Internalized insulin receptors are recycled to the cell surface in rat hepatocytes.
We have followed the fate of cell surface insulin receptors in isolated rat hepatocytes by both a biochemical and a morphological approach. Hepatocytes were labeled with the photoreactive and biologically active 125I-labeled insulin analogue, [2-nitro-4-azidophenylacetylB2]des-PheB1-insulin, under conditions that allow for minimal internalization (2 hr at 15 degrees C). Analysis of the cell-associated radioactivity by NaDodSO4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography revealed the specific labeling of a major insulin receptor subunit with Mr 130,000 and a minor degradation product with Mr 125,000. When the cells were exposed at 15 degrees C to trypsin at the end of the association period, these two bands were no longer observed, indicating that the labeled receptors were at the cell surface. This trypsin sensitivity of the receptor disappeared within 30-60 min of incubation of the cells at 37 degrees C, reflecting the internalization of the hormone-receptor complexes. Over the subsequent 4 hr of incubation, this was followed by a progressive reappearance of the receptor complexes at the cell surface, as indicated by the recovery of trypsin sensitivity of the labeled insulin receptors. An identical (both chronologically and quantitatively) journey of the insulin receptors was observed when the labeled material was studied by quantitative electron microscopic autoradiography. Thus, when the cells were incubated at 37 degrees C there was a rapid decrease (30-60 min) in the percentage of autoradiographic grains associated with the plasma membrane, followed by a progressive increase in this percentage over the subsequent 4 hr of incubation. In conclusion, using a biochemical and morphological approach to trace the photoaffinity-labeled insulin receptor, we have shown that the internalized hormone-receptor complex is recycled back to the cell surface.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D007328:Insulin; D007457:Iodine Radioisotopes; C035459:insulin, (2-nitro-4-azidophenylacetyl)(B2)-des-Phe(B1)-; D011972:Receptor, Insulin; D014357:Trypsin