CHARACTERIZATION OF TYPE-I AND TYPE-II INSULIN-LIKE GROWTH-FACTOR RECEPTORS IN AN INTESTINAL EPITHELIAL-CELL LINE

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. 1GF-I stimulates proliferation of IEC-6 cells at a lower concen-tration (ED60= 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 of125I-IGF-I and125I-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 of125I-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/106 cells. IGF-II was potent in displacing125I-IGF-I (K1= 8.1 ± 0.85 nM), but insulin had little effect. Affinity cross-linking with125I-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 of125I-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 BmM = 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 inhibiting125I-IGF-II binding. Affinity cross-linking with125I-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 with125I-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. © 1990 by The Endocrine Society.