Hot topic: New pharmacological tools reiterate lack of direct connection between Angiotensin 1–7 and the MAS1 GPCR

A new type of deorphanization conundrum confronted in pairing the GPCR, MAS1 with the hormonal peptide angiotensin 1–7 (Ang1-7) was emphasised in recent IUPHAR reviews (1, 2). More evidence for disconnection between Ang1-7 and MAS1 is presented in the recent paper by Gaidarov et al. (3). Ang1-7 is produced by ACE2 or neutral endopeptidase by cleavage of a single amino acid, phenylalanine 8, from angiotensin II (AngII), the major renin angiotensin system hormone. MAS1 was described as the primary receptor for Ang1-7 in regulating diverse biological activities, including vasodilatory, cardio-protective, antithrombotic, antidiuretic and antifibrotic effects (4). These activities are lost in tissues of MAS1-deficient animals, producing striking phenotypes observed in the cardiovascular, renovascular, nervous and reproductive systems. Vast physiological responses to Ang1-7 studied in MAS1-deficient animals serve as the most compelling argument in favor of Ang1-7 pairing with MAS1. However, support for a direct interaction of Ang1-7 with MAS1 lack demonstration of classical G protein signaling and desensitization response to Ang1-7, as well as a lack consensus on confirmatory molecular pharmacological analyses (1, 2).

MAS1-selective small molecule agonists and antagonists from Arena Pharmaceuticals have aided mechanistic study of signaling dynamics in recombinant MAS1 expressing cells. Gaidarov et al. systematically tested efficacy of MAS1-selective non-peptide agonists and antagonists in GPCR signaling and also in signaling-pathway independent assay platforms [3]. Arena Pharmaceutical’s non-peptide ligands modulated G protein-dependent and independent pathways through MAS1, including Gq and Gi pathways, 35S-GTPɣS binding, β-arrestin recruitment, Erk1/2 and Akt phosphorylation, arachidonic acid release, and receptor internalization. Moreover, non-peptide agonists produced robust responses in dynamic mass redistribution (DMR) assays that provide a pathway-agnostic cellular response. The Ang1-7 peptide obtained from multiple sources was inert. In the cell-free assay for G protein coupled MAS1, 35S-GTPɣS binding was undetected in the presence of Ang1–7 suggesting lack of direct interaction with MAS1.

To reject the in vivo analysis based MAS1 pairing with Ang1-7 would still be premature based on the conclusions of Gaidarov et al. (and similar papers cited in ref. #2). MAS1 pairing with Ang1-7 should be considered in the context of type 1 and type 2 errors originally described by Neyman and Pearson (5) and recently revisited (6). Type 1 errors are experiments causing rejection of a null hypothesis which may be true. Type 2 errors are experiments insufficient to reject a flawed null hypothesis. Experiments need to be designed to test the validity of MAS1 functions modulated in vivo by Arena Pharmaceutical ligands rather than simply failing to find pairing with Ang1-7. Involvement of additional GPCRs as “Ang1-7 receptors” or signalosome mechanisms that can link Ang1-7 with MAS1 deserve consideration (7).

Comments by Sadashiva S. Karnik (karniks@ccf.org.us) and Kalyan Tirupula

References

1. Karnik SS, Singh KD, Tirupula K, Unal H. (2017) Significance of angiotensin 1-7 coupling with MAS1 receptor and other GPCRs to the renin-angiotensin system: IUPHAR Review 22. Br J Pharmacol. 174: 737-753. doi: 10.1111/bph.13742. Review. PMID: 28194766
2. Karnik SS, Unal H, Kemp JR, Tirupula KC, Eguchi S et al. (2015) International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli [corrected]. Pharmacol Rev 67: 754-819. PMID: 26315714
3. Gaidarov I, Adams J, Frazer J, Anthony T, Chen X et al. (2018) Angiotensin (1-7) does not interact directly with MAS1, but can potently antagonize signaling from the AT1 receptor. Cell Signal 50: 9-24. PMID: 29928987
4. Bader M, Alenina N, Andrade-Navarro MA, Santos RA (2014). MAS and its related G protein-coupled receptors, Mrgprs. Pharmacol Rev. 66: 1080–1105. PMID: 25244929
5. Neyman, J. and Pearson, E.S. (1928). On the use and interpretation of certain test criteria for the purposes of statistical inference. Part I and Part II. Biometrika 20A, 175–240.
6. Lew MJ (2006). Principles: when there should be no difference–how to fail to reject the null hypothesis. Trends Pharmacol Sci. 27:274-278. PMID: 16595154
7. Tirupula KC, Zhang D, Osbourne A, Chatterjee A, Desnoyer R, Willard B et al. (2015). MAS C-terminal tail interacting proteins identified by mass spectrometry-based proteomic approach. PLoS One 10: e0140872. PMID: 26484771