SH2-CONTAINING PROTEINS AND PDGF SIGNALING

血小板衍生生长因子（PDGF）与其受体（PDGFR）结合 导致特异性PDGFR酪氨酸残基的磷酸化， 将含有src同源性2（SH 2）的蛋白募集到这些细胞中， 磷酸酪氨酸位点。 这些SH 2蛋白指导细胞内 介导PDGF趋化和促有丝分裂作用的反应，事件 与动脉粥样硬化和再狭窄有关。 SH 2蛋白结合到 PDGFR及其特异性靶酪氨酸（Y）位点是已知的。 之间 GT3激活蛋白（Y 771）、磷酸肌醇-3激酶 (740/751）、磷脂酶C γ 1021）和SHPTP 2（γ 1009）。 试图 了解PDGFR信号传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 转化到可能缺乏下游效应子的非表达细胞中， 由于内源性受体不能将受体引入天然细胞 背景，以及PDGF同种型和PDGFR亚基之间的重叠。 为了解决这些限制，申请人提出产生突变体 用于转染间充质细胞的嵌合PDGFR。 奇美拉会 由集落刺激因子-1（CSF-1）的胞外结构域组成， 受体融合到PDGFR的胞内结构域（CPDR）。 先前 工作已经建立了使用“加回”突变体在研究PDGF 发信号。 酪氨酸被取代的突变嵌合PDGFR（CPF 5 R） 在已知的SH 2结合位点（740，751，771，1009，1021）的苯丙氨酸将 生成。 从该突变体中，得到四个克隆，每个克隆具有一个酪氨酸位点， 将允许结合特定的SH 2蛋白。 通过使用CSF-1 作为配体，可以实现特异性突变体PDGFR激活，而不需要 内源性受体刺激 使用间充质细胞将确保 与正常PDGF通路的生理相关性。 转染这些 构建体、CSF-1刺激和下游效应（蛋白质）分析 研究，促有丝分裂，趋化性，细胞骨架重排）应 允许解剖这些复杂的信号通路。 数据的日期及时间为 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.