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Journal of Clinical Endocrinology & Metabolism Vol. 62, No. 4 692-699
doi:10.1210/jcem-62-4-692
Copyright © 1986 by the Endocrine Society.
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The Human Erythrocyte Insulin-Like Growth Factor I Receptor:Characterization and Demonstration of Ligand-Stimulated Autophosphorylation

VERONICA.M. CATANESE, FLORIN GRIGORESCU, GEORGE L. KING and C. RONALD KAHN

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 (ED50, 15 ng/ml), whereas insulin and proinsulin were 30- to 500-fold less potent. A monoclonal antibody to the IGF-I receptor ({alpha}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 [{gamma}32 P] ATP in the presence of Mn2+, and the receptor was identified by immuno-precipitation with {alpha}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 {alpha}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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