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