Abstract
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.
Highlights
From the Department of Medicine, University of California, San Diego and the Veterans Administration Medical Center, San Diego, California 92161 and the SDeutches Wollforschungsinstitut, Aachen,West Germany
Two lines of evidence suggest that occupancy of insulin receptors by the photoaffinity analog of insulin (NAPA-DP-insulin) is functionally similar to occupancy of receptors by noncovalently attached insulin. 1)The photoaffinity analog of insulin is an agonist which has 80-90% of insulin's biologic activity
The agonist activity is present even after photolysis and resultsin prolonged stimulation of lipogenesis in adipocytes [18]. 2 ) The kinetics of internalization andrecycling of photoaffinitylabeled insulin receptors in adipocytes are similar to that of unmodified receptors [9, 20]
Summary
Vol 261,No 19,h u e of July 5,pp. 8655-8659,1986 Printed in U.S.A. DISSOCIATION OF INSULIN-RECEPTOR COMPLEXES IS NOT REQUIRED FOR RECEPTOR RECYCLING*. We have used a photoreactive analog of insulin to covalently label cell-surface insulin receptors on isolated adipocytes and havemeasured the rate and extenotf insulin receptor recycling following internalization. Since internalization of native insulin-receptor complexes, whereas monensin, chloroquine, and Tris par- plexes is presumablyfollowed by ligand dissociation in aciditially inhibited this process. The intracellular receptors can either be sequestered withincethlle[6],degraded [7, 8], or recycled back to the plasma membrane (4, 5 , 9) These general pathways are applicable to a wide variety of surface-bound ligands (for review, see Ref. lo), the insulin, 2.5 mCi of Nalz5I,and 200 pl of 0.1 M phosphate buffer (pH 7.5) were added to a 12 X 75-mm tube previously coated with 40 pg of 1,3,4,6-tetrachloro-3cu,GcY-diphenylglycourc(IilODO-GEN, Pierce). Recycling of Insulin Receptors in Rat Adipocytes in 1% albumin Krebs-Ringer phosphate buffer with 10 mM Hepes
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