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

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

doi:10.1074/jbc.m307745200

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.

Highlights

Epidermal growth factor (EGF)1 is a small diffusible polypeptide first discovered and purified from rodent
Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface
Whereas the regulated shedding of transforming growth factor-␣, heparin binding EGF-like growth factor (HB-EGF), and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. 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

Summary

Introduction

Epidermal growth factor (EGF) is a small (about 6 kDa) diffusible polypeptide first discovered and purified from rodent. In the case of EGF, the cDNA analysis from human, mouse, and rat species predicted very large precursor molecules (respectively 1207, 1217, and 1133 amino acids) (10 –13) with an apparent molecular mass ranging from 150 to 185 kDa for the mature glycosylated precursors. These precursor molecules are predicted to be type I membrane proteins. Recent evidence suggests that proteolytic cleavage of cell surface proteins, i.e. ectodomain shedding, is an important mechanism whereby cells regulate the repertoire of proteins expressed on their surface, shifting molecular species from their membrane-bound forms to soluble ones with the potential modification of their biological properties [9, 23, 24]. The information concerning pro-EGF maturation and secretion does appear much more controversial

Methods

Results

Conclusion