Desensitization of Vascular Receptor Tyrosine Kinases

Summary

Principal Investigator: Neil Freedman
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 2005-08-01 - 2010-07-31
more information: NIH RePORT

Top Publications

  1. pmc Kruppel-like factor 15 is critical for vascular inflammation
    Yuan Lu
    J Clin Invest . 2013
  2. pmc Kalirin promotes neointimal hyperplasia by activating Rac in smooth muscle cells
    Jiao Hui Wu
    Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
    Arterioscler Thromb Vasc Biol 33:702-8. 2013
  3. pmc MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β(2)-adrenergic receptors
    Sang oh Han
    Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
    J Cell Biol 199:817-30. 2012
  4. pmc Vein graft neointimal hyperplasia is exacerbated by CXCR4 signaling in vein graft-extrinsic cells
    Lisheng Zhang
    Department of Medicine Cardiology, Duke University Medical Center, Durham, NC 27710, USA
    J Vasc Surg 56:1390-7. 2012
  5. pmc G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors
    Jiao Hui Wu
    Box 3187, Duke University Medical Center, Durham, NC 27710, USA
    Arterioscler Thromb Vasc Biol 32:308-16. 2012
  6. pmc Human umbilical cord blood-derived endothelial cells reendothelialize vein grafts and prevent thrombosis
    Melissa A Brown
    Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
    Arterioscler Thromb Vasc Biol 30:2150-5. 2010
  7. pmc Human umbilical cord blood endothelial progenitor cells decrease vein graft neointimal hyperplasia in SCID mice
    Shoukang Zhu
    Miller School of Medicine, University of Miami, FL 33101, USA
    Atherosclerosis 212:63-9. 2010
  8. pmc Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies
    Lisheng Zhang
    Department of Medicine Cardiology, Duke University Medical Center, Durham, NC, USA
    Hum Mol Genet 19:2754-66. 2010
  9. pmc Reciprocal regulation of the platelet-derived growth factor receptor-beta and G protein-coupled receptor kinase 5 by cross-phosphorylation: effects on catalysis
    Xinjiang Cai
    Departments of Medicine Cardiology, Duke University Medical Center, Durham, North Carolina, USA
    Mol Pharmacol 75:626-36. 2009
  10. pmc Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration
    Jihee Kim
    Department of Medicine Cardiology, Duke University Medical Center, Durham, NC 27710, USA
    Circ Res 103:70-9. 2008

Detail Information

Publications12

  1. pmc Kruppel-like factor 15 is critical for vascular inflammation
    Yuan Lu
    J Clin Invest . 2013
    ..These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation...
  2. pmc Kalirin promotes neointimal hyperplasia by activating Rac in smooth muscle cells
    Jiao Hui Wu
    Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
    Arterioscler Thromb Vasc Biol 33:702-8. 2013
    ..We therefore tested the hypothesis that Kalirin functions as a Rho-guanine nucleotide exchange factor in arterial smooth muscle cells (SMCs)...
  3. pmc MARCH2 promotes endocytosis and lysosomal sorting of carvedilol-bound β(2)-adrenergic receptors
    Sang oh Han
    Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA
    J Cell Biol 199:817-30. 2012
    ..In response to β blocker therapy with carvedilol, MARCH2 E3 ligase activity regulates cell surface β(2)AR expression and, consequently, its signaling...
  4. pmc Vein graft neointimal hyperplasia is exacerbated by CXCR4 signaling in vein graft-extrinsic cells
    Lisheng Zhang
    Department of Medicine Cardiology, Duke University Medical Center, Durham, NC 27710, USA
    J Vasc Surg 56:1390-7. 2012
    ....
  5. pmc G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors
    Jiao Hui Wu
    Box 3187, Duke University Medical Center, Durham, NC 27710, USA
    Arterioscler Thromb Vasc Biol 32:308-16. 2012
    ..This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis...
  6. pmc Human umbilical cord blood-derived endothelial cells reendothelialize vein grafts and prevent thrombosis
    Melissa A Brown
    Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA
    Arterioscler Thromb Vasc Biol 30:2150-5. 2010
    ....
  7. pmc Human umbilical cord blood endothelial progenitor cells decrease vein graft neointimal hyperplasia in SCID mice
    Shoukang Zhu
    Miller School of Medicine, University of Miami, FL 33101, USA
    Atherosclerosis 212:63-9. 2010
    ..We sought to determine whether exogenous endothelial progenitor cells could promote vein graft re-endothelialization, and thereby ameliorate neointimal hyperplasia...
  8. pmc Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies
    Lisheng Zhang
    Department of Medicine Cardiology, Duke University Medical Center, Durham, NC, USA
    Hum Mol Genet 19:2754-66. 2010
    ..We conclude that TNFR1 polymorphisms associate with aging-related CAD in humans, and TNFR1 contributes to aging-dependent atherosclerosis in mice...
  9. pmc Reciprocal regulation of the platelet-derived growth factor receptor-beta and G protein-coupled receptor kinase 5 by cross-phosphorylation: effects on catalysis
    Xinjiang Cai
    Departments of Medicine Cardiology, Duke University Medical Center, Durham, North Carolina, USA
    Mol Pharmacol 75:626-36. 2009
    ..We conclude that GRK5 tyrosyl phosphorylation is required for the activation of GRK5 by the PDGFRbeta, but not by the beta(2)-adrenergic receptor, and that by activating GRK5, the PDGFRbeta triggers its own desensitization...
  10. pmc Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration
    Jihee Kim
    Department of Medicine Cardiology, Duke University Medical Center, Durham, NC 27710, USA
    Circ Res 103:70-9. 2008
    ..These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty...
  11. ncbi Regulation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-5 in vascular smooth muscle cells involves the phosphatase Shp2
    Jiao Hui Wu
    Department of Medicine Cardiology, Duke University, Medical Center, Durham, North Carolina 27710, USA
    J Biol Chem 281:37758-72. 2006
    ....
  12. ncbi The platelet-derived growth factor receptor-beta phosphorylates and activates G protein-coupled receptor kinase-2. A mechanism for feedback inhibition
    Jiao Hui Wu
    Department of Medicine Cardiology, Duke University Medical Center, Durham, North Carolina 27710, USA
    J Biol Chem 280:31027-35. 2005
    ..We conclude that the activated PDGFRbeta itself phosphorylates GRK2 tyrosyl residues and thereby activates GRK2, which then serine-phosphorylates and desensitizes the PDGFRbeta...