Reversible change in thiol redox status of the insulin receptor alpha-subunit in intact cells.
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.
Garant
M J
MJ
Diabetes Section, Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, USA.
Kole
S
S
Maksimova
E M
EM
Bernier
M
M
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
D003470:Culture Media; D013438:Sulfhydryl Compounds; D011972:Receptor, Insulin; D005978:Glutathione; D000111:Acetylcysteine