On the enigmatic mechanism of signal transduction of

Abstract

The renin-angiotensin system (RAS) plays a central role in the maintenance of water and electrolyte balance in the body and is therefore one of the most important regulatory cascades for the control of physiological blood pressure. The system is based on a peptide cascade in which a long precursor protein is proteolytically cleaved to form the ten-amino-acid long angiotensin I (decapeptide), which is then converted to the octapeptide angiotensin II by the cleavage of two further amino acids. Angiotensin II (AngII) is the main effector of the classical RAS and mediates its effects mainly by binding to the angiotensin II type 1 receptor (AT1R), a G protein-coupled receptor (GPCR). AngII stimulation of the AT1R leads to the activation of G proteins, in particular the G&alpha;_q family, which then initiates signaling pathways that are ultimately responsible for the classic RAS effects of vasoconstriction and water/electrolyte reabsorption, and thus blood pressure elevation. Overactivation of this AngII/AT1R/G&alpha;_q axis has pathophysiological consequences, including increased blood pressure and cardiovascular remodeling processes that contribute to the development of diseases such as heart failure, atherosclerosis, etc. <br /> However, the discovery of other RAS peptides in the last 20 years has shown that the hormonal system is much more extensive and complex than the classical AngII/AT1R axis. A key role is played by the seven amino acid long heptapeptide angiotensin 1-7 (Ang1-7), which is predominantly formed from AngII (by removing the eighth amino acid). Interestingly, Ang1-7 exhibits effects opposite to the classical RAS axis with a variety of beneficial outcomes for the cardiovascular system. Some of these effects are related to interactions of Ang1-7 with different GPCRs (MAS/MrgD/AT2R), suggesting an alternative RAS axis in which Ang1-7 plays a pivotal role and is able to counteract the overactivation of the classical RAS axis. Notably, an imbalance between AngII and Ang1-7 has also been observed in patients with SARS-Cov2 virus infection and is currently being discussed as promoting a more severe disease progression. <br /> Recent publications have shown that Ang1-7 also acts through the AT1R but has a unique signaling mechanism: It was reported to not induce G protein activation (G protein-independent) but lead to recruitment of β-arrestins to the receptor and β-arrestin-mediated signaling (β-arrestin-biased signaling). Finally, the cardioprotective effects of Ang1-7 have been suggested to be mediated in part by G protein-independent, β-arrestin-dependent signaling at the AT1R. However, this mode of action contradicts recent findings from our group and others showing that β-arrestins are not required for the initiation of GPCR signaling, which instead was found to be entirely dependent on the presence of active Gα-proteins. Therefore, we decided to investigate whether the natural Ang1-7 peptide is a true G protein-independent, β-arrestin-biased agonist at the AT1R and how this may contribute to its beneficial effects on the cardiovascular system. <br /> We found that Ang1-7 signaling distal to the AT1R cannot be described as G protein- independent but is de facto G protein dictated. Remarkably, using pharmacological inhibition (FR900359, hereafter FR) and genome editing (CRISPR-Cas9), we identified that G&alpha;_q proteins, previously not associated with Ang1-7 activation, play a central role downstream of AT1Rsignaling. In addition, we were able to show that this G&alpha;_q-dependent signaling has a characteristic transient signaling profile that differs from that of the full agonist AngII. G&alpha;_qproteins, but not β-arrestins, are responsible for Ang1-7-driven pERK accumulation, the traditional readout for β-arrestin-dependent signaling. Ultimately, instead of β-arrestin-biased signaling, we found a unique β-arrestin-dependent trafficking mechanism for Ang1-7 at the AT1R, identifying the peptide as an efficacious "AT1 receptor internalizer". <br /> We believe that these two important findings, the promotion of a characteristic G protein-dependent transient signaling profile and effective β-arrestin-dependent internalization at the AT1R, may help to explain the reported beneficial effects of Ang1-7 through the AT1R in a variety of diseases.