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，主要涉及通过受体蛋白酪氨酸激酶（RPTKs）传递细胞信号。RPTKs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，也是血管形成的特征。RPTK可被丝氨酸/苏氨酸激酶负调控，而RPTK血小板衍生生长因子受体- b （PDGFRB）最近被G蛋白偶联受体激酶(GRKs) -2和-5磷酸化和脱敏，这是丝氨酸/苏氨酸激酶家族的成员，对七螺旋受体的脱敏很重要。该项目的目标是确定GRKs使RPTKs脱敏的机制。因此，该提案验证了以下假设：(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磷酸化是否会影响PDGFRB与重要调节蛋白的关联，该项目将使用免疫共沉淀方法，对表达生生性GRK水平、RNA干扰降低的GRK水平或不表达GRK的匹配细胞系进行免疫共沉淀。为了测试可能受GRKs调控的RPTKs的范围，本研究将使用GRK2-、GRK5-或grk6基因敲除小鼠或同源GRK+/+同胎对照小鼠的平滑肌细胞或成纤维细胞，研究激动剂诱导的对心血管生理重要的RPTKs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脱敏可以从grk缺陷细胞中RPTK自磷酸化的增强中推断出来。显示grk介导脱敏的RPTKs也将在体外用纯化的grk检测grk介导的磷酸化。为了确定哪些PDGFRB丝氨酸被GRKS磷酸化，本项目采用蛋白水解/质谱/微测序方法。本项目通过比较GRKS^VapoE“7”小鼠与同源GRK5+/+/apoE“A小鼠的动脉粥样硬化程度，来检验GRKS在动脉粥样硬化中的作用。心血管疾病的治疗意义可能源于确定grk如何调节RPTK信号，以及哪些RPTK受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.