Roles of the juxtamembrane and extracellular domains of angiotensin-converting enzyme in ectodomain shedding

Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cl eavage by a secretase. To investigate the requirements for ectodomain shedd ing, we replaced the glycosylphosphatidylinositol addition sequence in memb rane dipeptidase (MDP) - a membrane protein that is not shed - with the jux tamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resu lting construct, MDP STM,,,,, was targeted to the cell surface in a glycosy lated and enzymically active form, and was shed into the medium. The site o f cleavage in MDP-STMACE was identified by MS as the Arg(374)-Ser(375) bond , corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somat ic ACE. The release of MDP-STMACE and ACE from the cells was inhibited in a n identical manner by batimastat and two other hydroxamic acid-based zinc m etallosecretase inhibitors. In contrast, a construct lacking the juxtamembr ane stalk, MDP-TMACE, although expressed at the cell surface in an enzymica lly active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACE De ltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cle avable stalk, implying that in contrast with the C-terminal domain, the N-t erminal domain lacks a signal required for shedding. These data are discuss ed in the context of two classes of secretases that differ in their require ments for recognition of substrate proteins.