Cysteine 981 of the human insulin receptor is required for covalent cross-linking between beta-subunit and a thiol-reactive membrane-associated protein

Citation
Mj. Garant et al., Cysteine 981 of the human insulin receptor is required for covalent cross-linking between beta-subunit and a thiol-reactive membrane-associated protein, BIOCHEM, 39(24), 2000, pp. 7178-7187
Citations number
54
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
24
Year of publication
2000
Pages
7178 - 7187
Database
ISI
SICI code
0006-2960(20000620)39:24<7178:C9OTHI>2.0.ZU;2-J
Abstract
The cytoplasmic domain of the insulin receptor (IR) beta-subunit contains c ysteine (Cys) residues whose reactivity and function remain uncertain. In t his study, we examined the ability of the bifunctional cross-linking reagen t 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting prote ins that possess reactive thiols, Transfected Chinese hamster ovary cells e xpressing either the wild-type human IR, C-terminally truncated receptors, or mutant receptors with Cys --> Ala substitutions and mouse 3T3-L1 adipocy tes were used to compare the BMH effect. The results showed the formation o f a large complex between the wild-type human receptor beta-subunit and mol ecule X, a thiol-reactive membrane-associated protein, in both intact and s emipermeabilized cells in response to BMH. Prior cell stimulation with insu lin had only a modest effect in this process. Western blot analysis reveale d that the receptor alpha-subunit was not present in the beta-X complex. Th e BMH cross-linking did not inhibit in vitro tyrosine phosphorylation of th e receptor complexed with molecule X. Both the human IR Cys981Ala mutant an d murine IR, that lacks the equivalent of human Cys(981), failed to react, with BMH. Finally, Ilo covalent association between IR beta-subunit and IRS -1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treat ed cells expressing the wild-type human IR. These results demonstrate a str iking difference in reactivity among the cytoplasmic IR beta-subunit thiols and clearly show that Cys(981) of human IR beta-subunit is in close proxim ity to a thiol-reactive membrane-associated protein under basal and insulin -stimulated conditions.