Desensitization of Vascular Receptor Tyrosine Kinases

描述（由申请人提供）：动脉粥样硬化是工业化国家的主要死亡原因，其基本上涉及通过受体蛋白酪氨酸激酶（RPTK）的细胞信号传导。RPTKs是平滑肌细胞增殖和迁移所必需的，不仅是动脉粥样硬化的特征，也是血管发生的特征。RPTK可被丝氨酸/苏氨酸激酶负调控，并且RPTK血小板衍生生长因子受体-B（PDGFRB）最近已被证明被G蛋白偶联受体激酶（GRK）-2和-5磷酸化和脱敏，GRK-2和-5是对七螺旋受体脱敏重要的Ser/Thr激酶家族的成员。这个项目的目标是定义GRKs脱敏RPTKs的机制。因此，该提议测试了以下假设：（1）GRK介导的PDGFRP在特定丝氨酸上的磷酸化改变了PDGFRB与各种调节衔接蛋白、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GRK使RPTK脱敏，从而减少动脉粥样硬化。为了确定GRK介导的PDGFRB磷酸化是否会影响PDGFRB与重要调控蛋白的结合，本项目将使用免疫共沉淀方法与表达生理GRK水平、通过RNA干扰降低GRK水平或不表达GRK的匹配细胞系进行比较。为了测试RPTKs的范围，可能是由GRKs调节，这项工作将检查激动剂诱导的丝氨酰磷酸化和酪氨酰自磷酸化的RPTKs重要的心血管生理学，使用平滑肌细胞或成纤维细胞来源于GRK 2-，GRK 5-，或GRK 6-敲除小鼠，或同源GRK+/+同窝对照小鼠。GRK介导的RPTK脱敏将从GRK缺陷的RPTK自磷酸化的增强中推断，与GRK表达细胞相比。证明GRK介导的脱敏的RPTK也将在体外用纯化的GRK测定GRK介导的磷酸化。为了确定哪些PDGFRB丝氨酸被GRKS磷酸化，该项目采用蛋白水解/质谱/微测序方法。本项目通过比较GRK 5 +/+/apoE +/+小鼠与同类GRK 5 +/+/apoE +/+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。对心血管疾病的治疗意义可能来自于确定GRKs如何调节RPTK信号传导，以及哪些RPTKs受到GRK介导的调节。

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.