Insulin receptors on viable rat adipocytes were affinity-labeled using a biologically active and photosensitive analogue of insulin, 125I-B2(2-nitro, 4 azidophenylacetyl)-des-PheB1-insulin (125I-NAPA-DP-insulin). The radio-labeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of 125I-NAPA-DP-insulin (40 ng/ml) to rat adipocytes at 16°C, followed by photolysis, resulted in the specific labeling of essentially one protein with an apparent molecular weight of 430–450,000 daltons. When this radiolabeled protein was treated with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a 125,000-dalton protein and to a lesser extent in a 90,000-dalton protein. In addition, there was a minimal amount of specific labeling of a 115,000-dalton protein. Under certain experimental conditions, the nonreduced form of the photoaffinity-labeled receptor appeared as a heterogeneous population of proteins having apparent molecular weights of 430,000, 350,000, and 270,000 daltons.

Subsequent to photoaffinity labeling of insulin receptors at 16°C, adipocytes were incubated at 37°C for various periods of time to allow for internalization. This resulted in an initial rapid loss of radioactivity in the 430,000- and 125,000-dalton bands. At 60 min the amount of radioactivity in each of these bands was approximately 50% of that present before incubation at 37°C and stayed constant for 120 min. A first-order plot of the decline in receptor-associated radioactivity was biphasic with the initial phase having a half-life of 1.4 h. This loss of radiolabeled receptors was not observed at 16°C and was inhibited by chloroquine (200μM) and cycloheximide (3.5 μM). Under conditions in which approximately 6% of the receptors were photoaffinity-labeled, occupancy of the remaining receptors with insulin had no effect on the degradation of photolabeled receptors. The results suggest that photoaffinity-labeled insulin receptors on viable adipocytes are initially degraded via an endocytotic lysosomal pathway. This pathway of degradation is very rapid and appears to be different from that mediating the basal turnover of unoccupied receptors.

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