The studies proposed in this application will examine the effects of specific mutations in the human insulin receptor on insulin action. The ability of each mutation to evoke various insulin responses in an insulin-target cell (the adipocyte) will be examined by cell transfection experiments. The following mutations in the beta subunit of the insulin receptor will be studied: (1) Substitution of Phe for Tyr-960, (2) Deletion of the amino acid sequence, Ala-954 through Ala-965, (3) Deletion of 43 amino acids at the COOH terminus of the beta subunit, beginning at Ala-1301, (4) Substitution of Phe for Tyr-1146, (5) Substitution of Phe for Tyr-1316, (6) Substitution of Phe for Tyr-1322. These mutations were selected based on prior information that indicated a role for reach region in signal generation by the receptor. At present, the first three mutations are available for study, whereas the last three will be constructed during the grant period. The cDNA constructs that contain the mutations will be placed in an eukaroyte expression vector. This vector will be used to transfect preadipocyte fibroblasts from established mouse (3T3- F442A) and Chinese hampster embryo (CHEF/18) cell lines. Cells that express the wild-type and mutant human insulin receptors will be selected, cloned and used in studies of insulin action, both before and after the cells have been driven to differentiate to adipocytes. The insulin responses that will be examined include the activation of glucose transport and metabolism, the antilipolytic effect and the ability to mediate changes int he expression of mRNA levels for c-fos and c-myc. The ability of the various mutant insulin receptors to elicit these actions of insulin will be compared to any changes noted in the autophosphorylation of the receptors or phosphorylation of endogenous substrates. Results from these studies should provide more information of the structural features of the insulin receptor necessary for generating an insulin response. These studies will also help to determine if the diverse cellular processes regulated by insulin require the same or different signals from the insulin receptor. Overall, such information is needed in devising a more effective management program for diabetes and other states of altered insulin action.
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