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34 Benjafield, A.V. and Morris, B.J. (2000) Association analyses of endothelial nitric oxide synthase gene polymorphisms in essential hypertension. Am. J. Hypertens. 13, 994998 35 Sigusch, H.H. et al. (2000) Lack of association between 27-bp repeat polymorphism in intron 4 of the endothelial nitric oxide synthase gene and the risk of coronary artery disease. Scand. J. Clin. Lab. Invest. 60, 229235 36 Nakagami, H. et al. (1999) Coronary artery disease and endothelial nitric oxide synthase and angiotensin-converting enzyme gene polymorphisms. J. Thromb. Thrombolysis 8, 191195 37 Odawara, M. et al. (1998) Endothelial nitric oxide synthase gene polymorphism and coronary heart disease in Japanese NIDDM. Diabetologia 41, 365366 38 Hibi, K. et al. (1998) Endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction. Hypertension 32, 521526 39 Ichihara, S. et al. (1998) Association of a polymorphism of the endothelial constitutive nitric oxide synthase gene with myocardial infarction in the Japanese population. Am. J. Cardiol. 81, 8386 40 Burg, M. et al. (1997) Gene-polymorphisms of angiotensin converting enzyme and endothelial nitric oxide synthase in patients with primary glomerulonephritis. Clin. Nephrol. 48, 205211 41 Morita, T. et al. (1999) Effect of a polymorphism of endothelial nitric oxide synthase gene in Japanese patients with IgA nephropathy. Clin. Nephrol. 52, 203209

42 Yokoyama, K. et al. (1998). High accumulation of endothelial nitric oxide synthase (ecNOS): a gene polymorphism in patients with end-stage renal disease. Nephron 79, 360361 43 Yokoyama, K. et al. (1999). Relationship between erythropoietin administration and the endothelial nitric oxide synthase gene polymorphism in patients with hemodialysis. Nephron 82, 354355 44 Yahashi, Y. et al. (1998) The 27-bp repeat polymorphism in intron 4 of the endothelial cell nitric oxide synthase gene and ischemic stroke in a Japanese population. Blood Coagul. Fibrinolysis 9, 405409 45 Akar, N. et al. (1999) Endothelial nitric oxide synthase intron 4, 27 bp repeat polymorphism in Turkish patients with deep vein thrombosis and cerebrovascular accidents. Thromb. Res. 94, 6364 46 Yoshimura, M. et al. (2000) Genetic risk factors for coronary artery spasm: significance of endothelial nitric oxide synthase gene T786C and missense Glu298Asp variants. J. Invest. Med. 48, 367374 47 Hingorani, A.D. et al. (1999) A common variant of the endothelial nitric oxide synthase (Glu298Asp) is a major risk factor for coronary artery disease in the UK. Circulation 100, 15151520 48 Cai, H. et al. (1999) The Glu298Asp (G894T) mutation at exon 7 of the endothelial nitric oxide synthase gene and coronary artery disease. J. Mol. Med. 77, 511514 49 Yoshimura, M. et al. (2000) Genetic risk factors for coronary artery spasm: significance of endothelial

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nitric oxide synthase gene T786C and missense Glu298Asp variants. J. Invest. Med. 48, 367374 Kato, N. et al. (1999) Lack of evidence for association between the endothelial nitric oxide synthase gene and hypertension. Hypertension 33, 933936 Shoji, M. et al. (2000) Positive association of endothelial nitric oxide synthase gene polymorphism with hypertension in northern Japan. Life Sci. 66, 25572562 Poirier, O. et al. (1999) Polymorphisms of the endothelial nitric oxide synthase gene no consistent association with myocardial infarction in the ECTIM study. Eur. J. Clin. Invest. 29, 284290 Yoshimura, T. et al. (2000) Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with severe preeclampsia. J. Soc. Gynecol. Invest. 7, 238241 McNamara, D.M. et al. (1999) The Asp298 variant of endothelial nitric oxide synthase (eNOS) improves survival in patients with heart failure. Circulation 100, I507 Markus, H.S. et al. (1998) Endothelial nitric oxide synthase exon 7 polymorphism, ischemic cerebrovascular disease, and carotid atheroma. Stroke 29, 19081911 MacLeod, M.J. et al. (1999) No association between Glu/Asp polymorphism of NOS3 gene and ischemic stroke. Neurology 53, 418420 Akar, N. et al. (2000) No association between Glu/Asp polymorphism of NOS3 gene and ischemic stroke. Neurology 55, 460461

G-protein-coupled receptors and signaling networks: emerging paradigms

Maria Julia Marinissen and J. Silvio Gutkind
G-protein-coupled receptors (GPCRs) constitute the largest family of cellsurface molecules involved in signal transmission. These receptors play key physiological roles and their dysfunction results in several diseases. Recently, it has been shown that many of the cellular responses mediated by GPCRs do not involve the sole stimulation of conventional second-messenger-generating systems, but instead result from the functional integration of an intricate network of intracellular signaling pathways. Effectors for GPCRs that are independent of G proteins have now also been identified, thus changing the conventional view of the GPCRheterotrimeric-G-protein-associated effector. The emerging information is expected to help elucidate the most basic mechanism by which these receptors exert their numerous physiological roles, in addition to determining why the perturbation of their function results in many pathological conditions.

G-protein-coupled receptors (GPCRs) are the largest family of cell-surface molecules involved in signal transmission. These receptors are activated by a wide variety of ligands, including peptide and
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non-peptide neurotransmitters, hormones, growth factors, odorant molecules and light, and are encoded by the largest gene family in most animal genomes (e.g. 1% of total genes in Drosophila and >5% of all genes in Caenorhabditis elegans). Furthermore, >1% of the human genome encodes >1000 proteins with a heptahelical structure1. The large number of GPCRs and the importance of their physiological roles, which is directly supported by studies performed with GPCR knockout animals2 and their link to hereditary diseases3, have made the search for novel therapeutic drugs an important and constantly expanding activity in the pharmaceutical industry. Indeed, these receptors are the target of >50% of the current therapeutic agents on the market, including more than a quarter of the 100 top-selling drugs with benefits in the range of several billion US dollars1.

0165-6147/01/$ see front matter. Published by Elsevier Science Ltd. PII: S0165-6147(00)01678-3

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Biogenic amines Noradrenaline, dopamine, 5-HT, histamine, acetylcholine

Amino acids and ions Glutamate, Ca2+, GABA Lipids LPA, PAF, prostaglandins, leukotrienes, anandamine, S1P Peptides and proteins Angiotensin, bradykinin, thrombin, bombesin, FSH, LH, TSH, endorphins N Others
E1 E2 E3

Light, odorants, pheromones, nucleotides, opiates, cannabinoids, endorphins

I1 I2 I3

GDP C

Ion channels, PI3K, PLC-, adenylyl cyclases Biological responses Proliferation, differentiation, development, cell survival, angiogenesis, hypertrophy, cancer

G-protein-independent effector molecules i GTP Adenylyl cyclases, inhibition of cAMP production, ion channels, phosphodiesterases, phospholipases q GTP PLC-, DAG, Ca2+, PKC s GTP Adenylyl cyclases, increase in cAMP concentration 12 GTP RhoGEFs, Rho

P P

Gene expression regulation

Transcription factors

Nucleus

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Fig. 1. Diversity of G-protein-coupled receptors (GPCRs). A wide variety of ligands, including biogenic amines, amino acids, ions, lipids, peptides and proteins, use GPCRs to stimulate cytoplasmic and nuclear targets through heterotrimeric G-protein-dependent and -independent pathways. Such signaling pathways regulate key biological functions such as cell proliferation, cell survival and angiogenesis. Abbreviations: DAG, diacylglycerol; FSH, follicle-stimulating hormone; GEF guanine , nucleotide exchange factor; LH, leuteinizing hormone; LPA, lysophosphatidic acid; PAF platelet, activating factor; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PLC, phospholipase C; S1P sphingosine-1-phosphate; TSH, thyroid-stimulating hormone. ,

Maria Julia Marinissen e-mail: mmarinissen@dir.nidcr.ni h.gov J. Silvio Gutkind Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Building 30, Room 211, Bethesda, MD 208924340, USA. e-mail: sg39v@nih.gov

GPCRs owe their name to their extensively studied interaction with heterotrimeric G proteins (composed of an -, - and -subunit), which undergo conformational changes that lead to the exchange of GDP for GTP bound to the -subunit following receptor activation. Consequently, the G- and Gsubunits stimulate effector molecules, which include adenylyl and guanylyl cyclases, phosphodiesterases, phospholipase A2 (PLA2), phospholipase C (PLC) and phosphoinositide 3-kinases (PI3Ks), thereby activating or inhibiting the production of a variety of second messengers such as cAMP, cGMP, diacylglycerol, inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3], phosphatidyl inositol (3,4,5)trisphosphate [PtdIns(3,4,5)P3], arachidonic acid and phosphatidic acid, in addition to promoting increases in the intracellular concentration of Ca2+ and the opening or closing of a variety of ion channels (Fig. 1). However, beyond the dogma of GPCRG-protein, research conducted during the past few years suggests that the activation of GPCRs can lead to biochemical responses that are not mediated by heterotrimeric G proteins. Furthermore, it has been
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shown that a variety of cellular responses are mediated by the activation of effector molecules through novel molecular mechanisms, many of which do not involve the stimulation of classical second messengers, and that most biological responses mediated by GPCRs are not dependent on a single biochemical route, but result from the integration of the functional activity of an intricate network of intracellular signaling pathways. In this article, the classical and newly identified mechanisms through which GPCRs act will be summarized, with emphasis on their contribution to the regulation of cell growth.
G proteins and GPCRs in normal cell growth and cancer

Many potent mitogens such as thrombin, lysophosphatidic acid (LPA), bombesin, vasopressin, bradykinin, substance K, acetylcholine receptor agonists, angiotensin II and others stimulate cell proliferation by acting on their cognate GPCRs in a variety of cell types46. The discovery of the mas oncogene, whose protein product exhibits a typical heptahelical structure, provided a link between cellular transformation and GPCRs (Ref. 7). No activating mutations were found in mas but instead the observation that ectopic expression of 5-HT2C and muscarinic acetylcholine M1, M3 and M5 receptors could transform murine fibroblasts in an agonistdependent fashion8,9 provided evidence that wild-type GPCRs can be tumorigenic when exposed to an excess of locally produced or circulating agonists. However, if mutated, GPCRs might be rendered transforming

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Table 1. G proteins and G-protein-coupled receptors in tumorigenesis

Type of tumor Activating mutations: G proteins Gs Thyroid toxic adenomas, thyroid carcinomas, growth-hormone-secreting pituitary adenomas, McCuneAlbright syndrome Ovarian sex cord tumors, adrenal cortical tumors

Gi2 G-protein-coupled receptors TSH receptor FSH receptor LH receptor CCK2 receptor Ca2+-sensing receptor

Thyroid adenoma, thyroid carcinoma Ovarian sex cord tumors, ovarian small cell carcinoma Leydig cell hyperplasia, male precocious puberty Colorectal cancer Autosomal-dominant hypocalcemia, neoplasms

Autocrine and paracrine activation: Neuromedin B receptor Neurotensin receptor Gastrin receptor Cholecystokinin receptors Vasopressin receptors Small-cell lung carcinoma Prostate cancer, small-cell lung carcinoma Gastric cancer, small-cell lung carcinoma Pancreatic hyperplasia, pancreatic carcinoma, gastrointestinal cancer, small-cell lung carcinoma Small-cell lung carcinoma

Virally encoded G-protein-coupled receptors: Kaposis sarcoma-associated herpesvirus (KSHV) Herpes virus saimiri (HVS) Jaagsiekte sheep retrovirus (JSRV) Kaposis sarcoma Leukemias and lymphomas in non-human primates Ovine pulmonary carcinoma

even in an agonist-independent fashion as shown, for example, for 1B-adrenoceptors10. Such naturally occurring, constitutively activating mutations were found in thyroid-stimulating hormone receptors in 30% of hyperfunctioning human thyroid adenomas and in a minority of differentiated thyroid carcinomas11, which thus suggests a direct link between GPCRs and human cancer. Activating mutations have also been detected in other GPCRs (Table 1) such as leuteinizing hormone receptors (mutations result in the hyperplastic growth of Leydig cells that produces a form of familial male precocious puberty12) and Ca2+-sensing receptors (mutations cause autosomal dominant hypercalcemia13), which have a role in the regulation of some neoplasms14. Although activating mutations are infrequent in GPCRs, these receptors can often contribute to neoplasia when persistently stimulated by agonists released from tumors. For example, bombesin, gastrin-releasing peptide (GRP), neuromedin B (NMB), bradykinin, cholecystokinin (CCK), galanin, neurotensin and vasopressin are secreted by small-cell lung carcinoma cells, which also express their cognate GPCRs and are thus activated in an autocrine or paracrine fashion15. Similarly, several of these neuropeptide receptors and their agonists have been implicated in colon adenomas and carcinomas, gastric hyperplasia and cancer: GRP receptors in particular in
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prostate cancer, and CCK1 and CCK2 receptors in pancreatic hyperplasia and carcinoma6,15. Interfering with the function of these receptors can effectively prevent tumor growth in animal models15, which raises the possibility of developing novel agents that act on GPCRs for therapeutic intervention in cancer. Functional GPCRs are also encoded by the genome of several DNA viruses, including the human cytomegalovirus (HCMV)15, herpes virus saimiri (HVS)16 and Kaposis sarcoma-associated herpes virus (KSHV)17. These GPCRs are highly related to chemokine receptors18. The HCMV-encoded GPCRs might help elude the host immune system through the molecular mimicry of proteins that are normally involved in host defense mechanisms18. Expression of the HVS GPCR contributes to fatal lymphoproliferative diseases, including leukemias and lymphomas, caused by infection of several nonhuman primates with HVS (Ref. 16). The KSHV GPCR behaves as a constitutively active Gq-coupled receptor and is transforming when expressed in murine fibroblasts17. Recently, it has been shown that the KSHV-encoded GPCR can subvert proliferative pathways to stimulate the ectopic expression of vascular endothelial growth factor (VEGF)19, thus participating in the angiogenic response that characterizes Kaposis sarcoma lesions. In this regard, many GPCRs, including those for sphingosine-1phosphate, LPA, platelet-activating factor, thrombin, interleukin 8, growth regulated oncogene (GRO ), monocyte chemoattractant protein 1 (MCP-1) and stromal cell-derived factor (SDF-1), have been shown to play a key role in vasculogenesis and tumor-induced angiogenesis20. This feature has recently generated enormous attention because of the possibility of inhibiting tumor growth and spread by affecting the integrity of newly formed blood vessels21. Consistent with a role for GPCRs in normal and aberrant cell growth, at least ten of the 17 G-subunits cloned so far have been shown to harbor transforming potential22. Indeed, mutations that affect the intrinsic GTPase activity of two -subunits, Gs and Gi2, have been correlated with different types of tumors (Table 1). For example, the gsp oncogene encodes a GTPase-deficient mutant of Gs and is present in thyroid toxic adenomas (30%), thyroid carcinomas (10%), growth-hormone-secreting pituitary adenomas and McCuneAlbright syndrome (Table 1). Although the consequent increased concentrations of cAMP and activated PKA following expression of gsp can reverse the transformed phenotype in certain cells by inhibiting Raf1 (Ref. 23), the high level of production of this second messenger results in uncontrolled cell growth and transformation in other cell types. For example, in PC12 cells and thyroid cells, cAMP activates the mitogen-activated protein kinase (MAPK) pathway, which is intimately related to cell proliferation, by activating the small GTPases Rap1 and B-Raf, which in turn stimulate MAPK kinase (MEK) and MAPK (Ref. 24).

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The gip2 oncogene is the activated mutant of Gi2, and has been detected in human ovarian sex cord stromal tumors and adrenal cortical tumors25 (Table 1). Although the frequency of Gi2 mutations in these cancer types remains controversial, the expression of gip2 in fibroblasts leads to transformation, apparently by the parallel decreased concentration of cAMP and activity of PKA, which results in the activation of the MAPK pathway by repression of this otherwise inhibitory pathway in these cells26. Alternatively, Gi2 can activate the RasMAPK pathway by the recruitment to the membrane of the GTPase-activating protein Rap1GAPII and inhibition of GTP-bound Rap1 (Ref. 27), and by the release of the -dimers that act on a Ras- and PI3K-dependent pathway28. Recent results indicate that signal transducer and activator of transcription 3 (STAT3) also acts downstream of Gi proteins and mediates transformation in fibroblasts29. Naturally occurring mutations of the Gq family have not been identified in human tumors and studies carried out with activated mutant forms of these subunits have yielded contradictory results. Apparently low levels of activated Gq result in transformation whereas high levels lead to cell death in a cell-type-dependent manner22. The gep oncogene was simultaneously isolated from Ewings sarcoma and identified by virtue of its strong transforming activity in NIH 3T3 cells30,31. gep encodes a wild-type form of G12, a member of the G12/13 family of G-subunits. G12, as well as G13 and their activated mutants, can transform a variety of rodent fibroblasts. However, no activating mutations have yet been described in human cancers. Interestingly, breast, colon and prostate adenocarcinoma-derived cell lines were found to express high levels of wild-type G12/13 (Ref. 32), but whether overexpression of G12/13 is part of the mechanism that underlies the neoplastic conversion of these tumor-derived cells is still unclear. Interestingly, the use of bacterial toxins revealed that the small GTPase RhoA plays a key role in stress fiber formation, nuclear signaling and cell transformation induced by G12/13-subunits33,34. Furthermore, G12/13-subunits were recently shown to bind a family of Rho guanine nucleotide exchange factors (GEFs) that exhibits a structural domain highly related to that of regulators of G-protein signaling (RGS) and includes p115-RhoGEF (Ref. 35), PDZ-RhoGEF (Ref. 36) and leukemia-associated Rho GEF (LARG)37. Together, these exciting studies indicate that the G12/13 family of G proteins can act directly upstream from Rho by binding and activating a distinct family of RGScontaining RhoGEFs, and emphasize the important contribution of G12/13 and Rho GTPases to many of the cellular responses controlled by GPCRs (Ref. 38).
A network of MAPKs links GPCRs to the nucleus and beyond

proline-targeted serinethreonine kinases, collectively known as extracellular signal-regulated kinases (ERKs) or MAPKs, appears to play a central role. Following phosphorylation by their immediate upstream MEK, members of the MAPK family translocate to the nucleus where they phosphorylate transcription factors, thereby regulating the expression of genes that play a key role in normal and aberrant cell growth39. Indeed, efforts to understand the basic molecular processes by which GPCRs regulate the enzymatic activity of MAPKs has yielded fundamental clues about the biochemical routes used by these receptors to exert a wide range of biological functions. This article emphasizes the emerging concepts from this still highly active area of research.
MAPK

Recent work has revealed that multiple intracellular signaling pathways mediate the proliferative effects of GPCRs. Among them, a family of closely related
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Diverse GPCRs ligands can activate p42 and p44 MAPK in several cellular systems40 (Fig. 2). The search for the underlying molecular mechanism has produced some of the most fascinating observations about GPCRs in recent years. For example, the early observation that Gi receptors activate MAPK through -subunits in a PKC-independent but Rasdependent manner provided the first demonstration of a role for G in linking heterotrimeric G proteins to the Ras pathway41. By contrast, Gq-coupled receptors can use both PKC-dependent and PKCindependent pathways to stimulate the MAPK pathway (see http://www.stke.org/cgi/content/full/ OC_sigtrans;2000/40/re1 for an extensive review)42. The ability of tyrosine kinase inhibitors to reduce the activation of MAPK by GPCRs (Ref. 43) and the rapid tyrosine phosphorylation of Shc (Src homology and collagen) following GPCR stimulation with the consequent formation of ShcGrb2 (growth factor receptor-bound 2) complexes44 provided the first indication of a role for tyrosine kinases in the pathway linking GPCRs to RasMAPK. To date, several non-receptor tyrosine kinases (NRTKs) and receptor tyrosine kinases (RTKs) have been proposed to participate in this response. For example, Src or Src-like kinases can mediate the phosphorylation of Shc provoked by -adrenoceptors and -subunits45. In this case, Src stimulation appears to require the recruitment of -arrestin to GPCR kinase 2 (GRK2)phosphorylated -adrenoceptors, followed by receptor internalization and the consequent formation of molecular complexes between -arrestin and Src (Ref. 46). However, GPCR endocytosis might not be strictly required to activate MAPK (Ref. 47). Alternatively, Src can be activated by direct interaction with Gi and Gs (Ref. 48) or 3-adrenoceptors49. Other NRTKs, which include Csk, Lyn, Btk (Brutons tyrosine kinase) and two more distantly related molecules, proline-rich tyrosine kinase 2 (PYK2) and focal adhesion kinase (FAK), have also been proposed to mediate the activation of MAPK by Gi- and Gq-coupled receptors in a variety of cell types42. Their

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GPCR

RTK

GPCR

Arrestin

Src Shc Sos Grb2

Ras Rap

GDP

GDP i GTP q GTP

Raf1 PI3K Ca2+ PYK2 Ras GRF

B-Raf

Rap GAPII EPAC PKA

o GTP i GTP s GTP cAMP

MEK

PLC-

PKC

MAPK

P P

Gene expression regulation Nucleus

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Fig. 2. Multiple pathways link G-protein-coupled receptors (GPCRs) to mitogen-activated protein kinase (MAPK). Biochemical routes initiated by -subunits can stimulate Ras by the activation of receptor and non-receptor tyrosine kinases, which results in the recruitment of Sos to the membrane and the exchange of GDP for GTP bound to Ras. Activated Gq can stimulate Raf1 through protein kinase C (PKC) or by stimulating Ras by the Ca2+-dependent activation of RasGRF and tyrosine kinases acting on Sos. Gi, Go and Gs can also use tissue-restricted pathways regulating Rap, which can stimulate B-Raf and lead to the activation of MAPK. Activated MAPK translocates to the nucleus and phosphorylates nuclear proteins, including transcription factors, thereby regulating gene expression. Arrows represent positive stimulation and broken lines represent inhibition (see text for more details). Abbreviations: EPAC, exchange protein activated by cAMP; GAP GTPase-activating protein; , GRF guanine-nucleotide releasing factor; MEK, MAPK kinase; PI3K, phosphoinositide 3-kinase; , PKA, protein kinase A; PLC, phospholipase C; RTK, receptor tyrosine kinase.

contribution to MAPK activation is likely to be highly dependent on the cellular context. In other cases, activation of GPCRs can lead to the stimulation of RTKs, such as epidermal growth factor receptors (EGFRs)50, which thus creates docking sites for proteins that contain phosphotyrosine-binding domains and leads to the assembly of complexes that contain Sos (which in turn activates Ras). Several mechanisms have been proposed to mediate the transactivation of RTKs by GPCRs, including the activation of RTKs through NRTKs, the formation of complexes between GPCRs and RTKs, and the release of RTK ligands. Indeed, the activation of NRTK can lead to the phosphorylation of key tyrosine residues in EGFRs (Ref. 51), whereas the formation of stable molecular complexes between ligand-activated -adrenoceptors and EGFRs have been demonstrated following their co-internalization into clathrin-coated vesicles52. Furthermore, GPCRs can provoke the proteolytic cleavage and release of membrane-bound pro-hormones, such as heparin-binding epidermal growth factor (HB-EGF), by a yet unidentified metalloprotease that initiates an autocrineparacrine mechanism of activation of EGFRs (Ref. 53). Other RTKs are expected to be
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activated by GPCRs by similar mechanisms. Nonetheless, whether the activation of RTKs or NRTKs is strictly required for the stimulation of MAPK is not yet clear, and will surely be the focus of further investigation. PI3K isoforms are also required to link GPCRs to MAPK. For example, PI3K is activated following direct interaction with G-subunits and, independently of its lipid kinase activity, can act upstream of NRTKs and the ShcGrb2SosRas pathway leading to the activation of MAPK (Ref. 28). PI3K is also stimulated by GPCRs (Ref. 54), and the PI3K-dependent activation of Rac and p21-activated kinase (PAK) can cooperate with the stimulation of Ras to enhance the catalytic activity of Raf (Ref. 55), thus widening the complexity of putative mechanisms by which GPCRs can activate MAPK through the stimulation of PI3Ks. Another intriguing case is the activation of MAPK by Gq-coupled receptors, which can stimulate MAPK in a PKC-dependent and Ras-independent manner, in a PKC- and Ras-dependent manner, or in a PKCindependent but Ras-dependent manner, depending on the cell type and the receptor expression levels. For example, second messengers generated as a consequence of Gq stimulation can provoke Ras activation through RasGRF and RasGRP (also called CalDGEF), two tissue-restricted GEFs for Ras and Ras-related GTPases (http://www.stke.org/ cgi/content/full/OC_sigtrans;2000/40/re1). However, how PKC activates the MAPK pathway is still unclear because the direct phosphorylation of Raf might not be sufficient to activate MEK and MAPK (Ref. 56). Instead, PKC could facilitate the full activation of Raf by Ras through the stimulation of additional molecules involved in the RasRaf interaction.

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GPCR

Rho

GDP

Rac, Cdc42 GEFs

RGS, RhoGEFs

13 GTP

q GTP

Crk, PYK2 FAK, paxillin ASK1 MEKK MLKs

independently of the activation of Rac1 and Cdc42 (Ref. 60), and arrestin can bind a tissue-restricted isoform of JNK, JNK3, and cause its activation by the formation of a protein complex with ASK1 (Ref. 61). In this case, arrestin would be acting as a protein scaffold in the JNK pathway, similar to that described for JNK-interacting protein (JIP)62 (Fig. 3). Thus, as for MAPK, activation of JNK can proceed by several different mechanisms that are characteristic of each cell type.
p38 and ERK5

MEK5

MKK3/6

MKK4/7

ERK5

p38

ERK6

SAPK4

JNK
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Fig. 3. Novel signaling pathways that connect G-protein-coupled receptors (GPCRs) to Jun N-terminal kinase (JNK), p38 isoforms and big mitogen-activated kinase 1 (ERK5). Molecules that link GPCRs to the different members of the mitogen-activated protein kinase (MAPK) family are shown. Arrows represent positive stimulation and broken lines represent functional interactions where precise mechanisms are yet to be elucidated (see text for details). Abbreviations: FAK, focal adhesion kinase; GEF guanine nucleotide , exchange factor; MEK and MKK, MAPK kinase; MEKK, MAPK kinase kinase; MLK, mixed lineage kinase; PYK2, proline-rich tyrosine kinase 2; RGS, regulator of G-protein signaling; SAPK, stress-activated protein kinase.

As described above, both Gi and Gs can also regulate the MAPK pathway through the inactivation or activation, respectively, of the small GTP-binding protein Rap1. This Ras-related GTPase can inhibit MAPK in most cell types, but stimulates MAPK in other cells, primarily in those of neuronal origin, through the activation of B-Raf (Ref. 42). Rather than conflicting, these multiple examples discussed above stress the importance of the cellular context when defining the signal transduction pathways linking GPCRs to MAPKs. Indeed, many of the intervening molecules exhibit a very restricted tissue distribution, which is evidence of the evolution of highly specialized alternative mechanisms to activate the MAPK pathway by GPCRs in distinct cellular settings.
JNK

How GPCRs activate the Jun N-terminal kinase (JNK) is another unanswered question. This enzyme, also termed stress-activated protein kinase (SAPK), is structurally related to MAPK but the pathways used by GPCRs to activate these kinases are different. For example, activated forms of Rac and Cdc42 can initiate an independent MAPK cascade that regulates JNK activity57, analogous to that of Ras acting on MAPK. Free dimers and G12 and G13 are able to activate JNK in a Rac1Cdc42dependent manner42. However, the nature of the GEFs that connect and G12/G13 to Rac1 and Cdc42 is still unclear. Available data support a role for two GEFs for Rac1 and Cdc42 (Tiam1 and Dbl), as well as for two Ras GEFs (Ras-GRF1 and Ras-GRF2), which might stimulate Rac1 through their Dbl homology (DH) domain. In addition, PYK2 and FAK can also participate in signaling to JNK through the recruitment of the adaptor molecules Crk (Ref. 58) or paxillin59, which can activate GEFs for Rac and Cdc42. Interestingly, G12 can also activate JNK by activating the MEK kinase (MEKK), ASK1,
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The p38 MAPK family is activated by stress and surface receptors in a manner similar to that of JNK (Ref. 62). To date, four p38 MAPKs have been described, p38 (CSBP-1), p38, p38 (ERK6 or SAPK3) and p38 (SAPK4)63. The stimulation of the p38 family of MAPKs by GPCRs has been well documented, but their mechanism of activation is far from being fully understood. A few studies have shown that Gq and dimers activate p38 (Ref. 64), and two NRTKs, Btk (Ref. 65) and Src (Ref. 66), have been implicated in this mechanism. In addition, electrophysiological studies have implicated p38 isoforms downstream of G13 (Ref. 67), and Gq-coupled receptors have been shown to activate p38, as well as their more distantly related p38 and p38 isoforms68. The big mitogen-activated kinase 1 (BMK1 or ERK5; 80 kDa) has recently been characterized. It can be activated by oxidative stress and plays a role in early gene expression triggered by serum69. GPCRs can potently stimulate ERK5 (Ref. 68) through a mechanism that involves Gq and G13, independently of Rho, Rac1 and Cdc42 (Ref. 70). ERK5 appears to play an important role in the regulation of early gene expression through the phosphorylation of the transcription factor myocyte enhancer factor 2 (MEF2)68. These observations have raised considerable interest because the activation of MEF2 proteins by GPCRs might play an important role in neuronal survival71 and cardiac myocyte hypertrophy (Ref. 72). As for p38 isoforms, the mechanism of activation of ERK5 by GPCRs is still poorly understood and currently under investigation.
G-protein-independent signaling by heptahelical receptors

Several recent provocative reports have revealed that GPCRs can interact with a wide variety of intracellular molecules in addition to heterotrimeric G proteins, thus broadening the molecular mechanisms by which these receptors transduce environmental signals. For example, the adaptor molecule arrestin binds many phosphorylated GPCRs and is primarily involved in targeting these receptors for endocytosis. However, arrestin has also been shown to couple GPCRs to the activation of Src-like kinases and to facilitate the formation of multimolecular complexes, which includes

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components of the MAPK and JNK pathways61,73. Other structural features, which include PDZ, SH2 and SH3 domains, and polyproline-containing regions, also provide the molecular basis for direct interactions between GPCRs and several intracellular signaling molecules74. PDZ domains primarily bind proteins that harbor a C-terminal S/TxV(L/I) motif, which is frequently found in GPCRs. This proteinprotein interaction domain could be used by GPCRs to bypass the requirement of G proteins to activate intracellular signaling pathways, as illustrated by the ability of 2-adrenoceptors to associate with the Na+H+ exchanger regulatory factor, 5-HT2C receptors to interact with MUPP1, a multi-PDZ-domain protein with unknown functions, and Drosophila rhodopsin to bind InaD, a PDZ-containing scaffolding G protein that interacts with PLC-, PKC and the TRP ion channels74. Because many GPCRs include a C-terminal PDZ-binding region, the discovery of their binding partners should help elucidate many of the unique features that underlie their distinct biological responses. Some SH2-containing G proteins such as Grb2 have been reported to bind 2-adrenoceptors, and the tyrosine Janus-activated kinase JAK2 has been found to associate with angiotensin AT1A receptors through the SH2-containing tyrosine phosphatase SHP (Ref. 74). These interesting examples offer a glimpse of an emerging theme, which is the ability of GPCRs to be tyrosine phosphorylated and to subsequently recruit proteins that contain phosphotyrosine-binding domains, such as SH2 and PTB domains. GPCRs can also bind proteins that harbor polyproline-binding domains, such as SH3, WW and Enabled/VASP homology (EVH) domains75. For example, metabotropic glutamate (mglu) receptors bind a group of proteins containing EVH-like domains, Homer (1ac, 2 and 3), through a polyproline region (PPxxFP) in their C-terminal tails75. Homers can also homo- and heteromultimerize with other polyproline rich proteins, such as Ins(1,4,5)P3 receptors, which thus provides a functional link between mglu receptors and the generation of Ca2+ signals through the release of Ca2+ from the intracellular stores. Other GPCRs also contain polyproline stretches in their third intracellular domain or cytoplasmic tail, and their contribution to receptor signaling represents an active area of investigation74. The small G proteins Rho and Arf can be immunoprecipitated in an agonist-dependent fashion with AT1A and muscarinic acetylcholine M3
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receptors, and, although is not clear whether or not this represents a direct interaction, it is evident that GPCRs can form molecular complexes with some small G proteins without the heterotrimeric G proteins as obligatory intermediates76. These emerging findings, together with the observation that Frizzled, Smoothened and cAMP receptors in Dictyostelium induce several biological responses that are not coupled to G proteins, are changing the dogmatic idea of GPCRheterotrimeric-G-proteinassociated effector. In view of these new reports, some authors prefer to use designations other than GPCRs, such as serpentine, seven-transmembrane or heptahelical receptors, so as not to underrepresent the wide range of biochemical routes used by this large family of signal transducing receptors76.
Conclusions: unraveling the complexity of GPCRmediated signaling pathways

The complexity of the molecular mechanisms whereby GPCRs transduce environmental signals has just begun to be fully appreciated. For a long time, the study of GPCR signaling has been focused on classical second-messenger-generating systems. However, we now realize that these intracellular signaling molecules are not sufficient to explain their wide array of biological responses. Instead, each GPCR would be expected to stimulate not one but a large number of highly interconnected cytoplasmic signaling routes. In turn, this will lead to a temporally distinct pattern of activation of MAPKs, many of which will translocate to the nucleus and phosphorylate nuclear proteins, thereby affecting an intricate balance of regulatory molecules that control gene expression. Thus, the final biological outcome, including phenotypic differentiation, cell survival or death, hypertrophy, and normal and aberrant cell growth, will most probably result from the integration of a complex network of biochemical responses, which are highly dependent on receptor expression levels, coupling specificity and the repertoire of signaling molecules expressed in each cellular system. The recent advances in our understanding of how GPCRs regulate intracellular signaling networks should help elucidate the most basic mechanism by which these receptors exert their numerous physiological roles, as well as why the perturbation of their function results in many pathological conditions. It might also provide a golden opportunity to identify novel approaches for pharmacological intervention in several disease states.
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Proteomics post-genomic cartography to understand gene function

Soren Naaby-Hansen, Michael D. Waterfield and Rainer Cramer
The completion of the genomic sequences of numerous organisms from human and mouse to Caenorhabditis elegans and many microorganisms, and the definition of their genes provides a database to interpret cellular proteinexpression patterns and relate them to protein function. Proteomics technologies that are dependent on mass spectrometry and involve twodimensional gel electrophoresis are providing the main window into the world of differential protein-expression analysis. In this article, the limitations and expectations of this research field are examined and the future of the analytical needs of proteomics is explored.

levels, however, might not fully reflect their functional state, and protein expression profiling per se provides no data regarding subcellular site of action or interaction partners unless additional experimental steps are included. The scope of modern proteomics has therefore broadened considerably (Box 1).
The tools of proteomics

Soren Naaby-Hansen Michael D. Waterfield* Rainer Cramer Ludwig Institute for Cancer Research and Dept of Biochemistry and Molecular Biology, Royal Free and University College London Medical School, 91 Riding House Street, London, UK W1W 7BS. *e-mail: mike@ludwig.ucl.ac.uk

Biomedical research in the post-genomic era will be carried out against the backdrop of an information explosion about the ~30 000 genes whose sequence have been completed for mouse and humans. It is clear that high-throughput transcriptional profiling with chip technology will easily identify when, where and how much RNA is transcribed. However, there is usually no clear correlation between RNA transcription and protein expression1,2 and transcriptomics reveals no data regarding the activity of the product, leaving a huge information gulf about the regulation of protein expression, structure and function, which must be filled to allow fast-track exploitation of genomics. Proteomics is the large-scale study of gene expression at the protein level, which will ultimately provide direct measurement of protein expression levels and insight into the activity state of all relevant proteins. In an experimental context the term proteomics is derived from proteome, which by analogy to genome is defined as the entire protein complement expressed by a cell or tissue type. Study of the proteome dates back to the late 1970s. The key elements of classical proteomics are the separation of proteins in a sample using two-dimensional gel electrophoresis (2-DE) and their subsequent quantitation and identification. Protein expression
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The dynamic range of protein expression and modification makes the identification of the entire proteome a far bigger and more complex challenge than the sequencing of the genome. Although gene sequencing and expression analysis can be performed with high throughput and in an automated manner, major technical problems need to be resolved and new techniques developed before proteomics can become a
Box 1. Modern proteomics
Post-genomic proteomics includes areas such as: (1) determination of protein function; (2) characterization of post-translational modifications; (3) structural analysis; (4) analysis of the regulation of protein activity; (5) studies of protein interactions and complex formation; (6) analysis of protein trafficking and sequestration in subcellular compartments; (7) protein expression analysis; (8) analysis of signaling and metabolic pathways; (9) drug mode-of-action; and (10) toxicity studies. Bioinformatic integration of data from such conditional studies combined with data obtained by transcriptomic analysis will hopefully lead to a more comprehensive understanding of gene function and regulation, and to new approaches in the diagnosis, prevention and treatment of diseases.

0165-6147/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01663-1