Identification of an egf/tgf[alpha] receptor in primary cultures of
guinea pig gastric mucous epithelial cells.
Rutten, Michael J., Peter J. Dempsey, Cheryl A. Luttropp, Mitchell A.
Hawkey, Brett C. Sheppard, Richard A. Crass, Clifford W. Deveney, and
Robert J. Coffey, Jr.
V.A. Medical Center, Portland, OR 97201, Department of Surgery,
Oregon Health Sciences University, Portland, OR 97201; and
Departments of Medicine and Cell Biology, Vanderbilt University, and
VA Medical Center, Nashville, TN 37232
APStracts 2:0201G, 1995.
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 6-well plates with DMEM
+10% serum, then changed to serum-free media for 24 hr for binding
studies. The binding time course of 125I-EGF and 125I-TGF[alpha] in
GMEC cultures at 4oC was saturable reaching a plateau within 4-6 hr.
Competition binding curves found that the amount of unlabeled EGF and
TGF[alpha] to reduce 125I-EGF binding by 50% was 0.35 nM and 0.23 nM,
respectively. The amount of unlabeled EGF and TGF[alpha] to decrease
125I-TGF[alpha] binding by 50% was 0.30 nM and 0.21 nM, respectively.
A Scatchard analysis of the data disclosed that a single class of
high affinity binding sites (Kd = 0.24 nM) was present. The total
binding capacity (Bmax) was 20 fmoles/106 cells or 12,000 receptors
per cell. The binding of 125I-EGF and 125I-TGF[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 amount 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 the GMEC cultures for
EGF/TGF[alpha]-receptors showed increased fluorescence at the leading
edges and around the perimeter of cells. The detection of an
EGF/TGF[alpha] receptor was also confirmed by Western blotting. Our
findings demonstrate that guinea pig gastric mucous epithelial cells
possess a specific EGF/TGF[alpha] receptor, which further supports a
physiological role for EGF and TGF[alpha] as mitogens in these cells.
Received 17 November 1994; accepted in final form 28 September
1995.
APS Manuscript Number G454-4.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 November 95