One of the most challenging problems in biology is to understand how cell growth is controlled. The overall objective of the work proposed here is to understand how the peptide hormones insulin and insulin-like growth factor I (IGFI) modulate cellular effects such as cell proliferation by interacting with specific cell surface receptors. The receptors for insulin (IR) and IGFI(IGFIR) are structurally related cell surface receptors whose function is dependent upon hormone-activated intrinsic receptor tyrosine kinase activity. The second messenger pathways utilized by these receptors are undefined and it is equally unclear whether each receptor is coupled to similar or distinct signalling pathways. The molecular mechanisms by which hormone binding to the extracellular receptor subunit activates the cytoplasmic receptor tyrosine kinase and how this activation is coupled to later events are the key unsolved problems in the understanding of insulin and IGFI action. Clinically, problems related to insulin and the insulin receptor are involved in the pathogenesis of type I and II diabetes which affect 5% of the population in the United States. Excesses or deficiencies of IGFI lead to gigantism and small stature and IGFI have been implicated in the progression of some neoplastic tumors. This proposal asks three related questions, (1) How do the molecular structures of the extracellular portions of the IR and IGFIR determine hormone binding specificity and subsequent receptor kinase activation? (2) Are the overlapping biological effects of insulin and IGFI a reflection of intrinsic differences in signalling potential of the receptors linked to distinct functional pathways or due to differences in the distribution of receptors in different cell types? and (3) What specific receptor domains are involved in transducing and regulating the mitogenic signal from the activated kinase to the second messenger systems? To answer the first question, we will use receptor mutants and chimeras to precisely identify extracellular domains of the IR and IGFIR critical for hormone binding and signal transduction. To address the second question, cell lines which express only the IR or IGFIR and are mitogenically dependent upon either insulin or IGFI in serum-free medium will be transfected with the noncognate receptor cDNA to ask whether the mechanisms of insulin- and IGFI-mediated mitogenesis are similar or distinct. Secondly, to examine the potential for direct signalling between the IR and IGFIR, cell lines will be generated by cotransfection with specific combinations of receptor cDNAs and analyzed for inter-receptor hybrid formation and functional """"""""cross-talk"""""""" between the receptors. To address the third question, we will focus on the mitogenic effects of the hormones to assess the functions of mutant and chimeric receptors in specific cell backgrounds to localize regions of each receptor involved in the regulation of cell proliferation. Lastly, we will utilize cell lines whose growth in serum-free medium is dependent upon either insulin or IGFI to develop somatic cell genetic assays of receptor function. The studies proposed here should allow insight into fundamental mechanisms of receptor tyrosine kinase action and enhance the general understanding of growth control by receptor tyrosine kinases.
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