Heterodimerization of Endothelin-converting Enzyme-1 Isoforms Regulates the Subcellular Distribution of This Metalloprotease

Abstract

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.Endothelins (ET 1 -1, ET-2, and ET-3) are 21-residue peptides derived from three distinct genes (1). They are pleiotropic factors that play an important role in the regulation of the cardiovascular and endocrine systems (for review see Refs. 2 and 3). Endothelins are also crucial developmental factors as demonstrated by the targeted disruption of the genes coding for the precursors of ET-1 and ET-3, and of the genes coding for their receptors, ETA and ETB (4 -7). In addition, the expression of endothelin is associated with many pathological processes and with tumor growth (2, 8 -10). In order to fulfill such a wide spectrum of physiological functions, endothelins act through autocrine and paracrine mechanisms. Their biosynthesis thus requires tight local control. Endothelins are synthesized in the endoplasmic reticulum as precursors that undergo a two-step proteolytic maturation. Pro-endothelins are first processed at conserved multibasic sites by furin or a furin-like enzyme, in order to release an intermediate called big endothelin (big-ET) (11, 12), which is devoid of biological activity (13). Big-ET is then processed by endothelin converting enzyme (ECE) at a Trp-Val/Ile bond, which releases the biologically active peptide. This latter proteolytic step can occur in the extracellular medium and in the secretory pathway, so that cells secrete either big-ETs alone or together with endothelins (14, 15, 16). Endothelial cells co-express the precursor, the c...

Characterization of endothelin-1 and nitric oxide generating systems in corpus luteum-derived endothelial cells

“…Therefore, the identification of ECE-1 isoform distribution in luteal endothelial cells could elucidate the site of big ET-1 cleavage. The high levels of ECE-1d in luteal endothelial cells reported here favor an intracellular site of ET-1 activation (Muller et al 2003).…”

Endothelin-converting enzyme-1 is expressed in human cerebral cortex and protects against Alzheimer's disease

“…39 BACE-1 is also targeted to the endosomal system, 40 and the results of several groups support an important role for this intracellular compartment in Ab production. [41][42][43] In addition, endocytic pathway abnormalities have been shown to precede Ab deposition in the brains of patients affected by sporadic AD.…”

Isoforms of endothelin-converting enzyme-1 (ECE-1) have opposing effects on prostate cancer cell invasion

“…Endothelinconverting enzyme 1b has already been shown to form heterodimers (Muller et al, 2003) with other ECE-1 isoforms, and acts as a vehicle for regulating their distribution (Muller et al, 2003). Given the known signalling capability of NEP, a close homologue of ECE-1, it can be speculated that heterodimerisation of ECE-1 isoforms may trigger intracellular signalling.…”

“…They have similar catalytic properties but distinct subcellular localisation and tissue distribution (Schweizer et al, 1997). Endothelin-converting enzyme 1a and ECE-1c are localised at the cell surface, whereas ECE-1b and ECE-1d are intracellular with ECE-1b present in late endosomes or multivesicular bodies and ECE-1d concentrated in recycling endosomes (Muller et al, 2003). More recently, ECE-1 has been reported to regulate peptide receptor recycling in endosomes through metabolism of the peptide ligand (Roosterman et al, 2007).…”