Naturally Occurring Missense MRGPRX2 Variants Display Loss of Function Phenotype for Mast Cell Degranulation in Response to Substance P, Hemokinin-1, Human β-Defensin-3, and Icatibant

Abstract

Human mast cells (MCs) express a novel G protein-coupled receptor (GPCR) known as Mas-related GPCR X2 (MRGPRX2). Activation of this receptor by a diverse group of cationic ligands such as neuropeptides, host defense peptides, and Food and Drug Administration-approved drugs contributes to chronic inflammatory diseases and pseudoallergic drug reactions. For most GPCRs, the extracellular (ECL) domains and their associated transmembrane (TM) domains display the greatest structural diversity and are responsible for binding different ligands. The goal of the current study was to determine if naturally occurring missense variants within MRGPRX2's ECL/TM domains contribute to gain or loss of function phenotype for MC degranulation in response to neuropeptides (substance P and hemokinin-1), a host defense peptide (human β-defensin-3) and a Food and Drug Administration-approved cationic drug (bradykinin B2 receptor antagonist, icatibant). We have identified eight missense variants within MRGPRX2's ECL/TM domains from publicly available exome-sequencing databases. We investigated the ability of MRGPRX2 ligands to induce degranulation in rat basophilic leukemia-2H3 cells individually expressing these naturally occurring MRGPRX2 missense variants. Using stable and transient transfections, we found that all variants express in rat basophilic leukemia cells. However, four natural MRGPRX2 variants, G165E (rs141744602), D184H (rs372988289), W243R (rs150365137), and H259Y (rs140862085) failed to respond to any of the ligands tested. Thus, diverse MRGPRX2 ligands use common sites on the receptor to induce MC degranulation. These findings have important clinical implications for MRGPRX2 and MC-mediated pseudoallergy and chronic inflammatory diseases.

Figure 1. Naturally occurrence MRGPRX2 variants identified from the NHLBI GO ESP database

(A): Serpentine diagram of the secondary structure of human MRGPRX2. Each circle represents amino acid residue with one letter code. Solid background denotes the eight naturally occurring missense variants used in the present study. Extracellular (ECL), intracellular (ILC) and transmembrane (TM) domains are shown. (B): Amino acid change for each MRGPRX2 variant, PolyPhen-2 score, Minor allele frequency (MAF) and CADD score are shown.

Figure 2. Effects of naturally occurring MRGPRX2 mutations (L31I, V43I, F78L, G165E and D184H) on cell surface expression, SP-induced Ca²⁺ mobilization and degranulation in stably transfected RBL cells

(A): Cells were transfected with cDNA encoding wild-type (WT), N16H, W243R, or H253Y variant, transferred to 24 well plate and G418 was added to the culture medium 16 h after transfection. After 5 days, non-adherent cells were removed, adherent cells were lysed and total β-hexosaminidase content was determined. (B): Flow cytometry was performed with PE-anti-MRGPRX2 antibody to determine cell surface expression of WT and variants in stably transfected RBL cells. Representative histograms for WT/Variant (thick line) and control untransfected cells (thin line) are shown. (C): Cells expressing WT and MRGPRX2 variants were loaded with Indo-1 and intracellular Ca²⁺ mobilization in response to SP (1 μM) was determined. Data shown are representative of three independent experiments. (D): Cells were exposed to buffer (control) or SP (0. 3μM and 1 μM) for 30 min and β-hexosaminidase release was determined. All data points are expressed as mean ± SEM of three experiments performed in triplicate. Statistical significance was determined by two-tailed unpaired t-Test. *** indicates P value<.001, and ** indicates P value <.01.

Figure 3. Effects of naturally occurring MRGPRX2 mutations (L31I, V43I, F78L, G165E and D184H) on HK-1, hBD3 and icatibant-induced degranulation in stably transfected RBL cells

Cells stably expressing WT and MRGPRX2 variants were exposed to buffer (control) or stimulated with (A) hemokinin-1 (HK-1, 10 μM), (B) hBD3 (3 μM) or (C) icatibant (25 μg/ml) for 30 min and β-hexosaminidase release was determined. All the points expressed as a mean ± SEM of three experiments in triplicate. Statistical significance was determined by two tailed unpaired t-Test. *** indicates P value<.001, ** indicates and P value <.01.

Figure 4. FAM-SP-induced degranulation and binding in RBL cells overexpressing MRGPRX2

(A): RBL cells stably overexpressing MRGPRX2 (RBL-MRGPRX2) were exposed to PE-conjugated anti-MRGPRX2 or PE-conjugated isotype matched antibody and cell surface receptor expression was determined by flow cytometry. (B): RBL and RBL-MRGPRX2 cells were exposed to buffer (control), SP or FAM-SP for 30 min and β-hexosaminidase release was determined. (C): RBL or RBL-MRGPRX2 cells were exposed to FAM-SP (1 μM, 60 min, 4°C), washed and fluorescence was determined by flow cytometry. Data presented for flow are representative of 3 similar experiments and degranulation data are expressed as mean ± SEM of three experiments in triplicate. Statistical significance was determined by two tailed unpaired t-Test. *** indicates P value<.001

Figure 5. Effects of naturally occurring MRGPRX2 mutations (N16H, W243R and H259Y) on cell surface expression, Ca²⁺ mobilization and SP-induced degranulation in transiently transfected RBL cells

(A): Flow cytometry was performed using PE-anti-MRGPRX2 antibody to determine cell surface expression of wild-type (WT) and variants in transiently transfected in RBL cells. Representative histograms for WT/Variant (thick line) and control untransfected cells (thin line) are shown. (B): Cells expressing WT and MRGPRX2 variants were loaded with Indo-1 and intracellular calcium mobilization in response to SP (1 μM) was determined. Data shown are representative of three independent experiments. (C): Cells were exposed to buffer (control) or SP (1 μM) for 30 min and β-hexosaminidase release was determined. All data points are expressed as mean ± SEM of three experiments performed in triplicate. Statistical significance was determined by two tailed unpaired t-Test. ** indicates P value <.01.

Figure 6. Effects of MRGPRX2 mutations (N16H, W243R and H259Y) on degranulation in response to HK-1, hBD3 and icatibant

RBL cells transiently expressing WT, N16H, W243R and H259Y variants were stimulated with (A) HK-1 (10 μM), (B) hBD3 (3 μM) and (C) icatibant (25 μg/ml) and β-hexosaminidase release was determined. All the points expressed as a mean ± SEM of three experiments in triplicate. Statistical significance was determined by two tailed unpaired t-Test. *** indicates P value<.001, ** indicates P value <.01 and * indicates P value <.05.