title: Reversible change in thiol redox status of the insulin receptor alpha-subunit in intact cells.
abstract: In this study, we used maleimidobutyrylbiocytin to examine possible alteration that may occur in the redox state of the insulin receptor (IR) sulfhydryl groups in response to reduced glutathione (GSH) or N-acetyl-L-cysteine (NAC). Short-term treatment of intact cells expressing large numbers of IR with GSH or NAC led to a rapid and reversible reduction of IR alpha-subunit disulfides, without affecting the receptor beta-subunit thiol reactivity. The overall integrity of the oligomeric structure of IR was maintained, indicating that neither class I nor class II disulfides were targeted by these agents. Similar findings were obtained in cells transfected with IR mutants lacking cysteine524, one of the class I disulfides that link the two IR alpha-subunits. Membrane-associated thiols did not participate in GSH- or NAC-mediated reduction of IR alpha-subunit disulfides. No difference in insulin binding was observed in GSH-treated cells; however, ligand-mediated increases in IR autophosphorylation, tyrosine phosphorylation of cellular substrates, 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. GSH did not affect IR signaling in the absence of insulin. Our results provide 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.
authors.affiliation: Diabetes Section, Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Garant
authors.forename: S
authors.initials: S
authors.lastname: Kole
authors.forename: E M
authors.initials: EM
authors.lastname: Maksimova
authors.forename: M
authors.initials: M
authors.lastname: Bernier
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006528:Carcinoma, Hepatocellular; D006224:Cricetinae; D003470:Culture Media; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011972:Receptor, Insulin / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D003470:Culture Media; D013438:Sulfhydryl Compounds; D011972:Receptor, Insulin; D005978:Glutathione; D000111:Acetylcysteine