SH2-CONTAINING PROTEINS AND PDGF SIGNALING

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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.