The human erythrocyte insulin-like growth factor i receptor:Characterization and demonstration of ligand-stimulated autophosphorylation

Abstract

To characterize the insulin-like growth factor I (IGF-I) receptor on human erythrocytes, cells were purified from peripheral blood by Ficoll-Hypaque gradient centrifugation and incubated with [125I]IGF-I. Specific binding was maximal at pH 8.0 after 24 h at 4 C and increased linearly with cell number to 3.9 ± 0.2% (±SEM) for 3.0 × 109 cells/ml. The Scatchard plot of the binding data was linear, with 7 fmol [125I]IGF-I bound/109 cells and an affinity constant (K) of 1.8 × 109 M-1. Unlabeled IGF-I inhibited tracer binding half-maximally at 6 ng/ml. Multiplication- stimulating activity (or rat IGF-II) was 40% as potent (ED50, 15 ng/ml), whereas insulin and proinsulin were 30- to 500-fold less potent. A monoclonal antibody to the IGF-I receptor (aIR-3) inhibited IGF-I binding by 50% at a 1:1000 dilution and by 80% at a 1:250 dilution. Insulin binding was unaffected by the same dilutions. IGF-I receptor phosphorylation was studied in erythrocyte ghosts prepared by hypotonic lysis and solubilized in 1% Triton. The extract was preincubated with and without 100 ng/ml IGFI or porcine insulin and incubated with [γ-32P]ATP in the presence of Mn2+, and the receptor was identified by immunoprecipitation with αIR-3 antibody and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. IGF-I stimulated 4-fold the incorporation of 32P into a protein of 95, 000 mol wt, which was immunoprecipitated by αIR-3; insulin produced a 2-fold stimulation of this protein. This protein corresponds to the β-subunit of the IGF-I receptor. These data demonstrate that human erythrocytes have specific receptors for IGF-I, and that this IGF-I receptor, like the insulin receptor, undergoes ligand-stimulated autophosphorylation. Thus, analysis of erythrocyte IGF-I binding and receptor phosphorylation may be useful tools for the study of patients with a variety of growth disorders. © 1986 by The Endocrine Society.