Insulin-dependent covalent reassociation of isolated alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric disulfide-linked complex.
The purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Insulin treatment of the isolated alpha beta heterodimeric complex was observed to induce the complete reassociation to an alpha 2 beta 2 heterotetrameric state when analyzed by nondenaturing Bio-Gel A-1.5m gel filtration chromatography. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin affinity cross-linked and 32P-autophosphorylated alpha beta heterodimers demonstrated that the insulin-dependent reassociation to the alpha 2 beta 2 heterotetrameric state occurred both covalently and noncovalently under these conditions. Comparison by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the insulin-dependent covalent reassociation to an alpha 2 beta 2 heterotetrameric complex was due to the formation of a disulfide linkage(s) between the alpha beta heterodimers. beta subunit autophosphorylation of the control alpha 2 beta 2 heterotetrameric insulin receptor preparation was maximally stimulated within 5 min of insulin preincubation and occurred exclusively in the Mr = 400,000 alpha 2 beta 2 complex. Similarly, maximal insulin-stimulated beta subunit autophosphorylation of the alpha beta heterodimeric preparation occurred within 5 min of insulin pretreatment in the Mr = 210,000 alpha beta complex. However, 4 h of insulin pretreatment of the alpha beta heterodimer preparation induced the formation (6-fold) of a covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Maximal stimulation of substrate phosphorylation for the alpha 2 beta 2 heterotetrameric complex was also observed to occur within 5 min of insulin treatment, whereas maximal insulin-stimulated substrate phosphorylation of the alpha beta heterodimeric complex required greater than 4 h. These data demonstrate that (i) insulin treatment can induce the reassociation of the alpha beta heterodimeric complex into a covalent alpha 2 beta 2 heterotetrameric state, and (ii) insulin-dependent protein kinase activation of the alpha beta heterodimeric insulin receptor correlates with the covalent reassociation into a disulfide-linked alpha 2 beta 2 heterotetrameric complex.
Morrison
B D
BD
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Swanson
M L
ML
Sweet
L J
LJ
Pessin
J E
JE
D000478:Alkylation; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010920:Placenta / Q000032:analysis; D011108:Polymers; D011972:Receptor, Insulin / Q000378:metabolism*
D004220:Disulfides; D007328:Insulin; D046911:Macromolecular Substances; D011108:Polymers; D011972:Receptor, Insulin; D004229:Dithiothreitol