Table 1.
Binding affinities of [3H]SQ29,548 in HEK293 cells transiently expressing the WT or mutant human TP receptors.
Figure 1.
Agonist-induced total IP accumulation in HEK293 cells transiently expressing equal amounts of the WT (A) or E129V mutant (B) of human TP receptor.
Total IP accumulation was measured after incubation in the absence (basal) or presence of increasing concentrations of the indicated agonists for 30 min. Data are expressed as dpm/well. Error bars represent mean±SE of at least three independent experiments each performed in duplicates or triplicates (For the sake of clarity, in panel B, error bar direction of U46619 and I-BOP data is above and below, respectively). Curves are computer generated from the simultaneous analysis of at least three independent experiments. Values for EC50 and significant differences from WT are shown in Table 2.
Figure 2.
Agonist binding studies in HEK293 cells transiently expressing the WT (A) or E129V mutant (B) of human TP receptor.
For each construct, cold U46619, I-BOP, 8-isoPGF2α or 8-isoPGE2 was used in competition for 1 nM [3H]SQ29,548. Mixed type curves and heterologous competition curves were performed at 25°C with 30 min incubation. Binding is expressed as the ratio of bound ligand concentration to total ligand concentration (B/T, dimensionless) versus the logarithm of total unlabeled ligand concentration (Log T). Non-specific binding was calculated by computer as one of the unknown parameters of the model and was always <10% of total binding. Curves are computer generated from the simultaneous analysis of several independent mixed-type and heterologous competition experiments, each in duplicate. Values for Ki and significant differences from WT are shown in Table 3.
Table 2.
Total IP dose-response parameters for different agonists in HEK293 cells transiently expressing the WT or the E129V TP receptor.
Table 3.
Affinities of the agonists for the binding site of the receptor labelled by [3H]SQ29,548 in HEK293 cells transiently expressing the WT or the mutant human TP receptors.
Figure 3.
Analysis of basal and agonist induced total IP accumulation in HEK293 cells transiently expressing the WT or SAM mutants of human TP receptor in the absence and presence of Gαq overexpression.
TP and Gαq plasmids were added in a 1∶3 (3x) and 1∶5 ratio (5x), respectively. A. Total IP accumulation in basal conditions. B. Total IP accumulation induced by 30 min stimulation with 1 µM U46619. C. Fold increase over basal of total IP accumulation. D. Basal activity for WT and SAMs with increasing receptor expression in pmol/mg protein. Data are expressed as dpm/well. Error bars represent mean±SE of at least three independent experiments each performed in duplicates or triplicates.
Figure 4.
Functional analysis of basal and agonist induced total IP accumulation in HEK293 cells transiently expressing the double mutant E129V/E240V of human TP receptor without and following rescue with 1
µM SQ29,548 for 18 h. A. Total IP accumulation in basal condition and following 30 min stimulation with 1 µM U46619. B. Concentration-response curves of U46619. Error bars represent mean±SE of at least two independent experiments each performed in duplicates.
Figure 5.
Pharmacodynamic analysis of antagonists SQ29,548 and PTA2 in functional assay.
Total IP accumulation in HEK293 cells transiently expressing the WT or E129V mutant of human TP receptor was assayed in the absence and presence of Gαq overexpression at increasing concentrations of the antagonists for 30 min. TP and Gαq plasmids were added in a 1∶5 (5x) ratio. Data are expressed as dpm/well. Error bars represent mean±SE of at least three independent experiments each performed in duplicates or triplicates. Curves are computer generated from the simultaneous analysis of several independent experiments.
Figure 6.
BRET2 measurement of Gαqβ1γ2 complex activation in HEK293 living cells expressing equal amounts of the WT of human TP receptor or its E129V mutant.
A. BRET2 was measured between the donor Rluc8 and the acceptor GFP10 introduced at the residue 97 of the Gαq subunit and the N-terminal domain of the Gγ2 subunit, respectively. Agonist-induced coupling of TP receptor and Gq protein distances Gαq-Rluc8 and GFP10-Gγ2 giving rise to a decrease in the BRET signal. B. Protein expression levels of the constructs used for BRET experiments were set to be constant and able to assure the same level of basal BRET ratio in the presence of WT and E129V mutant of the human TP receptors. Total Gαq-Rluc8 luminescence was evaluated in HEK293 cells co-expressing Gαq-Rluc8 together with GFP10-Gγ2 and Gβ1 in the presence of WT or E129V mutant of the human TP receptor measuring the light emission in aliquots of the transfected cells incubated with 5 µM coelenterazine for 8 min. In the same cells stimulated with PBS, basal BRET ratio was calculated as the ratio of the light emitted by GFP10 (510–540 nm) over the light emitted by Rluc8 (370–450 nm). C. BRET was measured in HEK293 cells co-expressing Gαq-Rluc8 together with GFP10-Gγ2 and Gβ1 in the presence of WT (left) or E129V (right) mutant of the human TP receptor and stimulated with increasing concentrations of the indicated full and partial agonists. Results are the differences in the BRET signal measured in the presence and the absence of agonists, and are expressed as the mean value±SE of at least two independent determinations.
Table 4.
BRET concentration-response parameters for different agonists in HEK293 cells transiently expressing the WT or the E129V TP receptor.