Endothelins are peptide hormones with a potent vasoconstrictor activity that are also known to function as intercellular signaling molecules. The final step in the biosynthesis of endothelins is the proteolytic processing of precursor peptides by endothelin-converting enzymes (ECEs). ECE-1 is a zinc metalloendopeptidase related in amino acid sequence to neprilysin, a mammalian cell-surface peptidase involved in the metabolism of numerous biologically active peptides. Despite apparent structural similarities, ECE-1 and neprilysin have been considered to differ significantly in substrate specificity. In this study we have examined the activity of recombinant ECE-1 against a collection of biologically active peptides. ECE-1, unlike neprilysin, was found to have minimal activity against substrates smaller than hexapeptides, such as Leu-enkephalin. Larger peptides such as neurotensin, substance P, bradykinin, and the oxidized insulin B chain were hydrolyzed by ECE-1 as efficiently as big endothelin-1, a known in vivo substrate. Identification of the products of hydrolysis of six peptides indicates that ECE-1 has a substrate specificity similar to that of neprilysin, preferring to cleave substrates at the amino side of hydrophobic residues. The data indicate that ECE-1 possesses a surprisingly broad substrate specificity and is potentially involved in the metabolism of biologically active peptides distinct from the endothelins. Endothelins (ETs)1 are potent vasoconstrictive peptides of 21 amino acids produced by vascular endothelial cells (1). Three ET isoforms, ET-1, ET-2, and ET-3, encoded by distinct genes, are known to exist in humans (2). Endothelins are involved in the regulation of vascular tone and may also play roles in various cardiovascular and renal diseases (3). ETs are also required during embryonic development for the intercellular signaling necessary for the proper development of neural crestderived tissues (4). The final step in the biosynthesis of the endothelins is the conversion of 38 -41 residue precursors (big ETs) to the active hormones via the cleavage of a Trp 21 -Val/ Ile 22 bond by endothelin-converting enzymes (ECEs (5)). ECE-1 has been purified from vascular endothelium, endothelial cell lines, and lung microsomes (6 -8). ) is a Type II integral membrane protein expressed by endothelial cells in tissues such as aorta, lung, ovary, and testis. It has also been reported to be expressed by endocrine cells such as adrenal chromaffin cells and pancreatic ␤ cells (9). Targeted disruption of the ECE-1 gene has shown that ECE-1 is the physiologically relevant activating enzyme for both ET-1 and ET-3 in vivo (10).Molecular cloning of mammalian ECE-1 cDNAs has demonstrated the existence of three mRNAs transcribed from a single gene (11,12). The proteins encoded by these RNAs have identical catalytic domains but differ only in their NH 2 -terminal amino acid sequence. Two of the ECE-1 isoforms are expressed on the cell surface; the other is localized in the trans-Golgi network (12) An additional isof...

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Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and rats contain beta subunits that are type I integral membrane proteins and alpha subunits that are disulfide-linked to or noncovalently associated with membrane-anchored meprin subunits. Both alpha and beta subunits are synthesized with COOH-terminal domains predicted to be cytoplasmic, transmembrane, and epidermal growth factor-like; these domains are retained in beta subunits but are removed from alpha during maturation. The present studies establish that an inserted 56-amino acid domain (the "I" domain), present in alpha but not in beta, is necessary and sufficient for COOH-terminal proteolytic processing of the alpha subunit. This was demonstrated by expression of mutant meprin subunits (deletion mutants, chimeric alpha beta subunits, and beta mutants containing the I domain) in COS-1 cells. Mutations of two common processing sites present in the I domain (a dibasic site and a furin site) did not prevent COOH-terminal proteolytic processing, indicating that the proteases responsible for cleavage are distinct from those having these specificities. Deletion of the I domain from the alpha subunit resulted in accumulation of unprocessed subunits in a preGolgi compartment. Furthermore, COOH-terminal proteolytic processing of wild-type alpha subunits occurred before acquisition of endoglycosidase H resistance. Pulse-chase experiments and expression of an alpha subunit transcript containing a c-myc epitope tag, confirmed that proteolytic processing at the COOH terminus occurs in the endoplasmic reticulum. This work identifies the region of the alpha subunit that is essential for COOH-terminal processing and demonstrates that the differential processing of the evolutionarily-related subunits of meprin A that results in a structurally unique tetrameric protease begins in the endoplasmic reticulum.

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