Figure legends
Figure 1 . Time courses (A and C) and concentration-effect curves (B, D and E) in response to β-alanine in DMR (A, B and C) and FLIPR assays (D) in CHO cells expressing recombinant human MRGPRD or untransfected CHO cells (E).
Figure 2 . Time courses (A, B and C) and concentration-effect curves (D) and (E) in response to β-alanine in DMR (A, B, C and D) and FLIPR assays (E) in CHO cells expressing recombinant human MRGPRD. Cells were preincubated with either YM-254890 (10µM) (A) PTX (0.1µg/ml) (B) or PTX (0.1µg/ml) plus YM-254890 (10µM), (C) and concentration-response curves determined (D). The effects of YM-254890, PTX and PTX plus YM-254890 on FLIPR Ca2+ assays were also determined (D). Concentration-effect data are expressed as a percentage of the maximum response to agonist and are the mean ± SEM of a minimum of 5 replicates
Figure 3 . Time courses (A – D) for responses to GABA in the DMR assay in the presence of vehicle only (A), in presence of the Gq inhibitor YM254890 (10µM) (B), in the presence of PTX (0.1µg/ml) (C), or in the presence of both the indicated concentrations of YM-254890 and PTX (D). Concentration-effect curves (E) for these responses in the DMR assay at the 4-minute time point. Concentration-effect curves (F) in the FLIPR Ca2+assay. Concentration-effect data are expressed as a percentage of the maximum response to agonist and are the mean ± SEM of a minimum of 5 replicates.
Figure 4 . Time courses (A – D) for responses to Glycine in the DMR assay in the presence of vehicle only (A), in presence of the Gq inhibitor YM254890 (10µM) (B), in the presence of PTX (0.1µg/ml) (C), or in the presence of both the indicated concentrations of YM-254890 and PTX (D). Concentration-effect curves (E) for these responses in the DMR assay at the 4-minute time point. Concentration-effect curves (F) in Ca2+ FLIPR assay. Concentration-effect data are expressed as a percentage of the maximum response to agonist and are the mean ± SEM of a minimum of 5 replicates.
Figure 5 . Time course and concentration-responses to the Thioridazine hydrochloride response in the DMR assay in CHO cells expressing the human MRGPRD receptor in the absence (A), and presence (B) of PTX (0.1µg/ml) or in untransfected CHO cells (C). Concentration-effect responses in FLIPR Ca2+ assay in MRGPRD expressing CHO cells (D) and cell viability determined using the CyQUANT assay over 72 hours (E).
Figure 6 . Concentration-effect curves in MRGPRD expressing CHO cells in response to β-alanine (A, B), GABA (C, D) and Glycine (E, F) in DMR (A, C, E) and FLIPR Ca2+ (B, D, F) assays, in the presence of increasing concentrations of PD123,319. Data are expressed as a percentage of the maximum response to agonist and are the mean ± SEM of a minimum of 5 replicates
Figure 7 . Concentration-effect curves in MRGPRD expressing CHO cells in response to β-alanine (A, B), GABA (C, D) and Glycine (E, F) in DMR (A, C, E) and FLIPR Ca2+ (B, D, F) assays, in the presence of increasing concentrations of MU-6840. Data are expressed as a percentage of the maximum response to agonist and are the mean ± SEM of a minimum of 5 replicates
Figure 8 . Schild analyses (D, E, F) derived from concentration-effect curves in response to β-alanine (A, B, C) in FLIPR Ca2+ (A, B, D, E) and DMR (C, F) assays in the presence of increasing concentrations of PD123,319 or MU-6840 (B, E).
Figure 9. Supplementary data. Time and concentration-effect in response to increasing concentrations of PD123,319 (A) and MU-6840 (B) in MRGPRD expressing CHO cells in the DMR assay. Concentration-effect curves to increasing concentrations of Alamandine in FLIPR Ca2+ assay in MRGPRD expressing CHO cells (C) and in CyQUANT cell viability assay over 72 hours (D).