The purpose of these studies is to analyze molecular mechanisms by which insulin like growth factor 1 (IGF-1) stimulates smooth muscle cell (SMC) replication and migration. SMC synthesize IGF binding proteins (IGFBPs)2, 4 and 5 and these IGFBPs have been shown to modulate IGF-1 bioactivity. IGF-1 abundance in atherosclerotic lesions is determined by factors that regulate its synthesis but also by factors that regulate the abundance of the IGFBPs. These include proteolysis and adherence to extracellular matrix (ECM). Adherence to ECM results in a lowering of the IGFBP affinity for IGF-1 thus making it more readily accessible to receptors and this results in potentiation of IGF-1 actions. These studies will focus on defining how the GFBPs that are present in the SMC pericellular space control IGF-1 actions. An IGFBP-4 protease will be purified sequenced and cDNA clones prepared. The pure protease will be used to study how it functions to modify IGFBP-4 actions. The sites of proteolysis within IGFBP-4 will be identified and protease resistant mutants prepared. The mutants will be analyzed for their ability to inhibit IGF-1 effects on SMC replication. The specific sites in IGFBP-5 that mediate its binding to ECM proteins, such as plasminogen activator inhibitor-1, will be determined then these sites altered to create IGFBP-5 mutants that bind poorly to ECM. The ECM binding and proteolytic cleavage sites within IGFBP-5 will be mutated to create mutants that are selectively concentrated in the ECM or selectively increased in the culture media and the role of interstitial fluid IGFBP-5 as compared to ECM IGFBP-5 in modulating IGF-1 activity on this cell type will be analyzed. Since avB3 integrin receptor occupancy by ligands such as vitronectin appears to be required for the optimal cellular migration response to IGF-1, the role of binding of an IGF-1 stimulated transduction element (IRS-2) to avB3 in controlling SMC migration will be determined. The region of the avB3 subunit of the avB3 receptor that is necessary for IRS-2 binding will be identified and mutagenized to determine if SMC that have been transfected with this mutant can migrate in response to IGF-1. Similarly the sites in IRS-2 that are phosphorylated by the IGF-1 receptor will be determined and mutagenized to determine if blocking IRS-2 phosphorylation also results in a loss of the ability of IGF-1 to stimulate migration. The results of these studies should define molecular mechanisms by which IGF-1 functions coordinately with the IGFBPs and with the avBb3 receptor to stimulate SMC replication or migration and may suggest novel strategies for interfering with these processes to alter the progression of atherogenesis.
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