Identification, partial purification, and characterization of two guanosine triphosphate-binding proteins associated with insulin receptors

Abstract

We have previously suggested that at least two different G-proteins are involved in mediating insulin receptor functions. Here we identify and partially purify two G-proteins with apparent molecular masses of 41 and 67 kilodaltons (kDa) that interact with insulin receptors in rat adipocytes and human placenta. Treatment of isolated rat adipocytes with insulin inhibited pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa G-protein in subsequently isolated plasma membranes by 30.2 +/- 3.0% and in partially purified insulin receptor preparations by 35.6 +/- 5.7%. There was no associated decrease in the concentration of the 41-kDa G-protein in the plasma membranes, as determined by immunoblot with a common G alpha antibody. The common G alpha antibody also recognized a 67-kDa protein in the plasma membranes, the concentration of which was not affected by insulin. However, the 67-kDa protein was enriched in partially purified solubilized insulin receptor preparations. Two similar, 41- and 67-kDa G-proteins were identified in the wheat germ-purified insulin receptor preparations obtained from human placenta. Removal of these two G-proteins from insulin receptor preparations results in loss of the ability of insulin to stimulate receptor kinase activity. Addition of a fraction enriched with 41- and 67-kDa G-proteins to the G-protein-depleted insulin receptor restores the insulin sensitivity of the insulin receptor kinase activity. Furthermore, addition of G-protein-depleted insulin receptors to the fraction containing partially purified 41- and 67-kDa G-proteins enhances pertussis toxin-catalyzed ADP-ribosylation of the 41-kDa G-protein. These results indicate that either the 41- or 67-kDa G-protein, or both, interact with the insulin receptor mediating insulin receptor kinase activity. Such mutual interaction and regulation between the insulin receptor and G-proteins could be an important component of the signal transduction mechanism for insulin.