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

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.