Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation

The intrahelical salt bridge between $E/D^{3.49}$ and $R^{3.50}$ within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of $E^{3.49/6.30}$ in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes

Pharmacodynamic analysis of antagonists SQ29,548 and PTA₂ in functional assay.

<p>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.</p

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

<p> µ<b>M SQ29,548 for 18 </b><b>h.</b> 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.</p

Agonist-induced total IP accumulation in HEK293 cells transiently expressing equal amounts of the WT (A) or E129V mutant (B) of human TP receptor.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060475#pone-0060475-t002" target="_blank">Table 2</a>.</p

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.

<p>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.</p

BRET² measurement of Gα_qβ₁γ₂ complex activation in HEK293 living cells expressing equal amounts of the WT of human TP receptor or its E129V mutant.

<p>A. BRET² was measured between the donor Rluc8 and the acceptor GFP¹⁰ introduced at the residue 97 of the Gα_q subunit and the N-terminal domain of the Gγ₂ subunit, respectively. Agonist-induced coupling of TP receptor and Gq protein distances Gα_q-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 Gα_q-Rluc8 luminescence was evaluated in HEK293 cells co-expressing Gα_q-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 Gα_q-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.</p

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.

<p>Ki values were obtained by simultaneous analysis of at least 3 independent competition experiments analyzed with GraphPad Prism implemented with the LIGAND model (see Data and statistical analysis).</p>a<p>Affinity ratio was calculated as the ratio of the K_i for the WT TP receptor over the K_i of the E129V mutant.</p>**<p>p<0.01 vs. WT.</p

Binding affinities of [³H]SQ29,548 in HEK293 cells transiently expressing the WT or mutant human TP receptors.

<p>Binding affinities and capacities were obtained by simultaneous analysis of at least 3 independent mixed-type experiments each performed in duplicates, analyzed with GraphPad Prism implemented with the LIGAND model (see Data and statistical analysis).</p>a<p>In cotransfection experiments, TP and Gαq plasmids were added in a 1∶3 (3x) and 1∶5 (5x) ratio (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060475#s2" target="_blank">methods</a> for details).</p>b<p>Parameters refer to E129/240V double mutant before SQ29,548 rescue.</p

BRET concentration-response parameters for different agonists in HEK293 cells transiently expressing the WT or the E129V TP receptor.

<p>Values of EC₅₀ and E_max were obtained by simultaneous analysis with GraphPad Prism (see Data and statistical analysis) of at least two independent experiments each performed in duplicates.</p>a<p>Activity ratio was calculated as the ratio of the EC₅₀ for the WT TP receptor over the EC₅₀ of the E129V mutant.</p>*<p>p<0.05, ** p<0.01 vs. WT.</p