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PubMed · 3316225

Insulin-dependent intermolecular subunit communication between isolated alpha beta heterodimeric insulin receptor complexes.

L J Sweet·B D Morrison·P A Wilden·J E Pessin
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Abstract

The dissociation of the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex into an alpha beta heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the alpha beta heterodimeric complex, under conditions which preserved tracer insulin binding and protein kinase activities (pH 8.75 for 25 min followed by 2.0 mM DTT for 5 min) occurred with an approximate 50% efficiency. The resulting nondissociated alpha 2 beta 2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated alpha 2 beta 2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated alpha beta heterodimeric complex was stable to spontaneous reassociation for at least 72 h at pH 7.60. Kinetic analyses of the insulin receptor protein kinase activity demonstrated that the insulin stimulation of glutamic acid:tyrosine (4:1) synthetic polymer phosphorylation for both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes occurred via an increase in Vmax without any significant change in Km. Examination of beta subunit autophosphorylation of the alpha beta heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Further, the initial rate of insulin-stimulated beta subunit autophosphorylation in the isolated alpha beta heterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated alpha beta heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the beta subunit protein kinase domain with the covalent reassociation of the alpha beta heterodimeric complex into an alpha 2 beta 2 heterotetrameric disulfide-linked state.

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DithiothreitolHumansHydrogen-Ion ConcentrationInsulinKineticsMacromolecular SubstancesPlacentaProtein-Tyrosine KinasesReceptor, InsulinJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

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L J Sweet, B D Morrison, P A Wilden, J E Pessin. 1987-12-05. Insulin-dependent intermolecular subunit communication between isolated alpha beta heterodimeric insulin receptor complexes.. https://pubmed.ncbi.nlm.nih.gov/3316225/

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