The LONG-RANGE GOAL of this research is to understand the molecular mechanism by which the cell-surface insulin receptor, upon binding hormone, transmits a signal(s) to cellular end-targets, in particular the adipocyte/muscle-type glucose transport system. This work should provide insight into the cause of insulin resistance that often accompanies Non- Insulin Dependent Diabetes Mellitus. The SPECIFIC IMMEDIATE AIMS are to investigate and characterize: o the mechanism and role of insulin-activated autophos-phorylation and cellular (and model substrate phosphorylation by the insulin receptor's intrinsic tyrosine kinase. o the role of phosphorylation of p15 (422 protein) by the receptor kinase in insulin-stimulated glucose uptake and in other actions of insulin. We have obtained evidence that p15 is a cellular substrate (target) of the receptor kinase in 3T3-L1 adipocytes and that pp15 may be an intermediate in insulin-stimu-lated glucose uptake. o the mechanism of insulin-stimulated glucose uptake by the 3T3-L1 adipocyte system reconstituted in Xenopus oocytes. The steps in the signal transmission pathway will be delineated and their molecular mechanisms investigated. Intermediates in the insulin signalling pathway will be identified and their roles characterized using a combination of systems including:a) 3T3- L1 adipocytes in culture. These cells are exquisitely sensitive to insulin. We have purified several components implicated in the 3T3-L1 signal transmission pathway (eg. the insulin receptor, p15 and the """"""""insulin-responsive"""""""" glucose transporter) and we have cloned their corresponding cDNAs. These components and their mRNAs will be utilized to reconstitute the signalling pathway in Xenopus oocytes and in the 3T3-L1 preadipocyte; b) Xenopus oocytes; we propose to reconstitute the 3T3-L1 adipocyte insulin-stimulated signal transmission system in the oocyte by microinjection of mRNAs which encode these intermediates or by microinjection f the intermediates per se. To assess their roles in the signalling pathway, specific steps will be blocked/activated by microinjection of agents, including antibodies directed against putative intermediates, enzymes which may modify (dephosphorylate/phosphorylate) such intermediates, etc.; the initial steps in the pathway will be by- passed by microinjection of the """"""""activated"""""""" forms of putative intermediates, eg. pp15; c) Cell-free systems will be used to reconstruct segments of the signal transmission pathway from purified components in order to elucidate molecular mechanisms. Considerable progress has already been made by our group in characterizing the signal transmission system and certain of its components in 3T3-L1 adipocytes and Xenopus oocytes.
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