The purified human placental α2β2 heterotetrameric insulin-receptor complex was reduced and dissociated into functional αβ heterodimers by a combination of alkaline pH and dithiothreitol treatment. Wheat germ agglutinin (WGA) was observed to stimulate the β-subunit autophosphorylation of both the α2β2 heterotetrameric and αβ heterodimeric complexes in the absence of insulin. However, WGA was without effect on the insulin stimulation of β-subunit autophosphorylation in these insulin-receptor complexes. In contrast, monomeric WGA was unable to stimulate the basal exogenous substrate protein kinase activity in either the α2β2 heterotetrameric or αβ heterodimeric insulin receptor complexes. The stimulatory effect of WGA was biphasic, increasing the basal insulin receptor β-subunit autophosphorylation at low concentrations (250 ng/ml to 2.5 Î¼g/ml) and inhibiting at high concentrations (>25 Î¼g/ml). Similarly, equilibrium tracer insulin binding was not significantly altered by low concentrations of WGA (<1 Î¼g/ml) but was dramatically reduced at high WGA concentrations (Gt;2.5 Î¼g/ml). In contrast to the insulin-induced covalent association of the αβ heterodimeric insulin receptors to form a disulfidelinked α2β2 heterotetrameric complex, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the WGA stimulation of β-subunit autophosphorylation in the αβ heterodimer preparation occurred in the absence of covalent association. Nondenaturing Bio-Gel A-1.5m gel filtration chromatography and [125I]insulin affinity cross-linking demonstrated that WGA treatment of the αβ heterodimeric insulin receptors induced a noncovalent association into an α2β2 heterotetrameric state. These data support the hypothesis that the insulin receptor protein kinase activity, although dependent upon αβ heterodimeric subunit interactions, does not necessarily require covalent disulfide bond formation between the individual αβ heterodimeric species.
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