Structural requirements for the transmembrane activation of the insulin receptor kinase

Tetrameric insulin holoreceptor (α₂β₂) was reduced with dithiothreitol into αβ dimers such that they maintain up to 50% of insulin binding at tracer ligand concentrations. Scatchard analysis of insulin binding to dimers revealed that they had a reduced affinity for ligand by a factor of 3-6 compared to holoreceptor, whereas the maximum number of high affinity binding sites was not affected. The αβ dimers can be separated from holoreceptor by sucrose density gradient centrifugation, and hence, they are not associated by noncovalent interactions. Insulin-dependent autophosphorylation of αβ dimers isolated from low ionic strength sucrose density gradients was minimal and was always accompanied by reoxidation of dimers to the tetrameric holoreceptor. The reformed tetramer exhibited a strong insulin-dependent autophosphorylation reaction. Reoxidation was prevented by isolating αβ dimers in sucrose density gradients containing 0.15 M NaCl. Under these conditions, no insulin-dependent autophosphorylation was observed. When insulin receptor was first autophosphorylated and then reduced, receptor kinase activity, as assayed by histone phosphorylation, was not affected. Also, the insulin-independent, basal autophosphorylation was maintained after reduction into αβ dimers. We conclude that α-β interaction is not necessary for the maintenance of basal kinase activity or for insulin-activated kinase activity once autophosphorylation occurs. However, dimer-dimer interaction appears critical for the insulin-dependent activation of the receptor's intrinsic kinase activity.