraordinarily conservedthroughout evolution. Available data indicate that additionalmolecules might be also involved to organize these kinase cas-cades, including a protein designated Ste5, which binds yeast βγand plays a role as a platform or scaffold recruiting Ste11, Ste7,and Kss1 and Fus3 (209, 210). The Ste5, βγ, Ste11, Ste7, Kss1,Fus3 complex represents a prototypic MAPKKK-MAPKK-MAPK module. For another yeast MAPK pathway, the p38 os-mosensor system, the scaffolding capability lies within Pbs2, a

MAPKK that is able to link together the osmosensitive receptor,the most upstream MAPKKK, Ste11p, and the p38 MAPK,HOG1p (high-osmolarity glycerol response kinase) (211).

In the mammalian MAPK pathway, Ksr (212) and MP1(213) can bind both MAPK and its upstream activator MEK1,and have been proposed to act as scaffold polypeptides in thiscascade. For the JNK pathway, two proteins, JIP (JNK inhibito-ry protein) and JSAP (a novel JNK inhibitory protein), performthis function, bridging together MLKs, MKK7, and JNK (214,215), and MEKK1, SEK (stress-activated protein kinase kinaseor ERK kinase), and JNK (216), respectively. We can expectthat additional scaffolding molecules will soon be identified,thus providing the molecular basis for how the selectivity andspecificity can be maintained when transmitting signals fromthe membrane to the nucleus.

Conclusion: G Protein-Coupled Receptors andMAPKs: Signal Transduction or Signal Integration?Over the last few years we have begun to appreciate the complexi-ty of the signaling pathways by which cell surface GPCRs trans-mit signals to the nucleus. In the simplest possible model, GPCRsactivate G proteins, thereby causing their dissociation into α-GTPand βγ subunits. Each G protein subunit can then activate a specif-

Fig. 2. Novel signalingpathways link G protein-coupled receptors to thenucleus. Molecules linkingG protein-coupled receptorsto MAPK, JNK, p38s, andERK5 are depicted. See Fig. 1for a more detailed description ofmolecules acting on the MAPK path-way. Transcription factors binding tothe c-fos and c-jun promoters are usedas examples of gene expression regu-lation through these MAPK signalingpathways (see text for details). Arrows,positive stimulation; blocked lines, inhi-bition; dashed lines, interactions notwell established.

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ic set of small GTP-binding proteins of the Ras and Rho familieswhich, in turn, control the activity of parallel kinase cascades, re-sulting in the phosphorylation of key nuclear transcription factors.

However, the stimulation of GPCRs leads to the activationof a number of second messenger-generating systems and caneven affect the activity of other cell surface molecules, includ-ing integrins and growth factor tyrosine kinase receptors. Thiscan have a remarkable impact on signal transmission, as thesereceptors and their associated intracellular molecules can pro-vide docking sites for the assembly of distinct multimeric sig-naling complexes. Furthermore, classical second messengerscan also affect the duration and even the final biochemical orbiological response. For example, changes in intracellular[Ca2+] can activate the exchange activity of Ras-GRF (217-219), activate Pyk2 (106-108, 220) and a novel calcium-calmodulin-dependent PI3K (221), and can promote the de-phosphorylation of nuclear transcription factors through thecalmodulin-dependent phosphatase, calcineurin (222, 223).Thus, even limited changes in [Ca2+]i, caused either by Gq orby Gβγ subunits, might exert a profound effect on many as-pects of signal transmission. Similarly, many GPCRs can stim-ulate adenylyl cyclases (even if not coupled to Gs), and the ac-cumulation of cAMP can either inhibit or stimulate MAPKs,depending on the repertoire of signal transducing moleculesexpressed in each particular cellular setting (81, 224-229).Thus, changes in the intracellular concentrations of cAMP areexpected to initiate a complex series of events that influenceMAPK cascades.

While evidence of a possible cross-talk among MAPK cas-cades is already emerging (230), we are now also beginning tounderstand the complex interactions among molecules that bindto regulatory elements within the promoter region of growth-regulating genes. Activation of the Rac- or Cdc42-JNK pathwayby GPCRs through Gα12 or Gα13 and βγ dimers would be ex-pected to regulate the c-jun promoter through an AP1 (activat-ing protein 1) or ATF2 sites (231); however, the data suggestthat JNK, p38α, p38γ, and ERK5 all cooperate through the acti-vation of the AP1 and ATF2 sites and an adjacent MEF2 site toactivate the c-jun promoter (201). Thus, we can conclude that inthis case GPCRs control the expression of c-jun through severalindependent MAPK cascades that converge in the nucleus tostimulate transcription factors acting on its promoter (Fig. 2).Similarly, the c-fos (a transcription factor proto-oncogene) pro-moter contains a serum response element (SRE) that can bestimulated by either MAPK or JNK, which stimulates theternary complex factor (TCF) p62TCF (167, 232-234). Thep62TCF protein binds the SRE in cooperation with the serum re-sponse factor (SRF). SRF can also be stimulated by a yet to beidentified pathway initiated by Rho, thereby affecting the ex-pression of genes containing SRE elements within their promot-er region (235). However, the c-fos promoter also containsmany other sites adjacent to the SRE (236-238), suggesting thatthe regulation of the c-fos promoter may be more complex thananticipated, involving multiple interactions among its bindingproteins. Taken together, the emerging picture is that activationor repression of gene expression results from the activity of anumber of interlinked regulatory molecules and pathways,rather than from a single linear series of signaling events.

We conclude that the activation of GPCRs can affect the ac-tivity of a highly interconnected network of cytoplasmic signal-ing pathways. Depending on the G protein-coupling specificity

and the repertoire of available signaling molecules, this can leadto a temporally distinct pattern of activation of each member ofthe MAPK superfamily. Ultimately, these kinases can modifykey cytoplasmic molecules as well as translocate to the nucleuswhere they phosphorylate nuclear proteins, including transcrip-tion factors, thereby affecting an intricate balance of regulatorymolecules controlling gene expression. The recent explosion ofknowledge on the basic mechanisms regulating signal transmis-sion now affords the unique opportunity to begin unraveling theintricacies of how these signals are integrated in space and timeto elicit a final biological response. This knowledge may alsohelp us to understand how subtle perturbation of this signalingnetwork can result in pathological situations, thus providinggolden opportunities to identify novel molecular targets forpharmacological intervention in a variety of diseases.

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