The objective of this proposal is to identify and characterize GTP-binding regulatory proteins (G proteins) relevant to cell proliferation. G proteins serve often obligatory roles in signal transduction, the process by which information is conveyed from cell-surface receptors to target enzymes or channels. Several growth factors utilize G proteins as perhaps the sole means by which their mitogenic actions are exerted, while others act in synergy through conjoint use of both G protein-dependent and - independent pathways. Although G proteins serve fundamental roles in proliferation, the identities of those actually communicating with receptors for growth factors or those serving as targets for downstream modification are unclear. The functions of such G proteins, moreover, are poorly understood. Four experimental goals are proposed. First, G proteins that interact directly with, or in some fashion are activated by, receptors for growth factors will be identified. Strategies include co- immunoprecipitation of receptor.G protein complexes, photoaffinity labeling with [32P]gamma-azidoanilido GTP, and measurements of growth factor-altered GDP/GTP ratios. The focus in this and subsequent goals will be placed on G proteins relevant to thrombin and bombesin/gastrin- releasing peptide (GRP), two widely recognized mitogens that operate through heptahelical receptors and hence G proteins. Second, the subset of G proteins whose activation or modification is specifically required for cell replication will be identified. The experimental focus will be reinitiation of DNA synthesis by thrombin and GRP in quiescent cells in monolayer culture. Strategies include microinjection of antibodies that disrupt communication of G proteins with receptors or targets, the use of pertussis toxin-resistant analogues of G proteins, and dominant negative modulation. Third, the array of G proteins utilized by growth factors will be examined relative to those used by other agonists, and specific roles for the G proteins relevant to mitogenesis will be sought. Questions include what - as defined at the level of G proteins utilized and subsequent downstream events - identifies an agonist as mitogenic, and what are the molecular events by which G proteins achieve activation of MAP kinases. Fourth, the extent to which G proteins are subject to post- translational modification upon transformation and in relation to reinitiation of DNA synthesis will be examined. Two modifications recently demonstrated for G protein alpha subunits, tyrosine phosphorylation and palmitoylation, will constitute the focus. An understanding of G proteins in the context of growth factor action will provide substantial insight into the identities and coordination of pathways relevant to cell replication.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Pharmacology A Study Section (PHRA)
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University of Pennsylvania
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Siehler, Sandra; Manning, David R (2002) Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors. Biochim Biophys Acta 1582:94-9
Chen, C A; Manning, D R (2001) Regulation of G proteins by covalent modification. Oncogene 20:1643-52
Windh, R T; Lee, M J; Hla, T et al. (1999) Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins. J Biol Chem 274:27351-8
Shahrestanifar, M; Fan, X; Manning, D R (1999) Lysophosphatidic acid activates NF-kappaB in fibroblasts. A requirement for multiple inputs. J Biol Chem 274:3828-33
Cowen, D S; Molinoff, P B; Manning, D R (1997) 5-hydroxytryptamine1A receptor-mediated increases in receptor expression and activation of nuclear factor-kappaB in transfected Chinese hamster ovary cells. Mol Pharmacol 52:221-6
Barr, A J; Brass, L F; Manning, D R (1997) Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling. J Biol Chem 272:2223-9
Barr, A J; Manning, D R (1997) Agonist-independent activation of Gz by the 5-hydroxytryptamine1A receptor co-expressed in Spodoptera frugiperda cells. Distinguishing inverse agonists from neutral antagonists. J Biol Chem 272:32979-87
Cowen, D S; Sowers, R S; Manning, D R (1996) Activation of a mitogen-activated protein kinase (ERK2) by the 5-hydroxytryptamine1A receptor is sensitive not only to inhibitors of phosphatidylinositol 3-kinase, but to an inhibitor of phosphatidylcholine hydrolysis. J Biol Chem 271:22297-300
Butkerait, P; Zheng, Y; Hallak, H et al. (1995) Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits. J Biol Chem 270:18691-9