Reconstitution of Bovine Placental Insulin Receptor on Artificial Vesicles by Using Direct Protein Transfer Techniques

Abstract

Direct protein transfer with artificial boundary lipid, DDPC, has been used as a powerful isolation procedure for membrane proteins. In contrast to other methods using detergents as protein solubilizer, the target membrane proteins can be transferred directly from biomembrane to liposomes by this direct protein transfer method, without severe denaturation in the course of isolation. We have investigated the transfer of insulin receptor proteins from bovine placental plasma membrane into liposomes. All the subunits of insulin receptor was found reconstituted in liposomes. The reconstituted insulin receptor retained almost full activity to bind to insulin, leading to autophosphorylation as a result of signal transduction activity. It is quite important that the reconstituted insulin receptor can be isolated in liposomes, which can be served to further investigation of insulin receptor without any interferences from other enzymes in cells. Then we focused on the insulin binding activity. The reconstituted insulin receptor showed much higher binding affinity than that on plasma membrane or that solubilized in micelles, suggesting that insulin receptor protein isolated by a conventional method might catch a significant denaturation. A fully active and complete insulin receptor was isolated by this direct protein transfer method. This method is applicable extensively to other membrane proteins.