Insulin binding and insulin-dependent phosphorylation of the insulin receptor solubilized from human erythrocytes.

Abstract

Insulin receptors were extracted from human erythrocytes contained in 100 ml of blood with the nonionic detergent Triton X-100 with almost 100% yield. The solubilized receptor had binding characteristics similar to those of the intact cell. Using 125I-monoiodoinsulin as tracer and a computer-assisted statistical curve-fitting program, a cooperative model gave values of 1.7 X 10(9) M-1 for the Ke (affinity of the empty receptor) and of 1.6 X 10(8) M-1 for Kf (affinity of the filled receptor). Bovine desalanine-desasparagine insulin inhibited tracer binding with 3-6% the potency of porcine insulin. Serum (B-8) containing anti-insulin receptor antibodies inhibited binding by 70% at a dilution of 1:100. Receptor autophosphorylation reaction was studied by incubation of the Triton extract with [gamma-32P]ATP and Mn2+ in the presence or absence of insulin, and the receptor was identified by immunoprecipitation with anti-receptor antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Porcine insulin stimulated 4- to 5-fold the incorporation of 32P in a protein of Mr = 95,000, corresponding to the beta-subunit of the insulin receptor. Phosphoamino acid analysis revealed phosphorylation of the tyrosyl residues exclusively. The dose-response curve for insulin stimulation was sigmoidal; some effect of insulin was observed at 1 ng/ml but maximal effect was observed at 10 micrograms/ml. Bovine desalanine-desasparagine insulin, a noncooperative analogue of insulin, was able to fully stimulate the phosphate incorporation, but the dose-response curve was shifted to the right and steeper, consistent with the intrinsic affinity of this analogue for the insulin receptor. When insulin binding was performed under the same conditions as the phosphorylation, half-maximal stimulation of phosphate incorporation occurred with 6-29% of the fractional occupancy of the receptor. These data suggest that the insulin receptor of the human erythrocyte, as in other cells, is a tyrosine-specific protein kinase. Coupling between the receptor occupancy and kinase activation is complex. Furthermore, sufficient quantities of receptor can be easily obtained from a single individual to study the binding and kinase properties of the receptor opening the opportunity to a wide field of applications in human pathology.