Cysteine 981 of the Human Insulin Receptor Is Required for Covalent Cross-Linking between -Subunit and a Thiol-Reacti
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- 作者：Michael J. Garant ; Elena Maksimova ; Chahrzad Montrose-Rafizadeh ; Whaseon Lee-Kwon ; Sutapa Kole ; and Michel Bernier
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：June 20, 2000
- 年：2000
- 卷：39
- 期：24
- 页码：7178 - 7187
- 全文大小：381K
- 年卷期：v.39,no.24(June 20, 2000)
- ISSN：1520-4995
文摘
The cytoplasmic domain of the insulin receptor (IR) 
-subunit contains cysteine (Cys) residueswhose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctionalcross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins thatpossess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type humanIR, C-terminally truncated receptors, or mutant receptors with Cys 
Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complexbetween the wild-type human receptor -subunit and molecule X, a thiol-reactive membrane-associatedprotein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulinhad only a modest effect in this process. Western blot analysis revealed that the receptor 
-subunit wasnot present in the -X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylationof the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, thatlacks the equivalent of human Cys981, failed to react with BMH. Finally, no covalent association betweenIR -subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treatedcells expressing the wild-type human IR. These results demonstrate a striking difference in reactivityamong the cytoplasmic IR -subunit thiols and clearly show that Cys981 of human IR -subunit is inclose proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulatedconditions.
-subunit contains cysteine (Cys) residueswhose reactivity and function remain uncertain. In this study, we examined the ability of the bifunctionalcross-linking reagent 1,6-bismaleimidohexane (BMH) to covalently link IR with interacting proteins thatpossess reactive thiols. Transfected Chinese hamster ovary cells expressing either the wild-type humanIR, C-terminally truncated receptors, or mutant receptors with Cys Ala substitutions and mouse 3T3-L1 adipocytes were used to compare the BMH effect. The results showed the formation of a large complexbetween the wild-type human receptor -subunit and molecule X, a thiol-reactive membrane-associatedprotein, in both intact and semipermeabilized cells in response to BMH. Prior cell stimulation with insulinhad only a modest effect in this process. Western blot analysis revealed that the receptor -subunit wasnot present in the -X complex. The BMH cross-linking did not inhibit in vitro tyrosine phosphorylationof the receptor complexed with molecule X. Both the human IR Cys981Ala mutant and murine IR, thatlacks the equivalent of human Cys981, failed to react with BMH. Finally, no covalent association betweenIR -subunit and IRS-1, the protein tyrosine phosphatase LAR or SHP-2 was observed in BMH-treatedcells expressing the wild-type human IR. These results demonstrate a striking difference in reactivityamong the cytoplasmic IR -subunit thiols and clearly show that Cys981 of human IR -subunit is inclose proximity to a thiol-reactive membrane-associated protein under basal and insulin-stimulatedconditions.