Direct comparison of the rates of internalization and degradation of covalent receptor-insulin complexes in 3T3-L1 adipocytes. Internalization of occupied receptors is not the rate-limiting step in receptor-hormone complex degradation.
Insulin receptors on the surface of 3T3-L1 adipocytes were photolabeled using the iodinated analog, B29-lysine-substituted N-[N'-(2-nitro-4-azidophenyl)glycyl]insulin. Under optimal labeling conditions (below 15 degrees C), greater than 95% of the labeled receptor remained on the cell surface prior to incubation at 37 degrees C. When the labeled monolayers were returned to their normal culture environment (37 degrees C), the covalent receptor-insulin complexes were rapidly internalized at initial rates equivalent to 130-170% of labeled surface receptor/h. Internalization of the complexes proceeded to an equilibrium or end point distribution of 40% internal receptor and 60% cell-surface receptor. Under the several labeling conditions tested, covalent receptor-insulin complexes were degraded in an apparent first order process at 37 degrees C with half-lives between 5 and 7 h. This rate was equivalent to only 10% of the labeled receptor being degraded per h and was 13-17-fold slower than the initial rate of labeled receptor internalization. This study directly demonstrates that the initial rate of internalization of covalent receptor-insulin complexes is not the rate-limiting step in their degradation in 3T3-L1 adipocytes. Furthermore, 3T3-L1 adipocytes are unable to internalize all of the labeled surface receptor, suggesting that two classes of internalization competent and incompetent receptor may exist or that an equilibrium distribution of internal and cell-surface receptor is established by the relative rates of internalization and recycling of labeled receptor.
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D010782:Photolysis; D011972:Receptor, Insulin / Q000378:metabolism*; D013816:Thermodynamics
D001386:Azides; D007328:Insulin; D008623:Mercaptoethanol; C042015:insulin, (2-nitro-4-azidophenyl)-Gly(B29)-; D011972:Receptor, Insulin