Insulin receptor: reconstitution, lipid effects, and identification of its putative membrane substrates

In this study, the main objectives are (1) to reconstitute functional insulin receptors into liposomes with defined lipid compositions; (2) to study lipid effects on insulin binding and receptor kinase activities; and (3) to identify potential membrane substrates for the insulin receptor. Lectin-purified insulin receptors from human placenta were incorporated into PC/PE liposomes of different relative molar ratios. Prior to reconstitution, endogenous lipids of insulin receptor preparations were analyzed by thin layer, chromatography. Characterization of PC/PE proteoliposomes was achieved by freeze fracture electron microscopy. An affinity cross-linking procedure was performed to identify the insulin binding component. The receptor kinase activity was immunoprecipitated with human anti-receptor antibodies, 125I-insulin binding assays and phosphorylation assays were employed to study the lipid effects on the biochemical properties of the insulin receptor. The potential substrates for the insulin receptor were investigated by a double labelling technique. The insulin-dependent phosphorylation of membrane proteins was then examined by two dimensional gel electrophoresis. Finally, preliminary characterization of the membrane substrates was achieved by partial proteolysis and Western blot analysis