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© The Rockefeller University Press, 0021-9525/1997//747 $5.00
The Journal of Cell Biology, Volume 138, Number 4, , 1997 747-758

Article

Apical Enrichment of Human EGF Precursor in Madin-Darby Canine Kidney Cells Involves Preferential Basolateral Ectodomain Cleavage Sensitive to a Metalloprotease Inhibitor



Peter J. Dempsey*, Katherine S. Meise*, Yoshino Yoshitake{ddagger}, Katsuzo Nishikawa{ddagger}, and Robert J. Coffey

* Departments of Medicine and Cell Biology, Vanderbilt University School of Medicine and Veterans Affairs Medical Center, Nashville, Tennessee 37232-2279; and {ddagger} Department of Biochemistry, Kanazawa Medical University, Uchinada, Ishikawa 920-02, Japan

EGF precursor (proEGF) is a member of the family of membrane-anchored EGF-like growth factors that bind with high affinity to the epidermal growth factor receptor (EGFR). In contrast to human transforming growth factor-{alpha} precursor (proTGF{alpha}), which is sorted basolaterally in Madin-Darby canine kidney (MDCK) cells (Dempsey, P., and R. Coffey, 1994. J. Biol. Chem. 269:16878–16889), we now demonstrate that human proEGF overexpressed in MDCK cells is found predominantly at the apical membrane domain under steady-state conditions. Nascent proEGF (185 kD) is not sorted but is delivered equally to the apical and basolateral membranes, where it is proteolytically cleaved within its ectodomain to release a soluble 170-kD EGF form into the medium. Unlike the fate of TGF{alpha} in MDCK cells, the soluble 170-kD EGF species accumulates in the medium, does not interact with the EGFR, and is not processed to the mature 6-kD peptide. We show that the rate of ectodomain cleavage of 185-kD proEGF is fourfold greater at the basolateral surface than at the apical surface and is sensitive to a metalloprotease inhibitor, batimastat. Batimastat dramatically inhibited the release of soluble 170-kD EGF into the apical and basal medium by 7 and 60%, respectively, and caused a concordant increase in the expression of 185-kD proEGF at the apical and basolateral cell surfaces of 150 and 280%, respectively. We propose that preferential ectodomain cleavage at the basolateral surface contributes to apical domain localization of 185-kD proEGF in MDCK cells, and this provides a novel mechanism to achieve a polarized distribution of cell surface membrane proteins under steady-state conditions. In addition, differences in disposition of EGF and TGF{alpha} in polarized epithelial cells offer a new conceptual framework to consider the actions of these polypeptide growth factors.

Abbreviations used in this paper: BFA, brefeldin A; EGFR, epidermal growth factor receptor; endo H, endoglycosidase H; MDCK, Madin-Darby canine kidney cells; TGF{alpha}, transforming growth factor-{alpha}; TPCK, N-tosyl-L-phenylalanine chloromethylketone.

Address all correspondence to Dr. Peter J. Dempsey, Departments of Medicine and Cell Biology, GI Cancer Program, CC-2218 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232-2583. Tel.: (615) 343-0171. E-mail: peter.dempsey{at}mcmail.vanderbilt.edu


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