The development of a bioassay expressing the human MRGPRX2 receptor for ex vivo drug discovery
Jan Tytgat
A *
A
Abstract
Successful discovery of potent and selective ligands for therapeutically relevant receptors is indispensable for future clinical applications to treat a wide array of diseases. The mas-related G protein-coupled receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, governs to a significant extent the human immune response. A number of ligands for MRGPRX2 have been identified including human cathelicidin (LL-37) and antimicrobial peptides from plants (PAMPS). Future drug discovery to identify MRGPRX2 specific ligands undoubtedly will benefit from a continued screening of small molecule and peptide libraries. Mast cells (MCs) are generally difficult and expensive to maintain in vitro and, therefore, are not economical to use for screening large numbers of molecules. Herein, we present the development of a bioassay expressing the human MRGPRX2 receptor, linked to inwardly rectifying potassium (GIRK1/2) channels that are opened upon binding of β-γ subunits of a Gi/o-type protein following activation of MRGPRX2. This communication presents a method-focused study demonstrating a simple, yet robust and inexpensive electrophysiological approach (voltage clamp) using oocytes from Xenopus laevis to perform high-throughput screening of libraries. Unexpectedly, this bioassay also supports the claim that the MRGPRX2 receptor can couple efficiently to Gi/o-type proteins, a pathway not yet described. Furthermore, the approach allows structure-function research using mutated MRGPRX2 genes and can study reciprocal cross talks between MRGPRX2 and other co-expressed membrane-bound proteins, such as voltage- or ligand-gated ion channels, mimicking in vivo conditions.
Keywords: drug discovery, electrophysiology, G protein-coupled receptor, mast cells, MRGPRX2, oocytes, potassium channel.
References
1 Dziadowiec A, Popiolek I, Kwitniewski M, Porebski G. Modulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) by xenobiotic compounds and its relevance to human diseases. J. Xenobiot 2024; 14: 380-403.
| Crossref | Google Scholar | PubMed |
2 Quan PL, Sabaté-Brescó M, Guo Y, Martín M, Gastaminza G. The multifaceted Mas-related G protein-coupled receptor member X2 in allergic diseases and beyond. Int J Mol Sci 2021; 22: 4421.
| Crossref | Google Scholar | PubMed |
3 Tatemoto K, Nozaki Y, Tsuda R, Konno S, Tomura K, Furuno M, Ogasawara H, Edamura K, Takagi H, Iwamura H, Noguchi M, Naito T. Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors. Biochem Biophys Res Commun 2006; 349: 1322-1328.
| Crossref | Google Scholar | PubMed |
4 Kulka M, Sheen CH, Tancowny BP, Grammer LC, Schleimer RP. Neuropeptides activate human mast cell degranulation and chemokine production. Immunology 2008; 123: 398-410.
| Crossref | Google Scholar | PubMed |
5 Bakare OO, Gokul A, Fadaka AO, Wu R, Niekerk L-A, Barker AM, Keyster M, Klein A. Plant antimicrobial peptides (PAMPs): features, applications, production, expression and challenges. Molecules 2022; 27: 3703.
| Crossref | Google Scholar | PubMed |
6 Verjans E-T, Zels S, Luyten W, Landuyt B, Schoofs L. Molecular mechanisms of LL-37-induced receptor activation: an overview. Peptides 2016; 85: 16-26.
| Crossref | Google Scholar | PubMed |
7 Seldeslachts A, Peigneur S, Mebs D, Tytgat J. Unraveling the venom chemistry with evidence for histamine as key regulator in the envenomation by caterpillar Automeris zaruma. Front Immunol 2022; 13: 972442.
| Crossref | Google Scholar | PubMed |
8 Castells M, Madden M, Oskeritzian CA. Mast cells and Mas-related G protein-coupled receptor X2: itching for novel pathophysiological insights to clinical relevance. Curr Allergy Asthma Rep 2025; 25: 5.
| Crossref | Google Scholar | PubMed |
9 Facheris P, Jeffery J, Del Duca E, Guttman-Yassky E. The translational revolution in atopic dermatitis: the paradigm shift from pathogenesis to treatment. Cell Mol Immunol 2023; 20: 448-474.
| Crossref | Google Scholar | PubMed |
10 Kobayashi T, Ikeda K, Ichikawa T, Abe S, Togashi S, Kumanishi T. Molecular cloning of a mouse G-protein-activated K+ channel (mGIRK1) and distinct distributions of three GIRK (GIRK1, 2 and 3) mRNAs in mouse brain. Biochem Biophys Res Commun 1995; 208(3): 1166-1173.
| Crossref | Google Scholar | PubMed |
11 Bordon KCF, Cologna CT, Fornari-Baldo EC, Pinheiro-Júnior EL, Cerni FA, Amorim FG, Anjolette FAP, Cordeiro FA, Wiezel GA, Cardoso IA, Ferreira IG, de Oliveira IS, Boldrini-França J, Pucca MB, Baldo MA, Arantes EC. From animal poisons and venoms to medicines: achievements, challenges and perspectives in drug discovery. Front Pharmacol 2020; 11: 1132.
| Crossref | Google Scholar | PubMed |
12 Schemann M, Kugler EM, Buhner S, Eastwood C, Donovan J, Jiang W, Grundy D. The mast cell degranulator compound 48/80 directly activates Neurons. PLoS ONE 2012; 7(12): 52104.
| Crossref | Google Scholar | PubMed |
13 Oda T, Morikawa N, Saito Y, Masuho Y, Matsumoto S. Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leucocytes. J Biol Chem 2000; 275(47): 36781-36786.
| Crossref | Google Scholar | PubMed |
14 Pil J, Tytgat J. The role of the hydrophylic Asn230 residue of the µ-opioid receptor in the potency of various opioid agonists. Br J Pharmacol 2001; 134: 496-506.
| Crossref | Google Scholar | PubMed |
15 An D, Peigneur S, Hendrickx LA, Tytgat J. Targeting cannabinoid receptors: current status and prospects of natural products. Int J Mol Sci 2020; 21: 5064.
| Crossref | Google Scholar | PubMed |
16 Inoue A, Raimondi F, Kadji FMN, Singh G, Kishi T, Uwamizu A, Ono Y, Shinjo Y, Ishida S, Arang N, Kawakami K, Gutkind S, Aoki J, Russell RB. Illuminating G-protein-coupling selectivity of GPCRs. Cell 2019; 177(7): 1933-1947.
| Crossref | Google Scholar | PubMed |
17 Ivanina T, Rishal I, Varon D, Mullner C, Frohnwieser-Steinecke B, Schreibmayer W, Dessauer CW, Dascal N. Mapping the G βγ-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel. J Biol Chem 2003; 278(31): 29174-29183.
| Crossref | Google Scholar | PubMed |
18 Kuruma A, Hartzell C. Dynamics of calcium regulation of chloride currents in Xenopus oocytes. Am J Physiol – Cell Physiol 1999; 276: C161-C175.
| Crossref | Google Scholar | PubMed |
19 Seldeslachts A, Peigneur S, Tytgat J. Histamine receptors: ex vivo functional studies enabling the discovery of hits and pathways. Membranes 2023; 13: 897.
| Crossref | Google Scholar | PubMed |
20 Liman E, Tytgat J, Hess P. Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 1992; 9(5): 861-871.
| Crossref | Google Scholar | PubMed |
