CATALYTIC COMPETENCE AND REGULATION OF RECEPTOR KINASE

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The intact phosphorylated and unphosphorylated forms of insulin receptor kinase will be purified from human placenta by chromatography on immobilized anti-O-phosphotyrosyl antibody. The purified forms of the receptor will be used to characterize the multistep reaction pathway for the conversion of the unphosphorylated form of the receptor to the catalytically active phosphorylated form. In these studies the amino acid sequence around the reactive tyrosyl residue(s) in the receptor will be determined and compared to that of other tyrosine kinases. Rate and equilibrium constants will be evaluated for individual steps in the reaction pathway for receptor activation. Stoichiometry and equilibirum constants for the binding of insulin to the phosphorylated and unphosphorylated forms of the receptor will be measured to determine whether insulin binding might cause the unphosphorylated receptor to assume a conformation similar to that of the phosphorylated receptor. The inhibitory effect of zinc ion on individual steps in the reaction pathway for receptor activation will be characterized, since inhibition of autophosphorylation of the insulin receptor by zinc ion might serve to protect the islet cells and other tissues from deleterious effects of high concentrations of insulin at the site of normal or accidental release of the contents of insulin-containing granules. Another set of studies will be directed toward characterization of interactions of the receptor kinase with potential regulators of metabolism. Thus, the possibility will be assessed that insulin receptor kinase catalyzes phosphorylation of the glucose transporter protein and thereby decreases its exit rate from plasma membranes. The phosphorylation of insulin receptor by casein kinase I will be studied to determine whether casein kinase I catalyzed phosphorylation of insulin receptor alters its ability to bind insulin and undergo autophosphorylation, and whether casein kinase I catalyzed phosphorylations of the receptor might account for the phosphorylation at receptor seryl and thereonyl residues seen in whole cells. In a search for endogenous substrates for the insulin receptor kinase, anti-O-phosphotyrosyl antibody will be used to concentrate products of insulin promoted tyrosyl phosphorylations in whole cells. In an attempt to deduce the structural characteristics of natural substrates for the insulin receptor kinase, the substrate specificity of this enzyme toward nonpeptide and peptide substrates will be determined.