Biased Signaling of the Angiotensin II Type 1 Receptor Can Be Mediated through Distinct Mechanisms

Background: Seven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or beta-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of beta-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G protein-uncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit beta-arrestins. Since uncoupling of G proteins by increased ability to recruit beta-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms. Methodology/Principal Findings: We studied five AT1R mutants previously published to display pathway separation: D74N, DRY/AAY, Y292F, N298A, and Y302F (Ballesteros-Weinstein numbering: 2.50, 3.49-3.51, 7.43, 7.49, and 7.53). We find that D74N, DRY/AAY, and N298A mutants are more prone to beta-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit beta-arrestin in response to Sar(1)-Ile(4)-Ile(8) (SII) Ang II, a ligand solely activating the beta-arrestin pathway. Conclusions/Significance: Our analysis reveals that the underlying conformations induced by these AT1R mutants most likely represent principally different mechanisms of uncoupling the G protein, which for some mutants may be due to their increased ability to recruit beta-arrestin2. Hereby, these findings have important implications for drug discovery and 7TMR biology and illustrate the necessity of uncovering the exact molecular determinants for G protein-coupling and beta-arrestin recruitment, respectively.