Desensitization of Vascular Receptor Tyrosine Kinases

描述(由申请人提供)：动脉粥样硬化是工业化世界的主要死亡原因，从根本上讲，它涉及到通过受体蛋白酪氨酸激酶(RPTKs)传递细胞信号。RPTK是平滑肌细胞增殖和迁移所必需的，这不仅是动脉粥样硬化的特征，也是血管生成的特征。RPTKs可以被丝氨酸/苏氨酸激酶负性调节，而RPTK血小板衍生生长因子受体B(PDGFRb)最近被证明被G蛋白偶联受体激酶(GRKs)-2和-5磷酸化和脱敏，GRKs-2和-5是丝氨酸/苏氨酸激酶家族的成员，在七螺旋受体的脱敏中起重要作用。该项目的目标是确定GRK使RPTK脱敏的机制。因此，这一建议检验了以下假设：(1)GRK介导的PDGFRP在特定丝氨酸上的磷酸化(S)改变了PDGFRb与各种调节接头蛋白、磷酸酶或酶之间的相互作用；(2)GRK介导的RPTK磷酸化是RPTK脱敏的一般机制；(3)平滑肌细胞GRKs使RPTKs脱敏，从而减少动脉粥样硬化。为了确定GRK介导的PDGFRb磷酸化是否影响PDGFRb与重要调控蛋白的联系，该项目将使用免疫共沉淀方法，与表达生理性GRK水平、通过RNA干扰降低GRK水平或不表达GRK的匹配细胞株进行比较。为了测试可能受GRKs调控的RPTK的范围，这项工作将使用来自GRK2-、GRK5-或GRK6-基因敲除小鼠的平滑肌细胞或成纤维细胞，或来自GRK+/+的同源对照小鼠，检测激动剂诱导的RPTK对心血管生理学重要的RPTK的丝氨酸磷酸化和酪氨酸自磷酸化。与表达GRK的细胞相比，GRK介导的RPTK脱敏作用将从GRK缺陷细胞中RPTK自身磷酸化的增强来推断。证明GRK介导的脱敏的RPTKs也将在体外用纯化的GRKs检测GRK介导的磷酸化。为了确定哪些PDGFRb丝氨酸(S)被GRKS磷酸化，该项目采用了蛋白质分解/质谱学/显微测序的方法。本项目通过比较GRKS、VapoE“7”小鼠和GRK5+/+/apoE“A小鼠的动脉粥样硬化程度来测试GRKS在动脉粥样硬化中的作用。通过确定GRKS如何调节RPTK信号以及哪些RPTK受到GRK介导的调节，可能对心血管疾病的治疗有意义。

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.