Angiotensin II Type 2 Receptor–Bradykinin B ₂ Receptor Functional Heterodimerization

Abstract

Abstract-Angiotensin II type 2 (AT 2 R) or bradykinin B 2 (B 2 R) receptor activation enhances NO production. Recently, we demonstrated enhancement of NO production when AT 2 R and B 2 R are simultaneously activated in vivo. However, the mechanism involved in this enhancement is unknown. Using confocal fluorescence resonance energy transfer microscopy, we report the distance between the AT 2 R and B 2 R in PC12W cell membranes to be 50Ϯ5 Å, providing evidence and quantification of receptor heterodimerization as the mechanism for enhancing NO production. The rate of AT 2 R-B 2 R heterodimer formation is largely a function of the degree of AT 2 R-B 2 R expression. The physical association between the dimerized receptors initiates changes in intracellular phosphoprotein signaling activities leading to phosphorylation of c-Jun terminal kinase, phosphotyrosine phosphatase, inhibitory protein B␣, and activating transcription factor 2; dephosphorylation of p38 and p42/44 mitogen-activated protein kinase and signal transducer inhibitor of transcription 3; and enhancing production of NO and cGMP. Controlling the expression of AT 2 R-B 2 R, consequently influencing their biologically active dimerization, presents a potential therapeutic target for the treatment of hypertension and other cardiovascular and renal disorders. Key Words: receptors, bradykinin Ⅲ nitric oxide Ⅲ angiotensin Ⅲ bradykinin T he angiotensin II type 2 receptor (AT 2 R) mediates a vasodilator 1-3 cascade that includes bradykinin (BK), NO, and cGMP. BK, the major effector hormone of the kallikreinkinin system, acts mainly through the BK B 2 receptor (B 2 R) to mediate most of its cardiovascular and renal actions. Our previous data demonstrated that, compared with individual receptor contributions, simultaneous activation of AT 2 R and B 2 R led to a 70% increase in NO production, 1 suggesting interaction between these 2 receptors. However, the molecular mechanism involving the AT 2 R-B 2 R-NO pathway is unknown.Receptor-receptor crosstalk is an essential process for plasma membrane-localized receptors, including those involving the superfamily of the 7 trans-membrane G proteincoupled receptors (GPCRs). 4 It is well established that a variety of cell surface receptors interact with each other to form dimers and that this is essential for their activation. Recent studies provided evidence for the existence of GPCR homodimers and heterodimers. 5 Heterodimerization has effects on ligand binding, receptor activation, desensitization, and trafficking, as well as receptor signaling different from those of the homodimer or oligomer. 6,7 Heterodimerization provides a newly recognized means of modulation of receptor function, as well as cross-talk between GPCRs. We hypothesized that AT 2 R and B 2 R heterodimerize to enhance NO production. In this study, fluorescence resonance energy transfer (FRET) microscopy was used to measure the molecular proximity between AT 2 R and B 2 R in PC12W in vivo and to provide evidence for and quantification of receptor heterodime...

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