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J. Biol. Chem., Vol. 278, Issue 46, 45255-45268, November 14, 2003

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Regulated Cell Surface Pro-EGF Ectodomain Shedding Is a Zinc Metalloprotease-dependent Process^*

Sylvain M. Le Gall, Rodolphe Auger, Catherine Dreux, and Philippe Mauduit

From the UMR 8619, Institut de Biochimie Biophysique Moléculaire et Cellulaire, Université Paris XI, 91405 Orsay Cedex, France

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-, 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- 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.

Received for publication, July 17, 2003 , and in revised form, August 21, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Doctoral contract from the Ministère de la jeunesse, de l'éducation et de la recherche.

To whom correspondence should be addressed. Tel.: 33-1-69158033; Fax: 33-1-69853715; E-mail: philippe.mauduit{at}bbmpc.u-psud.fr.

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