Tissue distribution and subcellular localization of an endogenous substrate (ppl20) for the insulin receptor-associated tyrosine kinase

The β-subunit of the insulin receptor possesses a tyrosine-specific protein kinase activity which may play a role in coupling insulin binding to insulin action. Previously, we have identified a substrate for the receptor-associated protein kinase in a cell-free system. This endogenous substrate (ppl20), which appeared to be a glycoprotein with an apparent mol wt of 120,000, was detected in rat liver microsomes. In the present work, we have demonstrated that ppl20 is localized to a highly purified preparation of rat liver plasma membranes (Neville preparation). Moreover, ppl20 appears to be specific to liver, having been detected in liver from rat, monkey, and rabbit, but not in rat brain, skeletal muscle, heart, kidney, or adipocytes. As a preliminary to addressing the question of whether insulin stimulates phosphorylation of ppl20 in intact cells, we have sought to identify tissue culture cell lines that contain both insulin receptors and ppl20. We have succeeded in identifying ppl20 in two cell lines derived from rat liver: 1) H35 hepatoma cells (Reuber hepatoma) and 2) rat hepatocytes transformed with a temperature-sensitive mutant form of SV-40 (cultivated at both permissive and nonpermissive temperatures). In conclusion, ppl20 appears to be a liver-specific plasma membrane glycoprotein which serves as a substrate for phosphorylation by the insulin receptor-associated protein kinase in a soluble cell-free system. The presence of ppl20 in cultured cell lines will facilitate investigation of whether the phosphorylation of ppl20 in intact cells is physiologically regulated in response to insulin. © 1986 by The Endocrine Society.