Recycling of photoaffinity-labeled insulin receptors in rat adipocytes. Dissociation of insulin-receptor complexes is not required for receptor recycling.
We have used an iodinated, photoreactive analog of insulin, 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, to covalently label insulin receptors on the cell surface of isolated rat adipocytes. Following internalization of the labeled insulin-receptor complexes at 37 degrees C, we measured the rate and extent of recycling of these complexes using trypsin to distinguish receptors on the cell surface from those inside the cell. The return of internalized photoaffinity-labeled receptors to the cell surface was very rapid at 37 degrees C proceeding with an apparent t 1/2 of 6 min. About 95% of the labeled receptors present in the cell 20 min after the initiation of endocytosis returned to the cell surface by 40 min. Recycling was slower at 25 and 16 degrees C compared to 37 degrees C and essentially negligible at 12 degrees C or in the presence of energy depleters. Addition of excess unlabeled insulin had no effect on the recycling of photoaffinity-labeled insulin receptor complexes, whereas monensin, chloroquine, and Tris partially inhibited this process. These data indicate that dissociation of insulin from internalized receptors is not necessary for insulin receptor recycling. Furthermore, agents which have been shown to prevent vesicular acidification inhibit the recycling of insulin receptors by a mechanism other than prevention of ligand dissociation.
D000273:Adipose Tissue / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D010782:Photolysis; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics; D014325:Tromethamine / Q000494:pharmacology; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D007328:Insulin; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D014325:Tromethamine; C035459:insulin, (2-nitro-4-azidophenylacetyl)(B2)-des-Phe(B1)-; D002738:Chloroquine; D008985:Monensin; D011972:Receptor, Insulin; D014357:Trypsin