PHORBOL ESTER-INDUCED JUXTAMEMBRANE CLEAVAGE OF ANGIOTENSIN-CONVERTING ENZYME IS NOT INHIBITED BY A STALK CONTAINING INTRACHAIN DISULFIDES

Specialized proteases, referred to as sheddases, secretases, or membra ne-protein-solubilizing proteases (MPSPs), solubilize the extracellula r domains of diverse membrane proteins by catalyzing a specific cleava ge in the juxtamembrane stalk regions of such proteins. A representati ve MPSP (tumor necrosis factor-alpha convertase) was cloned recently a nd shown to be a disintegrin metalloprotease that is inhibited by pept ide hydroxamates including the compound TAPI. Substrate determinants t hat specify cleavage by MPSPs remain incompletely characterized, bur m ay include the physicochemical properties of the stalk or unidentified recognition motifs in the stalk or the extracellular domain. We const ructed a mutant angiotensin-converting enzyme (ACE) in which the stalk has been replaced with an epidermal growth factor (EGF)-like domain ( ACE-JMEGF), to test the hypothesis that MPSP cleavage requires an open , comparatively unfolded or extended stalk. Wild-type ACE is a type I transmembrane (TM) ectoprotein that is efficiently solubilized by a ty pical MPSP activity. We found that ACE-JMEGF was solubilized inefficie ntly and accumulated in a cell-associated form on transfected Chinese hamster ovary (CHO) cells; cleavage was stimulated by phorbol ester an d inhibited by TAPI, features typical of MPSP activity. Determination of the C-terminus of soluble ACE-JMEGF revealed that, surprisingly, cl eavage occurred at a Gly-Phe bond between the fifth and sixth cysteine s within the third disulfide loop of the EGF-like domain. Reduction of intact CHO cells with tributylphosphine resulted in the rapid release of ACE-JMEGF (but not wild-type ACE) into the medium, suggesting that a proportion of membrane-bound ACE-JMEGF is cleaved but remains cell- associated via disulfide tethering, The mechanism for the release of A CE-JMEGF in the absence of chemical reduction is unclear. We conclude that the presence of a compact, disulfide-bridged domain does not per se inhibit cleavage by an MPSP activity, but ectodomain release is pre vented by disulfide tethering to the TM domain.