Internalization and molecular processing of insulin receptors in isolated rat adipocytes.

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Abstract
The cellular fate of insulin receptors in isolated rat adipocytes was studied by using a biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin), to photoaffinity label the insulin receptors. Insulin receptors specifically labeled with 125I-labeled NAPA-DP-insulin were identified by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Under nonreducing conditions, specific bands of Mr [molecular ratio] 330,000, 295,000 and 260,000 were identified; under disulfide reducing conditions, these were converted into Mr 125,000 and 90,000 subunits. When cells labeled at 16.degree. C were immediately trypsinized, all of the receptor bands were degraded into lower MW fragments, indicating that the labeled receptors were all on the cell surface. However, when the labeled cells were incubated at 37.degree. C for 1 h prior to trypsin exposure, .apprxeq. 30% of the receptors were trypsin insensitive, indicating that this fraction was translocated intracellularly. Processing of the insulin receptors appeared to occur; incubation at 37.degree. C (but not at 16.degree. C) resulted in generation of a Mr 115,000 component from the Mr 125,000 subunit as well as in the disappearance of the Mr 330,000 and 295,000 species. Inclusion of chloroquine during photoaffinity labeling at 16.degree. C and during the subsequent incubation at 37.degree. C showed that this agent: increased the trypsin-insensitive (intracellular) receptor pool, blocked conversion of the Mr 125,000 subunit into the Mr 115,000 component and prevented the disappearance of the Mr 330,000 and 295,000 species. These studies show that insulin-receptor complexes are internalized and processed intracellularly at a chloroquine-sensitive site(s).