SH2-CONTAINING PROTEINS AND PDGF SIGNALING

血小板衍生生长因子与其受体结合 导致特定的PDGFR酪氨酸残基的磷酸化和 含有(SH2)蛋白的src同源2在这些细胞中的募集 磷酸酪氨酰基位置。这些SH2蛋白引导细胞内 介导PDGF趋化和有丝分裂作用的反应、事件 与动脉粥样硬化和再狭窄有关。与SH2结合的蛋白 PDGFR及其特定的目标酪氨酸(Y)位点是已知的。其中 它们是GTP酶激活蛋白(Y 771)、磷酸肌醇-3激酶 (740/751)，磷脂酶CGamma 1021)和SHPTP2(Gamma 1009)。试图 了解PDGFR信号转导受到几个因素的限制； 这些次级信号分子的重叠效应，转染法 转化为可能缺乏下游效应器的非表达细胞， 由于内源性受体而不能在天然细胞中引入受体 背景，以及PDGF亚型和PDGFR亚基之间的重叠。 为了解决这些限制，申请人提议产生突变 嵌合PDGFRs在骨髓间充质细胞中的转染。嵌合体将会 由克隆刺激因子-1(CSF-1)的胞外区组成 受体融合到PDGFR的胞内区(CpdR)。上一首 研究已经确定了在研究血小板衍生生长因子时使用“加回”突变体 发信号。酪氨酸取代的突变嵌合PDGFR(CPF5R) 已知SH2结合位点(740、751、771、1009、1021)的苯丙氨酸将 将被生成。在这个突变体中，有四个克隆，每个克隆都有一个酪氨酸位点 添加回来，将允许结合特定的SH2蛋白。通过使用CSF-1 作为配体，特异性突变体PDGFR的激活可以在没有 内源性受体刺激。使用间充质细胞将确保 生理上与正常的PDGF通路相关。这些基因的转染法 构建、CSF-1刺激和下游效应分析(蛋白质 研究、有丝分裂、趋化性、细胞骨架重排)应 允许解剖这些复杂的信号通路。调查结果将是 应用于动脉粥样硬化和再狭窄动物模型的研究。

Platelet-derived growth factor (PDGF) binds to its receptor (PDGFR) resulting in phosphorylation of specific PDGFR tyrosine residues and recruitment of src homology 2 containing (SH2) proteins to these phosphotyrosyl sites. These SH2 proteins direct the intracellular reactions mediating PDGF's chemotactic and mitogenic effects, events implicated in atherosclerosis and restenosis. The SH2 proteins binding to the PDGFR and their specific target tyrosine (Y) sites are known. Among them are GTPase activating protein (Y 771), phosphoinositol-3 kinase (740/751), phosplipase CGamma 1021), and SHPTP2 (Gamma 1009). Attempts to understand PDGFR signaling have been limited by several factors; overlapping effects of these secondary signaling molecules, transfection into nonexpressing cells potentially lacking downstream effectors, inability to introduce receptors in native cells due to endogenous receptor background, and overlap between PDGF isoforms and PDGFR subunits. To resolve these limitations, the applicant proposes to generate mutant chimeric PDGFRs for transfection in mesenchymal cells. The chimera will consist of the extracellular domain of colony stimulating factor-1 (CSF-1) receptor fused to the intracellular domain of the PDGFR (CPDR). Previous work has established the use of "add back" mutants in studying PDGF signaling. A mutant chimeric PDGFR (CPF5R) with tyrosine replaced by phenylalanine at known SH2 binding sites (740, 751, 771, 1009, 1021) will be generated. From this mutant, four clones, each with one tyrosine site added back, will allow binding of a specific SH2 protein. By using CSF-1 as the ligand, specific mutant PDGFR activation can be achieved without endogenous receptor stimulation. Use of mesenchymal cells will ensure physiologic relevance to normal PDGF pathways. Transfection of these constructs, CSF-1 stimulation and analyses for downstream effects (protein studies, mitogenicity, chemotaxis, cytoskeletal rearrangements) should allow dissection of these complex signal pathways. Findings will be applied to studies in animal models of atherosclerosis and restenosis.