The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT(1) receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B-1 and B-2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT(1)-B-2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT(1) agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT(1) antagonist, losartan, inhibits endogenous B-2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1), Ile(4), Ile(8)]-AngII require the AT(1) receptor and result from arrestin-dependent co-internalization of AT(1)-B-2 heterodimers. BRET50 measurements indicate that AT(1) and B-2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B-2 receptor activation of G(q/11)-dependent intracellular calcium influx and G(i/o)-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B-2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT(1)-B-2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B-2 receptor signaling by binding to the orthosteric ligand binding site of the AT(1) receptor and promoting co-sequestration of AT(1)-B-2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT(1) receptor ligands may translate into different pharmacologic actions.