title: The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2.
abstract: Angiotensin-converting enzyme (ACE)-2 is a homolog of the well-characterized 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 coronavirus. We have recently shown that like ACE, ACE2 undergoes ectodomain shedding and that this shedding event is up-regulated by phorbol esters. In the present study, we 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 glutathione-S-transferase-calmodulin, but not glutathione-S-transferase alone, in pull-down assays using cell lysates. Finally, to investigate whether 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 and calmidazolium. Both trifluoperazine (25 micromol/liter) and calmidazolium, (25 micromol/liter) 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 analyzed 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.
authors.forename: Zon W
authors.initials: ZW
authors.lastname: Lai
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Lew
authors.forename: Michael A
authors.initials: MA
authors.lastname: Yarski
authors.forename: Fi-Tjen
authors.initials: FT
authors.lastname: Mu
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
authors.forename: A Ian
authors.initials: AI
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000085962:Angiotensin-Converting Enzyme 2; D000818:Animals; D016466:CHO Cells; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2