Apical enrichment of human EGF precursor in Madin-Darby canine kidney cells involves preferential basolateral ectodomain cleavage sensitive to a metalloprotease inhibitor.
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 (proTGFalpha), 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 TGFalpha 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 TGFalpha in polarized epithelial cells offer a new conceptual framework to consider the actions of these polypeptide growth factors.
Dempsey
P J
PJ
Department of Medicine, Vanderbilt University School of Medicine,p5 Veterans Affairs Medical Center, Nashville, Tennessee 37232-2279, USA. peter.dempsey@mcmail.vanderbilt.edu
Meise
K S
KS
Yoshitake
Y
Y
Nishikawa
K
K
Coffey
R J
RJ
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D016764:Cell Polarity; D004285:Dogs; D004815:Epidermal Growth Factor / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008970:Molecular Weight; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D011498:Protein Precursors / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012995:Solubility; D013876:Thiophenes / Q000494:pharmacology
D011498:Protein Precursors; D013876:Thiophenes; D010649:Phenylalanine; D004815:Epidermal Growth Factor; C080985:batimastat; D008666:Metalloendopeptidases