Rescue and activation of a binding-deficient insulin receptor. Evidence for intermolecular transphosphorylation

M. Taouis, R. Levy-Toledano, P. Roach, S. I. Taylor, P. Gorden

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

Binding of insulin to the α subunit of the insulin receptor (IR) leads to autophosphorylation of the β subunit. The reaction proceeds as intramolecular transphosphorylation between αβ half-receptors of the heterotetrameric receptor dimer (α2β2). Since IRs are mobile in the plane of the plasma membrane, it is also possible that transphosphorylation may occur between adjacent holoreceptors (α2β2) by an intermolecular reaction. To address this question, we cotransfected NIH-3T3 cells with two IR cDNA constructs: a truncated but functionally normal IR lacking the C- terminal 43 amino acids (Δ43) and a full-length Leu323 mutant receptor that is expressed on the cell surface but that does not bind insulin. A clonal cell line was selected from cells cotransfected with a 1/5 ratio of Δ43 cDNA/Leu323 cDNA. The two homodimers (Leu323 and Δ43) were expressed without detectable formation of hybrid receptors. By using specific antibodies, we demonstrate that in cells coexpressing both homodimers, the Leu323 mutant receptor was phosphorylated in vivo by the Δ43 IR in an insulin-dependent manner. However, when the Leu323 mutant receptor was expressed alone, no phosphorylation was detected. In addition, we demonstrate the association of the phosphorylated Leu323 mutant receptor with insulin receptor substrate-1 and with phosphatidylinositol 3-kinase. These findings indicate that insulin binding is not required for phosphorylation of the Leu323 mutant receptor, that the phosphorylation of the Leu323 mutant receptor occurs by an intermolecular transphosphorylation mechanism, and, finally, that the Leu323 mutant receptor, once phosphorylated, can associate with insulin receptor substrate-1 and phosphatidylinositol 3- kinase.

Original languageEnglish (US)
Pages (from-to)27762-27766
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number44
StatePublished - Jan 1 1994
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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