The chief cause of death in the industrialized world, atherosclerosis fundamentally involves cellular signaling through receptor protein tyrosine kinases (RPTKs). RPTKs are required for the smooth muscle cell proliferation and migration that characterize not only atherosclerosis, but also vasculogenesis. RPTKs can be negatively regulated by serine/threonine kinases, and the RPTK platelet-derived growth factor receptor-B (PDGFRB) has recently been shown to be phosphorylated and desensitized by G protein-coupled receptor kinases (GRKs) -2 and -5, members of a Ser/Thr kinase family important for desensitization of heptahelical receptors. The goal of this project is to define mechanisms by which GRKs desensitize RPTKs. Accordingly, this proposal tests the following hypotheses: (1) that GRK-mediated phosphorylation of the PDGFRP on specific serine(s) alters the interaction between the PDGFRB and various regulatory adaptor proteins, phosphatases, or enzymes;(2) that GRK-mediated RPTK phosphorylation is a general mechanism for RPTK desensitization;(3) that smooth muscle cell GRKs desensitize RPTKs and thereby reduce atherosclerosis. To determine whether GRK-mediated phosphorylation of the PDGFRB affects the association of the PDGFRB with important regulatory proteins, this project will use a co-immunoprecipitation approach with matched cell lines expressing either physiologic GRK levels, GRK levels reduced by RNA interference, or no GRK. To test the range of RPTKs that may be regulated by GRKs, this work will examine agonist-induced seryl phosphorylation and tyrosyl autophosphorylation in RPTKs important to cardiovascular physiology, using smooth muscle cells or fibroblasts derived from GRK2-, GRK5-, or GRK6-knockout mice, or from cognate GRK+/+ littermate control mice. GRK-mediated RPTK desensitization will be inferred from augmentation of RPTK autophosphorylation in GRK-deficient, compared with GRK-expressing cells. RPTKs that demonstrate GRK-mediated desensitization will also be assayed for GRK-mediated phosphorylation, with purified GRKs in vitro. To determine which PDGFRB serine(s) is (are) phosphorylated by GRKS, this project employs a proteolysis/mass spectrometry/microsequencing approach. This project tests the role of GRKS in atherosclerosis by comparing the extent of atherosclerosis in GRKS^VapoE""""""""7"""""""" mice with that in congenic GRK5+/+/apoE""""""""A mice. Therapeutic implications for cardiovascular diseases may derive from determining how GRKs regulate RPTK signaling, and which RPTKs are subject to GRK-mediated regulation.
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