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The Human Erythrocyte Insulin-Like Growth Factor I Receptor:Characterization and Demonstration of Ligand-Stimulated Autophosphorylation

Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital Harvard Medical School Boston, Massachusetts 02215

Address correspondence and requests for reprints to:Dr. C. Ronald Kahn, Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02215.

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 x 10⁽⁹⁾cells/ml. The Scatchard plot of the binding data was linear, with 7 fmol [¹²⁵I]IGF-I bound/10⁹cells and an affinity constant (K) of 1.8 x 10⁹ M^–1.Unlabele d IGF-I inhibited tracer binding half-maximally at 6 ng/ml. Multiplication-stimulating activity (or rat IGF-II) was 40% as potent (ED_50, 15 ng/ml), whereas insulin and proinsulin were 30- to 500-fold less potent. A monoclonal antibody to the IGF-I receptor (IR-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 erythrocyteghosts prepared by hypotonic lysis and solubilized in 1% Triton. The extract was preincubated with and without 100 ng/ml IGF-I or porcine insulin and incubated with [–³² P] ATP in the presence of Mn²⁺, and the receptor was identified by immuno-precipitation with IR-3 antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IGF-I stimulated 4-fold the incorporation of ³²P into a protein of 95,000 mol wt, which was immunoprecipitated by IR-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 autophosphoryla-tion. 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. (J Clin Endocrinol Metab62: 692,1986)

Received August 5, 1985.

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