The insulin receptor is a membrane-spanning tyrosine kinase that phosphorylates itself in response to insulin binding. Autophosphorylation activates the receptor as a substrate kinase, leading to its direct phosphorylation of additional proteins within the cell, including IRS-1 and Shc. Once phosphorylated on specific tyrosine residues, these cytoplasmic substrates engage and activate SH2 domain-containing enzymes which propagate the signals into the cell. Since the insulin receptor and its interactions with cellular substrates provide the initiating events in insulin action cascades, a detailed understanding of these interactions should provide insights into normal cellular metabolism and the pathophysiology of NIDDM, and these insights may translate into therapeutic interventions. Both of these substrates contain a new type of phosphotyrosine binding (PTB) domain distinct from the well characterized SH2 domain. The applicant showed that PTB domains of Shc and IRS-1 bind to the insulin receptor at a site just within the membrane, and it is hypothesized that this positions the substrates for efficient phosphorylation by the kinase. Studies are proposed to (1) analyze binding characteristics of IRS-1 and Shc PTB domains in detail, (2) determine three-dimensional structures of the PTB domains, (3) analyze the role of PTB domains in insulin receptor-catalyzed phosphorylations of IRS-1 and Shc, and (4) as a long term goal, to determine the crystallographic structure of an insulin receptor kinase/PTB domain signaling complex. Results from these studies should provide a detailed structural basis for understanding the initiating events in insulin action and transmembrane signaling by the insulin receptor. Since related or identical mechanisms likely occur in additional receptor signaling pathways, proposed studies should also interest those working in many areas of oncology, endocrinology and immunology.
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