Insulin-dependent intermolecular subunit communication between isolated αβ heterodimeric insulin receptor complexes

L. J. Sweet, B. D. Morrison, P. A. Wilden, Jeffrey E. Pessin

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

The dissociation of the purified human placental α2β2 heterotetrameric insulin receptor complex into an αβ heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the αβ 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 α2β2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5 m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated α2β2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated αβ 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 α2β2 heterotetrameric and αβ heterodimeric complexes occurred via an increase in V(max) without any significant change in K(m). Examination of β subunit autophosphorylation of the αβ heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled α2β2 heterotetrameric complex. Further, the initial rate of insulin-stimulated β subunit autophosphorylation in the isolated αβ hterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated αβ heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the β subunit protein kinase domain with the covalent reassociation of the 2aβ heterodimeric complex into an α2β2 heterotetrameric disulfide-linked state.

Original languageEnglish (US)
Pages (from-to)16730-16738
Number of pages9
JournalJournal of Biological Chemistry
Volume262
Issue number34
StatePublished - 1987
Externally publishedYes

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Insulin Receptor
Dithiothreitol
Communication
Insulin
Protein Kinases
Gels
Phosphorylation
Chromatography
Disulfides
Dilution
Gel Chromatography
Tyrosine
Glutamic Acid
Carrier Proteins
Polymers
Chemical activation
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Insulin-dependent intermolecular subunit communication between isolated αβ heterodimeric insulin receptor complexes. / Sweet, L. J.; Morrison, B. D.; Wilden, P. A.; Pessin, Jeffrey E.

In: Journal of Biological Chemistry, Vol. 262, No. 34, 1987, p. 16730-16738.

Research output: Contribution to journalArticle

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abstract = "The dissociation of the purified human placental α2β2 heterotetrameric insulin receptor complex into an αβ heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the αβ 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 α2β2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5 m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated α2β2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated αβ 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 α2β2 heterotetrameric and αβ heterodimeric complexes occurred via an increase in V(max) without any significant change in K(m). Examination of β subunit autophosphorylation of the αβ heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled α2β2 heterotetrameric complex. Further, the initial rate of insulin-stimulated β subunit autophosphorylation in the isolated αβ hterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated αβ heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the β subunit protein kinase domain with the covalent reassociation of the 2aβ heterodimeric complex into an α2β2 heterotetrameric disulfide-linked state.",
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