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. 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 K_D = 3.1 ± 0.35 nM and B_max = 50.7 ± 6 fmol/10⁶cells. IGF-II was potent in displacing ¹²⁵I-IGF-I (K_I = 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 M_r 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, K_D = 0.87 ± 0.08 nM and B_max = 28 ± 2.5 fmol/10⁶ cells; low affinity, high capacity sites, K_D = 60 = ± 8.8 nM and B_max = 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 M_r 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.