ACE inhibitors (ACEI) elicit an ACE-dependent signaling cascade that affects endothelial gene expression via phosphorylation of ACE on Ser1270 and activation of the JNK/c-Jun-pathway. To clarify the mechanism of signal initiation, we determined whether ACE forms dimers and whether dimerization is required for signaling. Native gel electrophoresis revealed that ACE exists as monomer and as dimer in endothelial cells. ACE dimerization was confirmed using the split-ubiquitin assay in yeast and by chemical crosslinking. ACEI elicited a concentration

and time-dependent increase in dimerization, comparable with that of ACEI-induced ACE phosphorylation. Both the basal, as well as the ACEI-stimulated dimerization of ACE were unaffected by mutation of the N-domain active site, but were abolished by inactivation of the C-domain active site following mutation of the two essential Zn2+-complexing histidine residues. Moreover, the ramiprilat-induced phosphorylation of ACE as well as the subsequent JNK-activation, although apparent in N-domain mutated ACE-expressing cells, were not observed in cells expressing the C-domain mutant. These data suggest that the initial step in the ACE signaling pathway is the ACEI-induced dimerization of the enzyme, which is dependent on interactions involving the C-domain active site of ACE.