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-␣, 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.

P2Y2 Nucleotide Receptors Enhance α-Secretase-dependent Amyloid Precursor Protein Processing

“…TAPI-2 inhibited UTP-stimulated sAPP␣ release with an estimated IC 50 of 1.8 M (Fig. 6B), comparable with a value reported previously for the inhibition of ADAM17/ TACE (59,60). These results confirm that APP processing due to P2Y 2 R activation requires metalloprotease activity and that ADAM17/TACE is likely involved.…”

ADAM10 Mediates Ectodomain Shedding of the Betacellulin Precursor Activated by p-Aminophenylmercuric Acetate and Extracellular Calcium Influx

“…Similar sequential modification events have been observed with other EGF-like growth factors (39,41,46,47,72,73). Release of the BTC ectodomain by C-terminal processing appears to be preferred over N-terminal cleavage events, a preference also observed with amphiregulin and EGF processing (41,43,72).…”

“…By contrast, other EGFlike factors, including TGF␣ (39), neuregulin (42), amphiregulin (41), HB-EGF (40), and possibly EGF (43,44), all undergo activated shedding in response to this phorbol ester. PMAinduced shedding of these EGF-like growth factors and other transmembrane molecules has predominantly involved ADAM17 (75).…”

Phosphorylation of TNF-α converting enzyme by gastrin-releasing peptide induces amphiregulin release and EGF receptor activation

“…PI3-K also has been implicated in GPCR and EGFR crosstalk (17)(18)(19). In addition to intracellular molecules involved in GPCR-induced EGFR activation, growing evidence suggests that transmembrane metalloproteases mediate EGFR proligand shedding in response to GPCR ligands (8,(20)(21)(22)(23). Cumulative results indicate that the metalloproteinase involved in EGFR proligand cleavage is both cell type-and GPCR ligand-specific.…”