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

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous αβ heterodimeric insulin receptors in addition to the disulphide-linked α₂β₂ 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 M(r) 400,000 and 200,000 bands, corresponding to the α₂β₂ heterotetrameric and αβ heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the α₂β₂ 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 α₂β₂ heterotetrameric insulin receptor complex, whereas in the absence of IAN both α₂β₂ and αβ species were observed. Turkey erythrocyte α₂β₂ heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of β subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte αβ 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 αβ heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte αβ 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 α₂β₂ heterotetrameric human placenta insulin receptors. The αβ heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the α₂β₂ holoreceptor complexes during membrane preparation.