Elsevier

Cellular Signalling

Volume 50, October 2018, Pages 9-24
Cellular Signalling

Angiotensin (1–7) does not interact directly with MAS1, but can potently antagonize signaling from the AT1 receptor

https://doi.org/10.1016/j.cellsig.2018.06.007Get rights and content

Highlights

  • Small molecule synthetic MAS1 agonists activate Gq, Gi and β-arrestin pathways.

  • Synthetic MAS1 agonists activate Erk and Akt and promote release of arachidonic acid.

  • Synthetic MAS1 agonists induce MAS1 internalization and elicit DMR assay responses.

  • Ang (1–7) does not initiate detectable signaling through MAS1 in recombinant cells.

  • Ang (1–7) antagonizes concentration-biased signaling by Ang II in endothelial cells.

Abstract

Angiotensin (1–7) has been reported to be a ligand for the GPCR MAS1. Small molecule MAS1 modulators have also been recently characterized. Aside from convincing evidence for MAS1 activation of Gq signaling, little is known about MAS1 mediated signaling pathways initiated by these ligands, especially Ang (1–7). We performed a comprehensive characterization of recombinant MAS1 signaling induced by Ang (1–7) and small molecule ligands through numerous G protein-dependent and independent pathways, and in a signaling pathway agnostic approach. We find that small molecule ligands modulate numerous G protein-dependent and independent pathways through MAS1, including Gq and Gi pathways, GTPγS binding, β-arrestin recruitment, Erk1/2 and Akt phosphorylation, arachidonic acid release, and receptor internalization. Moreover, in dynamic mass redistribution (DMR) assays that provide a pathway-agnostic readout of cellular responses, small molecule agonists produced robust responses. In contrast, Ang (1–7) failed to induce or block signaling in any of these assay platforms. We detected specific binding of radiolabeled Ang (1–7) to rat aortic endothelial cell (RAEC) membranes, but not to recombinant MAS1. Biphasic, concentration-dependent biased signaling responses to Ang II were detected in RAEC. These phases were associated with vastly different DMR characteristics and this likely provides a molecular basis for previously observed concentration-dependent divergent physiological actions of Ang II. Both phases of Ang II signaling in RAECs were potently inhibited by Ang (1–7), providing a plausible molecular mechanism for Ang (1–7) as counter regulator of the Ang II- AT1 axis, responsible at least in part for Ang (1–7) physiological activities.

Introduction

MAS1 is a class 1 G protein-coupled receptor, originally cloned 30 years ago as proto-oncogene, based on its ability to transform NIH-3T3 cells [1]. More recently, large numbers of MAS related receptors have been identified, forming a family of MAS-related G protein-coupled receptors (Mrgpr) [2, 3]. MAS1 is ubiquitously expressed, with the highest expression detected in testis and many regions of the brain, followed by kidney, heart, blood vessels, spleen, retina, liver, tongue, lung and others [4, 5].

Since its discovery, MAS1 has been extensively studied, and numerous inroads into understanding its physiological function have been made. Most of our knowledge of MAS1 function is derived from studies of MAS1-deficient animals. In the CNS, MAS1 deficiency influenced memory formation related long-term potentiation (LTP) in the hippocampus [6], and led to sex-specific changes in anxiety related behaviors [7, 8]. In testis, although no gross changes in fertility or organ morphology were observed,MAS1 deficiency led to changes in spermatogenesis and erectile function, and affected genes involved in steroidogenesis [7, 9]. MAS1 deficiency led to a metabolic syndrome phenotype, either when deleted alone, or in addition to ApoE, depending on the host mouse strain [[10], [11], [12]]. Striking phenotypes resulting from MAS1 deficiency have been observed in the cardiovascular system, with both beneficial and deleterious effects observed upon MAS1 ablation. MAS1 deficiency leads to impairment of cardiomyocyte function and deleterious effects on overall cardiac function, accompanied by profibrotic changes [[13], [14], [15]]. MAS1 deficiency was also shown to lead to endothelial dysfunction, vascular resistance and high blood pressure, albeit in a mouse strain-dependent fashion [12, 16, 17]. In contrast, MAS1 deficiency provided protection from salt-induced hypertension [18]. Moreover, MAS1 ablation provides protection from ischemia-reperfusion injury in both kidney and heart [19, 20]. In heart, reduced infarct size has been observed in MAS1 deficient mice subjected to ischemia-reperfusion injury, an effect mirrored by pharmacological inhibition of MAS1 function in wild type animals with small molecule inverse agonists [19]. Cardioprotective effects of MAS1 inverse agonists were also observed in pigs upon ischemia-reperfusion injury [21]. In isolated rat and mouse hearts, MAS1 inhibition was cardioprotective, leading to improved coronary flow and reduced arrhythmias [19]. The reasons for opposing cardiovascular actions of MAS1 deficiency seen in different studies are unclear, but may be related to the genetic backgrounds of the animals used and the different adaptive morphological and/or compensatory changes induced by developmental/continual MAS1 deficiency in certain genetic backgrounds versus others. However, the cardioprotective effect of both MAS1 ablation and pharmacological inhibition in multiple species provides convincing evidence that these effects are mediated by inhibition of MAS1 function [19].

Angiotensin (1-7) (Ang (1-7)), a derivative of angiotensin II (Ang II), has been described as an endogenous ligand for MAS1 [22, 23]. Ang (1-7) is produced by cleavage of a single amino acid, phenylalanine, from Ang II, mediated by ACE2 or neutral enteropeptidase. Ang (1-7) has diverse biological activities, including vasodilatory, cardioprotective, antithrombotic, antidiuretic, antifibrotic and other effects [[24], [25], [26], [27], [28], [29], [30], [31], [32], [33]]. Many of these activities are lost in MAS1-deficient animals or tissues, and this serves as the most compelling argument implicating Ang (1-7) as a potential MAS1 ligand [4, 22, [34], [35], [36], [37], [38], [39]]. Published data on the interaction of Ang (1-7) with MAS1 consists mostly of radio- and fluorescent-ligand binding [22, 23, [40], [41], [42]]. Specific labeling of tissues and primary cells by radiolabeled Ang (1-7) has been demonstrated, and appears to be lost in MAS1-deficient animals [22, 34, 39]. However, evidence for a direct interaction of Ang (1-7) with MAS1 is very limited. Ang (1-7) binding studies performed in well controlled experiments in a gain-of-function mode in recombinantly expressing cells are mostly lacking. Moreover, in several additional reports, Ang (1-7) failed to activate Gq-mediated signaling through recombinantly expressed MAS1 [19, [43], [44], [45]]. Thus, there is significant uncertainty surrounding the role of Ang (1-7) as an endogenous MAS1 ligand. The inability of Ang (1-7) to initiate classical G protein signaling and induce MAS1 desensitization, in conjunction with the somewhat inconclusive nature of available pharmacological and radioligand binding data led the IUPHAR nomenclature committee to refrain from formally designating MAS1 as an Ang (1-7) receptor [46].

Neuropeptide FF (NPFF) has also been shown to activate MAS1 in recombinant systems, leading to engagement of the Gq pathway [2, 43]. NPFF is not potent (EC50 approx. 0.4 μM) [43], and given its low systemic and tissue concentrations it is unlikely to be an endogenous ligand for the receptor. Finally, highly selective, small molecule MAS1 agonists and inverse agonists capable of modulating both Gq and Gi signaling have been reported [19].

The signaling properties of MAS1 have not been thoroughly characterized. It has been suggested that Ang (1-7) may be a biased MAS1 ligand, exerting its biological activities through MAS1 mediated activation of G protein-independent pathways, which lead to arachidonic acid release and Akt/eNOS activation [4]. Signaling by small molecule MAS1 agonists, outside of the Gq and Gi pathways, has not been evaluated. We have performed a comprehensive evaluation of MAS1 signaling through numerous G protein-dependent and independent signaling pathways. In addition, we employed a signaling pathway agnostic approach, using label-free, dynamic mass redistribution (DMR) technology. Our goal was to identify MAS1 dependent signaling fingerprints for small molecule agonists and Ang (1-7), and to investigate the possibility of signaling bias between the two classes of ligands. In addition, we have investigated additional, MAS1-independent molecular mechanisms for Ang (1-7) action as a counter regulator of the Ang II- AT1 receptor axis.

Section snippets

Materials and methods

Rat aortic endothelial cells (Cat. # R304-05a) and neonatal rat ventricular myocytes (Cat. # R357-75) were purchased from Cell Applications (San Diego, CA). HEK-293 (Cat. # CRL-1573) and CHO-K1 (Cat.# CCL-61) were obtained from ATCC (Manassas, VA). PathHunter HEK293 (Cat. # 93-0165) and CHO-K1 (Cat. # 93-0164) β-Arrestin parental cell lines were from DiscoveRx (Fremont, CA). Test compounds were synthetized at Arena Pharmaceuticals or purchased from Sigma-Aldrich (St. Louis, MO) or Tocris

Signaling through Gq and Gi pathways and modulation of GTPγS binding

To evaluate MAS1 receptor signaling we employed several previously-described, prototypical small molecule agonists and inverse agonists [19], and their close analogs (Fig. 1A). Ang (1-7) was included in all studies, with the Ang (1-7) nonpeptide mimetic AVE-0991 [23] and the Ang (1-7) analog A-779, a reported Ang (1-7) antagonist [49], also tested in some studies. Initially, we explored in detail if and how MAS1 agonists, inverse agonists and Ang (1-7) signal through heterotrimeric G proteins.

Discussion

The identity of Ang (1-7) as a potential MAS1 agonist originated primarily from observations of loss of numerous Ang (1-7) physiological activities in MAS1 deficient animals and tissues. However, there is limited evidence for a direct interaction of MAS1 with Ang (1-7), which lacks the ability to activate MAS1-mediated Gq signaling [19, [43], [44], [45]]. The discovery of selective, small molecule MAS1 agonists and inverse agonists provided us with critically important chemical tools to

Acknowledgments

We thank Sunny Al-Shamma and Carleton Sage for critical reading of the manuscript and for valuable scientific discussions.

Declaration of interest

All contributing authors are shareholders and/or employees of Beacon Discovery and/or Arena Pharmaceuticals, who sponsored these studies.

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