Title: 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. 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. endothelin adrenocorticotropic hormone big endothelin endothelin-converting enzyme neutral endopeptidase trans-Golgi network phosphate-buffered saline Dulbecco's modified Eagle's medium 1,4-piperazinediethanesulfonic acid Endothelins (ET1-1, ET-2, and ET-3) are 21-residue peptides derived from three distinct genes (1Inoue A. Yanagisawa M. Kimura S. Kasuya Y. Miyauchi T. Goto K. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2863-2867Crossref PubMed Scopus (2556) Google Scholar). They are pleiotropic factors that play an important role in the regulation of the cardiovascular and endocrine systems (for review see Refs. 2Rubanyi G.M. Polokoff M.A. Pharmacol Rev.;. 1994; 46: 325-415PubMed Google Scholar and 3Masaki T. Endocr. Rev.;. 1993; 14: 256-268Crossref PubMed Scopus (195) Google Scholar). 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 (4Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W.H. Kamada N. et al.Nature. 1994; 368: 703-710Crossref PubMed Scopus (886) Google Scholar, 5Baynash A.G. Hosoda K. Giaid A. Richardson J.A. Emoto N. Hammer R.E. Yanagisawa M. Cell. 1994; 79: 1277-1285Abstract Full Text PDF PubMed Scopus (825) Google Scholar, 6Hosoda K. Hammer R.E. Richardson J.A. Baynash A.G. Cheung J.C. Giaid A. Yanagisawa M. Cell. 1994; 79: 1267-1276Abstract Full Text PDF PubMed Scopus (887) Google Scholar, 7Clouthier D.E. Hosoda K. Richardson J.A. Williams S.C. Yanagisawa H. Kuwaki T. Kumada M. Hammer R.E. Yanagisawa M. Development. 1998; 125: 813-824Crossref PubMed Google Scholar). In addition, the expression of endothelin is associated with many pathological processes and with tumor growth (2Rubanyi G.M. Polokoff M.A. Pharmacol Rev.;. 1994; 46: 325-415PubMed Google Scholar, 8Haynes W.G. Webb D.J. J. Hypertens. 1998; 16: 1081-1098Crossref PubMed Scopus (325) Google Scholar, 9Egidy G. Juillerat-Jeanneret L. Jeannin J.F. Korth P. Bosman F.T. Pinet F. Am. J. Pathol. 2000; 157: 1863-1874Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10Egidy G. Baviera E. Ciuffo G. Corvol P. Pinet F. Hypertension. 2001; 38: 1137-1142Crossref PubMed Scopus (10) Google Scholar). 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) (11Denault J.B. Claing A. D'Orleans-Juste P. Sawamura T. Kido T. Masaki T. Leduc R. FEBS Lett. 1995; 362: 276-280Crossref PubMed Scopus (85) Google Scholar, 12Blais V. Fugere M. Denault J.B. Klarskov K. Day R. Leduc R. FEBS Lett. 2002; 524: 43-48Crossref PubMed Scopus (36) Google Scholar), which is devoid of biological activity (13Okada K. Miyazaki Y. Takada J. Matsuyama K. Yamaki T. Yano M. Biochem. Biophys. Res. Commun. 1990; 171: 1192-1198Crossref PubMed Scopus (149) Google Scholar). 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 (14Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 15Harrison V.J. Barnes K. Turner A.J. Wood E. Corder R. Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6344-6348Crossref PubMed Scopus (95) Google Scholar, 16Parnot C., Le Moullec J.M. Cousin M.A. Guedin D. Corvol P. Pinet F. Hypertension. 1997; 30: 837-844Crossref PubMed Scopus (32) Google Scholar). Endothelial cells co-express the precursor, the converting enzyme and the receptor, thus implying autocrine function of endothelin. On the other hand, luteal cells (17Levy N. Gordin M. Mamluk R. Yanagisawa M. Smith M.F. Hampton J.H. Meidan R. Endocrinology. 2001; 142: 5254-5260Crossref PubMed Scopus (43) Google Scholar) and neurons (18Schmidt-Ott K.M. Tuschick S. Kirchhoff F. Verkhratsky A. Liefeldt L. Kettenmann H. Paul M. J. Cardiovasc. Pharmacol. 1998; 31: S364-S366Crossref PubMed Scopus (17) Google Scholar) express only the converting enzyme and the receptor, and three different cell types express the precursor, the converting enzyme and the receptor in seminiferous tubules (19Tripiciano A. Peluso C. Morena A.R. Palombi F. Stefanini M. Ziparo E. Yanagisawa M. Filippini A. J. Cell Biol. 1999; 145: 1027-1038Crossref PubMed Scopus (32) Google Scholar), demonstrating the paracrine function of endothelin through extracellular processing of big-ET. The cellular distribution of ECE thus plays a central role in controlling the biosynthetic pathway of endothelins.ECE is a type II membrane protein of the neutral endopeptidase (NEP) family, with an N-terminal cytosolic tail and a catalytic ectodomain. Unlike other proteases of this family, ECE is expressed as covalent dimers (20Schmidt M. Kroger B. Jacob E. Seulberger H. Subkowski T. Otter R. Meyer T. Schmalzing G. Hillen H. FEBS Lett. 1994; 356: 238-243Crossref PubMed Scopus (183) Google Scholar, 21Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike H. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar, 22Shimada K. Takahashi M. Turner A.J. Tanzawa K. Biochem. J. 1996; 315: 863-867Crossref PubMed Scopus (86) Google Scholar). Two genes coding for ECE-1 and ECE-2 have been cloned (20Schmidt M. Kroger B. Jacob E. Seulberger H. Subkowski T. Otter R. Meyer T. Schmalzing G. Hillen H. FEBS Lett. 1994; 356: 238-243Crossref PubMed Scopus (183) Google Scholar, 23Shimada K. Takahashi M. Tanzawa K. J. Biol. Chem. 1994; 269: 18275-18278Abstract Full Text PDF PubMed Google Scholar, 14Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 24Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). ECE-1 has a broader tissue distribution and is always expressed at higher levels than ECE-2. The targeted disruption of the ECE-1 gene resulted in a lethal phenotype that combined both the phenotypes of the inactivation of ET-1 or ETA and of ET-3 or ETB, thus demonstrating the central role of ECE-1 in the endothelin system (25Yanagisawa H. Yanagisawa M. Kapur R.P. Richardson J.A. Williams S.C. Clouthier D.E. de Wit D. Emoto N. Hammer R.E. Development. 1998; 125: 825-836Crossref PubMed Google Scholar). On the other hand, the inactivation of the ECE-2 gene did not result in significant modification of the mouse phenotype unless it was combined with the ECE-1 gene inactivation (26Yanagisawa H. Hammer R.E. Richardson J.A. Emoto N. Williams S.C. Takeda S. Clouthier D.E. Yanagisawa M. J. Clin. Invest. 2000; 105: 1373-1382Crossref PubMed Scopus (167) Google Scholar). ECE-1 is expressed in the endothelium of all organs as well as in nonvascular cells of many tissues including brain and neuroendocrine tissues (Refs. 14Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 21Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike H. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar, 18Schmidt-Ott K.M. Tuschick S. Kirchhoff F. Verkhratsky A. Liefeldt L. Kettenmann H. Paul M. J. Cardiovasc. Pharmacol. 1998; 31: S364-S366Crossref PubMed Scopus (17) Google Scholar, and 27Korth P. Bohle R.M. Corvol P. Pinet F. J. Histochem. Cytochem. 1999; 47: 447-462Crossref PubMed Scopus (76) Google Scholar; for review see Ref. 28Barnes K. Turner A.J. Neurochem Res. 1997; 22: 1033-1040Crossref PubMed Scopus (44) Google Scholar). Four isoforms of ECE-1 have been identified (Refs. 29Valdenaire O. Rohrbacher E. Mattei M.G. J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar and 30Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R. Tougard C. Michel J.B. Eur J Biochem. 1999; 264: 341-349Crossref PubMed Scopus (148) Google Scholar; for review see Ref. 31Turner A.J. Barnes K. Schweizer A. Valdenaire O. Trends Pharmacol Sci. 1998; 19: 483-486Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and a related gene organization has recently been described for ECE-2 (32Lorenzo M.N. Khan R.Y. Wang Y. Tai S.C. Chan G.C. Cheung A.H. Marsden P.A. Biochim. Biophys. Acta. 2001; 1522: 46-52Crossref PubMed Scopus (26) Google Scholar, 33Ikeda S. Emoto N. Alimsardjono H. Yokoyama M. Matsuo M. Biochem. Biophys. Res. Commun. 2002; 293: 421-426Crossref PubMed Scopus (28) Google Scholar). ECE-1 isoforms result from the use of alternate promoters located upstream of specific exons (29Valdenaire O. Rohrbacher E. Mattei M.G. J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 30Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R. Tougard C. Michel J.B. Eur J Biochem. 1999; 264: 341-349Crossref PubMed Scopus (148) Google Scholar, 34Orzechowski H.D. Richter C.M. Funke-Kaiser H. Kroger B. Schmidt M. Menzel S. Bohnemeier H. Paul M. J. Mol. Med. 1997; 75: 512-521Crossref PubMed Scopus (47) Google Scholar). Thus, the 4 ECE-1 isoforms share the same catalytic domain and only differ in their N terminus, which codes for their cytosolic tails (Fig. 1 A). As these domains are responsible for their targeting, the specificity of ECE isoforms resides in their subcellular distribution. Considering the importance of the intracellular and extracellular biosynthesis of endothelins, the subcellular distribution of ECE isoforms may play a central role in the regulation of the endothelin system and constitutes an important factor for the efficient inhibition of ECE in pathological conditions.Extensive work aimed to define the intracellular localization of ECE-1 has been done in endothelial cells (21Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike H. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar, 35Barnes K. Shimada K. Takahashi M. Tanzawa K. Turner A.J. J. Cell Sci. 1996; 109: 919-928PubMed Google Scholar, 36Russell F.D. Skepper J.N. Davenport A.P. Circ. Res. 1998; 83: 314-321Crossref PubMed Scopus (103) Google Scholar). Most of these studies were however non relevant to isoform specificity due to the lack of antibodies that can distinguish between ECE-1b, 1c, and 1d. More recent work was based on the expression of ECE-1 isoforms in transfected fibroblasts or epithelial cells. In these models, ECE-1a and ECE-1c were targeted to the plasma membrane, whereas ECE-1b was localized to intracellular organelles, and ECE-1d displayed an intermediate distribution (30Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R. Tougard C. Michel J.B. Eur J Biochem. 1999; 264: 341-349Crossref PubMed Scopus (148) Google Scholar, 37Schweizer A. Valdenaire O. Nelbock P. Deuschle U. Dumas Milne Edwards J.B. Stumpf J.G. Loffler B.M. Biochem. J. 1997; 328: 871-877Crossref PubMed Scopus (192) Google Scholar, 38Azarani A. Boileau G. Crine P. Biochem. J. 1998; 333: 439-448Crossref PubMed Scopus (38) Google Scholar, 39Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). Neuroendocrine cells constitute another major cell type expressing ECE-1 in vivo (28Barnes K. Turner A.J. Neurochem Res. 1997; 22: 1033-1040Crossref PubMed Scopus (44) Google Scholar, 27Korth P. Bohle R.M. Corvol P. Pinet F. J. Histochem. Cytochem. 1999; 47: 447-462Crossref PubMed Scopus (76) Google Scholar). In these cells, precursors and peptides, as well as processing enzymes, are sorted between the constitutive and the regulated secretory pathways. We used the neuroendocrine AtT-20 cells as a model. These are corticotrope pituitary cells that are specialized in the synthesis and regulated secretion of adrenocorticotropic hormone (ACTH). In these cells, we investigated the subcellular distribution and the heterodimerization of ECE-1 isoforms, as the function of intracellular ECE-1 is a matter of debate, and the role of dimerization is still unknown.DISCUSSIONECE-1 belongs to the family of NEP metalloproteases. Among these enzymes, ECE-1 has two unique characteristics: first, it is present and active both at the cell surface and in intracellular organelles; second, it exists as covalent dimers. In the present paper, we have investigated these two aspects of ECE-1 cell biology. Our experiments provided the first extensive analysis of the intracellular distribution of the 4 ECE-1 isoforms in the secretory pathway and in the endosomal system. The two isoforms localized in intracellular compartments, ECE-1b and 1d, displayed distinct but complementary patterns of distribution in the endosomal system: ECE-1d was present in the recycling endosomes and ECE-1b in the late endosomes/multivesicular bodies. Using biochemical methods, we could show that ECE-1 isoforms exist as heterodimers. The discovery of such heterodimers suggested a novel role for the isoform-specific targeting of ECE-1. We showed that the targeting signal responsible for the intracellular distribution of ECE-1b could redirect the plasma membrane ECE-1a to the endosomal system. These data demonstrated that the specific N terminus of ECE-1b constitutes a regulatory domain that is able to modify the distribution of other ECE-1 isoforms. Such a regulatory mechanism could be physiologically relevant as many cell types, including endothelial cells, co-express endogenously several ECE-1 isoforms (37Schweizer A. Valdenaire O. Nelbock P. Deuschle U. Dumas Milne Edwards J.B. Stumpf J.G. Loffler B.M. Biochem. J. 1997; 328: 871-877Crossref PubMed Scopus (192) Google Scholar, 30Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R. Tougard C. Michel J.B. Eur J Biochem. 1999; 264: 341-349Crossref PubMed Scopus (148) Google Scholar).Our study was undertaken in the AtT-20 cell line due to the important function of endothelins in regulating the activity of neuroendocrine cells through autocrine and paracrine mechanisms (Ref. 49Kanyicska B. Lerant A. Freeman M.E. Endocrinology. 1998; 139: 5164-5173Crossref PubMed Scopus (27) Google Scholar; for review see Ref. 3Masaki T. Endocr. Rev.;. 1993; 14: 256-268Crossref PubMed Scopus (195) Google Scholar). Endothelins have been detected in the secretory granules of neuroendocrine cells (50Suzuki H. Yamamoto T. Kikuyama S. Uemura H. Gen. Comp. Endocrinol. 1997; 107: 12-22Crossref PubMed Scopus (18) Google Scholar), thus raising the possibility that ECE-1 was targeted to the secretory granules, like common neuroendocrine endopeptidases. Our results rejected the possibility that big-ET was processed in secretory granules, as none of the intracellular isoforms were detected in the secretory granules. Our data thus suggested that the mature endothelin detected in the secretory granules might result from processing in the TGN. Processing of pro-endothelin by furin in this compartment is the first step that is required for ECE-1 to release endothelin from its precursor (51Kido T. Sawamura T. Hoshikawa H. D'Orleans-Juste P. Denault J.B. Leduc R. Kimura J. Masaki T. Eur J Biochem. 1997; 244: 520-526Crossref PubMed Scopus (27) Google Scholar). Even though we did not find that ECE-1 was concentrated in the TGN, like furin is (45Molloy S.S. Thomas L. Van Slyke J.K. Stenberg P.E. Thomas G. EMBO J. 1994; 13: 18-33Crossref PubMed Scopus (419) Google Scholar), ECE-1 is transported through this compartment during biosynthesis. The intracellular processing of big-ET in the TGN would thus not be a specialized function for any ECE-1 isoform. Indeed, we have already shown that ECE-1a, a plasma membrane isoform, could generate endothelin in the secretory pathway as well as in the extracellular medium (16Parnot C., Le Moullec J.M. Cousin M.A. Guedin D. Corvol P. Pinet F. Hypertension. 1997; 30: 837-844Crossref PubMed Scopus (32) Google Scholar). Whereas ECE-1b and ECE-1d could generate endothelin in the secretory pathway, these isoforms should not be considered more specific than ECE-1a or 1c for this processing. In support of this, big-ET is the main species secreted from cells that co-express pre-pro-endothelin and an intracellular isoform of ECE-1, thus indicating the low efficiency of intracellular processing of big-ET by ECE-1 (14Xu D. Emoto N. Giaid A. Slaughter C. Kaw S. deWit D. Yanagisawa M. Cell. 1994; 78: 473-485Abstract Full Text PDF PubMed Scopus (856) Google Scholar). On the other hand, ECE-2 could specifically fulfill this role as it displays the acidic optimum pH required for efficient conversion of precursors in the lumen of the TGN and the secretory vesicles (24Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar), and as 2 of the 4 recently identified isoforms of ECE-2 were found in intracellular compartments (33Ikeda S. Emoto N. Alimsardjono H. Yokoyama M. Matsuo M. Biochem. Biophys. Res. Commun. 2002; 293: 421-426Crossref PubMed Scopus (28) Google Scholar).Previous studies aimed at identifying the intracellular compartments containing ECE-1b have produced controversial results. ECE-1b was detected in the Golgi apparatus, where it was co-localized with the Golgi marker wheat germ agglutinin in endothelial cells (35Barnes K. Shimada K. Takahashi M. Tanzawa K. Turner A.J. J. Cell Sci. 1996; 109: 919-928PubMed Google Scholar) and with TGN38 in transfected fibroblasts (37Schweizer A. Valdenaire O. Nelbock P. Deuschle U. Dumas Milne Edwards J.B. Stumpf J.G. Loffler B.M. Biochem. J. 1997; 328: 871-877Crossref PubMed Scopus (192) Google Scholar, 39Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). In addition, the presence of ECE-1b in late endosomes/multivesicular bodies has been described in transfected epithelial cells and fibroblasts (38Azarani A. Boileau G. Crine P. Biochem. J. 1998; 333: 439-448Crossref PubMed Scopus (38) Google Scholar, 39Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). Our results were in agreement with these latter studies. In addition, we demonstrated that ECE-1d was also present in the endosomal system, but concentrated in the recycling endosomes. We showed that ECE-1b and 1d were cycling between the plasma membrane and endosomes using three different lines of evidence: first, extracellular activity was detected from cells expressing ECE-1b and 1d; second, cells expressing these isoforms were able to capture antibodies directed against the ectodomain; third, expression of a dominant negative mutant of dynamin, which blocks internalization of proteins, retained these ECE-1 isoforms at the plasma membrane, but had no effect on furin and on TGN38. In agreement with these results, ECE-1b was co-localized with rab5, a marker for early endosomes, which is the first intracellular compartment where endocytic vesicles fuse (38Azarani A. Boileau G. Crine P. Biochem. J. 1998; 333: 439-448Crossref PubMed Scopus (38) Google Scholar).The ECE-1b-specific di-leucine motif was responsible for the intracellular retention of this isoform (52Cailler F. Zappulla J.P. Boileau G. Crine P. Biochem. J. 1999; 341: 119-126Crossref PubMed Scopus (13) Google Scholar, 39Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). In the present study, we demonstrated that the intracellular localization of ECE-1d also relies on the presence of a specific leucine-based motif (Val8-Leu9). We had shown that a second leucine-based motif (Leu31-Val32), common to ECE-1b, 1c and 1d, was also involved in ECE-1b intracellular retention (39Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F. Corvol P. Tougard C. J. Cell Sci. 1999; 112: 3115-3125Crossref PubMed Google Scholar). Here we found that this signal is not involved in ECE-1b degradation. On the other hand, the ECE-1b specific di-leucine motif (Leu12-Leu13) was responsible for the rapid degradation of this isoform. This motif alone was however not sufficient. Even though leucine-based motifs have been implicated at multiple steps of intracellular sorting in the TGN, the endosomes and the plasma membrane (for review see Ref. 53Sandoval I.V. Bakke O. Trends Cell Biol. 1994; 4: 292-297Abstract Full Text PDF PubMed Scopus (258) Google Scholar), the determinants responsible for the specificity of the transport steps involved remain to be identified (54Sandoval I.V. Martinez-Arca S. Valdueza J. Palacios S. Holman G.D. J. Biol. Chem. 2000; 275: 39874-39885Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Several lines of evidence suggested that the sorting of ECE-1b relied on additional signal to the LL motif: first, the mutation of the ECE-1b LL motif only slightly increased the plasma membrane localization, compared with the mutation of ECE-1d VL motif; second, exchanging the VL and LL motifs modified ECE-1d degradation and localization, but not that of ECE-1b; third, the chimera containing the N-terminal residues of ECE-1b upstream of the VL motif of ECE-1d resulted in the rapid degradation of this construct. Our results suggested that ECE-1b N terminus encodes another complementary signal that does not reside in the primary structure. The additional signal could correspond to a secondary structure or to a post-translational modification that would increase the affinity of the di-leucine motif for an associated protein responsible for sorting to the degradation pathway. In the case of bovine isoforms of ECE-1, Emoto et al. (55Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) have provided evidence for the targeting of bovine ECE-1b to the plasma membrane, and for the sorting of bovine ECE-1a to lysosomes (55Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It should be noted that bovine ECE-1b corresponds to human ECE-1c, which we also found targeted to the plasma membrane, and that bovine ECE-1a is poorly conserved with human ECE-1a. Indeed, the targeting signals responsible for bovine ECE-1a sorting are not present in human ECE-1.The detection of ECE-1 isoforms in the endosomal system could correspond to some novel physiological function of this enzyme, as big-ET is not present in these compartments. Endosomal ECE-1 could constitute an intracellular pool that could be rapidly translocated to the plasma membrane upon stimulation of the cells, as suggested by the cycling of ECE-1b and ECE-1d between endosomes and plasma membrane. Alternatively, the intracellular retention of some ECE isoform could regulate the distribution of other isoforms through direct protein interaction. ECE-1 is expressed as disulfide-linked dimers, but the role of ECE-1 dimerization has not yet been found (20Schmidt M. Kroger B. Jacob E. Seulberger H. Subkowski T. Otter R. Meyer T. Schmalzing G. Hillen H. FEBS Lett. 1994; 356: 238-243Crossref PubMed Scopus (183) Google Scholar, 21Takahashi M. Fukuda K. Shimada K. Barnes K. Turner A.J. Ikeda M. Koike H. Yamamoto Y. Tanzawa K. Biochem. J. 1995; 311: 657-665Crossref PubMed Scopus (108) Google Scholar, 22Shimada K. Takahashi M. Turner A.J. Tanzawa K. Biochem. J. 1996; 315: 863-867Crossref PubMed Scopus (86) Google Scholar). We have already showed that ECE-1 is present only as dimers on the plasma membrane of endothelial cells (56Muller L. Valdenaire O. Barret A. Korth P. Pinet F. Corvol P. Tougard C. J. Cardiovasc. Pharmacol. 2000; 36: S15-S18Crossref PubMed Google Scholar). The present analysis of ECE-1 biosynthesis demonstrated that the complete dimerization of ECE-1 is a feature common to the 4 isoforms. Dimerization occurs rapidly after synthesis, most probably in the endoplasmic reticulum as we could observe the transient appearance of dimer intermediates that most likely corresponded to incompletely glycosylated or unfolded ECE-1. The Cys412 residue, present in the catalytic domain, is responsible for the dimerization (22Shimada K. Takahashi M. Turner A.J. Tanzawa K. Biochem. J. 1996; 315: 863-867Crossref PubMed Scopus (86) Google Scholar). The role of ECE-1 dimerization has been investigated using site-directed mutagenesis of this cysteine. Mutation into a serine did not modify the glycosylation and the subcellular distribution of ECE-1. However, this mutation slightly modified the enzymatic characteristics of ECE-1 (22Shimada K. Takahashi M. Turner A.J. Tanzawa K. Biochem. J. 1996; 315: 863-867Crossref PubMed Scopus (86) Google Scholar, 57Savage P., De Lombaert S. Shimada K. Tanzawa K. Jeng A.Y. J. Cardiovasc. Pharmacol. 1998; 31: S16-S18Crossref PubMed Scopus (3) Google Scholar).Here, we provided evidence for the formation of heterodimers in endothelial cells endogenously co-expressing several isoforms. The investigation of the formation of heterodimers in these cells was however limited due to the low level of expression of ECE-1a, and to the lack of antibodies specific for either ECE-1b or 1c or 1d. Therefore, we generated AtT-20 cell lines that co-expressed ECE-1a, the isoform with the higher plasma membrane expression, and ECE-1b, which displays the more stable intracellular localization. In double transfected cells we could confirm the presence of heterodimers. Both isoforms were colocalized in intracellular vesicles and the extracellular activity of ECE-1a was decreased. In addition, triple labeling experiments demonstrated that ECE-1a was translocated to the late endosomes/multivesicular bodies that contained ECE-1b. Importantly, heterodimerization of these isoforms did not increase the expression of ECE-1b at the plasma membrane, thus demonstrating the dominant role of