Over the past decade, it has become apparent that many G protein-coupled receptors (GPCRs) transmit signals that influence cellular differentiation and growth, including stimulation of Ras family GTPases and activation of mitogen-activated protein (MAP) kinase pathways. Many common clinical conditions, incIuding pressure overload cardiac hypertrophy, myocardial fibrosis, neointimal hyperplasia of vascular smooth muscle, anabolic bone remodeling, and prostate hypertrophy have been shown to involve these GPCR-mediated signals. Prior study of the mechanisms that GPCRs use to control the activity of tyrosine protein kinases and regulate the ERK1/2 MAP kinase cascade has revealed that these responses are often the result of novel signaling events, such as the formation of complexes between GPCRs, Src family tyrosine kinases, and beta-arrestin-bound MAP kinases, and cross talk between GPCRs and classical receptor tyrosine kinases. Emerging data suggest that different signaling mechanisms lead to the formation of spatially separate and functionally distinct MAP kinase pools. This application has two broad goals. The first is to characterize in detail the molecular mechanisms underlying GPCR-mediated ERK activation via beta-arrestin scaffolds and transactivated epidermal growth factor (EGF) receptors. The second goal is understand how these novel signals are integrated to determine the cellular response to GPCR activation. The first specific aim of the proposal is to determine, using wild type and mutant GPCRs in cellular models systems, the mechanism of ERK activation on beta-arrestin scaffolds, including the role of heterotrimeric G protein subunits. G protein effectors, Src kinases, and Ras GTPases. The second specific aim is to determine, using a novel mixed cell assay system consisting of ligand donor and ligand accepter cell populations, as well as direct assays of GPCR-stimulated heparin-binding EGF release, the mechanisms used by GPCRs to control matrix metalloprotease-dependent release of EGF receptor ligands, and to characterize mechanisms of metalloprotease-independent EGF receptor transactivation.
The third aim i s to test the hypothesis that the consequences of GPCR-stimulated ERK kinase activation are determined by the mechanism of activation. These experiments will employ endogenously-expressed GPCRs in beta-arrestin knockout fibroblasts, into which beta-arrestin expression has or has not been stably restored, to determine the role of beta-arrestin scaffolds and EGF receptor transactivation in controlling the phosphorylation of specific cytosole, membrane and nuclear ERK substrates, and the transcriptional response to GPCR stimulation.
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