Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes.

Abstract

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analog (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15.degree. C. At this temperature, only limited (.apprx. 20%) internalization of the ligand occurred. In a 2nd step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37.degree. C. After 2 h at 37.degree. C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15% of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after EM revealed that .apprx. 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. Sodium dodecyl sulfate polyacrylamide-gel electrophoresis analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37.degree. C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. In hepatocytes the insulin receptor is internalized with insulin.