Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

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Abstract
Cell surface and cryptic insulin receptors were solubilized from the particulate fraction of murine 3T3-L1 adipocytes with buffer containing 1% Triton X-100. Solubilized receptors were cross-linked with 125I-labeled insulin and disuccinimidyl suberate and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography after specific immunoprecipitation. Two insulin-binding polypeptides were identified: the more abundant protein had a MW of 130,000, corresponding to the size of the hormone-binding subunit of insulin receptors on the surface of target cells; the 2nd polypeptide exhibited a MW of 200,000 and appears to be a component of the latent pool because it was unaffected when 3T3-L1 adipocytes were exposed to trypsin under conditions that result in a 95% reduction in cell surface insulin-binding activity and the loss of the MW 130,000 polypeptide in cross-linking experiments. Unexpectedly, the population of MW 200,000 molecules in intact cells was accessible for limited cleavage by chymotrypsin, yielding a MW 195,000 insulin-binding polypeptide. When 3T3-L1 adipocytes received a 15-min pulse of [35S]methionine, the predominant immunoprecipitated polypeptide had a MW of 180,000. During a 1.5-h chase, radioactivity in the MW 180,000 species rapidly declined while the latent MW 200,000 polypeptide became intensely labeled. After a 5-h chase period, broad protein bands with MW of 130,000 and 90,000 were visualized as the major immunoprecipitated radioactive polypeptides. The MW 180,000 species may be a very early biosynthetic precursor that may be subsequently processed to a MW 200,000 form and one or both of the smaller receptor subunits at the cell surface.

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