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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL077185-05
Application #
7652397
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Srinivas, Pothur R
Project Start
2005-08-01
Project End
2012-01-31
Budget Start
2009-08-01
Budget End
2012-01-31
Support Year
5
Fiscal Year
2009
Total Cost
$369,790
Indirect Cost
Name
Duke University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Wu, Jiao-Hui; Fanaroff, Alexander C; Sharma, Krishn C et al. (2013) Kalirin promotes neointimal hyperplasia by activating Rac in smooth muscle cells. Arterioscler Thromb Vasc Biol 33:702-8
Lu, Yuan; Zhang, Lisheng; Liao, Xudong et al. (2013) Kruppel-like factor 15 is critical for vascular inflammation. J Clin Invest 123:4232-41
Zhang, Lisheng; Brian, Leigh; Freedman, Neil J (2012) Vein graft neointimal hyperplasia is exacerbated by CXCR4 signaling in vein graft-extrinsic cells. J Vasc Surg 56:1390-7
Wu, Jiao-Hui; Zhang, Lisheng; Fanaroff, Alexander C et al. (2012) G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors. Arterioscler Thromb Vasc Biol 32:308-16
Han, Sang-oh; Xiao, Kunhong; Kim, Jihee et al. (2012) MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound ?(2)-adrenergic receptors. J Cell Biol 199:817-30
Zhang, Lisheng; Connelly, Jessica J; Peppel, Karsten et al. (2010) Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies. Hum Mol Genet 19:2754-66
Brown, Melissa A; Zhang, Lisheng; Levering, Vrad W et al. (2010) Human umbilical cord blood-derived endothelial cells reendothelialize vein grafts and prevent thrombosis. Arterioscler Thromb Vasc Biol 30:2150-5
Zhu, Shoukang; Malhotra, Anuj; Zhang, Lisheng et al. (2010) Human umbilical cord blood endothelial progenitor cells decrease vein graft neointimal hyperplasia in SCID mice. Atherosclerosis 212:63-9
Cai, Xinjiang; Wu, Jiao-Hui; Exum, Sabrina T et al. (2009) Reciprocal regulation of the platelet-derived growth factor receptor-beta and G protein-coupled receptor kinase 5 by cross-phosphorylation: effects on catalysis. Mol Pharmacol 75:626-36
Kim, Jihee; Zhang, Lisheng; Peppel, Karsten et al. (2008) Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration. Circ Res 103:70-9

Showing the most recent 10 out of 12 publications