In the first step in insulin action, insulin binds to its receptor on the surface of the target cell. The insulin receptor is a transmembrane protein that possesses tyrosine-specific protein kinase activity. When insulin binds to the extracellular domain of the receptor, this activates the receptor tyrosine kinase activity. A growing body of evidence suggests that the activation of the receptor's tyrosine kinase is a necessary step in initiating the biological actions of insulin. Accordingly, we have embarked upon a search for intracellular proteins that are substrates for phosphorylation by the receptor-associated tyrosine kinase. We have identified one such substrate in rat liver plasma membranes: a glycoprotein with an apparent molecular weight of 120,000 (pp 120). In addition to being a substrate for the insulin receptor, ppl2O can be phosphorylated by the receptors for epidermal growth factor and insulin-like growth factor I. pp 120 is present in liver from several species, but has not been identified in other tissues. The glycoprotein (ppl2O) was immunoaffinity-purified using monoclonal antibody HA4. Based on partial amino acid sequence data, pp)20 has been tentatively identified as ectoATPase - an enzyme associated with hepatocyte plasma membranes. Studies are underway to express ectoATPase by transfection of cDNA in tissue culture cell lines. This will facilitate answering the question of whether tyrosine phosphorylation regulates the enzymatic activity of ectoATPase.
