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Rho Gtpase-Dependent Transformation by G Protein-Coupled Receptors

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Rho Gtpase-Dependent Transformation by G Protein-Coupled Receptors Oncogene (2001) 20, 1547 ± 1555 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Rho GTPase-dependent transformation by G protein-coupled receptors Ian P Whitehead*,1, Irene E Zohn2 and Channing J Der3 1Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey, NJ 07103- 2714, USA; 2The Laboratory of Molecular Embryology, The Rockefeller University, 1230 York Avenue, Box 32, New York, NY 10021, USA; 3University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Department of Pharmacology, Chapel Hill, North Carolina, NC 27599-7295, USA G protein coupled receptors (GPCRs) constitute the amino terminus, and are often reversibly palmitylated largest family of cell surface receptors, with more than on a conserved carboxy terminal cysteine. 1000 members, and are responsible for converting a Ligands that have been shown to interact with diverse array of extracellular stimuli into intracellular members of the GPCR family include neurotransmit- signaling events. Most members of the family have ters, hormones, phospholipids, photons, odorants and de®ned roles in intermediary metabolism and generally purine nucleotides (Dohlman et al., 1987). At least four perform these functions in well-dierentiated cells. distinct mechanisms have been described through However, there is an increasing awareness that some which ligands interact with their corresponding GPCRs can also regulate proliferative signaling path- GPCRs. Biogenic amines bind within hydrophilic ways and that chronic stimulation or mutational pockets that are formed by coordinated interactions activation of receptors can lead to oncogenic transforma- between the extracellular loops of their receptors. tion. Activating mutations in GPCRs are associated with Peptide hormones also bind within such pockets but several types of human tumors and some receptors require additional interactions with extracellular resi- exhibit potent oncogenic activity due to agonist over- dues. Ligands such as glycoproteins, hormones or expression. Additionally, expression screening analyses glutamate bind directly to the amino terminus of the for novel oncogenes identi®ed GPCRs whose expression receptor, and then the ligand receptor complex folds causes the oncogenic transformation of NIH3T3 mouse into the hydrophilic pocket. Finally, thrombin cleaves ®broblasts. These include Mas, G2A, and the PAR-1 the amino terminus of its receptor, Par-1, thus thrombin receptor. In this review we summarize the releasing a tethered ligand that causes receptor signaling and transforming properties of these GPCR activation. Regardless of the mechanism of ligand oncoproteins. What has emerged from these studies is the stimulation, engagement of a GPCR by its ligand delineation of a GTPase cascade where transforming generally leads to activation of an associated member GPCRs cause aberrant growth regulation via activation of the heterotrimeric G protein family. of Rho family small GTPases. Oncogene (2001) 20, GPCRs associate with members of the heterotrimeric 1547 ± 1555. G protein family that are anchored to the intracellular surface of the plasma membrane (Bourne, 1997). These Keywords: gene transfer assays; Dbl family proteins G proteins consist of an a subunit (Ga) that binds guanine nucleotides, and a dimer that consists of a b and g subunit (Gbg). The intracellular signaling events GPCRs and heterotrimeric G proteins that are triggered by the activation of heterotrimeric G proteins is determined by the particular combination of Members of the GPCR family are characterized by a Ga and Gbg subunits that are present in the complex, tertiary structure that consists of seven transmembrane and the availability of speci®c eectors and regulatory domains that are connected by a series of extracellular proteins. The GPCR interacts with the G protein and intracellular loops (Dohlman et al., 1987). The through multiple contacts, with the most critical amino terminus of the receptor is extracellular and residues being located in the third cytoplasmic loop often contributes to ligand recognition and binding, and in the membrane proximal portion of the while the intracellular carboxy terminus contributes to cytoplasmic tail. eector binding and to the propagation of signaling In the absence of ligand, the GPCR associates with events. The transmembrane regions, which represent an inactive GDP-bound complex. Upon ligand engage- the most highly conserved domains within the ment, the GPCR assumes an active con®guration and structure, consist of 20 ± 25 amino acid stretches that functions as a guanine nucleotide exchange factor are predicted to form alpha helices. Typically, GPCRs (GEF), catalyzing the exchange of GDP for GTP on contain at least one N-linked glycosylation site in the the Ga subunit. Upon conversion to the active GTP- bound form, the Ga subunit will dissociate from the Gbg subunit and each component is free to elicit a distinct array of eector functions. For example, *Correspondence: IP Whitehead activated Ga subunits have been shown to be both G protein-couple receptor oncoproteins and Rho GTPases IP Whitehead et al 1548 activators and inhibitors of adenylyl cyclases, or have ®broblasts (Gupta et al., 1992; Pace et al., 1991). The been shown to directly activate phospholipase C. Gbg wild type Ga12 subunit (gep oncogene) was isolated in subunits have also been shown to activate adenylyl a screen of a soft tissue sarcoma library for proteins cyclases or phospholipase C, but there is also evidence whose expression cause focus formation in NIH3T3 that they can activate Ras-mediated signaling pathways cells (Chan et al., 1993). However, subsequent analysis (Crespo et al., 1994; Koch et al., 1994), although the has failed to establish that Ga12 overexpression is nature of this activating event remains somewhat responsible for the original malignancy. obscure. Experimental mutation of Ga subunits that render them GTPase-de®cient (with Q to L mutations analogous to the oncogenic Q61L mutation of Ras Oncogenic potential of GPCRs and heterotrimeric G proteins) has also revealed the growth-promoting proteins activity of Ga12,Ga13,Gai2,Gaq, and Gao (reviewed in Dhanasekaran et al. (1998)). Of these, the QL Although GPCRs have been best characterized for the mutants of the Ga12/Ga13 family have exhibited the speci®c role that many play in regulating diverse greatest transforming potency (Jiang et al., 1993; Vara metabolic processes, it is becoming increasingly clear Prasad et al., 1994; Voyno-Yasenetskaya et al., 1994; that members of this family can regulate proliferative Xu et al., 1993). Taken together, these examples signaling pathways (Coso et al., 1995a; Gutkind, 1998; provide a persuasive body of evidence linking the van Biesen et al., 1996). These include the serotonin 1C deregulated activation of GPCRs, and their corre- receptors (Julius et al., 1989) and the M1,M3 and M5 sponding G proteins, to human malignancies. subtypes of the muscarinic acetylcholine receptors (Gutkind et al., 1991) that cause agonist-dependent transformation of NIH3T3 cells. Point mutations Expression cloning for novel oncogenes: identi®cation of render the a1B-adrenergic receptor activated and Mas, G2A, and PAR-1 transforming in a ligand-independent fashion (Allen et al., 1991). Activating mutations in GPCRs are also Adding to the body of evidence that members of the known to be associated with several types of human GPCR family may represent important oncoproteins tumors. Mutations in the thyroid-stimulating hormone has been the identi®cation of GPCRs in biological receptor have been identi®ed in 30% of hyperfunction- screens for proteins whose expression can cause the ing thyroid adenomas and similar mutations in the oncogenic transformation of murine ®broblasts. We luteinizing hormone receptor are associated with male describe the identi®cation and characterization of three precocious puberty (Parma et al., 1993; Shenker et al., transforming GPCRs, Mas, G2A, and Par-1, that were 1993). Activation of both of these receptor types leads identi®ed in these gene transfer analyses. to increased cellular concentrations of cAMP, and they The identi®cation of the mas oncogene by Wigler are transforming in cell types in which cAMP functions and colleagues provided the ®rst direct evidence for the as a mitogen. oncogenic potential of GPCRs (Young et al., 1986). There is also evidence that GPCRs may contribute The mas oncogene was originally isolated from a to tumorigenesis via paracrine and autocrine mechan- human epidermoid carcinoma genomic DNA library isms. For example, small cell lung carcinomas secrete and identi®ed by its ability to render NIH3T3 cells high levels of gastrin-reducing peptide (GRP) and GRP tumorigenic in nude mice. The mas gene isolated in this receptor antagonists can block the growth of these cells screen contained rearrangements in its 5' noncoding both in vitro and in vivo (Cuttitta et al., 1985; sequences, but not in its coding sequence. Thus, mas Mahmoud et al., 1991). Gastric cancers also secrete transforming activity is mediated by overexpression GPCR ligands such as GRP and gastrin, colon and and not mutational activation. Mas transcripts and ovarian cancer patients exhibit elevated levels of protein are expressed primarily in the central nervous gastrin and lysophosphatidic acid (LPA) in their serum system. High levels of mas transcripts were detected in (Seitz et al., 1991; Xu et al., 1995). the cerebral cortex,
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