The insulin receptor is one of the more studied molecules of modern biochemistry because it plays a key role in organismal glucose homeostasis; it initiates the cellular response(s) that allows glucose to be cleared from the circulation. Moreover, as a ligand-regulated, tyrosine-specific protein kinase, it is representative of a relatively large class of similar molecules, all of which appear to play extremely central roles in the regulation of metabolism, cell growth and differentiation. Thus, it will be very important to understand the detailed structure of the insulin receptor and its close and distant relatives, and to understand how this structure relates to biological function. Such knowledge will almost certainly yield information that will guide the design of more rational and specific treatments for diseases such as diabetes. In this proposal, we will continue to elucidate the biochemical steps by which insulin binding leads to receptor autophosphorylation and subsequent activation of the receptor's exogenous kinase activity. These steps include further identification of the receptor sequence(s) involved in insulin binding, and an examination of the likely differences induced in the individual beta subunits by asymmetric ligand binding. We will perform ultra structural analysis of the ligand bound and unoccupied receptor, and of site-specific antibody-receptor complexes by electron microscopy (EM.). We will identify receptor-associated proteins from rat fat cells under conditions where this can be correlated with the physiologically relevant insulin response of the adipocyte. We will employ two novel approaches towards these specific aims. First, we have selected cells that overexpress the insulin receptor to a great degree such that several hundred micrograms can be easily purified in 100% functional yield with regard to kinase activity. This will greatly facilitate the needed preparation of highly purified insulin receptor in large amounts. Secondly, we are using a novel insulin derivative, BBpa- insulin, (L-benzoylphenylalanine B25, B29epsilon-biotin insulin). This insulin analog can be covalently linked to the receptor in exceptionally high yields (>70%) with respect to receptor bound ligand thus allowing examination of receptor-ligand complexes with little or no background of unoccupied receptors. Moreover, the biotin group allows detection of these complexes by avidin derivatives even after fixation for E.M. or SD- PAGE. Thus, we feel confident that a substantial amount of novel information relevant to insulin action and diabetes will result from the proposed studies.
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