ATP-dependent desensitization of insulin binding and tyrosine kinase activity of the insulin receptor kinase. The role of endosomal acidification.
Incubating endosomes with ATP decreased binding of 125I-insulin but not 125I-labeled human growth hormone. Increasing ATP concentrations from 0.1 to 1 mM increased beta-subunit tyrosine phosphorylation and insulin receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations beta-subunit tyrosine phosphorylation and IRK activity were markedly decreased. This was not observed with nonhydrolyzable analogs of ATP, nor with plasma membrane IRK, nor with endosomal epidermal growth factor receptor kinase autophosphorylation. The inhibition of endosomal IRK tyrosine phosphorylation and activity was completely reversed by bafilomycin A1, indicating a role for endosomal proton pump(s). The inhibition of IRK was not due to serine/threonine phosphorylation nor was it influenced by the inhibition of phosphotyrosyl phosphatase using bisperoxo(1,10-phenanthroline)oxovanadate anion. Prior phosphorylation of the beta-subunit with 1 mM ATP did not prevent the inhibition of IRK activity on incubating with 5 mM ATP. To evaluate conformational change we incubated endosomes with dithiothreitol (DTT) followed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Without DTT the predominant species of IRK observed was alpha2 beta2. With DTT the alpha beta dimer predominated but on co-incubation with 5 mM ATP the alpha2 beta2 form predominated. Thus, ATP-dependent endosomal acidification contributes to the termination of transmembrane signaling by, among other processes, effecting a deactivating conformational change of the IRK.
Polypeptide Hormone Laboratory, McGill University, Montreal, Quebec H3A 2B2, Canada.
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D018942:Macrolides; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism*; D015398:Signal Transduction; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D007328:Insulin; D018942:Macrolides; D013439:Sulfhydryl Reagents; C057620:bafilomycin A; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol