Oncogene (1998) 17, 1331 ± 1342 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Cell growth control by G protein-coupled receptors: from signal transduction to signal integration J Silvio Gutkind Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, National Institutes of Health Bethesda, Maryland 20892, USA Keywords: G proteins; G protein-coupled receptors; G protein-coupled receptors and cell proliferation Ras; MAP kinases; Rho; cell growth The use of Pertussis toxin (Ptx), that catalyses the ADP-ribosylation of G protein a subunits of the ai family (Ui and Katada, 1990) thereby preventing their interaction with receptors, provided the ®rst clue that a Many growth factors are known to bind and activate number of mitogens act on this family of cell surface either receptors possessing an intrinsic protein-tyrosine receptors. For example, Pouyssegur and colleagues kinase activity (Yarden et al., 1986), or those that noted that DNA-synthesis in CHO cells in response to transmit signals to the cytoplasm through the thrombin was blocked by Ptx treatment, whereas the interaction with heterotrimeric GTP-binding proteins proliferative response to PDGF was Ptx-insensitive (G proteins). The latter are collectively known as G (Chambard et al., 1987; Pouyssegur et al., 1988). A protein-coupled receptors (GPCRs) and comprise the similar approach led to the discovery that one of the largest group of cell surface receptors. With more than most potent mitogens present in serum, lysopho- 1000 members, they represent more than 1% of the sphatidic acid (LPA), a simple naturally occurring *100 000 proteins encoded by the human genome. phospholipid, was also acting on the G protein-linked The best known family of GPCRs exhibit a common class of receptors (van Corven et al., 1989). Many structural motif consisting of seven membrane-span- other Ptx sensitive mitogens were subsequently ning regions (Dohlman et al., 1987) (Figure 1). These identi®ed (see van Biesen et al., 1996 for a review). receptors can be activated by a diverse array of However, biochemical studies provided evidence that external stimuli, including growth factors, vasoactive many mitogens also acted on GPCRs, albeit their polypeptides, chemoattractants, neurotransmitters, hor- biological eects were mediated by Ptx-insensitive G mones, phospholipids, photons, odorants, and taste proteins and therefore were resistant to Ptx treatment. ligands. Activation of GPCRs by these agents elicits a These included thrombin acting on smooth muscle profound change in the transmembrane a helices, thus cells, bombesin on Swiss 3T3 cells and bronchial aecting the conformation of intracellular loops epithelium, vasopressin, bradykinin and endothelin on uncovering previously masked G protein binding sites Swiss 3T3 cells, endothelin on human mesangial cells, (Altenbach et al., 1996; Bourne, 1997; Wess, 1997). substance K on human skin ®broblasts, acetylcholine This causes the exchange of GDP for GTP bound to receptor agonists on embryonic astrocytes, angiotensin the G protein a subunit, and a conformational change on smooth muscle cells, and many others (see in three ¯exible `switch regions' of Ga, activating Ga Moolenaar, 1991). Thus, it became clear that many and causing its dissociation from the bg heterodimers ligands acting via GPCRs could elicit a mitogenic (Lambright et al., 1994, 1996; Sondek et al., 1994, response in a variety of cell types (reviewed in 1996). In turn, GTP-bound G protein a subunits or bg Rozengurt, 1986; van Biesen et al., 1996) and that complexes initiate intracellular signaling responses by these receptors can transduce proliferative signals when acting on eector molecules such as adenylyl cyclases, acting on Ptx sensitive or insensitive heterotrimeric G phosphodiesterases, phospholipases, or regulating the proteins. Furthermore, recent gene knock-out studies activity of ion channels, ion transporters, and a indicated that certain GPCRs are essential for cell growing number of kinases. To date, 16 distinct growth under physiological conditions (Nagata et al., mammalian G protein a subunits have been identi- 1996). ®ed, and divided into four families based upon sequence similarity: as, ai, aq, and a12 (Wilkie et al., 1992). In addition, 11-G protein g subunits and ®ve G protein b subunits have been cloned so far. Taken Oncogenic potential of G protein-coupled receptors together, it is becoming increasingly clear that GPCRs represent one of the most diverse signal transduction Constitutive activation of receptor-protein tyrosine systems in eukaryotic cells. The biochemical and kinases, either by structural alteration of the receptor biological consequences of this diversity in subunit itself or by deregulated presentation of the ligand, can composition have just begun to be appreciated. In this induce mouse ®broblasts to acquire a fully transformed review, we will describe the role of GPCRs in normal phenotype (Aaronson and Tronick, 1985; Kraus et al., and aberrant cell growth and will then focus on recent 1988; Tronick and Aaronson, 1988). Thus, it has been eorts aimed to elucidate their downstream intracel- suggested that unrestricted activation of proliferative lular signaling pathways controlling cell proliferation. pathways contribute to the malignant state. Discovery Cell growth control by G protein coupled receptors JS Gutkind 1332 Figure 1 Divergent kinase cascades link G protein-coupled receptors to the nucleus. (see text for details) of the mas oncogene (Young et al., 1986) provided the cally expressed in NIH3T3 cells and activated by the ®rst link between cellular transformation and GPCRs. serotonin present in the culture medium (Julius et al., The mas oncogene, which encodes a putative GPCR, 1989). Using a more de®ned experimental system, was initially cloned using standard transfection assays muscarinic m1, m3, and m5 receptors were shown to by virtue of its ability to induce tumors in mice (Young transform NIH3T3 cells in a strictly agonist-dependent et al., 1986). The natural agonist for the mas oncogene manner (Gutkind et al., 1991). As the natural agonist product is still unknown, but is likely to be a serum for these receptors, the neurotransmitter acetylcholine, component. Similarly, serotonin 1C receptors were is not present in serum, it was possible to correlate shown to harbor transforming potential when ectopi- biochemical responses with the biological eects Cell growth control by G protein coupled receptors JS Gutkind 1333 elicited by an exogenously added cholinergic agonist. lymphomas, in several non-human primates (Nicholas In addition, this approach established that while Gq et al., 1992). The protein product of one of its genes, coupled receptors, m1, m3, and m5, were highly ECRF3, behaves as a functional chemokine receptor, transforming, Gi coupled receptors, m2 and m4, where with a ligand speci®city identical to that of the IL8 without focus-forming activity, under those experi- receptor (Nicholas et al., 1992). Furthermore, the mental conditions. Together, these ®ndings demon- HVS encoded GPCR responds biochemically to IL8, strated that certain GPCRs could behave as potent suggesting a role for chemokines in the molecular agonist-dependent oncogenes, and raised the possibility pathogenesis of viral infection. Interestingly, the that activating mutations in GPCRs may render them KSHV-encoded GPCR is also highly related to the transforming. Indeed, this was subsequently shown for chemokine receptor family, and has been recently the a1b G protein-linked receptor to noradrenaline shown to behave as a constitutively active Gq-coupled (Allen et al., 1991). In this case, speci®c point receptor and, as predicted, to be sucient to subvert mutations in the C-terminal juxtamembrane region of normal growth control when expressed in cultured the third intracellular loop of the a1b receptor relieved ®broblasts (Arvanitakis et al., 1997). In addition, the requirement of ligand activation for its transform- recent evidence has indicated that the KSHV-GPCR ing ability. Furthermore, the recent identi®cation of can eectively activate signaling pathways typical of constitutively activating mutations in TSH receptors in transforming receptors, and play an unsuspected role 30% of thyroid adenomas (Parma et al., 1993) in angiogenesis (Bais et al., in press). provided now a direct link between this class of Consistent with a role for GPCRs in normal and receptors and human cancer. Similarly, mutationally aberrant cell growth, constitutively active mutants of activated LH receptors have been identi®ed in a form Gai,Gaq,Ga0,Ga12 and Ga13 were also shown to of familial male precocious puberty that results from behave as transforming genes in a variety of cell types, hyperplastic growth of Leydig cells (Shenker et al., and mutationally activated Ga proteins were identi®ed 1993). in several disease states (reviewed in Dhanasekaran et Even more prevalent than activating mutations, al., 1995). For example, mutationally activated Gas, paracrine and autocrine stimulation of multiple referred to as the gsp oncogene (Landis et al., 1989), GPCRs for neuropeptides and prostaglandins has results in hyperplasia of endocrine cells, and are been implicated in a number of human neoplasias. present in human thyroid and pituitary tumors Particularly, bombesin, gastrin-releasing peptide (reviewed in Dhanasekaran et al., 1995), and in the (GRP), neuromedin B, bradykinin, cholecystokinin McCune-Albright syndrome,