The rat liver insulin receptor

Abstract

Using insulin affinity chromatography, we have isolated higly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 .alpha.-subunit, the Mr 90,000 .beta.-subunit, and varying proportions of the Mr 45,000 .beta.''-subunit. The specific insulin binding of the purified receptor was 25-30 .mu.g of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the .beta.-subunit to .beta.'' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact .beta.-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing .beta.-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors. In summary, rat liver and human placental receptors differ functionally in both .alpha.- and .beta.-subunits. Insulin binding to the .alpha.-subunit of the purified rat liver receptor communicates a signal that activates the .beta.-subunit; however, major proteolytic destruction of the .beta.-subunit does not affect insulin binding to the .alpha.-subunit. This suggests that communication does not occur in the reverse direction, i.e., .beta. .fwdarw. .alpha.