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 I-125-insulin but n ot I-125-labeled human growth hormone. Increasing ATP concentrations f rom 0.1 to 1 mM increased P-subunit tyrosine phosphorylation and insul in receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations P-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 activit y was completely reversed by bafilomycin A(1), indicating a role for e ndosomal proton pump(s), The inhibition of IRK was not due to serine/t hreonine phosphorylation nor was it influenced by the inhibition of ph osphotyrosyl phosphatase using bisperoxo(1,10-phenanthroline)oxovanada te 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 dithio threitol (DTT) followed by SDS-polyacrylamide gel electrophoresis unde r nonreducing conditions. Without DTT the predominant species of IRK o bserved was alpha(2)beta(2). With DTT the alpha beta dimer predominate d but on co-incubation with 5 mM ATP the alpha(2)beta(2) form predomin ated. Thus, ATP-dependent endosomal acidification contributes to the t ermination of transmembrane signaling by, among other processes, effec ting a deactivating conformational change of the IRK.