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 microM) 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/10(6) cells. 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 greater than or equal to 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 alpha-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/10(6) cells; low affinity, high capacity sites, KD = 60 = +/- 8.8 nM and Bmax = 1780 +/- 230 fmol/10(6) 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.
Park
J H
JH
Department of Pediatrics, Creighton University, Omaha, Nebraska.
Vanderhoof
J A
JA
Blackwood
D
D
Macdonald
R G
RG
D001667:Binding, Competitive; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017451:Receptors, Somatomedin
D007328:Insulin; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II