Metabolism of covalent receptor-insulin complexes by 3T3-L1 adipocytes. Synthesis and use of photosensitive insulin analogs to study insulin receptor metabolism in cell culture

To facilitate labeling cell surface insulin receptors and analyzing their metabolism by 3T3-L1 adipocytes, a characterization of both the interaction of photosensitive insulin analogs with 3T3-L1 adipocytes and the conditions for photocross-linking these derivatives to the insulin receptor are described. The synthesis and purification of two photoaffinity analogs of insulin are presented. Both B29-lysine- and A1-glycine-substituted N-(2-nitro-4-azidophenyl)glycyl insulin compete with 125I-insulin for binding to 3T3-L1 adipocytes, and the B29-derivative retains a biological activity similar to that for native insulin. An apparatus developed for these studies permits photolysis of cells in monolayer culture using the visible region of the lamp emission spectrum. Activation of the photoderivative by this apparatus occurs with a half-life of approximately 15 s and permits rapid photolabeling of a single species of receptor of 300,000 Da. The conditions for photolabeling permit a measurement of the turnover of covalent receptor-insulin complexes by 3T3-L1 adipocytes in monolayer culture. Degradation of this complex occurs as an apparent first order process with a half-life of 7 h. A comparison with previous studies (Reed, B. C., Ronnett, G. V., Clements, P. R., and Lane, M. D. (1981) J. Biol. Chem 256, 3917-3925; Ronnett, G. V., Knutson, V. P., and Lane, M. D. (1982) J. Biol. Chem. 257, 4285-4291) indicates that in a "down-regulated" state, 3T3-L1 adipocytes degrade covalent receptor-hormone complexes with kinetics similar to those for the degradation of dissociable receptor-hormone complexes.