AJP - Gastrointestinal and Liver Physiology, Vol 270, Issue 4 604-G612, Copyright © 1996 by American Physiological Society

ARTICLES

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

M. J. Rutten, P. J. Dempsey, C. A. Luttropp, M. A. Hawkey, B. C. Sheppard, R. A. Crass, C. W. Deveney and R. J. Coffey Jr
Department of Surgery, Oregon Health Sciences University, Portland, USA.

Binding and localization of transforming growth factor-alpha (TGF-alpha) 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-alpha in GMEC cultures at 4 degrees C was saturable, reaching a plateau within 4-6 h. Competition-binding curves revealed that the amount of unlabeled EGF and TGF-alpha to reduce 125I-EGF binding by 50% was 0.35 and 0.23 nM, respectively. The amount of unlabeled EGF and TGF-alpha to decrease 125I-TGF-alpha 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 approximately 20 fmol/10(6) cells or approximately 12,000 receptors per cell. The binding of 125I-EGF and 125I-TFG-alpha to GMEC cultures was maximal between pH 7.0 and 8.5. No specific binding of EGF or TGF-alpha could be detected below pH 5.0. The half-maximal pH dissociation value for EGF and TGF-alpha was pH 5.89 and pH 6.83, respectively. We found no difference in the final amounts of membrane-bound or internalized 125I-EGF and 125I-TGF-alpha. 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-alpha. Immunofluorescence microscopy of GMEC cultures for EGF/TGF-alpha receptors showed increased fluorescence at the leading edges and around the perimeter of cells. Detection of an EGF/TGF-alpha receptor was also confirmed by Western blotting. Our findings demonstrate that guinea pig GMEC possess a specific EGF/TGF-alpha receptor, which further supports a physiological role for EFG and TFG-alpha as mitogens in these cells.

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