The endogenous functional turkey erythrocyte and rat liver insulin receptor is an alpha 2 beta 2 heterotetrameric complex.
Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.
Treadway
J L
JL
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Morrison
B D
BD
Wemmie
J A
JA
Frias
I
I
O'Hare
T
T
Pilch
P F
PF
Pessin
J E
JE
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004910:Erythrocyte Membrane / Q000737:chemistry*; D005033:Ethylmaleimide; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007460:Iodoacetamide; D008099:Liver / Q000737:chemistry*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D010920:Placenta / Q000737:chemistry; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D014422:Turkeys / Q000097:blood
D003432:Cross-Linking Reagents; D007328:Insulin; D046911:Macromolecular Substances; D011494:Protein Kinases; D011972:Receptor, Insulin; D005033:Ethylmaleimide; D007460:Iodoacetamide