MRGPR-X1 induce EGR-1 via ERK-1/2 and CCR2 via NFAT in primary DRG neurons.

<p>BAM8-22-induced (2 µM) calcium signals in rat DRG neurons co-expressing MRGPR-X1 and aequorin or solely aequorin are presented in (A). BAM8-22-induced (2 µM, 8 h) activation of the NFAT (B) or TCF/SRF (C) reporter is shown in rat DRG neurons transiently co-expressing MRGPR-X1. RTQ-PCR experiments were performed with cDNAs derived from serum-starved MRGPR-X1 expressing rat DRG neurons stimulated or not with BAM8-22 (2 µM) for 6 h (D) or 40 min (E). CsA (1 µM, 30 min) was used to block calcineurin in (B and D) or PD-184352 (10 µM, 30 min) to inhibit ERK-1/2 activity in (C and E). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

B2R do not induce CCR2 despite NFAT activation.

<p>(A) BAM8-22- (2 µM) or BK-induced (1 µM) calcium signals were determined in single fura2-loaded F11-MRGPR-X1 cells by calcium imaging. Data of ∼300 cells were compiled and expressed as the mean ± S.E.M. RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BK (1 µM) for 1 h in (B) or 6 h in (E). Relative BK-induced gene expression was normalized to β-actin, calculated using the ΔΔCp method, and expressed as the mean ± S.E.M. (C) Serum-starved F11-MRGPR-X1 cells were stimulated or not with BK (1 µM) for the indicated period of time and expression of EGR-1 was determined by western-blotting. Afterwards blots were stripped and re-probed with an antibody against ERK-2 (t-ERK-2). One representative blot is shown. Ligand-induced EGR-1 expression was quantified by densitometry and is given normalized to not stimulated cells as the mean ± S.E.M. (D) BK-induced (1 µM, 6 h) activation of the NFAT reporter is shown in F11-MRGPR-X1 cells. Data are expressed as the mean ± S.E.M. PD-184352 (10 µM, 30 min) was used to inhibit ERK-1/2 activity in (B) or CsA (1 µM, 30 min) to block calcineurin in (D and E). In (B–E) 4 independent experiments were conducted, respectively. In (F) CCR2 protein expression in F11-MRGPR-X1 cells was assessed by CCL2-promoted (100 ng/ml) inhibition of FSK-induced (5 µM) cAMP accumulation after pre-stimulation of the cells with BAM8-22 or BK (1 µM, 20 h). In (F, left panel) one representative experiment is shown and in (F, right panel) data from 5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BK-stimulated inhibitor-treated and untreated cells or between BK- and BAM8-22-stimulated cells.</p

MRGPR-X1 induce CCR2 via NFAT in F11-MRGPR-X1 cells.

<p>RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BAM8-22 (1 µM) for 6 h (A-C) using specific primers for 5 distinct genes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058756#pone-0058756-t001" target="_blank">table 1</a>. The name of the analyzed gene is listed beneath the corresponding bar. Genes with a p value <0.05 are show in black bars. In (B and C) cells were treated or not with CsA (1 µM, 30 min). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. BAM8-22-induced (1 µM, 20 h) CCR2 protein expression in F11-MRGPR-X1 cells was assessed by flow cytometry (D) or by CCL2-promoted (100 ng/ml) inhibition of FSK-induced (5 µM) cAMP accumulation (E). In (D) one representative experiment is shown. Accumulation of the data from 5 independent experiments revealed an increase in the number of CCR2 positive cells by 10.8±1.4% after BAM8-22 stimulation. In (E, left panel) one representative experiment is shown and in (E, right panel) data from 5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

MRGPR-X1 stimulate TCF/SRF- and NFAT-dependent reporter in HEK293- and F11-MRGPR-X1 cells.

<p>TCF/SRF-, NFAT- or CREB-dependent reporter gene constructs were transfected into HEK293-MRGPR-X1 (A–C) or in F11-MRGPR-X1 (D–F) cells. Cells were stimulated with 1 µM BAM8-22 for 6 h or as indicated. 5 µM FSK was used as a control for the CREB reporter. CsA (1 µM, 30 min) was used to block the NFAT activator calcineurin and PD-184352 (10 µM, 30 min) to block ERK-1/2 activity. Data from 3–5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate significant differences to not stimulated cells. Dagger signs indicate significant differences between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

MRGPR-X1 induce EGR-1 via ERK-1/2 in F11-MRGPR-X1 cells.

<p>RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BAM8-22 (1 µM) for 1 h (A, C and D) using specific primers for 15 distinct genes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058756#pone-0058756-t001" target="_blank">table 1</a>. The name of the analyzed gene is listed beneath the corresponding bar. Genes with a p value <0.05 are show in black bars. In (C and D) cells were treated or not with PD-184352 (30 min, 10 µM). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. In (B) serum-starved F11-MRGPR-X1 cells were stimulated or not with BAM8-22 (1 µM) for the indicated period of time and expression of EGR-1 was determined by western-blotting. Afterwards blots were stripped and re-probed with an antibody against ERK-2 (t-ERK-2). One representative blot is shown. Ligand-induced EGR-1 expression was quantified by densitometry and is given normalized to not stimulated cells. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. Asterisk indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

MRGPR-X1-induced signaling in DRG neurons and CTMCs.

<p>A cartoon illustrating the signaling circuit by which MRGPR-X1 affect chemokine signaling in DRG neurons and connective tissue mast cells is given.</p

MRGPR-X1-induced CCL2 release in LAD-2 mast cells.

<p>(A) MRGPR-X1 (40 cycles) or β-actin (30 cycles) mRNA expression was determined in LAD-2 cells by RT-PCR. As a negative control, RT-PCR was conducted without addition of cDNA (H₂O). (B) Calcium signals in fura2-loaded LAD-2 cells are shown after injection of BAM8-22 (5 µM) or ionomycin (5 µM) or HBS as positive or negative control, respectively. (C) CCL2 release in LAD-2 cells after stimulation with BAM8-22 (5 µM, 18 h) was determined by ELISA. Data from 4 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells.</p

Effect of glutamate on intracellular calcium concentrations in wild-type and Tas1r1/mCherry knock-in mice.

<p>To evaluate the effect of MSG on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in sperm lacking the Tas1r1 receptor, capacitated cells were loaded with Fura-2/AM and subsequently fluorescence intensity of sperm populations was determined in a plate reader. Therefore, 90 µl of a capacitated sperm suspension (450,000–900,000 cells) were stimulated with different concentrations of MSG (1 mM MSG, 10 mM MSG, 50 mM MSG) by injecting 10 µl of a concentrated tastant stock solution. The concentration of the cation ionophore ionomycin used as a positive control was 5 µM; HS/NaHCO₃ buffer alone served as negative control. Fura-<i>2</i> fluorescence was recorded with excitation wavelengths of 340 and 380 nm; subsequently data were calculated as ratio (<i>F340/F380</i>) and plotted against the time in seconds. Presented data are mean values ± SD of sperm of wild-type [<i>+/+</i>] and Tas1r1-deficient [<i>−/−</i>] mice measured in triplicates, which were representative for 3 experiments per genotype.</p

Capacitation and acrosome reaction in Tas1r1 null sperm from the Tas1r1/mCherry mouse line.

<p>[<b>A</b>] Capacitation dependent efflux of cholesterol in Tas1r1-deficient mice. To quantify capacitation dependent cholesterol release in isolated epididymal sperm of wild-type and Tas1r1 null mutant animals, equal amounts of a homogeneous sperm suspension were incubated for different time periods (0 min, 30 min, 60 min, 90 min, 120 min) in HS/BSA/NaHCO₃ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032354#s4" target="_blank">Materials and Methods</a>. At the indicated time points, aliquots of the supernatant were collected and used to measure cholesterol release using a fluorometric-based quantification kit. Obtained data were calculated as cholesterol efflux per cell after subtracting basal cholesterol content at the beginning of the incubation (0 min: [<i>+/+</i>]: 42±3 ng cholesterol/10⁶ sperm; [<i>−/−</i>]: 37±2 ng cholesterol/10⁶ sperm). Time-dependent sterol release in sperm of both genotypes increased over time and showed no significant difference (p≤0.05). Data, presented as mean values ± SEM, are the average of nine independent sperm preparations of C57BL/6wild-types and Tas1r1-deficient animals from the same colony. [<b>B</b>] A23187 and <i>zona pellucida</i> induced acrosomal secretion in Tas1r1 null sperm. To assess whether Tas1r1-deficient sperm show a defect in the acrosomal exocytotic machinery or in recognizing the egg's coat, respectively, <i>in vitro</i> capacitated spermatozoa of wild-type and Tas1r1 null mutant littermates were either treated with 10 µM A23187 [<i>A23187</i>] or alternatively with solubilised <i>zona pellucida</i> [<i>ZP</i>] at 37°C for 30 min. Subsequently, aliquots of sperm were stained with Commassie blue G.250 and acrosomal status was quantified by counting at least 200 cells for each condition. Data, calculated as absolute percentages of acrosome reacted sperm represent mean values ± SEM of independent experiments with different mouse sperm preparations ([<i>A23187</i>], n = 15; [<i>ZP</i>], n = 7). Spontaneously occurring secretion rates were determined incubating sperm in corresponding buffer used to dilute the stimulating compounds [buffer with DMSO: wild-type [<i>+/+</i>]: 28.1±2.2%; Tas1r1 [<i>−/−</i>]: 35.2±2.5%; ZP buffer alone: wild-type [<i>+/+</i>]: 33.1±3.5%; Tas1r1 [<i>−/−</i>]: 37.7±3.0%). Statistical analysis was done using a Student's t-test comparing acrosome reacted sperm of both genotypes.</p

Detection of Tas1r-transcripts from cDNA of murine vallate papillae and testicular tissue using RT-PCR.

<p>Primer sets specific for the murine Tas1r1 and Tas1r3 yielded amplification products with the expected size ([<i>Tas1r1</i>]; 468 bp; ([<i>Tas1r3</i>]; 510 bp) from cDNA derived from taste [VP] as well as from testicular tissue ([<i>Te</i>]), whereas the primer pair for the Tas1r2 only resulted in the generation of an amplification product in taste cDNA ([<i>Tas1r</i>2]; 403 bp [<i>VP</i>]), but not in testicular cDNA ([<i>Te</i>]). cDNA quality was assured determining amplification products with a primer pair against the housekeeping gene beta-actin (right panel, [<i>actin</i>]; 425 bp]). Negative controls present samples in which water was used instead of cDNA ([<i>H₂O</i>]). The identities of amplified taste receptor subtypes are indicated on the top of each panel. The corresponding 500 bp DNA size marker is shown on the left of both panels.</p