The role of insulin receptor sulphydryl groups in insulin binding and cellular response in rat adipocytes.
Phenylarsine oxide (PAO), an agent which reacts with vicinal sulphydryl groups and dithiothreitol (DTT), a disulphide reducing agent, inhibited insulin binding to intact adipocytes with half maximal inhibition occurring at 28 microM and 340 microM, respectively. Pretreatment of adipocytes with DTT (2mM) prevented insulin stimulation of glucose uptake by approximately 50%. The marked inhibition of insulin binding to adipocytes by PAO and DTT is consistent with the involvement of the receptor cysteine-rich region of hormone binding. Furthermore, DTT inhibition of insulin binding suggests that the integrity of disulphide bridges is critical for insulin binding. The inhibitory effect of DTT and PAO on insulin binding were not additive, instead addition of DTT to PAO-treated adipocytes effected 15% reversal of binding inhibition. The marked inhibition of insulin binding by addition of low concentrations of DTT (0.2-2.0mM) to intact adipocytes is in contrast to the previously reported biphasic response for the effect of DTT on insulin binding to isolated plasma membranes from rat adipocytes (Schweitzer et al. Proc. Natl. Acad. Sci. U.S.A. 77, 4692-4696, 1980). Scatchard plots for 125I-iodoinsulin binding to adipocytes in the basal state were linear. In contrast, Scatchard analysis of insulin binding to plasma membranes prepared from both basal and insulin-stimulated adipocytes yielded severely curvilinear plots. The data suggests that (i) fundamental differences exist between the receptor state in intact cells and isolated plasma membranes and (ii) that a disulphide-rich region within the insulin receptor, other than the previously reported class I and class II disulphide bridges, is critical for insulin binding and cellular response.
Douen
A G
AG
Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, U.K.
Jones
M N
MN
D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism* / Q000494:pharmacology
D001152:Arsenicals; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; C029341:oxophenylarsine; D011972:Receptor, Insulin; D005947:Glucose; D004229:Dithiothreitol