Characterization of type I and type II insulin-like growth factor receptors in an intestinal epithelial cell line

Jung H.Y. Park, Jon A. Vanderhoof, Darcy Blackwood, Richard G. Macdonald

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54 Scopus citations

Abstract

Insulin-like growth factors (IGFs) and insulin stimulate DNA and protein synthesis in IEC-6 cells (an intestinal epithelial cell line) grown in a chemically defined medium. IGF-I stimulates proliferation of IEC-6 cells at a lower concentration (ED50 = 1.6 nM) than either insulin or IGF-II. To gain insight into the mechanisms by which IGFs stimulate IEC-6 cell growth, we have examined the characteristics of specific IGF receptors on IEC-6 cells. Binding of 125I-IGF-I and 125I-IGF-II to IEC-6 monolayers was analyzed by incubation with various concentrations (0.2 nM to 0.5 μM) of radiolabeled IGFs for 16 h at 3 C. Scatchard plots of 125I-IGF-I binding were linear, suggesting a single class of binding sites with KD = 3.1 ± 0.35 nM and Bmax = 50.7 ± 6 fmol/106cells. IGF-II was potent in displacing 125I-IGF-I (KI = 8.1 ± 0.85 nM), but insulin had little effect. Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed three bands with Mr of 270,000, 245,000, and 133,000, and the major band was the 133,000 species. Labeling of the 133,000 and 270,000 bands was ≥80% inhibited by 10-7 M unlabeled IGF-I, less potently inhibited by IGF-II and not at all by insulin. These results suggest that the 133,000 band represents the α-subunit of the type I IGF receptor. Scatchard plots of 125I-IGF-II binding to IEC-6 cell monolayers were curvilinear, suggesting two classes of binding sites: high affinity, low capacity sites, KD = 0.87 ± 0.08 nM and Bmax = 28 ± 2.5 fmol/106 cells; low affinity, high capacity sites, KD = 60 = ± 8.8 nM and Bmax = 1780 ± 230 fmol/ 106 cells. Neither IGF-I nor insulin was effective in inhibiting 125I-IGF-II binding. Affinity cross-linking with 125I-IGF-II labeled predominantly a 245,000 band, suggesting that this species is the type II receptor. A band with Mr 131,000 was barely detectable with 125I-insulin. These results indicate that IEC-6 cells have abundant quantities of the type I and II IGF receptors and few insulin receptors, suggesting that the mitogenic effect of IGFs is mediated through the type I IGF receptor.

Original languageEnglish (US)
Pages (from-to)2998-3005
Number of pages8
JournalEndocrinology
Volume126
Issue number6
StatePublished - Jun 1990

ASJC Scopus subject areas

  • Endocrinology

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