Properties of the Insulin Receptor Isolated from Liver and Fat Cell Membranes

Abstract The insulin-binding proteins isolated from membranes of rat liver and of fat cells with the nonionic detergent, Triton X-100, appear to have similar or identical physicochemical and kinetic properties. The denaturation of the soluble receptor by reagents, such as sodium dodecyl sulfate, urea, and guanidine·HCl, is reversible if moderately low concentrations of the reagents are used. The binding protein is relatively stable to storage at 4 or -20°. Digestion of the water-soluble receptor with phospholipase C and neuraminidase does not affect its capacity to bind 125I-insulin. High concentrations of NaCl (2 m) are virtually without effect on the insulin-receptor-binding interaction. The specific insulin-binding activity of the receptor is completely destroyed by digesting with concentrations of trypsin which are too low to modify the native receptor in the intact membrane but which are equally effective in destroying the receptor of phospholipase-treated membranes. Gel filtration experiments on columns of Sepharose 6B calibrated with several standard [14C]acetyl proteins indicate that the soluble insulin receptor has a molecular radius (Stokes radius) of about 70 A. This large molecular size is not changed by delipidation of the membranes before extracting the receptor, by phospholipase digestion of the soluble receptor, or by performing the chromatography in buffers containing varying amounts of detergent, 2 m NaCl, or 10% sucrose. In the absence of detergent highly aggregated forms of the receptor are observed. Sedimentation in 5 to 20% sucrose gradients containing 0.1 to 1.0% Triton X-100 reveals that the water-soluble receptor has a sedimentation constant of 11 S, as judged by the behavior of various standard 14C-labeled proteins under similar conditions. As in the gel filtration experiments, no change in sedimentation occurs after delipidation procedures or by varying the detergent concentration of the sucrose solutions. On the basis of gel filtration and sedimentation experiments, the receptor has a molecular weight of about 300,000, a frictional ratio of about 1.5, and an axial ratio (prolate ellipsoid) of about 9. The highly asymmetrical character of the protein is probably not explained by unusually high values of solvation or of partial specific volume (v). The insulin-receptor sediments in cesium chloride solutions having densities of 1.228 and 1.298, consistent with the view that it is not a lipoprotein. Scatchard plots indicate the presence of a kinetically homogeneous binding function with a dissociation constant near 10-10 m. The rate constants of insulin-receptor complex formation and dissociation are 2 to 3 x 106 mole-1 sec-1 and about 4 x 10-4 sec-1, respectively. Complex formation does not result in detectable alteration of the insulin molecule. No specific insulin-binding activity is detected in the cytosol of fat cell homogenates, and after homogenization of fat cells treated with trypsin or agarose-trypsin, it is possible to show that intracellular membranes or particles have no significant and specific insulin-binding activity. These results argue strongly for the exclusive localization of the receptor to the cell surface.