Calmodulin interacts with angiotensin-converting enzyme-2 (ACE2) and inhibits shedding of its ectodomain - PubMed (original) (raw)

Calmodulin interacts with angiotensin-converting enzyme-2 (ACE2) and inhibits shedding of its ectodomain

Daniel W Lambert et al. FEBS Lett. 2008.

Abstract

Angiotensin-converting enzyme-2 (ACE2) is a regulatory protein of the renin-angiotensin system (RAS) and a receptor for the causative agent of severe-acute respiratory syndrome (SARS), the SARS-coronavirus. We have previously shown that ACE2 can be shed from the cell surface in response to phorbol esters by a process involving TNF-alpha converting enzyme (TACE; ADAM17). In this study, we demonstrate that inhibitors of calmodulin also stimulate shedding of the ACE2 ectodomain, a process at least partially mediated by a metalloproteinase. We also show that calmodulin associates with ACE2 and that this interaction is decreased by calmodulin inhibitors.

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Figures

Figure 1

Figure 1

ACE2 interacts with calmodulin. (A) Software analysis of the cytoplasmic domain of ACE2 (

http://calcium.uhnres.utoronto.ca

) reveals the presence of a calmodulin binding motif (bold, highlighted in red) proximal to the transmembrane domain (italics). The sequence is highly conserved in humans, mice and rats. (B) Cell lysates collected from untransfected HEK293 cells or cells stably transfected with ACE2 (HEK‐ACE2) were immunoprecipitated with an anti‐calmodulin antibody or IgG (referred to as IP) and immunoblotted for ACE2 (referred to as WB). (C) HEK‐ACE2 cells were treated with the calmodulin inhibitor W‐7 (25 μM) for 30 min, lysates collected and immunoprecipitated with an anti‐calmodulin antibody before being immunoblotted for ACE2. A blot representative of the results of three experiments is shown together with the results of densitometrical analysis performed on the blot, ±S.E.M. ∗ P< 0.05.

Figure 2

Figure 2

Inhibition of calmodulin increases ACE2 ectodomain shedding. HEK‐ACE2 cells (A) or Huh7 cells (B) were incubated in the presence or absence of calmidazolium (25 μM; CaMi) for 30 min in OptiMem. HEK‐ACE2 cells were incubated in the presence (closed circles) or absence (open circles) of CaMi (25 μM) for the times indicated (C), or for 30 min at the concentrations indicated (D). Media was collected, concentrated and assayed for ACE2 activity as described in Section 2. The results represent data collected from at least three independent experiments, ±S.E.M. ∗ P< 0.05.

Figure 3

Figure 3

CAMi‐mediated ectodomain shedding is blocked by metalloproteinase inhibitor. HEK‐ACE2 cells were pre‐incubated with either the sheddase inhibitor GM6001 (25 μM); (A and C) or the cysteine proteinase inhibitors E‐64 and leupeptin, the serine proteinase inhibitor 3,4‐dichloroisocoumarin or the aspartic proteinase inhibitor pepstatin A (B) for 30 min before the addition of CaMi (25 μM) for 30 min. Media from these and from untreated control cells was collected, concentrated and assayed for ACE2 activity as described in Section 2. As an additional control, media were collected from untreated cells and incubated in the presence or absence of 50 μM GM6001 before being assayed for ACE2 activity as described. The results are presented relative to untreated control flasks and represent data collected from four (A), two (B) or three (C) independent experiments. ∗ P< 0.1.

Figure 4

Figure 4

The ACE2 homologue collectrin contains a putative calmodulin binding motif. Alignment of human somatic ACE, ACE2 and collectrin functional domains. Stars indicate catalytic sites, black rectangles the transmembrane domains and white rectangles denote observed (∗) or predicted calmodulin binding domains. The ACE2 and collectrin peptide sequence alignment shows conserved residues (highlighted) and predicted calmodulin binding domain (outlined). There is no homology between ACE and ACE2 or collectrin in this region. The transmembrane hydrophobic regions of all three proteins are in italics.

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