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-cal- modulin, 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 endo- genously 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 jutmol/liter) and calmidazolium, (25 jutmol/liter) significantly increased the release of ACE2 into the medium (44.1 ± 10.8%, P < 0.05, Student's ttest; 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 showthat 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 shed- dase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding. Copyright © 2009 by The Endocrine Society.