Human ACE and bradykinin B ₂ receptors form a complex at the plasma membrane

Abstract

To investigate how angiotensin-I converting enzyme (ACE) inhibitors enhance the actions of bradykinin (BK) on B 2 receptors independent of blocking BK inactivation, we expressed human somatic ACE and B 2 receptors in CHO cells. Bradykinin and its ACE-resistant analogue were the receptor agonists. B 2 fused with green fluorescent protein (GFP) and ACE were coprecipitated with antisera to GFP or ACE shown in Western blots. Immunohistochemistry of fixed cells localized ACE by red color and B 2 -GFP by green. Yellow color on plasma membranes of coexpressing cells also indicated enzyme-receptor complex formation. Using ACE-fused cyan fluorescent protein donor and B 2 -fused yellow fluorescent protein acceptor, we registered fluorescence resonance energy transfer (FRET) by the enhanced fluorescence of donor upon acceptor photo-bleaching, establishing close (within 10 nm) positions of B 2 receptors and ACE. Bradykinin stimulation cointernalized ACE and B 2 receptors. We expressed ACE fused to N-terminus of B 2 receptors, anchoring only receptors to plasma membranes. Here, in contrast to cells where both ACE and B 2 receptors are separately anchored, ACE inhibitors neither enhance activation of chimeric B 2 , nor resensitize desensitized B 2 receptors. Heterodimer formation between ACE and B 2 receptors can be a mechanism for ACE inhibitors to augment kinin activity at cellular level.

Carboxypeptidase M and Kinin B1 Receptors Interact to Facilitate Efficient B1 Signaling from B2 Agonists

“…However, there is emerging evidence that analogous protein assemblies may occur on the cell surface, possibly in lipid microdomains (41)(42)(43). Although interactions between peptide receptors and related peptidases have not been extensively studied, recent evidence indicates that ACE and B2 kinin receptor do interact on the cell surface (44,45). Because ACE readily inactivates bradykinin, it was previously thought that ACE inhibitors enhance B2R signaling solely by blocking degradation of the B2 agonist (46).…”

“…with ACE-resistant B2 agonists) (47). Subsequent studies showed that ACE and B2Rs form heterodimers on the cell membrane and are co-localized in the same membrane microdomain (44,45), indicating that conformational changes in ACE upon inhibitor binding are transmitted to the B2R, which results in allosteric modification of its signaling properties.…”

Ace inhibitors - activators of kinin receptors

“…Формирование комплексов АПФ-Б 2 Р на клеточной мембране в культуре СНО-клеток позднее было подтверждено методом иммуногистохимии с использованием флуоресцентной микроскопии [83]. В качестве стимуляторов Б 2 Р были взяты Бк и его аналог, устойчивый к действию АПФ.…”

Bradykinine et néphroprotection

“…L'implication de la BK dans l'effet des IEC reste une source de débats contradictoires, souvent explicables par les méthodologies de mesure de la BK, celles des prélèvements sanguins, ainsi que par les conditions physiopathologiques et expérimentales (âge, régimes alimentaires, imprégnation hormonale, hypertension) qui induisent un niveau d'activité variable du SKK. Un nouveau mécanisme additionnel d'action des IEC reposerait sur la formation d'un complexe membranaire entre l'enzyme de conversion de l'angiotensine (ECA) et le RB2 (complexe ECA/RB2) qui augmenterait la sensibilité du RB2 pour la bradykinine [3]. Par ailleurs, le traitement par les IEC entraîne l'accumulation d'angiotensine I dégradée en angiotensine 1-7 par plusieurs enzymes.…”