Angiotensin-Converting Enzyme (ACE) Dimerization Is the Initial Step in the ACE Inhibitor-Induced ACE Signaling Cascade in Endothelial Cells

Abstract

The binding of angiotensin-converting enzyme (ACE) inhibitors to ACE initiates a signaling cascade that involves the phosphorylation of the enzyme on Ser1270 as well as activation of the c-Jun NH 2 -terminal kinase (JNK) and leads to alterations in gene expression. To clarify how ACE inhibitors activate this pathway, we determined their effect on the ability of the enzyme to dimerize and the role of ACE dimerization in the initiation of the ACE signaling cascade. In endothelial cells, ACE was detected as a monomer as well as a dimer in native gel electrophoresis and dimerization/oligomerization was confirmed using the split-ubiquitin assay in yeast. ACE inhibitors elicited a rapid, concentration-dependent increase in the dimer/ monomer ratio that correlated with that of the ACE inhibitorinduced phosphorylation of ACE. Cell treatment with galactose and glucose to prevent the putative lectin-mediated self-association of ACE or with specific antibodies shielding the N terminus of ACE failed to affect either the basal or the ACE inhibitor-induced dimerization of the enzyme. In ACE-expressing Chinese hamster ovary cells, ACE inhibitors elicited ACE dimerization and phosphorylation as well as the activation of JNK with similar kinetics to those observed in endothelial cells. However, these effects were prevented by the mutation of the essential Zn 2ϩ -complexing histidines in the C-terminal active site of the enzyme. Mutation of the N-terminal active site of ACE was without effect. Together, our data suggest that ACE inhibitors can initiate the ACE signaling pathway by inducing ACE dimerization, most probably via the C-terminal active site of the enzyme.Two isoforms of the angiotensin-converting enzyme (ACE) exist in mammals: somatic or tissue ACE and testicular ACE. These enzymes are both type I transmembrane proteins consisting of an extended extracellular domain and a short cytoplasmic tail and are derived from a single gene by virtue of alternative use of transcription initiation sites (Hubert et al., 1991). The extracellular portion of testicular ACE has one active site, whereas that of somatic ACE has two active sites (the N and C domains), each of which contains the amino acid sequence HEMGH that is crucial for Zn 2ϩ binding (Wei et al., 1991). Soluble forms of ACE can also be detected in plasma and other body fluids and are generated by the enzymatic cleavage of the C-terminal extracellular portion, in the socalled juxtamembrane stalk region. All of the ACE isoforms hydrolyze circulating peptides and catalyze the extracellular conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, which is a potent vasopressor. ACE also inactivates the vasodilator peptides bradykinin and kallidin, which are derived from kininogen by the action of kallikreins (for review, see Bernstein et al., 2005). Inhibition of ACE is expected to prevent the formation of angiotensin II and to potentiate the hypotensive response to bradykinin, which would lead to the lowering of blood pressure. Somatic ACE also...

Angiotensin-converting enzyme gene polymorphism predicts the time-course of blood pressure response to angiotensin converting enzyme inhibition in the AASK trial

“…In the case of ACE, ACE-inhibitors have been shown to convert ACE monomers to homodimers [24], and this dimerization process may be important for disrupting ACE activity, or perhaps intracellular signaling by membrane-spanning ACE in endothelial cells. Finally, heterosis is best documented during association with a three-state diploid genotype, while attempts at association based solely on the two-state presence or absence of an allele may not detect the phenomenon [11]; thus previous attempts at two-state 'allelic' association at ACE may partially explain the inconclusive findings on ACE-I reported to date [10].…”

Hereditary Determinants of Human Hypertension

“…Either of 2 homozygous genotypes at G12269A responded significantly faster than G/A heterozygotes; similar associations were seen comparing homozygous to heterozygous ACE haplotypes, suggesting that the genetic phenomenon of heterosis may be an important determinant of responsiveness to an ACE inhibitor. Because the ACE enzyme may typically function as a homodimer, 56 the possibility of ACE variant heterodimerization arises as a potential explanation for phenotypic heterosis in this setting.…”

Renin-angiotensin system in the kidney: What is new?

“…Binding of ACE substrates or inhibitors to this enzyme can stimulate intracellular signaling pathways: ACE inhibitors (perindoprilat and ramiprilat), like the ACE substrate (bradykinin), could also increase COX-2 expression, ACE phosphorylation at Ser1270 and activation of JNK in endothelial cells [25] . The modulation of gene expression in endothelial cell by ACE inhibitors and JNK/c-Jun pathway requires ACE dimerization through the C domain of the enzyme [26] . This indicates that, although ACE is not a cell surface receptor, it is involved in cell functions.…”

“…New functions for well known members of the RAS have been found. For example, ACE, known for its catalytic action on Ang Ⅰ, also functions as a signal transduction molecule, initiating a series of intracellular events when stimulated [25,26] . Besides increasing catalytic activity of renin and prorenin, the renin/(Pro)renin receptor (PRR), cloned in 2002 [27] , can also induce an intracellular signaling pathway generating effects in an angiotensinindependent manner [6,27] .…”