The endogenous functional turkey erythrocyte and rat liver insulin receptor is an α2β2 heterotetrameric complex

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Abstract
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 resutled 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 inuslin-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-sensitive. 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.

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