1. The identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2.
- Author
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Lai ZW, Lew RA, Yarski MA, Mu FT, Andrews RK, and Smith AI
- Subjects
- Amino Acid Sequence, Angiotensin-Converting Enzyme 2, Animals, CHO Cells, Calmodulin-Binding Proteins chemistry, Calmodulin-Binding Proteins genetics, Calmodulin-Binding Proteins metabolism, Cells, Cultured, Cricetinae, Cricetulus, Cytoplasm metabolism, Humans, Molecular Sequence Data, Peptidyl-Dipeptidase A genetics, Protein Processing, Post-Translational, Sequence Homology, Amino Acid, Transfection, Calmodulin metabolism, Peptidyl-Dipeptidase A chemistry, Peptidyl-Dipeptidase A metabolism, Protein Interaction Domains and Motifs genetics
- 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.
- Published
- 2009
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