Cytoplasmic juxtamembrane region of the insulin receptor: a critical role in ATP binding, endogenous substrate phosphorylation, and insulin-stimulated bioeffects in CHO cells

Abstract

We have expressed in CHO cells a mutant receptor (IRA960) from which 12 amino acids in the juxtamembrane region (A954-D965), including Tyrm have been deleted. The mutant receptor bound insulin normally but exhibited an increased K,,, for ATP during autophosphorylation. Upon prolonged incubation in vitro, or at high ATP concentrations such as those observed in vivo, autophosphorylation of IRA960 was similar to wild type, and the in vitro phosphotransferase activity of the autophosphorylated IRAW was normal. These results suggest that the deletion did not cause a nonspecific structural disruption of the catalytic domain of IRA960. In vivo autophosphorylation of the IRA960 receptor was reduced by 30% after 2 min of insulin stimulation and was similar to the wild-type receptor after 30 min of insulin stimulation. However, the mutant receptor was defective in insulin-stimulated tyrosyl phosphorylation of the endogenous substrate pp185. In additon, IRA960 was deficient in mediating insulin stimulation of glycogen and DNA synthesis. Thus, autophosphorylation of the insulin receptor is necessary but not sufficient for signal transmission. These data extend the hypothesis that the cytoplasmic juxtamembrane region of the insulin receptor is important for its interactions with ATP, intracellular substrates, and other proteins and is broadly necessary for biological signal transmission.