Reversible change in thiol redox status of the insulin receptor alpha-subunit in intact cells

In this study, we used maleimidobutyrylbiocytin to examine possible alterat ion that may occur in the redox state of the insulin receptor (IR) sulfhydr yl groups in response to reduced glutathione (GSH) or N-acetyl-L-cysteine ( NAC). Short-term treatment of intact cells expressing large numbers of IR w ith GSH or NAC led to a rapid and reversible reduction of IR alpha-subunit disulfides, without affecting the receptor beta-subunit thiol reactivity. T he overall integrity of the oligomeric structure of IR was maintained, indi cating that neither class I nor class II disulfides were targeted by these agents. Similar findings were obtained in cells transfected with IR mutants lacking cysteine(524), one of the class I disulfides that link the two IR alpha-subunits. Membrane-associated thiols did not participate in GSH- or N AC-mediated reduction of IR alpha-subunit disulfides. No difference in insu lin binding was observed in GSH-treated cells; however, ligand-mediated inc reases in IR autophosphorylation, tyrosine phosphorylation of cellular subs trates, and dual phosphorylation of the downstream target mitogen-activated protein kinase were inhibited at concentrations of GSH (10 mM or greater) that yielded a significant increase in IR alpha-subunit thiol reactivity. G SH did not affect IR signaling in the absence of insulin. Our results provi de the first evidence that the IR alpha-subunit contains a select group of disulfides whose redox status can be rapidly altered by the reducing agents GSH and NAC.