Reagents and peptides
CXC motif chemokine 14 (CXCL14) was purchased from BIOZOL (Eching, Germany). Proadrenomedullin N-terminal 20 peptide (PAMP-20) was obtained from Tocris Bioscience (Bristol, UK), and cortistatin-14 was from GenScript (NJ, USA). Custom synthesis of CXCL14 analogs was performed by JPT Peptide Technologies (Berlin, Germany) (see Supplementary Fig. 6). Stock solutions were prepared either in dimethyl sulfoxide (DMSO) or in RNase-, DNase- and protease-free water from Carl Roth GmbH & Co (Karlsruhe, Germany). The MRGPRX2 antagonist PSB-172656 (synonymous designations: B-40 and CB70) was synthesized as descibed68,69.
Plasmids
The constructs pLXSN-MRGPRX2 (NM_001303615.2), pLVX MRGPRB2-IRES-mCherry (NM_175531.4), and pCMV-ARMS1-ProLink2TM-MRGPRX3 (NM_054031.3) had been generated as previously described69,70. All plasmids were verified by sequencing (Eurofins Genomics Germany, Ebersberg, Germany).
Cell lines and transfection
The cell lines CHEM1 and CHEM1-MRGPRX2 are adherent rat cell lines (HTSCHEM-1RTA, HTS058RTA, Merck-Millipore, Darmstadt, Germany); which were cultured under standard cell culture conditions.
LN229 glioblastoma cells ATCC®, CRL-2611TM, were a kind gift from Prof. Dr. B. Scheffler (University of Bonn, Germany). LN229 cells stably expressing MRGPRX2 were generated by retroviral transfection. Briefly, GP+envAM-12 packaging cells (LGC Standards GmbH, Wesel, Germany), derived from mouse fibroblasts, were seeded at a density of 1.5 × 106 cells per T25-flask in 5 mL of Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal calf serum (FCS), 100 U/mL of penicillin (PAN-Biotech) and 100 µg/mL of streptomycin (PAN-Biotech). Packaging cells were transfected with pLXSN-MRGPRX2 and pcDNA3-VSV-G using Lipofectamine 2000 according to the manufacturer’s instructions (Thermo Fisher Scientific, Darmstadt, Germany). The medium was exchanged for DMEM supplemented with 10% FCS, 100 U/mL of penicillin, 100 µg/mL of streptomycin and 3 mM of sodium butyrate (stock solution: 500 mM in water, sterile-filtered) to enhance virus production. To produce retroviral particles, cells were cultivated for 48 h at 32°C and 5% CO2. On day 4, the target cells LN229 were seeded into T25 cell culture flasks (5 × 105 cell). The supernatants of the packaging cells (3 mL) containing infectious retroviral particles were sterile-filtered on the next day using Filtropur S 0.2 µm non-pyrogenic sterile filters (Sarstedt AG & Co. KG, Nümbrecht). Polybrene (hexadimethrine bromide, a cationic polymer, stock solution 4 mg/mL in water, sterile-filtered), was added to the supernatant (final concentration: 0.008 mg/mL). The medium of the LN229 cells was removed and exchanged for the supernatant containing the polybrene solution. After 150 min, the medium was changed back to DMEM medium containing 10% FCS, 100 U/mL of penicillin and 100 µg/mL of streptomycin. Cells were selected after 48 h by the addition of G418 (200 µg/mL).
1321N1 Astrocytoma cells were purchased from Sigma Aldrich-Merck KGaA (Darmstadt, Germany, 86030402). The cells were transfected with MRGPRB2 using lentiviral transfection. 1.5 × 106 Lenti-X 293 T cells were seeded into each T25 cell culture flask in 5 mL of DMEM, supplemented with 10% FCS, 100 U/mL of penicillin and 100 µg/mL of streptomycin. After 20 h of incubation at 37°C and 5% CO2, when the cells were grown to 80–90% confluence, the medium was exchanged for 5 mL DMEM with 10% FCS without antibiotics and incubated for 4 h. Subsequently, transfection took place using Xfect polymer according to the manufacturer’s protocol (Clontech, Takara Bio Europe, France). The pLVX MRGPRB2-IRES-mCherry-Xfect mixture was added to the packaging cells and they were incubated overnight. Two days post-transfection, the supernatant from transfected Lenti-X cells containing the lentiviral particles were harvested, filtered, and then added to the target cells, 1321N1 astrocytoma cells, with 6 µL polybrene solution (4 mg/mL in water, final concentration: 0.008 mg/mL). After 150 min of lentiviral transfection the virus containing medium was removed and replaced for 5 mL of DMEM, supplemented with 10% FCS and 100 U/mL of penicillin and 100 µg/mL of streptomycin. Cells were incubated for 72 h at 37°C and 10% CO2. When the cells showed 80% confluence, they were seeded into Nunc™ 177399 Lab-Tek® Chamber Slide™ Systems. To know how many astrocytoma cells expressed the target receptor, cells were analyzed under the fluorescence microscope at the m-cherry excitation wavelength of 587 nm using its emission wavelength of 610 nm. Subsequently, fluorescent single clones were isolated by fluorescence-activated cell sorting.
Chinese hamster ovary (CHO) β-arrestin cells had been engineered by Eurofins DiscoverX (Fremont, CA, USA) to stably express β-arrestin-2 fused to a large fragment of β-galactosidase (called enzyme acceptor, or EA). These commercially available parental CHO-K1-β-arrestin-2 cells (93-0164) were purchased. Additionally, CHO β-arrestin cells expressing MRGPRX1, MRGPRX2 and MRGPRX4 fused to a small enzyme donor fragment ProLink™ (PK) were purchased (93-0919C2, 93-0309C2 and 3-0541C2A, respectively). Parental CHO-β-arrestin cells were cultured in a humidified incubator with 5% CO2 at 37°C in Gibco F-12 Ham Nutrient Mixture medium (Life Technologies) supplemented with 10% FCS, 100 U/mL of penicillin, 100 µg/mL of streptomycin, and 300 μg/mL of hygromycin. CHO-β-arrestin cells expressing MRGPRX3 were established by transfecting the parental CHO-K1-β-arrestin-2 cells with a pCMV-ARMS1-ProLink2TM-MRGPRX3 plasmid using LipofectamineTM 2000 (Thermo Fischer Scientific, Schwerte, Germany) according to the manufacturer’s recommendation, and stably expressing cells were obtained by antibiotic selection with gentamicin (PAN-Biotech). Cells expressing the desired receptor were then maintained in 800 μg/mL gentamicin.
Calcium mobilization assays using CHEM-1 cells
A cell-based fluorometric assay was employed for the determination of intracellular calcium release using Fluo-8. Cells were seeded into microtiter plates and incubated under standard cell culture conditions. After 24 h the medium was removed, and the cells were washed with phosphate-buffered saline (PBS). The cells were incubated with loading buffer (40 ml PBS with 2 mM Ca2+, 800 µl of brilliant black solution (2%), 732 µl of probenecid solution (0.5 M) in 1 N NaOH, 400 µL of pluronic (10%) in water, and 152 µl of Fluo-8 (5.7 ng/ml in water)) for 30 min. For fluorescence readout and compound testing a FLIPR tetra system was used according to the manufacture’s protocol (Molecular Devices, CA 94089- 1136, USA).
Calcium mobilization assays using LN229 cells
On the day before the assay, glioblastoma LN229-MRGPRX2 stably expressing MRGPRX2 (produced by retroviral transfection) were seeded into black sterile 96-well microplates at a density of 2.5 × 105 cells/mL per well in 200 µL of DMEM/F12- Nutrient Mixture medium, supplemented with 10% FCS, 100 U/mL of penicillin 100 µg/mL of streptomycin, and 200 µg/mL of gentamicin, and incubated overnight. Fluo-4 AM solution (1 mM) (Thermo Fisher Scientific) was prepared by diluting 50 µg in 45.9 µL of DMSO. On the day of the assay, the cell medium was removed and exchanged for 40 µL of dye solution per well, consisting of 4970 µL of Hank’s Buffered Saline Solution (HBSS), 15 µL of 20% Pluronic-F and 15 µL of 1 mM Fluo-4 AM (final concentration of Fluo-4 AM: 3 µM). After an incubation time of 1 h at room temperature in the dark, the solution of each well was replaced by 190 µL of HBSS. Agonist solution (10 µL) in HBSS, namely carbachol (100 µM), or PAMP-20 (5 µM), was pipetted using a FlexStation® 3 Multi-Mode Microplate Reader. For the determination of baseline fluorescence, HBSS buffer without additives was used. Fluorescence was measured at 525 nM (excitation 485 nM) for 120 intervals of 1 s (number of flashes: 10 each) by utilizing the SoftMax® Pro5.1 Microplate Data Acquisition & Analysis Software. Three to four independent experiments were performed in duplicates unless otherwise indicated. Measurements are given in relative fluorescent units as the maximum response minus the minimum response.
For testing the MRGPRX2/B2 antagonist PSB-172656, similar steps were carried out except that the dye solution was replaced by 189 µL of HBSS, and the MRGPRX2/B2 antagonist was diluted in DMSO, then added manually (1 µL, final concentration 100 nM) 30 min before agonist addition as described above. The final DMSO concentration did not exceed 0.5% (v/v).
Calcium mobilization assays using 1321N1 astrocytoma cells
1321N1 astrocytoma cells recombinantly expressing MRGPRB2-mCherry were used. All other steps were as described above for MRGPRX2, except that the fluorescence was measured at 525 nM (excitation 485 nM) for 60 intervals of 1 s (number of flashes: 10 each) by utilizing the SoftMax® Pro5.1 Microplate Data Acquisition & Analysis Software.
β-Arrestin recruitment assays
β-Arrestin recruitment assays were based on the enzyme complementation technology. The assays were carried out as previously described56. Briefly, cells were seeded into white 96-well plates (Nunclon Delta surface plates, Thermo Fisher Scientific) in 90 µl Opti-MEM medium (Thermo Fisher Scientific) supplemented with 2% fetal bovine serum, 100 U/mL of penicillin, 100 µg/mL of streptomycin, 800 μg/mL of gentamicin, and 300 μg/mL of hygromycin B. On the next day, the assays were performed. Peptides were diluted, and 10 µl of solution were added to the cells, which were subsequently incubated for 90 min. The final DMSO concentration did not exceed 1% (v/v). Then, 50 µL of detection reagent (DiscoverX) was added. After 60 min of incubation in the dark at room temperature, chemiluminescence was measured by using a Mithras LB 940 plate reader (Berthold Technologies, Bad Wildbad, Germany). The results are means ± SDs of three to five independent experiments, each in duplicates.
GPCR panel screening
Screening the GPCRs panels was performed by DiscoverX (Eurofins) using β-arrestin recruitment assays unless otherwise indicated (for details, see Supplementary Data 2).
Immunostaining of MRGPRX3-expressing cells
One day before staining, β-arrestin-CHO-MRGPRX3 cells were seeded onto sterile coverslips in a 6 well-plate (Sarstedt, Nümbrecht, Germany) and cultured overnight. The next day, the cells were fixed with 4% paraformaldehyde (pre-heated at 37 °C) for 20 min at room temperature. The cells were then washed with phosphate-buffered saline (PBS, pH 7.4) and blocked for 15 min with 1% bovine serum albumin/PBS solution. Cells were incubated in the dark for 60 min with the primary antibody, and then 30 min with the secondary antibody both diluted in 1% BSA/PBS. The primary antibody was diluted at 1:1000, and the secondary antibody at 1:500). PBS was used to wash cells between and after antibody incubations. In addition, the nuclei of the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1 mg/μl diluted 1:1000 in 1% BSA/PBS) for 5 min in the dark. Finally, coverslips were mounted using FluoromountTM Aqueous Mounting medium (Sigma-Aldrich-Merck KGaA, Darmstadt, Germany) and stored in the dark at 4°C. The mouse monoclonal anti-PK/PL antibody (DiscoverX, Fremont, CA, USA) was used as the primary antibody and goat anti-mouse-AlexaFluor488 (Jackson Immuno Research, Hamburg, Germany) as the secondary antibody to stain the Prolink tag on MRGPRX3. A Nikon A1 spectral confocal microscope operating with an argon laser, and the NIS Element Advanced Research software 4.0 were used for image acquisition and analysis. Each staining was repeated two to three times and at least ten squares (60× objective), each containing 3–15 cells, were imaged for each sample. Representative pictures are shown (Supplementary Fig. 1b).
Molecular modeling
The cryoEM structure of the human MRGPRX2 in complex with the peptide ligand cortistatin-14 was selected for molecular docking experiments using AutoDock PDB: 7S8L50,71. A model of the human MRGPRX2 was prepared using the protein preparation tool, and protonated according to Protonate 3D implemented in Molecular Operating Environment (MOE 2019.01)72. The AutoDockTools were applied to add the atomic partial charges and for computing the three-dimensional energy scoring grid for a box of 60 × 60 x 60 points with a spacing of 0.375 Å71. The prepared structure of the human MRGPRX2 was applied for flexible molecular docking using AutoDock4.2. The selected peptide CXCL14(57-65) was docked into the binding site of the peptide agonist cortistatin-14, previously identified by cryoEM of MRGPRX247,50 Fifty independent docking calculations were performed using the varCPSO-ls algorithm from PSO@Autodock implemented in AutoDock4.271. The docking calculations were computed by setting the termination criteria as 50,000 evaluation steps. The parameters “cognitive coefficient (c1)” and “social coefficient (c2)” of the varCPSO-ls algorithm was set at 6.05 and the swarm size at 60 individual particles. The default values were applied for the remaining parameters of the algorithm. The poses obtained for CXCL14(57-65) from docking studies were explored by visual inspection on the basis of the docking score and the top-ranked pose was selected as the putative binding pose. For sequence alignment, the Clustal Omega online tool was used73. The snake plot of MRGPRX2 was generated using the GPCR database74.
G protein activation assay using BRET2 (TRUPATH)
MRGPRX2-induced G protein heterotrimer dissociation was measured in LN229 cells using a BRET2 assay (TRUPATH, Addgene kit no. 1000000163)45, in analogy to a previously described procedure with minor modifications75. On the first day, parental LN229 cells, or LN229 cells stably overexpressing MRGPRX2, cultivated in a growth medium, were detached from cell culture flasks by trypsination. Cells were seeded into 6-well plates at a density of approximately 700,000 cells per well in a volume of 2 mL, and incubated at 37 °C for 4 h before transfection. Transient transfection was performed using Lipofectamine 2000 according to the manufacturer’s recommendation (ThermoFisher, Waltham, MA, USA). The cells were then transfected with the biosensors (100 ng of each pcDNA5/FRT/TO-Gαi1-RLuc8, pcDNA3.1-Gβ3, and pcDNA3.1-Gγ9-GFP2 per well) and incubated at 37 °C for 18 h. On the second day, media were removed, and cells were gently detached by adding 0.1 M phosphate-buffered saline, pH 7.4, containing 0.5 mM ethylenediaminetetraacetic acid (EDTA), and transferred to 96-well white bottom plates (Greiner BioOne, Frickenhausen, Germany) at a density of 30,000 cells per well in 60 μL of growth medium. On the third day, the assay was performed by careful aspiration of the medium and by gently washing the cells with assays buffer (HBSS and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4). Subsequently, assay buffer was added (60 µL) followed by 10 µL of a freshly prepared 50 µM coelenterazine 400a solution (Biomol, Hamburg, Germany). After a 5 min equilibration period, 30 μL of agonist solution was added to the cells and the mixture was incubated for 5 min. The final DMSO concentration in the assay buffer was 1%. Next, luminescence and fluorescence were measured in a Mithras LB940 plate reader, using 395 and 510 nm emission filters for the RLuc8 and GFP2 signals, respectively. The BRET ratio between the GFP2 and the RLuc8 signal intensity was computed and corrected for the baseline signal to obtain ΔBRET values. Results were visualized by GraphPad PRISM 8 (GraphPad, San Diego, CA, USA).
Statistics and reproducibility
Data were analyzed using Prism 10.0 (GraphPad Software Inc., San Diego, CA, USA). All data are presented as means ± SD from three to five independent experiments performed in duplicates unless otherwise indicated. The graphs were generated using the same software. Sigmoidal dose-response (variable slope) parameters were used for analyzing the data to calculate EC50 and Emax values. To determine the level of significance, data were analyzed using one-way ANOVA with Bonferroni’s multiple comparisons test and multiple t-tests with Welch correction were used for pairwise comparisons between two groups (Prism 10.0). A p-value of less than or equal to 0.05 was considered to be significant and exact p-values are disclosed in a Source Data file (Supplementary Data 3). The NIS Element Advanced Research software 4.0 was used for microscopy image acquisition and analysis.
Reporting summary
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