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(Hypertension. 1998;31:3.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Endothelin-Converting Enzyme

Ultrastructural Localization and Its Recycling From the Cell Surface

Kay Barnes; Carolyn Brown; Anthony J. Turner

From the Department of Biochemistry and Molecular Biology, University of Leeds, UK.

	Abstract

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Abstract—The potent vasoconstrictor endothelin-1 (ET-1) is secreted constitutively by endothelial cells and has been implicated in the pathophysiology of several cardiovascular diseases. It is generated from its inactive intermediate, big ET-1, through the action of endothelin-converting enzyme (ECE). Using several complementary techniques, we have demonstrated that ECE is present at the cell surface and on intracellular vesicles and that it recycles from the cell surface in endothelial cells. This is the first ultrastructural localization of ECE in lung and the first time big ET-1 and ECE have been colocalized by immunogold in a vesicular population, 50 to 100 nm in diameter. In addition, by double immunogold staining of ultrathin cryosections, we have localized ECE together with angiotensin-converting enzyme on the luminal membrane of endothelial cells. With cell surface biotinylation of a transformed rat endothelial cell line and of human umbilical vein endothelial cells, we have confirmed the presence of ECE on the plasma membrane. After treatment of endothelial cells with chloroquine, ECE and trans-Golgi network 38 protein were shown by immunofluorescence staining to localize to the same intracellular compartment.

Key Words: endothelin-1 • immunoelectron microscopy • constitutive secretion • trans-Golgi network

	Introduction

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The endothelins are a family of potent vasoconstrictor peptides that were originally isolated from the supernatant of cultured porcine aortic endothelial cells.¹ They have been implicated in the pathophysiology of cardiac diseases including hypertension, vascular hypertrophy, and atherosclerosis.^{2 3} The precursor proendothelins are cleaved by furin,⁴ a prohormone convertase of the constitutive secretory pathway, to produce the inactive intermediates, big ETs, from which mature ETs are generated by the proteolytic action of ECE.^{5 6} Furin, which is involved in the proteolytic processing of many proproteins, is mainly resident in the TGN, but it also cycles between the cell surface and the TGN/endosomal system.^{7 8} ECE, however, is unique to proendothelin processing in endothelial, smooth muscle, and other cell types, and its precise subcellular locations, which have been a matter of some controversy (see Turner and Tanzawa⁶ for discussion), are critical for the design of ECE inhibitors acting as therapeutic agents.

After purification and cDNA cloning studies,^{9 10 11 12 13} ECE (subsequently named ECE-1) was revealed as a highly glycosylated type II, integral membrane, neutral metalloprotease. ECE-1 exists in two isoforms, ECE-1 and ECE-1ß, with differing N-terminal cytoplasmic domains but showing similar enzymatic properties and tissue distribution.¹⁴ A homolog, designated ECE-2, has also been cloned and characterized.¹⁵ ECE-1 and ECE-2 are homologous with neutral endopeptidase-24.11 (E-24.11; neprilysin) and with the Kell blood group protein, both resident plasma membrane ectoproteins.^{11 15} Unlike E-24.11 however, ECE-1 exists as a disulfide-linked homodimer.^{8 10 16}

ECE-1 has been localized to endothelial cells by immunohistochemical analysis in a variety of tissues and to several endothelial, neuronal, and glial cell lines.^{8 17 18} In rat lung, from which ECE was first purified,¹⁹ the immunoreactivity was observed in the endothelial cells of the arteries and veins.⁸ At the subcellular level, using cultured cells, ECE has been shown to be clustered along the plasma membrane^{8 17} and to be upregulated and redistributed to an intracellular compartment, probably the Golgi, after treatment with the metalloprotease inhibitors phosphoramidon or thiorphan.¹⁷ It was proposed that this phenomenon may be due to the inhibition of the activity of a novel metalloprotease involved in the turnover of the ECE protein.¹⁷

Immunoelectron microscopical studies have demonstrated the presence of big ET-1 and ET-1 in a subcellular vesicular fraction prepared from bovine aortic endothelial cells,²⁰ but no ultrastructural studies of ECE localization have followed. In the present study, using immunogold staining and ultrathin cryosections of pig and rat lung, we have colocalized ECE with big ET-1 on intracellular vesicles and with ACE on the luminal surface of endothelial cells.

To establish unequivocally the presence of ECE on plasma membranes, we have used cell surface biotinylation of a transformed rat lung endothelial cell line (TRLEC-03)⁸ and human umbilical vein endothelial cells (HUVEC), detecting the biotinylated protein by immunoblotting after sedimentation with streptavidin-agarose. In parallel, we have compared the distribution of the cell surface metallopeptidase ACE with a protein (rat TGN38/human TGN46) which, like furin, resides predominantly in the TGN and cycles between this compartment and the cell surface.^{21 22} By these criteria, ECE appears to be intermediate in its subcellular distribution compared with these two protein markers. After chloroquine treatment of endothelial cells, ECE was then shown by immunofluorescence staining to redistribute and be highly localized to an intracellular compartment, probably endosomal, a phenomenon previously observed with both furin and TGN38.²¹

	Methods

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Simian virus 40–transformed TRLEC-03 cells were a gift from Dr S. Tsurufuji, Institute of Cytosignal Research, Tokyo, Japan. Phosphoramidon, peptstatin A, and leupeptin were from the Peptide Institute Inc, Osaka, Japan. The peptide DIIWFNTPEHVVPYGLGNH₂ was synthesized by the Multiple Sclerosis Peptide Laboratory, Oxford Brookes University, Oxford, UK. The ECL western blotting kit, anti-mouse biotin, streptavidin fluorescein isothiocyanate (FITC) conjugate, anti-rabbit tetramethylrhodamine isothiocyanate (TRITC), and immunogold conjugates were from Amersham International plc. Cell culture reagents were purchased from GIBCO BRL, Life Technologies Ltd. All other inhibitors and reagents were purchased from Sigma Chemical Co.

Antibody Specificity
The specificities of the monoclonal antibodies AEC27-121 and AEC32-236 to rat and human ECE have been demonstrated previously.⁸ The antibodies recognize an epitope in the C-terminal domain of ECE-1.¹⁴ To check the specificity of AEC32-236 against pig lung, membranes were immunoblotted with the antibody, and a single 120-kDa band was visualized (results not shown). This antibody was selected, therefore, for routine immunogold staining of pig lung cryosections. The antibodies to ACE were affinity-purified,²³ and the specificity of the antibodies to TGN38 has been reported.²⁴

Cell Culture
TRLEC-03 cells were cultured in RPMI-1640 medium containing 10% (vol/vol) fetal calf serum.⁸ HUVEC were grown in a supplemented endothelial cell basal medium according to the manufacturer’s instructions (TCS Biologicals Ltd). For some biotinylation experiments, the TRLEC-03 and HUVEC were incubated with 0.1 mmol/L phosphoramidon for 48 hours before biotinylation and immunoblotting.

Biotinylation of Endothelial Cell Monolayers
All stages of the procedure were performed at 4°C. The medium was removed from the flasks, and each monolayer of cells was washed three times with 10 mL of ice cold Dulbecco’s PBS. After a rinsing with 40 mmol/L bicarbonate buffer (pH 8.6), 50 µL of biotinamidocaproate N-hydroxysuccinimide ester in dimethylformamide (40 mmol/L) was added in 5 mL of the bicarbonate buffer. After 15 minutes, this solution was removed and replaced by a fresh solution of the biotinylation reagent. The cells were washed twice with PBS and lysed with 0.5 or 1 mL of 1% Triton X-100 (vol/vol) in PBS, which included 0.2 mmol/L PMSF, 0.002 mmol/L pepstatin A, and 0.01 mmol/L leupeptin. After the cells were scraped from the flask, the collected lysate was centrifuged at 100 000g for 30 minutes to pellet the debris. The clear lysate (1 mg protein) was rotated for 2 hours with 400 µL of streptavidin/agarose beads suspended in 0.01 mol/L sodium phosphate (0.15 mol/L NaCl, pH 7.2). The beads were recovered by centrifugation for 1 minute at 10 000g and washed twice in PBS by rotation and centrifugation. To elute the biotinylated proteins, 50 µL SDS-PAGE sample buffer was added to the beads and then heated at 100°C for 4 minutes. These samples and aliquots of the nonbiotinylated supernatant fractions were stored at -20°C. On some occasions, to check that removal of biotinylated proteins was complete, a second aliquot of streptavidin-agarose beads was rotated with the lysate and removed for analysis.

Electrophoresis and Blotting
Before gel electrophoresis, SDS-PAGE sample buffer was added to the clear thawed lysates from the biotinylation experiments or to the pig lung membranes. All samples were treated with 5% mercaptoethanol, separated on 7.5% gels, and immunoblotted as in Barnes et al.¹⁷ Proteins were visualized by using the Amersham ECL Western blotting kit. The primary antibodies, AEC27-121 (4 mg/mL) and AEC32-236 (4 mg/mL), to ECE were diluted 1/200 or 1/400. The monoclonal supernatant 2F7.1 to rat TGN38 was diluted 1/10 and the polyclonal antibody P12 to human TGN46²⁵ was diluted 1/500. The polyclonal antibody to human ACE, RP179 (5 mg/mL), was diluted 1/5000.

Enzyme and Protein Assays
ECE was assayed by a high-performance liquid chromatography method, using the synthetic peptide DIIWFNTPEHVVPYGLG- amide as substrate, in which cleavage occurs exclusively at the W-F bond.²⁶ Protein was determined by the bicinchoninic acid method with bovine serum albumin (1 mg/mL) as standard.²⁷

Preparation of Lung Tissue for Cryosectioning
Piglet lung was prepared for sectioning from animals (three large white cross-strain) that had been perfused with 4% paraformaldehyde and 0.01% glutaraldehyde, as described previously.²⁸ Small pieces of tissue <1 mm³ were postfixed for 4 hours in the same fixative and embedded for ultracryotomy according to the method of Tokuyasu.²⁹ Specimens were then sectioned by using a Reichert Jung FC4E Ultracut E. Rat lung tissue from a Wistar rat (250 to 350 g) that had been given a lethal dose of sodium phenobarbitone (120 mg/kg IP) was perfused with saline through the pulmonary artery followed by 8% paraformaldehyde in 0.1 mmol/L phosphate buffer (pH 7.4) for 30 minutes. After excision, the rat lung was treated as above. All experiments were performed under license in accordance with the regulations of the UK Animals (Scientific Procedures) Act of 1986.

Immunostaining of Cryosections
Ultracryosections were immunostained on grids as in Barnes et al.²⁸ For single- and double-labeling experiments, the primary antibodies were used at the following dilutions: 1:5 for AEC32-236, AEC27-121 to ECE, and anti-big ET-1; and 1:10 for RP183 to porcine ACE and immunogold conjugates (5, 15, and 30 nm). In all double-labeling experiments, the primary and secondary antibodies were added simultaneously. For control sections, the primary antibodies were omitted or replaced by preimmune serum.

Immunofluoresence of Chloroquine-Treated Cells
Monolayers of TRLEC-03 were treated by the addition to the culture medium of either 0.1 mmol/L chloroquine for 1 hour or 0.3 mmol/L chloroquine for 3 hours. The cells were then fixed with methanol:acetone [1:1 (vol/vol)] and immunostained as in Barnes et al.¹⁷ For double immunostaining, AEC 32-236 to ECE (1/10 dilution) and a polyclonal antibody to TGN38 (1918, 1/500 dilution) were added concomitantly. After incubation with anti-mouse biotin, the anti-rabbit TRITC and streptavidin FITC conjugates were applied simultaneously.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Localization of ECE and ACE on Luminal Membranes of Porcine and Rat Lung Endothelial Cells
To visualize the localizations of ECE, ACE, and big ET-1 on lung endothelial cells in situ, ultrathin cryosections were cut from prepared pig and rat tissue and immunostained by the relevant primary and secondary gold conjugates. A cocktail of the antibodies AEC27-121 and AEC32-236 to ECE was applied to rat lung sections to enhance detection, and immunogold was observed mainly at the luminal surface of endothelial cells (Fig 1a through 1c). The antibodies RP183 to ACE and AEC 32-236 to ECE were applied together to pig lung cryosections as described in "Methods." The ACE antigen was seen distributed evenly along the luminal membrane (Fig 2a and b), whereas the immunogold decorating the ECE antigen was less abundant and appeared to be localized heterogeneously in clusters (Figs 1a through 1c and 2a and 2b). Single immunogold labeling also revealed ACE and ECE on or near the luminal membranes of the endothelial cells (results not shown).

View larger version (121K): [in this window] [in a new window]   Figure 1. Rat lung cryosections immunostained for ECE. The primary antibodies, AEC32-236 and AEC27-121 to ECE, were applied simultaneously to cryosections. ECE protein in rat lung endothelial cells (a, b, and c) is marked by 15 nm gold particles. C indicates collagen fiber. Bars: a, b=0.15 µm; c=0.25 µm.

View larger version (174K): [in this window] [in a new window]   Figure 2. Double-labeling of pig lung ultracryosections for ACE and ECE. In all sections, primary antibodies AEC32-236 to ECE and RP183 to ACE were applied simultaneously to cryosections. Sites for ACE on the luminal endothelial membranes are denoted by 30-nm (a) and 5-nm (b) immunogold particles (straight arrows). ECE protein is marked by 15-nm gold particles (a and b; curved arrows). ACE and ECE are colocalized on or near the luminal endothelial membranes. A cluster of 15-nm gold particles (b; arrowheads) decorate ECE located adjacent to the nucleus (N). At the higher magnification (b), small vesicles (approximately 50 to 100 nm; curved arrows) are visible immunostained for ECE. Bars: a=0.15 µm; b=0.25 µm.

Dual Localization of ECE and Big ET-1 on Pig Lung Cryosections
ECE was additionally revealed by immunogold particles on vesicles varying from 50 to 100 nm (Fig 2b) at or near the surface of the cells or, on some occasions, in a perinuclear position (Fig 2b). Single immunogold labeling revealed big ET-1 in vesicles of approximately 50 to 100 nm (Fig 3a and 3b). To ascertain whether ECE colocalized with big ET-1, cryosections were double-labeled with two sizes of immunogold (Fig 3c through 3e). The gold particles labeling ECE and big ET-1 were found in close proximity to each other (Fig 3c through 3e) and within the same vesicle (Fig 3d and 3e).

View larger version (134K): [in this window] [in a new window]   Figure 3. Single- and double-labeling of ultrathin cryosections for big ET-1 and ECE. The primary antibody to big ET-1 was applied to cryosections alone (a and b) or concomitantly with AEC32-236 to ECE (c, d, and e). Big ET-1 is marked by 30 nm immunogold in (a), (c), and (e), and by 5 nm immunogold in (b) and (d). ECE protein is visualized with 15 nm gold particles (c, d, and e). Big ET-1 (a and b; arrows) is seen associated with vesicles (50 to 100 nm). Some examples of colocalization of big ET-1 and ECE are shown (c, d, and e; curved arrows). RBC indicates red blood cell. Bars: a, b, and e=0.25 µm; c=0.15 µm; d=0.20 µm.

Cellular Locations of ECE, TGN38/46, and ACE Compared by Biotinylation of Endothelial Cell Monolayers
To compare the relative cell surface and intracellular locations of ECE, TGN38/46, and ACE, cultured endothelial cell monolayers were biotinylated, and the biotinylated cell surface proteins were isolated by using streptavidin-agarose. Subsequent Western blot analysis of the isolated fractions was used to reveal the proteins. In parallel experiments, cells were pretreated with 100 µmol/L phosphoramidon, which previously has been shown to upregulate ECE and cause its accumulation intracellularly.¹⁷ Biotinylated cell surface ECE protein was present in both untreated and phosphoramidon-treated TRLEC-03 (Fig 4a) and HUVEC (Fig 4b) cells. ACE was also demonstrated in the cell surface fraction from HUVEC consistent with its known plasmalemma location (Fig 4b). In contrast, cell surface TGN38 protein was not detectable in the biotinylated cell surface fraction in TRLEC-03 cells (Fig 4a) and was barely detectable in HUVEC (Fig 4b). When the cell lysates were treated with a second aliquot of streptavidin-agarose beads, no biotinylated proteins were detectable in the sedimented bead fraction, indicating that their removal had been complete at the first precipitation (results not shown).

View larger version (23K): [in this window] [in a new window]   Figure 4. Immunoblot of biotinylated cell surface and intracellular protein fractions from (a) TRLEC-03 cells, and (b) HUVEC cells. TRLEC-03 cells and HUVEC were grown in the presence or absence of 0.1 mmol/L phosphoramidon (PR). After biotinylation of the cell surface, the surface-labeled proteins were solubilized, isolated on streptavidin-agarose beads by centrifugation, separated by SDS-PAGE, and immunoblotted in (a) with AEC27-121 to ECE and 2F7.1 to TGN38 as described in "Methods." Lanes 1 and 2, eluant (16 µL) from the streptavidin-agarose bead fraction; lanes 3 and 4, protein (10 µg) from the supernatant fraction. The fractions in (b) were immunoblotted with AEC32-236 to ECE, P12 to TGN46, and RH179 to ACE. Lanes 1 and 2, eluant (35µL) from the streptavidin-agarose bead fraction (surface-labeled proteins); lanes 3 and 4, 10 µg protein from the supernatant fractions.

To detect intracellular protein, the supernatant fractions of the cell lysates, after removal of surface proteins with streptavidin-agarose, were subjected to immunoblotting. Equivalent amounts of lysate protein were compared from TRLEC-03 and HUVEC cells. The intracellular fractions revealed both rat and human ECE (Fig 4a and 4b), rat TGN38 (Fig 4a), and human TGN46 (Fig 4b), but intracellular ACE was undetectable (Fig 4b). The level of intracellular ECE in the lysate supernatant from TRLEC-03 cells (Fig 4a) and HUVEC (Fig 4b) was greater in phosphoramidon-pretreated cell monolayers, approximately 3- and 2-fold, respectively (as quantified by densitometry [Scanmaster 3, Howtek]; three experiments), and some decrease was detected in the surface labeling. Immunoblotting of the total cell lysate from HUVEC confirmed the substantial upregulation of ECE levels, whereas no changes were seen in the levels of ACE or TGN46 (results not shown). No increase in the level of rat TGN38 was observed in the cell lysate from phosphoramidon-treated TRLEC-03 cells (Fig 4a).

Specific Activity of ECE on TRLEC-03 Cell Membranes Prepared From Untreated and Phosphoramidon-Treated Cells
Membranes prepared from TRLEC-03 cells that had been grown in the presence or absence of phosphoramidon were assayed for ECE in duplicate from three separate membrane preparations. The specific activity of ECE in membranes from phosphoramidon-treated cells was approximately 2-fold greater than in membranes from untreated cells, being 2.4±0.15 (duplicates from three experiments with treated cells) and 1.3±0.04 (duplicates from three experiments with untreated cells) nmol · min^-1 · mg^-1, respectively. This correlates well with the increase estimated by immunoblotting, establishing that the increased ECE protein is functionally active.

Localization of ECE and TGN38 on Chloroquine-Treated Cells
To compare the localization of ECE with that of TGN38, chloroquine-treated and untreated TRLEC-03 cells (which express only ECE-1¹² ) were stained by immunofluorescence. TGN38, which in untreated cells is predominantly located to the TGN, redistributed to endosomes after 3 hours (Fig 5b, d, and f), as has been demonstrated previously.²¹ ECE-1 redistributed to the Golgi after a 1-hour treatment with 0.1 mmol/L chloroquine and to similar endosome-like organelles near the surface after 3 hours incubation with 0.3 mmol/L chloroquine (Fig 5a, 5c, and 5e). In double-immunostaining experiments, colocalization of ECE and TGN in the same vesicles was sometimes observed after treatment (Fig 5g and h).

View larger version (111K): [in this window] [in a new window]   Figure 5. Immunofluorescence staining for ECE and TGN38 of chloroquine-treated TRLEC-03. In the left-hand panel, the TRLEC-03 cells are immunostained for ECE, and in the right-hand panel for TGN38. Cells in (a) and (b) are untreated; in (c) and (d), cells were treated for 1 hour with 0.1 mmol/L chloroquine, and in (e), (f), (g), and (h) for 3 hours with 0.3 mmol/L chloroquine. The cells in (g) and (h) were double-labeled for ECE (FITC) and TGN38 (TRITC), respectively. Organelles stained for both ECE and TGN are marked by arrows. Bar: (a) through (h)=20 µm.

	Discussion

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The endothelial cells in lung are an important source of vasoactive substances that maintain vascular tone. The most potent vasoconstrictor, ET-1, is constitutively secreted by lung endothelial cells, and expression of ECE mRNA is particularly high in human and bovine lung tissue.^{9 10} Although ECE has been localized by immunohistochemical light microscopy in endothelial cells of lung tissue,⁸ this is the first study to present immunological ultrastructural data. ECE has been visualized by immunogold particles in rat and porcine lung tissue. Its distribution has been compared with that of its substrate, big ET-1, and with the ectoenzyme, ACE, which is known to be concentrated along the luminal surface of the vascular endothelium where it metabolizes circulating vasoactive peptides, especially angiotensin I and bradykinin.^{30 31} In this study, ACE has functioned as a useful marker of cell surface membranes of endothelial cells in the lung tissue and in cell lines. From these ultrastructural studies together with biotinylation studies and from the effect of chloroquine treatment on cells, we propose ECE-1 cycles between the TGN and the cell surface of endothelial cells.

The previous observations of a plasmalemma location for ECE on cultured endothelial cells^{8 17} is confirmed by the observation that ECE and ACE are detected together on membranes of the lung cryosections. In the present study, a proportion of the total ECE also occurs with ACE in the cell surface fraction of biotinylated TRLEC-03 cells and HUVECs. A small amount of the TGN46 protein was detected in the cell surface fraction of the biotinylated HUVEC cells, and it has been shown that in the steady state, this corresponds to <1% of total TGN46.³² The biotinylation of cultured monolayers has been used by other authors to establish the subcellular location of proteins. For example, metargidin, a membrane-anchored metalloprotease-disintegrin protein, has recently been localized to the plasma membrane by cell surface-biotinylation.³³ The cell surface ECE-1 visualized in this study may, therefore, be involved in a postsecretory processing role analogous to that of ACE.^{8 34 35} Indeed, in coculture experiments with independent cell lines expressing ECE-1 and prepro–ET-1, exogenous secreted big ET-1 was converted to the mature peptide.¹³

From the present data, however, there is also strong evidence to suggest that a proportion of ECE-1 is present inside the cell. In the ultrastructural studies, ECE-1 immunostaining was seen associated with membranes of vesicles that were immunopositive for big ET-1, suggesting that at least a proportion of the intermediate peptide is processed intracellularly and delivered to the cell surface through the constitutive secretory pathway. This is supported by previous ultrastructural studies demonstrating the presence of ET-1 and big ET-1 on vesicles prepared from bovine aortic endothelial cells that are of a similar size to those in the present immunohistological study (approximately 50 to 100 nm),²⁰ and by endogenous conversion of big ET-1 in double transfection experiments.¹³ Furthermore, in the biotinylation experiments, although ECE-1 and TGN38/46 were detected in the intracellular fraction, the cell surface protein, ACE, was not present. The rat TGN38 and the human isoform TGN46 are type I integral membrane proteins that are localized predominantly to the TGN but cycle between the TGN and the cell surface.^{36 37}

An accumulation of ECE in an intracellular compartment seen after treatment of cell monolayers with the lysosomotropic drug, chloroquine, also implies a significant amount of ECE-1 is located inside the cell. Chloroquine neutralizes the pH of acidic cell organelles, and it has been shown previously that in cells treated with this compound, the recycling of rat TGN38/41 and human TGN46 is interrupted, and that TGN38/46 gradually accumulates throughout the cell in non-Golgi structures.^{21 22 37} The prohormone convertase, furin, which processes the proendothelin to big ET-1, is located in the same Golgi compartment as TGN38/46. Furin, like TGN38/46, recycles through the cell surface and, after chloroquine treatment, is predominantly located in endosomes.³⁸ As ECE-1 was shown to be located in the Golgi and eventually accumulate in endosome compartments in chloroquine-treated TRLEC-03 cells, it is possible that it is cycling through the cell in a similar mode to furin and to TGN38/46 proteins consistent with a role as an intracellular processing enzyme. Thus, furin and ECE can act consecutively in proendothelin processing in transport vesicles and at the cell surface to generate active ET.

The cycling of furin is probably dependent on two types of targeting motifs present in its cytoplasmic tail³⁹ (Fig 6). The C-terminal cytoplasmic domain of furin contains a tyrosine-based motif, YKGL (amino acids 758 to 761), and a leucine-based motif, LI (amino acids 755 and 756), similar to those found in the N-terminal cytoplasmic tail of ECE-1 (Fig 6). TGN38 also has a tyrosine motif YQRL (amino acids 309 to 312) (Fig 6), which is believed to be responsible for its rapid retrieval from the cell surface.⁴⁰ In ECE-1, the tyrosine-based motif is YKRA, rather than the YKGL present in furin and YQRL in TGN38. Mutagenesis studies of tyrosine motifs have shown that the presence of an alanine in the fourth position substantially reduces the TGN localization of a protein and favors surface localization. This reflects the steady-state situation with ECE in which its localization is seen to be intermediate between that of ACE (plasma membrane) and TGN38/46 (predominantly TGN). The leucine-based motif in ECE-1, LV (amino acids 15 and 16), could aid in directing the protein from the cell surface to intracellular compartments including endosomes, lysosomes, the TGN, and the basolateral surface of polarized epithelial cells.⁴¹ Unlike ECE-1, ECE-1ß (Fig 6) only contains the di-leucine motif, LL (amino acids 32 and 33). When ECE-1 is expressed at high levels in transfected COS-1 cells, intracellular staining located predominantly in the Golgi is seen.⁸ Because in the TRLEC-03 cells, ECE-1ß was not detected,¹² the predominant ECE-1, like furin, appears to be cycling in transport vesicles between the cell surface and the TGN. This would allow the consecutive processing of proendothelin during transit as well as at the cell surface. Unlike the furin and TGN38/46 protein, however, the present data indicate that, in steady state, a much greater proportion of ECE-1 is located at the cell surface. However, upregulation and relocation to a perinuclear site does occur in certain circumstances, for example, after treatment with low concentrations of metalloprotease inhibitors,^{10 17} and in this study we demonstrate that this ECE still has catalytic activity.

View larger version (19K): [in this window] [in a new window]   Figure 6. Cytoplasmic sequences of ECE-1, ECE 1ß, E-24.11, furin, and TGN38. ECE 1, ECE 1ß, and E-24.11 are type I transmembrane proteins with cytoplasmic N-termini; furin and TGN38 are type II transmembrane proteins with cytoplasmic C-termini. Boxed sequences are phosphorylation sites for CK II, underlined sequences are tyrosine-based motifs, and double-underlined sequences determine TGN localization (see "Discussion" for details).

ECE-1 also contains several sequences for phosphorylation by casein kinase II (CK II) (Fig 6). In a recent study,⁴² it has been shown that CK II phosphorylates the ECE homolog, E-24.11, at two sites in its cytoplasmic domain, Ser⁵ and Thr²⁴ (Fig 6). These authors have suggested that this CK II-mediated phosphorylation may promote internalization of this enzyme. The pattern of phosphorylation sequences in close proximity to a leucine-based motif also occurs in furin (Fig 6), and these motifs show a marked similarity to those found in the N-terminus of ECE-1. Potential PK-A and PK-C sites are also present in this region, which might also play a role in internalization.

From several criteria, including electron microscopical colocalization studies, cell surface biotinylation of protein and chloroquine treatment of cells, we suggest that, following synthesis, ECE-1 is directed on constitutive vesicles to the plasma membrane during which precursor processing occurs and from where it can be internalized to endosomes and recycled to the TGN. The similarity of both internalization and phosphorylation sequence motifs to other cycling proteins involved in protein-processing events endorses this hypothesis.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme ECE = endothelin-converting enzyme ET = endothelin HUVEC = human umbilical vein endothelial cells SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis TGN = trans-Golgi network TRLEC = transformed rat lung endothelial cells

	Acknowledgments

 
We thank the British Heart Foundation for financial support. C.B. is a recipient of a Biotechnology and Biological Sciences Research Council research studentship. We thank George Banting and Sreenivasan Ponnambalam for the kind gift of antibodies to TGN38 and TGN46, respectively, and Kazuhiko Tanzawa for the antibodies to ECE.

	Footnotes

 
Reprint requests to Kay Barnes, Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

Received June 4, 1997; first decision June 20, 1997; accepted July 14, 1997.

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