The overall goal is to elucidate at the molecular level the pathways of signaling from the insulin receptor to the known cellular responses of insulin. The insulin receptor is a protein tyrosine (tyr) kinase, and there is evidence to support the hypothesis that signaling proceeds by the phosphorylation of target proteins. Consequently, the general approach will be to purify and characterize those proteins that are rapidly phosphorylated on tyr in response to insulin and to determine the roles of these proteins in signal transduction from the insulin receptor. The project will mainly involve the use of the 3T3-L1 adipocyte, a cultured cell that is very insulin-responsive. In preliminary studies, the major insulin-elicited phosphotyrosyl (Ptyr) protein in this cell, one of 160 kDa (designated pp160), has been purified and the amino acid sequences of peptides from it obtained. Additionally, four other polypeptides, of 55, 44, 40 and 27 kDa, also phosphorylated on tyr in response to insulin, have been partially purified.
Specific aims are: (i) to clone and sequence the cDNA encoding pp160, (ii) to determine the role of pp160 in insulin action in vivo, both by preventing its action, through overexpression upon transfection with DNA encoding it and through direct introduction or purified pp160, (iii) to characterize pp160 with regard to possible function, subcellular location, associated polypeptides, interaction with the insulin receptor, and sites of phosphorylation, and (iv) to purify completely each of the other four Ptyr polypeptides and investigate these as described for pp160 under aims i-iii. The proposed research is of direct relevance to diabetes. For both type I and II diabetes a knowledge of he signaling pathways from the insulin receptor may serve as the basis for the design of better therapeutic agents and/or regimes. Moreover, in the case of type II diabetes, where the basic cause(s) are not yet known, a modification in one of these proteins may prove to be a cause.
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