The Pathways Connecting G Protein-coupled Receptors to the Nucleus through Divergent Mitogen-activated Protein Kinase Cascades

Abstract

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), 1The 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. currently comprising more than 1000 members, exhibits a common structural motif consisting of seven membrane-spanning regions (1Dohlman H.G. Caron M.G. Lefkowitz R.J. Biochemistry. 1987; 26: 2657-2664Crossref PubMed Scopus (419) Google Scholar) (Fig. 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 α subunit and apparently the dissociation of the βγ heterodimers. In turn, GTP-bound G protein α subunits or βγ 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 α subunits have been molecularly cloned and are divided into four families based upon sequence similarity: αs, αi, αq, and α12. Similarly, eleven G protein γ subunits and five G protein β 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. Proliferative signaling has generally been associated with polypeptide growth factor receptors that possess an intrinsic protein tyrosine kinase activity (2Yarden Y. Escobedo J.A. Kuang W.J. Yang-Feng T.L. Daniel T.O. Tremble P.M. Chen E.Y. Ando M.E. Harkins R.N. Francke U. Fried V.A. Ullrich A. Williams L.T. Nature. 1986; 323: 226-232Crossref PubMed Scopus (769) Google Scholar). A variety of oncogenes have been found to code for mutated forms of these receptors (3Sherr C.J. Rettenmier C.W. Sacca R. Roussel M.F. Look A.T. Stanley E.R. Cell. 1985; 41: 665-676Abstract Full Text PDF PubMed Scopus (982) Google Scholar) and their ligands (4Doolittle R.F. Hunkapiller M.W. Hood L.E. Devare S.G. Robbins K.C. Aaronson S.A. Antoniades H.N. Science. 1983; 221: 275-277Crossref PubMed Scopus (983) Google Scholar) or for molecules that participate in their growth-promoting pathways (5Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4611) Google Scholar). On the other hand, GPCRs have been traditionally linked to tissue-specific, fully differentiated cell functions (1Dohlman H.G. Caron M.G. Lefkowitz R.J. Biochemistry. 1987; 26: 2657-2664Crossref PubMed Scopus (419) Google Scholar). However, GPCRs are also expressed in proliferating cells, and they have been implicated in embryogenesis, tissue regeneration, and growth stimulation (reviewed in Ref. 6Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar). In this regard, many ligands acting via GPCRs, including thrombin, bombesin, bradykinin, substance P, endothelin, serotonin, acetylcholine, gastrin, prostaglandin F2α, and lysophosphatidic acid, are known to elicit a mitogenic response in a variety of cell types (reviewed in Refs. 6Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar and 7van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar), and recent gene knock-out studies indicate that certain GPCRs are essential for cell growth under physiological conditions (8Nagata A. Ito M. Iwata N. Kuno J. Takano H. Minowa O. Chihara K. Matsui T. Noda T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11825-11830Crossref PubMed Scopus (219) Google Scholar). 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 (9Young D. Waitches G. Birchmeier C. Fasano O. Wigler M. Cell. 1986; 45: 711-719Abstract Full Text PDF PubMed Scopus (328) Google Scholar). Subsequently, serotonin 1C (10Julius D. Livelli T.J. Jessell T.M. Axel R. Science. 1989; 244: 1057-1062Crossref PubMed Scopus (287) Google Scholar), muscarinic m1, m3, and m5 (11Gutkind J.S. Novotny E.A. Brann M.R. Robbins K.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4703-4707Crossref PubMed Scopus (262) Google Scholar), and adrenergic α1 (12Allen L.F. Lefkowitz R.J. Caron M.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11354-11358Crossref PubMed Scopus (292) Google Scholar) 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 α subunits. In recent studies, constitutively active mutants of Gαi, Gαq, Gα0, Gα12, and Gα13 were shown to behave as transforming genes in a variety of cell types (reviewed in Ref. 13Dhanasekaran N. Heasley L.E. Johnson G.L. Endocr. Rev. 1995; 16: 259-270Crossref PubMed Scopus (178) Google Scholar). 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 Gαs results in hyperplasia of endocrine cells and has been found in human thyroid and pituitary tumors (reviewed in Ref. 13Dhanasekaran N. Heasley L.E. Johnson G.L. Endocr. Rev. 1995; 16: 259-270Crossref PubMed Scopus (178) Google Scholar) and in the McCune-Albright syndrome, a disease in which multiple endocrine glands exhibit autonomous hyperproliferation (14Weinstein L.S. Shenker A. Gejman P.V. Merino M.J. Friedman E. Spiegel A.M. N. Engl. J. Med. 1991; 325: 1688-1695Crossref PubMed Scopus (1399) Google Scholar). Interestingly, activated Gαs contributes significantly to hyperplasia only in tissues where cAMP stimulates proliferation, thus acting as an oncogene referred to as the gsp oncogene (15Landis C.A. Masters S.B. Spada A. Pace A.M. Bourne H.R. Vallar L. Nature. 1989; 340: 692-696Crossref PubMed Scopus (1225) Google Scholar). Activating mutations have also been identified for Gαi2, referred to as the gip2 oncogene, in a subset of ovarian sex cord stromal tumors and adrenal cortical tumors (16Lyons J. Landis C.A. Harsh G. Vallar L. Grunewald K. Feichtinger H. Duh Q.Y. Clark O.H. Kawasaki E. Bourne H.R. McCormick F. Science. 1990; 249: 655-659Crossref PubMed Scopus (927) Google Scholar). On the other hand, Gα12, referred as the gep oncogene (17Xu N. Voyno-Yasenetskaya T. Gutkind J.S. Biochem. Biophys. Res. Commun. 1994; 201: 603-609Crossref PubMed Scopus (88) Google Scholar,18Xu N. Bradley L. Ambdukar I. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6741-6745Crossref PubMed Scopus (174) Google Scholar), was isolated as a transforming gene from a soft tissue sarcoma-derived cell (19Chan A.M. Fleming T.P. McGovern E.S. Chedid M. Miki T. Aaronson S.A. Mol. Cell. Biol. 1993; 13: 762-768Crossref PubMed Scopus (145) Google Scholar), although its role in tumorigenesis remains unclear. Naturally occurring activated mutations in members of the Gαq 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 (20Parma J. Duprez L. Van Sande J. Cochaux P. Gervy C. Mockel J. Dumont J. Vassart G. Nature. 1993; 365: 649-651Crossref PubMed Scopus (851) Google Scholar) 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 (21Shenker A. Laue L. Kosugi S. Merendino Jr., J.J. Minegishi T. Cutler Jr., G.B. Nature. 1993; 365: 652-654Crossref PubMed Scopus (653) Google Scholar). 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 (22Cuttitta F. Carney D.N. Mulshine J. Moody T.W. Fedorko J. Fischler A. Minna J.D. Nature. 1985; 316: 823-826Crossref PubMed Scopus (1149) Google Scholar), colon adenomas and carcinomas (23Hoosein N.M. Kiener P.A. Curry R.C. Rovati L.C. McGilbra D.K. Brattain M.G. Cancer Res. 1988; 48: 7179-7183PubMed Google Scholar), and gastric hyperplasia and cancer (24Tahara E. J. Cancer Res. Clin. Oncol. 1990; 116: 121-131Crossref PubMed Scopus (185) Google Scholar). Sequences encoding functional GPCRs have also been found in the genome of transforming DNA viruses, including herpesvirus saimiri (25Nicholas J. Cameron K.R. Honess R.W. Nature. 1992; 355: 362-365Crossref PubMed Scopus (155) Google Scholar) and Kaposi's sarcoma-associated herpesvirus (26Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (575) Google Scholar). 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 (6Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (852) Google Scholar). However, recent studies using mutant tyrosine kinase receptors suggested that PIP2hydrolysis is neither necessary nor sufficient for mitogenesis (27Mohammadi M. Dionne C.A. Li W. Li N. Spivak T. Honegger A.M. Jaye M. Schlessinger J. Nature. 1992; 358: 681-684Crossref PubMed Scopus (336) Google Scholar,28Coughlin S.R. Escobedo J.A. Williams L.T. Science. 1989; 243: 1191-1194Crossref PubMed Scopus (288) Google Scholar). Furthermore, a number of GPCR agonists induce the PIP2turnover pathway but fail to stimulate growth when added alone to quiescent cells (29Moolenaar W.H. Cell Growth Differ. 1991; 2: 359-364PubMed Google Scholar). 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. 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 (30Pages G. Lenormand P. L'Allemain G. Chambard J.C. Meloche S. Pouyssegur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (925) Google Scholar). 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 (31Mansour S.J. Matten W.T. Hermann A.S. Candia J.M. Rong S. Fukasawa K. Vande Woude G.F. Ahn N.G. Science. 1994; 265: 966-970Crossref PubMed Scopus (1260) Google Scholar, 32Schlessinger J. Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (343) Google Scholar). 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 p21 ras, 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. 32Schlessinger J. Trends Biochem. Sci. 1993; 18: 273-275Abstract Full Text PDF PubMed Scopus (343) Google Scholar). MEKs ultimately phosphorylate p44 mapk and p42 mapk, 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. 33Davis R.J. J. Biol. Chem. 1993; 268: 14553-14556Abstract Full Text PDF PubMed Google Scholar). 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. 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 (34Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar, 35Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar, 36Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar). However, under identical experimental conditions, activated forms of Gαi2, Gαq, Gs, or G12 were not able to induce MAPK activation (35Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar). The failure of activated Gα subunits to mimic receptor stimulation of MAPK activity and the accumulating evidence supporting an active role for the Gβγ dimers in signal transmission (37Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar) prompted exploration of the role of βγ complexes in signaling to the MAPK pathway. This led to the observation that membrane-bound forms of βγ heterodimers can directly elicit signaling pathways leading to MAPK activation (35Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar) and prompted the search for molecules acting downstream of Gβγ in this biochemical route. In a variety of experimental conditions, it was shown that MAPK activation by βγ subunits required neither PLC-β nor PKC activation but was blocked by dominant interfering mutants of the GTP-binding protein Ras (34Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar, 35Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar) and that βγ subunits can induce the accumulation of Ras in the GTP-bound, active form (34Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar). Taken together, these findings indicated that signaling from GPCRs to MAPK involves βγ 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. 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 (38Hordijk P.L. Verlaan I. van Corven E.J. 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Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar) have recently obtained evidence that Src, or a Src-like kinase, links βγ 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 (42Ptasznik A. Traynor-Kaplan A. Bokoch G.M. J. Biol. Chem. 1995; 270: 19969-19973Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 43Wan Y. Kurosaki T. Huang X.Y. Nature. 1996; 380: 541-544Crossref PubMed Scopus (258) Google Scholar) and a novel Ca2+ and PKC-dependent protein tyrosine kinase, Pyk2 (44Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar, 45Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (879) Google Scholar, 46Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). 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 (47Gutkind J.S. Robbins K.C. Biochem. Biophys. Res. Commun. 1992; 188: 155-161Crossref PubMed Scopus (41) Google Scholar, 48Rankin S. Morii N. Narumiya S. Rozengurt E. FEBS Lett. 1994; 354: 315-319Crossref PubMed Scopus (131) Google Scholar) 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 (49Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 50Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1324) Google Scholar) 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 Gβγ and the Ras-MAPK pathway have been recently identified. They include the protein tyrosine phosphatase SH-PTP1 (51Gaits F. Li R.Y. Bigay J. Ragab A. Ragab-Thomas M.F. Chap H. J. Biol. Chem. 1996; 271: 20151-20155Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) 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 Gβγ (52Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar). In addition, several groups observed that wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, can diminish MAPK activation by GPCRs (see Ref. 53Hawes B.E. Luttrell L.M. van Biesen T. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 12133-12136Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), and a novel PI3K isotype, termed PI3Kγ, that is activated by Gβγ complexes (54Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. et al.Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar) was found to play a critical role in linking Gi-coupled receptors and Gβγ to the MAPK signaling pathway (55Lopez-Ilasaca M. Crespo P. Pelicci P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (629) Google Scholar). In this case, PI3Kγ was found to act downstream from Gβγ 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 (56Pace A.M. Faure M. Bourne H.R. Mol. Biol. Cell. 1995; 6: 1685-1695Crossref PubMed Scopus (65) Google Scholar,57Takahashi T. Kawahara Y. Okuda M. Ueno H. Takeshita A. Yokoyama M. J. Biol. Chem. 1997; 272: 16018-16022Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), 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 (56Pace A.M. Faure M. Bourne H.R. Mol. Biol. Cell. 1995; 6: 1685-1695Crossref PubMed Scopus (65) Google Scholar), 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 Gαi2, the gip2 oncogene, which can be observed in only a limited number of cell types (58Gupta S.K. Gallego C. Johnson G.L. Heasley L.E. J. Biol. Chem. 1992; 267: 7987-7990Abstract Full Text PDF PubMed Google Scholar). 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 (59Thomas S.M. DeMarco M. D'Arcangelo G. Halegoua S. Brugge J.S. Cell. 1992; 68: 1031-1040Abstract Full Text PDF PubMed Scopus (503) Google Scholar, 60Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). 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 (60Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), fully PKC-independent (61Charlesworth A. Rozengurt E. Oncogene. 1997; 14: 2323-2329Crossref PubMed Scopus (34) Google Scholar), or partially PKC-dependent (62Crespo P. Xu N. Daniotti J.L. Troppmair J. Rapp U.R. Gutkind J.S. J. Biol. Chem. 1994; 269: 21103-21109Abstract Full Text PDF PubMed Google Scholar). We can conclude that multiple molecules may mediate MAPK activation by GPCRs and Gβγ. The expression of some of these molecules follows a restricted tissue distribution (44Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1253) Google Scholar, 52Mattingly R.R. Macara I.G. Nature. 1996; 382: 268-272Crossref PubMed Scopus (156) Google Scholar, 54Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. et al.Science. 1995; 269: 690-693Crossref PubMed Scopus (642) Google Scholar), 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. 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 (64Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In particular, activation of GPCRs but not tyrosine kinase receptors for PDGF led, in NIH 3T3 cells, to a remarkable expression of c-jun (64Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). This response did not correlate with MAPK activation (64Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), 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) (65Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar) or stress-activated protein kinases (SAPKs) (66Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2414) Google Scholar), 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 (64Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), 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 (64Coso O.A. Chiariello M. Kalinec G. Kyriakis J.M. Woodgett J. Gutkind J.S. J. Biol. Chem. 1995; 270: 5620-5624Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 66Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2414) Google Scholar). Soon, it was found that the Ras-related small GTP-binding proteins Rac1 and Cdc42 initiate an independent kinase cascade regulating JNK activity (67Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1567) Google Scholar) and that Rac and Cdc42 are an integral part of the signaling route linking many cell surface receptors, including GPCRs, to JNK (68Coso O.A. Teramoto H. Simonds W.F. Gutkind J.S. J. Biol. Chem. 1996; 271: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). 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. 69Fanger G.R. Gerwins P. Widmann C. Jarpe M.B. Johnson G.L. Curr. Opin. Genet. Dev. 1997; 7: 67-74Crossref PubMed Scopus (297) Google Scholar). Further examination of the G protein subunits linking GPCRs to JNK provided evidence that free βγ dimers (68Coso O.A. Teramoto H. Simonds W.F. Gutkind J.S. J. Biol. Chem. 1996; 271: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and, in some cellular systems, Gα12 (70Prasad M.V. Dermott J.M. Heasley L.E. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: 18655-18659Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) 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 (71Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar), and a recent study suggests that both G12 and Gβγ might connect GPCRs to Rho and to serum response factor (72Fromm C. Coso O. Montaner S. Xu N. Gutkind J.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10098-10103Crossref PubMed Scopus (196) Google Scholar). Those molecules linking GPCRs and heterotrimeric G proteins to Rho remain undefined. The emerging picture from recent reports is that in mammalian cells, βγ 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 βγ 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 β 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 (73Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (865) Google Scholar). An additional scaffolding protein, Ste5, binds yeast βγ and several components of this MAPK cascade. In mammalian cells a number of sequentially acting molecules are required instead to connect GPCRs and Gβγ 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 Gαq in the visual system of the fruit fly (63Tsunoda S. Sierralta J. Sun Y. Bodner R. Suzuki E. Becker A. Socolich M. Zuker C.S. Nature. 1997; 388: 243-249Crossref PubMed Scopus (552) Google Scholar). Thus, it is conceivable that still unidentified scaffolding proteins might also participate in the mammalian pathway connecting heterotrimeric G proteins to MAPK cascades.