Endothelin-converting enzyme (ECE-1) is a type II integral membrane protein which plays a key role in the biosynthetic pathway of the vasoconstricting

Endothelin (ET), the most potent vasoconstricting substance known so far, was first identified in cultured endothelial cells (Yanagisawa et al., 1988). The ET family includes three peptides, ET-1, ET-2 and ET-3, encoded by distinct genes (Inoue et al., 1989) and present in a wide variety of cells and tissues (see Rubanyi and Polokoff, 1994, for review). Maturation of the ET peptides occurs in two steps. After removal of their signal peptides (Fabbrini et al., 1991), the ET precursors (or preproETs) are processed by dibasic pairspecific endopeptidases into the inactive big ETs (Yanagisawa et al., 1988). ETs are then generated from these polypeptides via the hydrolysis of a Trp-Val bond (Trp-Ile in big ET-3) by a specific endothelin-converting enzyme (ECE; Yanagisawa et al., 1988). Purification of ECE (Ohnaka et al., 1993; Takahashi et al., 1993) enabled the cloning of two enzymes termed ECE1 (Shimada et al., 1994; Xu et al., 1994) and ECE-2 (Emoto and Yanagisawa, 1995). Both enzymes are type II integral membrane proteins and belong to the family of zinc metalloproteases related to enkephalinase or neutral endopeptidase (NEP). ECE-1 and ECE-2 are able to cleave big ETs, yet with different pH optima (7 for ECE-1, 5.5 for ECE2). However, the recent targeted disruption of the ECE-1 gene in mice (Yanagisawa et al., 1998) suggests that ECE-1 is the main enzyme responsible for the cleavage of big endothelins. The potential use of ECE-1 inhibitors in the treatment of a number of vascular pathologies underlines the importance of identifying the subcellular site of ET processing. Conflicting results have been produced with respect to this problem, demonstrating either an intracellular or a plasma membrane localization for ECE activity (see Barnes et al., 1996, for a review). Immunofluorescence studies have shown that, in contrast to NEP or angiotensin-converting enzyme, ECE-1 in endothelial cells is localized both intracellularly and at the cell surface (Barnes et al., 1996; Schweizer et al., 1997). These different subcellular localizations could be due to the existence of several isoforms for ECE-1. Indeed, we and others have shown that three distinct human ECE-1 isoproteins were produced by a single gene through the use of alternate promoters (Valdenaire et al., 1995; Orzechowski et al., 1997; Schweizer et al., 1997). The three ECE-1 isoforms, termed ECE-1a, -1b and -1c, differ in the first part of their short cytoplasmic N-terminal tail and are identical in the rest of their sequence (Fig. 1A). They cleave big ETs with comparable efficiency, but, when expressed in CHO cells, they differ with respect to their subcellular localization. Thus ECE-1a is strongly expressed at the cell surface, ECE-1b remains 3115 Journal of Cell Science 112, 3115-3125 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0308