Identification of an EGF/TGF-a receptor in primary cultures of guinea pig gastric mucous epithelial cells

Michael J. Rutten, Peter J. Dempsey, Cheryl A. Luttropp, Mitchell A. Hawkey, Brett C. Sheppard, Richard A. Crass, Clifford W. Deveney, Robert J. Coffey

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Binding and localization of transforming growth fac- tor-a (TGF-α) and epidermal growth factor (EGF) were assessed using in vitro primary cultures of guinea pig gastric mucous epithelial cells (GMEC). GMEC were isolated and cultured in six-well plates with Dulbecco's modified Eagle's medium + 10% serum and then changed to serum-free medium for 24 h for binding studies. The binding time course of 125I-labeled EGF and 125I-TGF-α in GMEC cultures at 4°C was saturable, reaching a plateau within 4-6 h. Competitionbinding curves revealed that the amount of unlabeled EGF and TGF-α to reduce 125I-EGF binding by 50% was 0.35 and 0.23 nM, respectively. The amount of unlabeled EGF and TGF-α to decrease 125I-TGF-α binding by 50% was 0.30 and 0.21 nM, respectively. A Scatchard analysis of the data disclosed that a single class of high-affinity binding sites (dissociation constant = 0.24 nM) was present. The maximal binding capacity was ∼20 fmol/106 cells or ∼12,000 receptors per ceU. The binding of 125I-EGF and 125I-TGF-α to GMEC cultures was maximal between pH 7.0 and 8.5. No specific binding of EGF or TGF-α could be detected below pH 5.0. The half-maximal pH dissociation value for EGF and TGF-α was pH 5.89 and pH 6.83, respectively. We found no difference in the final amounts of membrane-bound or internalized 125IEGF and 125I-TGF-α. However, there was a significant difference (P < 0.05) at 5-30 min in the rate of dissociated and internalized 125I-EGF and 125I-TGF-α. Immunofluorescence microscopy of GMEC cultures for EGF/TGF-α-receptors showed increased fluorescence at the leading edges and around the perimeter of cells. Detection of an EGF/TGF-α receptor was also confirmed by Western blotting. Our findings demonstrate that guinea pig GMEC possess a specific EGF/ TGF-α receptor, which further supports a physiological role for EGF and TGF-α as mitogens in these cells.

Original languageEnglish (US)
Pages (from-to)G604-G612
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume270
Issue number4 33-4
DOIs
StatePublished - Jan 1 1996

Keywords

  • Epidermal growth factor
  • Mitogen
  • Repair
  • Signal transduction
  • Stomach
  • Transforming growth factor-α
  • Ulcers

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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