Fig 2: Activation of MRGPRD mediates IL-6 release. HeLa cells transiently expressing empty vector (pCMV-GS) or MRGPRD-HA were stimulated with either vehicle control or β-alanine (100 µM; final concentration/well). The normalized IL-6 release is represented in ng/mg. β-alanine- or vehicle-treated cells expressing MRGPRD released high and intermittent levels of IL-6, respectively. Whereas vehicle- or β-alanine-treated cells expressing the empty vector did not release significant amounts of IL-6. On the right, a representative Western blot of whole cell lysates from HeLa cells transiently transfected with either empty vector (pCMV-GS) or MRGPRD-HA plasmids is displayed. Expression of the MRGPRD-HA receptor (~37 kDa) was observed in both vehicle and β-alanine (100 µM)-treated MRGPRD-transfected cells, whereas cells transfected with the empty vector showed no expression (i.e., absence of a protein band of ~37 kDa). The graph represents the mean ± s.e.m values from three independent experiments. Statistical significance was determined using one-way analysis of variance (ANOVA), and Sidak’s post hoc test was applied for multiple comparisons. * p ≤ 0.05 was considered significant and ‘ns’ is non-significant.

Fig 3: IL-6 assay optimization. To achieve a high dynamic window for the IL-6 detection, the experiment timeline was optimized. (A) At 24 h post-transfection, medium was replaced by FBS-free medium, and cells were treated with vehicle or β-alanine (100 µM). At 24 h post-treatment, i.e., at 48 h post-transfection, samples were collected for IL-6 detection. IL-6 levels from β-alanine-treated MRGPRD-expressing cells were 2-fold higher when compared to those from vehicle-treated MRGPRD-expressing cells. (B) Similarly, at 24 h post-transfection, medium was replaced by FBS-free medium for another 24 h, and at 48 h, the medium was once again replaced by FBS-free medium, and cells were treated with vehicle or β-alanine (100 µM). Samples were collected for IL-6 detection after 24 h of treatment, i.e., at 72 h post-transfection. IL-6 levels from β-alanine-treated MRGPRD-expressing cells were ~5-fold higher when compared to those from only vehicle-treated MRGPRD-expressing cells, which is substantially higher than for the protocol used in (A). The cells expressing empty vector (pCMV-GS), treated with β-alanine or vehicle, did not show IL-6 release above the basal level (A,B). Below the graphs are representative Western blots of cell lysates from HeLa cells transiently transfected with empty vector (pCMV-GS) or MRGPRD-HA plasmid, showing receptor expression in vehicle or β-alanine (100 µM)-treated MRGPRD-expressing cells subjected to the experiment timeline protocols illustrated in (A,B), respectively. Each graph represents the mean ± s.e.m values from three independent experiments. Statistical significance was determined using one-way ANOVA, and Sidak’s post hoc test was applied for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 were considered significant and ‘ns’ is non-significant.

Fig 4: β-alanine-stimulated MRGPRD-induced IL-6 release is dependent on NF-kB activation. (A) HeLa cells stably expressing MRGPRD-NLuc were pre-treated with either vehicle control or the Gαq inhibitor (YM-254890; 10 µM), the pan-PLC inhibitor (U-73122; 10 µM) or the IKK inhibitor (IKK-16; 5 µM) for 1 h before being stimulated with β-alanine (1mM). β-alanine-stimulated MRGPRD-expressing HeLa cells induced NF-κB phosphorylation, whereas no induction was observed in cells pre-treated with either of the three applied inhibitors. (B) Similarly, HeLa cells stably expressing MRGPRD-NLuc were pre-treated with either vehicle control or Gαq (YM-254890; 10 µM) or pan-PLC (U-73122; 10 µM) or IKK (IKK-16; 5 µM) inhibitors for 1 h before being subjected to β-alanine (100 µM). MRGPRD-expressing cells upon stimulation with β-alanine showed a strongly reduced IL-6 release after being pretreated with either YM-254890 or IKK-16 and a less prominent but still significantly reduced IL-6 release compared to β-alanine stimulated MRGPRD expressing cells that were not pre-exposed with the inhibitor. Each graph represents the mean ± s.e.m values from three independent experiments. The comparison between the two groups was analyzed by one-way ANOVA with Sidak’s post hoc test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 were considered significant and ‘ns’ is non-significant.

Fig 5: Concentration–effect curves of β-alanine obtained in the presence or absence of FBS. HeLa cells expressing the empty vector (pCMV-GS; square symbol) and MRGPRD-HA (triangle symbol) were treated with increasing concentrations of β-alanine in the absence or presence of FBS. Cells expressing MRGPRD induced the release of IL-6 in a concentration-dependent manner when treated with β-alanine with FBS (A) or without FBS (B). A higher basal activity of MRGPRD was observed in cells treated under FBS conditions as compared to non-FBS conditions. No significant IL-6 release was observed from cells expressing the empty vector (pCMV-GS). The dynamic range obtained when cells were treated in FBS (A) conditions was 2.85, whereas it increased to 4.62 when cells were treated in FBS-free conditions (B). Each graph represents the mean ± s.e.m values from at least three independent experiments.