Endothelin-Converting Enzyme

Barnes, Kay; Brown, Carolyn D.; Turner, Anthony J.

doi:10.1161/01.hyp.31.1.3

Cited by 50 publications

(24 citation statements)

References 42 publications

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“…A vascular endothelial cell line originally established from rat lung (TRLEC) (17,18) was kindly donated by the Institute of Cytosignal Research Inc. (Tokyo, Japan), and was cultured in DMEM containing 10% FBS. Human adult dermal microvascular endothelial cells (hADM) and human aortic smooth muscle cells (hASM) were purchased from Cell Systems (Kirkland, USA), and were cultured in a half-Nutrient Mixture F-12 Ham/half-DMEM mixture containing endothelial cell growth supplement, heparin, glutamine, and 10% FBS.…”

Section: Cell Culturementioning

confidence: 99%

Laminar Shear Stress Up-Regulates Inducible Nitric Oxide Synthase in the Endothelium

et al. 2004

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Section: Cell Culturementioning

confidence: 99%

Laminar Shear Stress Up-Regulates Inducible Nitric Oxide Synthase in the Endothelium

et al. 2004

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“…Although the carboxyl terminus containing the CEVW sequence is identical in each of the isoforms, the amino-terminal sequences may be responsible for differences in tissue specificity of expression as well as differences in subcellular localization. The different isoforms are localized either to the cell surface or an intracellular compartment, possibly the Golgi (5,19,(21)(22)(23)(24)(25)(26).…”

Section: In Vitro Prenylation Of the Hexapeptide Hkcevw-thementioning

confidence: 99%

Conserved Cysteine and Tryptophan Residues of the Endothelin-converting Enzyme-1 CXAW Motif Are Critical for Protein Maturation and Enzyme Activity

MacLeod¹,

Fuller²,

Scholten³

et al. 2001

Journal of Biological Chemistry

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The neprilysin (NEP)/endothelin-converting enzyme (ECE) family of metalloproteases contains a highly conserved carboxyl-terminal tetrapeptide sequence, CXAW, where "C" is cysteine, "X" is a polar amino acid, "A" is an aliphatic residue, and "W" is tryptophan. Although this sequence strongly resembles a prenylation motif, human ECE-1 did not appear to be prenylated when labeled in vivo using various isoprenoid precursors in cell lines expressing ECE-1. We used site-directed mutagenesis to investigate the role of the CXAW motif and determined that the conserved cysteine residue of the CXAW motif in ECE-1, Cys 755 , is critical for proper folding of the enzyme, its export from the endoplasmic reticulum, and its maturation in the secretory pathway. In addition, site-directed mutagenesis revealed that the conserved tryptophan residue of the sequence CEVW appears to be important for endoplasmic reticulum export and is essential for enzyme activity. Deletion of Trp 758 or substitution with alanine greatly slowed maturation of the enzyme, and resulted in more than a 90% loss of enzyme activity relative to the wild type. Conservative substitution of the tryptophan with phenylalanine did not reduce activity, whereas replacement with tyrosine, methionine, or leucine reduced enzyme activity by 50%, 75%, and 85%, respectively. Together, these data indicate that the conserved CEVW sequence does not serve as a prenylation signal and that both the conserved cysteine and tryptophan residues are necessary for proper folding and maturation of the enzyme. Furthermore, the conserved tryptophan appears to be critical for enzyme activity.

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“…Phosphorylation of the single mutants was greatly reduced and was nearly abolished in the double mutants in both in vivo and in vitro labeling assays. Analysis by MALDI-MS of 32 P-labeled proteolytic peptides derived from wild type 1c and the 1c mutants supported both Ser 18 and Ser 20 as phosphorylated residues. These data demonstrate the first finding that hECE-1 is constitutively phosphorylated within its cytoplasmic domain in an isoform-specific manner.…”

mentioning

confidence: 90%

“…Isoforms 1b, 1c, and 1d but not 1a, were constitutively phosphorylated in vivo when expressed in Chinese hamster ovary cells and casein kinase I readily phosphorylated the immunopurified isoforms in vitro. Site-directed mutagenesis established that two conserved serine residues, Ser 18 and Ser…”

mentioning

confidence: 99%

Constitutive Phosphorylation of Human Endothelin-converting Enzyme-1 Isoforms

MacLeod¹,

Husain

Gage

et al. 2002

Journal of Biological Chemistry

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To investigate the phosphorylation of human endothelin-converting enzyme-1 (hECE-1) and identify potential residues involved, both in vivo and in vitro phosphorylation labeling assays of hECE-1 isoforms were performed in combination with site-directed mutagenesis and mass spectrometric analyses. Initial studies found that endogenous hECE-1 was constitutively phosphorylated in a primary endothelial cell line. The four known isoforms of hECE-1 expressed in this cell line (1a, 1b, 1c, and 1d) were then cloned by reverse transcription-PCR to determine which isoform(s) may be phosphorylated. The isoforms differ only in the first portion of their short aminoterminal cytoplasmic domains whereas their transmembrane domains and ectodomains of the proteins are identical. Isoforms 1b, 1c, and 1d but not 1a, were constitutively phosphorylated in vivo when expressed in Chinese hamster ovary cells and casein kinase I readily phosphorylated the immunopurified isoforms in vitro. Site-directed mutagenesis established that two conserved serine residues, Ser 18 and Ser 20, (numbering based on isoform 1c) form at least one phosphorylation site in these three isoforms. Mutant forms of 1b, 1c, and 1d were constructed in which a single alanine was introduced at either serine residue and a double mutant for each isoform was constructed as well in which both serines were replaced with alanine. Phosphorylation of the single mutants was greatly reduced and was nearly abolished in the double mutants in both in vivo and in vitro labeling assays. Analysis by MALDI-MS of 32 P-labeled proteolytic peptides derived from wild type 1c and the 1c mutants supported both Ser 18 and Ser 20 as phosphorylated residues. These data demonstrate the first finding that hECE-1 is constitutively phosphorylated within its cytoplasmic domain in an isoform-specific manner.

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Endothelin-Converting Enzyme

Abstract: 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

Cited by 50 publications

References 42 publications

Laminar Shear Stress Up-Regulates Inducible Nitric Oxide Synthase in the Endothelium

Laminar Shear Stress Up-Regulates Inducible Nitric Oxide Synthase in the Endothelium

Conserved Cysteine and Tryptophan Residues of the Endothelin-converting Enzyme-1 CXAW Motif Are Critical for Protein Maturation and Enzyme Activity

Constitutive Phosphorylation of Human Endothelin-converting Enzyme-1 Isoforms

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