The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2

Zon Weng Lai, Rebecca Ann Lew, Michael A Yarski, Fi-Tjen Mu, Robert Keith Andrews, Alexander Ian Smith

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Angiotensin-Converting Enzyme 2 (ACE2) is a homologue of the well-characterised plasma membrane-bound angiotensin-converting enzyme. ACE2 is thought to play a critical role in regulating heart function, and in 2003, ACE2 was identified as a functional receptor for severe acute respiratory syndrome (SARS) coronavirus. We have recently shown that like Angiotensin Converting Enzyme (ACE), ACE2 undergoes ectodomain shedding and that this shedding event is upregulated by phorbol esters. In the present study, we have used gel shift assays to demonstrate that calmodulin, an intracellular calcium-binding protein implicated in the regulation of other ectodomain shedding events, binds a 16 amino acid synthetic peptide corresponding to residues 762-777 within the cytoplasmic domain of human ACE2, forming a calcium-dependent calmodulin-peptide complex. Furthermore, we have demonstrated that ACE2 expressed in Chinese hamster ovary cells specifically binds to GST-calmodulin, but not GST alone, in pull-down assays using cell lysates. Finally, to investigate if calmodulin has any effect on ACE2 ectodomain shedding in cells that endogenously express the enzyme, cells from a human liver cell line (Huh-7) expressing ACE2 were incubated with calmodulin specific inhibitors; trifluoperazine (TFP) and calmidazolium. Both TFP (25 micromol/L) and calmidazolium, (25 micromol/L), significantly increased the release of ACE2 into the medium (44.1+/-10.8 p <0.05, Student s t-test; unpaired, two-tailed, and 51.1+/-7.4 p <0.05, One-way ANOVA, respectively;), as analysed by an ACE2-specific quenched fluorescence substrate assay. We also show that the calmodulin specific inhibitor -stimulated shedding of ACE2 is independent from phorbol ester-induced shedding. In summary, we have demonstrated that calmodulin is able to bind ACE2 and suggest that the ACE2 ectodomain shedding and/or sheddase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding.
Original languageEnglish
Pages (from-to)2376 - 2381
Number of pages6
JournalEndocrinology
Volume150
Issue number5
DOIs
Publication statusPublished - 2009

Cite this

Lai, Zon Weng ; Lew, Rebecca Ann ; Yarski, Michael A ; Mu, Fi-Tjen ; Andrews, Robert Keith ; Smith, Alexander Ian. / The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2. In: Endocrinology. 2009 ; Vol. 150, No. 5. pp. 2376 - 2381.
@article{5db3718e04e04cf39b38c4bf770b5bf6,
title = "The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2",
abstract = "Angiotensin-Converting Enzyme 2 (ACE2) is a homologue of the well-characterised plasma membrane-bound angiotensin-converting enzyme. ACE2 is thought to play a critical role in regulating heart function, and in 2003, ACE2 was identified as a functional receptor for severe acute respiratory syndrome (SARS) coronavirus. We have recently shown that like Angiotensin Converting Enzyme (ACE), ACE2 undergoes ectodomain shedding and that this shedding event is upregulated by phorbol esters. In the present study, we have used gel shift assays to demonstrate that calmodulin, an intracellular calcium-binding protein implicated in the regulation of other ectodomain shedding events, binds a 16 amino acid synthetic peptide corresponding to residues 762-777 within the cytoplasmic domain of human ACE2, forming a calcium-dependent calmodulin-peptide complex. Furthermore, we have demonstrated that ACE2 expressed in Chinese hamster ovary cells specifically binds to GST-calmodulin, but not GST alone, in pull-down assays using cell lysates. Finally, to investigate if calmodulin has any effect on ACE2 ectodomain shedding in cells that endogenously express the enzyme, cells from a human liver cell line (Huh-7) expressing ACE2 were incubated with calmodulin specific inhibitors; trifluoperazine (TFP) and calmidazolium. Both TFP (25 micromol/L) and calmidazolium, (25 micromol/L), significantly increased the release of ACE2 into the medium (44.1+/-10.8 p <0.05, Student s t-test; unpaired, two-tailed, and 51.1+/-7.4 p <0.05, One-way ANOVA, respectively;), as analysed by an ACE2-specific quenched fluorescence substrate assay. We also show that the calmodulin specific inhibitor -stimulated shedding of ACE2 is independent from phorbol ester-induced shedding. In summary, we have demonstrated that calmodulin is able to bind ACE2 and suggest that the ACE2 ectodomain shedding and/or sheddase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding.",
author = "Lai, {Zon Weng} and Lew, {Rebecca Ann} and Yarski, {Michael A} and Fi-Tjen Mu and Andrews, {Robert Keith} and Smith, {Alexander Ian}",
year = "2009",
doi = "10.1210/en.2008-1274",
language = "English",
volume = "150",
pages = "2376 -- 2381",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "Endocrine Society",
number = "5",

}

The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2. / Lai, Zon Weng; Lew, Rebecca Ann; Yarski, Michael A; Mu, Fi-Tjen; Andrews, Robert Keith; Smith, Alexander Ian.

In: Endocrinology, Vol. 150, No. 5, 2009, p. 2376 - 2381.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2

AU - Lai, Zon Weng

AU - Lew, Rebecca Ann

AU - Yarski, Michael A

AU - Mu, Fi-Tjen

AU - Andrews, Robert Keith

AU - Smith, Alexander Ian

PY - 2009

Y1 - 2009

N2 - Angiotensin-Converting Enzyme 2 (ACE2) is a homologue of the well-characterised plasma membrane-bound angiotensin-converting enzyme. ACE2 is thought to play a critical role in regulating heart function, and in 2003, ACE2 was identified as a functional receptor for severe acute respiratory syndrome (SARS) coronavirus. We have recently shown that like Angiotensin Converting Enzyme (ACE), ACE2 undergoes ectodomain shedding and that this shedding event is upregulated by phorbol esters. In the present study, we have used gel shift assays to demonstrate that calmodulin, an intracellular calcium-binding protein implicated in the regulation of other ectodomain shedding events, binds a 16 amino acid synthetic peptide corresponding to residues 762-777 within the cytoplasmic domain of human ACE2, forming a calcium-dependent calmodulin-peptide complex. Furthermore, we have demonstrated that ACE2 expressed in Chinese hamster ovary cells specifically binds to GST-calmodulin, but not GST alone, in pull-down assays using cell lysates. Finally, to investigate if calmodulin has any effect on ACE2 ectodomain shedding in cells that endogenously express the enzyme, cells from a human liver cell line (Huh-7) expressing ACE2 were incubated with calmodulin specific inhibitors; trifluoperazine (TFP) and calmidazolium. Both TFP (25 micromol/L) and calmidazolium, (25 micromol/L), significantly increased the release of ACE2 into the medium (44.1+/-10.8 p <0.05, Student s t-test; unpaired, two-tailed, and 51.1+/-7.4 p <0.05, One-way ANOVA, respectively;), as analysed by an ACE2-specific quenched fluorescence substrate assay. We also show that the calmodulin specific inhibitor -stimulated shedding of ACE2 is independent from phorbol ester-induced shedding. In summary, we have demonstrated that calmodulin is able to bind ACE2 and suggest that the ACE2 ectodomain shedding and/or sheddase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding.

AB - Angiotensin-Converting Enzyme 2 (ACE2) is a homologue of the well-characterised plasma membrane-bound angiotensin-converting enzyme. ACE2 is thought to play a critical role in regulating heart function, and in 2003, ACE2 was identified as a functional receptor for severe acute respiratory syndrome (SARS) coronavirus. We have recently shown that like Angiotensin Converting Enzyme (ACE), ACE2 undergoes ectodomain shedding and that this shedding event is upregulated by phorbol esters. In the present study, we have used gel shift assays to demonstrate that calmodulin, an intracellular calcium-binding protein implicated in the regulation of other ectodomain shedding events, binds a 16 amino acid synthetic peptide corresponding to residues 762-777 within the cytoplasmic domain of human ACE2, forming a calcium-dependent calmodulin-peptide complex. Furthermore, we have demonstrated that ACE2 expressed in Chinese hamster ovary cells specifically binds to GST-calmodulin, but not GST alone, in pull-down assays using cell lysates. Finally, to investigate if calmodulin has any effect on ACE2 ectodomain shedding in cells that endogenously express the enzyme, cells from a human liver cell line (Huh-7) expressing ACE2 were incubated with calmodulin specific inhibitors; trifluoperazine (TFP) and calmidazolium. Both TFP (25 micromol/L) and calmidazolium, (25 micromol/L), significantly increased the release of ACE2 into the medium (44.1+/-10.8 p <0.05, Student s t-test; unpaired, two-tailed, and 51.1+/-7.4 p <0.05, One-way ANOVA, respectively;), as analysed by an ACE2-specific quenched fluorescence substrate assay. We also show that the calmodulin specific inhibitor -stimulated shedding of ACE2 is independent from phorbol ester-induced shedding. In summary, we have demonstrated that calmodulin is able to bind ACE2 and suggest that the ACE2 ectodomain shedding and/or sheddase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding.

UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19164471

U2 - 10.1210/en.2008-1274

DO - 10.1210/en.2008-1274

M3 - Article

VL - 150

SP - 2376

EP - 2381

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

IS - 5

ER -