title: Regulated cell surface pro-EGF ectodomain shedding is a zinc metalloprotease-dependent process.
abstract: Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.
authors.affiliation: UMR 8619, Institut de Biochimie Biophysique Moléculaire et Cellulaire, Université Paris XI, 91405 Orsay Cedex, France.
authors.forename: Sylvain M
authors.initials: SM
authors.lastname: Le Gall
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Auger
authors.forename: Catherine
authors.initials: C
authors.lastname: Dreux
authors.forename: Philippe
authors.initials: P
authors.lastname: Mauduit
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D051381:Rats; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry*
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D000939:Epitopes; D007476:Ionophores; D014409:Tumor Necrosis Factor-alpha; C076715:pervanadate; D014638:Vanadates; D004815:Epidermal Growth Factor; D011493:Protein Kinase C; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606464:Adam17 protein, rat; D015032:Zinc; D002118:Calcium