ATP-dependent Desensitization of Insulin Binding and Tyrosine Kinase Activity of the Insulin Receptor Kinase

Abstract

Incubating endosomes with ATP decreased binding of 125 I-insulin but not 125 I-labeled human growth hormone. Increasing ATP concentrations from 0.1 to 1 mM increased ␤-subunit tyrosine phosphorylation and insulin receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations ␤-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 A 1 , 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 ␤-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 ␣ 2 ␤ 2. With DTT the ␣␤ dimer predominated but on co-incubation with 5 mM ATP the ␣ 2 ␤ 2 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. Following insulin binding to its receptor in intact cells the insulin receptor kinase (IRK)1 undergoes tyrosine autophosphorylation and kinase activation (1, 2). Equally rapidly there is internalization of activated insulin-IRK complexes into ENs (3, 4). IRK signaling appears largely to involve tyrosine phosphorylation of adaptor proteins, including insulin receptor substrate-1 (5), insulin receptor substrate-2 (6, 7), and SHC (8), which function as docking entities to entrain the insulin signaling sequence. The observation that the activated IRK is internalized to ENs is consistent with the occurrence of transmembrane signaling intracellularly (9, 10). Indeed studies in liver parenchyma have shown that the accumulation of activated IRKs exclusively in ENs is sufficient to promote insulin receptor substrate-1 tyrosine phosphorylation (11). In adipocytes it has also been shown that internal membranes are the principal sites where insulin receptor substrate-1 phosphorylation and phophatidylinositol 3-kinase activation occur (12). Given the above, understanding the mechanisms controlling receptor function in ENs is important for understanding the regulation of insulin signaling. Studies have shown that the level of IRK tyrosine phosphorylation and hence activity is altered and ultimately reduced by an IRK-associated phosphotyrosine phosphatase in ENs (4, 13). Discovery of the endosomal acidic insulinase (14) has demonstrated a mechanism by which intraendosomal insulin concentrat...

Effect of Inhibiting Vacuolar Acidification on Insulin Signaling in Hepatocytes

“…4c). In previous work we showed that endosomal IRK is inactivated consequent to a conformational change induced by acidic pH (13). Thus, neutralizing vacuolar pH with bafilomycin could suppress this acidification-dependent conformational change leading to increased IRK activity.…”

“…Furthermore, a reduced intraendosomal pH (pH Յ 6) promotes dissociation of internalized insulin-IRK complexes and degradation of insulin (10,11) by an acidic endosomal insulinase recently identified as cathepsin D (12). The acidic pH of endosomes also effects a conformation-dependent inactivation of the IRK (13). Thus, the acidic pH of the endocytic compartment and the IRK-associated phosphotyrosine phosphatase(s) therein play fundamental roles in attenuating insulin signaling.…”

Dissection of the multifunctional “receptor-recycling” endocytic compartment of hepatocytes

“…Recently, it was shown that endosome acidification also contributes to insulin receptor tyrosine kinase inactivation. 18 One of the major problems regarding the integrated understanding of the intracellular sorting and trafficking is that the biochemistry of different endosome fractions is largely unresolved. In view of the complexity of the mammalian endocytic compartment, it is not surprising that isolation and biochemical characterization of this pleiomorphic organelle from tissue homogenates is a major task.…”

Role of Endosomal Trafficking Dynamics on the Regulation of Hepatic Insulin Receptor Activity: Models for Fao Cells

“…The endosomal acidic environment (pH ∼6) promotes ligand-receptor dissociation, 13 as well as a conformational dependent inactivation of the IR. 17 Insulin is degraded within the endosome by an acidic insulinase, 2,19 and the IR is either sent to lysosomes for degradation or recycled to the plasma membrane for another round of binding, activation, and internalization. 31 Receptor-mediated endocytosis is a common cellular mechanism for adjusting the amount of insulin in plasma or the number of receptors on the cell surface.…”