Functional enhancement of AT1R potency in the presence of the TPαR is revealed by a comprehensive 7TM receptor co-expression screen.

BACKGROUND: Functional cross-talk between seven transmembrane (7TM) receptors can dramatically alter their pharmacological properties, both in vitro and in vivo. This represents an opportunity for the development of novel therapeutics that potentially target more specific biological effects while causing fewer adverse events. Although several studies convincingly have established the existence of 7TM receptor cross-talk, little is known about the frequencey and biological significance of this phenomenon. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the extent of synergism in 7TM receptor signaling, we took a comprehensive approach and co-expressed 123 different 7TM receptors together with the angiotensin II type 1 receptor (AT1R) and analyzed how each receptor affected the angiotensin II (AngII) response. To monitor the effect we used integrative receptor activation/signaling assay called Receptor Selection and Amplification Technology (R-SAT). In this screen the thromboxane A2α receptor (TPαR) was the only receptor which significantly enhanced the AngII-mediated response. The TPαR-mediated enhancement of AngII signaling was significantly reduced when a signaling deficient receptor mutant (TPαR R130V) was co-expressed instead of the wild-type TPαR, and was completely blocked both by TPαR antagonists and COX inhibitors inhibiting formation of thromboxane A2 (TXA2). CONCLUSIONS/SIGNIFICANCE: We found a functional enhancement of AT1R only when co-expressed with TPαR, but not with 122 other 7TM receptors. In addition, the TPαR must be functionally active, indicating the AT1R enhancement is mediated by a paracrine mechanism. Since we only found one receptor enhancing AT1R potency, our results suggest that functional augmentation through 7TM receptor cross-talk is a rare event that may require specific conditions to occur.

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 ß-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 ß-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 ß-arrestins. Since uncoupling of G proteins by increased ability to recruit ß-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 ß-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit ß-arrestin in response to Sar1-Ile4-Ile8 (SII) Ang II, a ligand solely activating the ß-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 ß-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 ß-arrestin recruitment, respectively.

The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation.

The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensin AT(1) receptor signalling is illustrated by the common use of angiotensin AT(1) receptor-inverse agonists in clinical practice. It is well established that rodent orthologues of the angiotensin AT(1) receptor can selectively signal through G protein-dependent and -independent mechanisms in recombinant expression systems, primary cells and in vivo. The in vivo work clearly demonstrates profoundly different cellular consequences of angiotensin AT(1) receptor signalling in the cardiovascular system, suggesting pharmacological potential for drugs which specifically affect a subset of angiotensin AT(1) receptor actions. However, it is currently unknown whether the human angiotensin AT(1) receptor can signal through G protein-independent mechanisms - and if so, what the physiological impact of such signalling is. We have performed a detailed pharmacological analysis of the human angiotensin AT(1) receptor using a battery of angiotensin analogues and registered drugs targeting this receptor. We show that the human angiotensin AT(1) receptor signals directly through G protein-independent pathways and supports NIH3T3 cellular proliferation. The realization of G protein-independent signalling by the human angiotensin AT(1) receptor has clear pharmacological implications for development of drugs with pathway-specific actions and defined biological outcomes.