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The Identification of a Calmodulin-Binding Domain within the Cytoplasmic Tail of Angiotensin-Converting Enzyme-2

Peptide Biology Laboratory (Z.W.L., R.A.L., A.I.S.), Department of Biochemistry and Molecular Biology, and Cardiovascular Biology Laboratory (F.-T.M., R.K.A.), Department of Immunology (Alfred Medical Research and Education Precinct), Monash University, Victoria 3800, Australia; and Epigenetics in Human Health and Disease Laboratory (M.A.Y.), Baker Heart Research Institute, 3004 Victoria, Australia

Address all correspondence and requests for reprints to: A. Ian Smith, Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia. E-mail: ian.smith{at}med.monash.edu.au.

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 µmol/liter) and calmidazolium, (25 µmol/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.