Thromboxane A2 G Protein-Coupled Receptor Production and Crystallization for Structure Studies.

G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands.

Refined pharmacophore features for virtual screening of human thromboxane A2 receptor antagonists.

For a long time, the structural basis of TXA2 receptor is limited due to the lack of crystal structure information, till the release of the crystal structure of TXA2 receptor, which deepens our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor. In this research, we report the successful implementation in the discovery of an optimal pharmacophore model of human TXA2 receptor antagonists through virtual screening. Structure-based pharmacophore models were generated based on two crystal structures of human TXA2 receptor (PDB entry 6IIU and 6IIV). Docking simulation revealed interaction modes of the virtual screening hits against TXA2 receptor, which was validated through molecular dynamics simulation and binding free energy calculation. ADMET properties were also analyzed to evaluate the toxicity and physio-chemical characteristics of the hits. The research would provide valuable insight into the binding mechanisms of TXA2 receptor antagonists and thus be helpful for designing novel antagonists.

Structural basis for ligand recognition of the human thromboxane A2 receptor.

Stimulated by thromboxane A2, an endogenous arachidonic acid metabolite, the thromboxane A2 receptor (TP) plays a pivotal role in cardiovascular homeostasis, and thus is considered as an important drug target for cardiovascular disease. Here, we report crystal structures of the human TP bound to two nonprostanoid antagonists, ramatroban and daltroban, at 2.5 Å and 3.0 Å resolution, respectively. The TP structures reveal a ligand-binding pocket capped by two layers of extracellular loops that are stabilized by two disulfide bonds, limiting ligand access from the extracellular milieu. These structures provide details of interactions between the receptor and antagonists, which help to integrate previous mutagenesis and SAR data. Molecular docking of prostanoid-like ligands, combined with mutagenesis, ligand-binding and functional assays, suggests a prostanoid binding mode that may also be adopted by other prostanoid receptors. These insights into TP deepen our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor.

Expression of the TPα and TPß isoforms of the thromboxane prostanoid receptor (TP) in prostate cancer: clinical significance and diagnostic potential.

The prostanoid thromboxane (TX)A2 plays a central role in haemostasis and is increasingly implicated in cancer progression. TXA2 signals through two T Prostanoid receptor (TP) isoforms termed TPα and TPß, with both encoded by the TBXA2R gene. Despite exhibiting several functional and regulatory differences, the role of the individual TP isoforms in neoplastic diseases is largely unknown.This study evaluated expression of the TPα and TPß isoforms in tumour microarrays of the benign prostate and different pathological (Gleason) grades of prostate cancer (PCa). Expression of TPß was significantly increased in PCa relative to benign tissue and strongly correlated with increasing Gleason grade. Furthermore, higher TPß expression was associated with increased risk of biochemical recurrence (BCR) and significantly shorter disease-free survival time in patients post-surgery. While TPα was more variably expressed than TPß in PCa, increased/high TPα expression within the tumour also trended toward increased BCR and shorter disease-free survival time. Comparative genomic CpG DNA methylation analysis revealed substantial differences in the extent of methylation of the promoter regions of the TBXA2R that specifically regulate expression of TPα and TPß, respectively, both in benign prostate and in clinically-derived tissue representative of precursor lesions and progressive stages of PCa. Collectively, TPα and TPß expression is differentially regulated both in the benign and tumourigenic prostate, and coincides with clinical pathology and altered CpG methylation of the TBXA2R gene. Analysis of TPß, or a combination of TPα/TPß, expression levels may have significant clinical potential as a diagnostic biomarker and predictor of PCa disease recurrence.

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/106 cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

Biochemical assay of G protein-coupled receptor oligomerization: adenosine A1 and thromboxane A2 receptors form the novel functional hetero-oligomer.

G protein-coupled receptors (GPCRs) are classified into a family of seven transmembrane receptors. Receptor oligomerization may be the key to the expression and function of these receptors, for example, ligand binding, desensitization, membrane trafficking, and signaling. The accumulation of evidence that GPCRs form an oligomerization with a functional alternation may change the strategy for the discovery of novel drugs targeting GPCRs. Identification of the oligomer is essential to elucidate GPCR oligomerization. GPCR oligomerizations have been demonstrated using various biochemical approaches, which include the coimmunoprecipitation method, fluorescence resonance energy transfer assay, and bioluminescence RET assay. Thus, various assays are useful for the study of GPCR oligomerization, and we should choose the best method to match the purpose. We previously targeted adenosine A1 and thromboxane A2 (TP) receptors to form a functionally novel hetero-oligomer, since both receptors function in the same cells. This chapter describes the methods used to detect GPCR oligomerization and alterations in the signaling pathways, principally according to our findings on oligomerization between adenosine A1 and TPα receptors.

Human thromboxane A2 receptor genetic variants: in silico, in vitro and "in platelet" analysis.

Thromboxane and its receptor have emerged as key players in modulating vascular thrombotic events. Thus, a dysfunctional hTP genetic variant may protect against (hypoactivity) or promote (hyperactivity) vascular events, based upon its activity on platelets. After extensive in silico analysis, six hTP-α variants were selected (C(68)S, V(80)E, E(94)V, A(160)T, V(176)E, and V(217)I) for detailed biochemical studies based on structural proximity to key regions involved in receptor function and in silico predictions. Variant biochemical profiles ranged from severe instability (C(68)S) to normal (V(217)I), with most variants demonstrating functional alteration in binding, expression or activation (V(80)E, E(94)V, A(160)T, and V(176)E). In the absence of patient platelet samples, we developed and validated a novel megakaryocyte based system to evaluate human platelet function in the presence of detected dysfunctional genetic variants. Interestingly, variant V80E exhibited reduced platelet activation whereas A160T demonstrated platelet hyperactivity. This report provides the most comprehensive in silico, in vitro and "in platelet" evaluation of hTP variants to date and highlightscurrent inherent problems in evaluating genetic variants, with possible solutions. The study additionally provides clinical relevance to characterized dysfunctional hTP variants.

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.

New insights into structural determinants for prostanoid thromboxane A2 receptor- and prostacyclin receptor-G protein coupling.

G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA(2)) receptor (TP) and prostacyclin (PGI(2)) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease.

Involvement of lipid rafts in multiple signal transductions mediated by two isoforms of thromboxane A2 receptor: dependency on receptor isoforms and downstream signaling types.

Lipid rafts, microdomains in the plasma membrane, are known to be involved in G protein-coupled receptor signal transduction; however, their involvement in thromboxane A(2) receptor (TP) signaling remains to be clarified. We examined whether two isoforms of TP, TPα and TPß, utilize lipid rafts for multiple G protein signal transduction. Sucrose density gradient centrifugation followed by western blotting of HEK cells expressing TPα or TPß revealed the localization of both TPα and TPß in lipid rafts. Furthermore, methyl-ß-cyclodextrin, which destroys lipid raft structure by depleting cholesterol, influenced G protein signaling elicited by TPα and TPß to varying degrees. Phosphatidylinositol hydrolysis and cAMP accumulation induced by TPα or TPß stimulation was markedly inhibited by methyl-ß-cyclodextrin. In contrast, treatment with methyl-ß-cyclodextrin partially inhibited RhoA activation induced by TPα stimulation, but failed to affect TPß stimulation. Furthermore, the inhibitory action of methyl-ß-cyclodextrin on cAMP accumulation was specific to TPα and TPß, because methyl-ß-cyclodextrin enhanced forskolin and ß-adrenergic stimulation-induced cAMP accumulation. These results indicate that TP isoforms depend on lipid rafts during G(q) and G(s) signaling, while G(13) signaling mediated by TP isoforms does not. Moreover, TPα seems to be more lipid raft-dependent with respect to RhoA activation than TPß. These results indicate that the two isoforms of the TP mediate multiple signal transductions with varying degrees of lipid raft dependency. Moreover, our results provide a deeper understanding of the function of lipid rafts in G protein signaling and the physiological meaning of TP isoforms.

A novel antibody targeting the ligand binding domain of the thromboxane A2 receptor exhibits antithrombotic properties in vivo.

In efforts to define new targets for antithrombotic purposes, there is interest in utilizing antibodies targeting ligand binding domains of platelet receptors. To this end, we have recently shown that an antibody (designated C-EL2Ab), which targets the C-terminus of the 2nd extracellular loop (C-EL2) of the thromboxane A(2) receptor (TPR), selectively blocks TPR-mediated platelet aggregation, under both in vitro and ex vivo experimental conditions. In the current studies we sought to determine whether C-EL2Ab exhibits in vivo antithrombotic activity, by employing a carotid artery injury thrombosis model. It was found that mice treated with C-EL2Ab, exhibited a significant increase in time for occlusion, when compared to controls such as normal rabbit IgG, or an antibody which targets a region separate from the ligand binding site (i.e., EL1). We next examined the effect of C-EL2Ab on hemostasis, and found no increase in tail bleeding times in C-EL2Ab treated mice, compared to the aforementioned controls. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, and is devoid of increased bleeding risk. Moreover, the identification of a functionally active TPR sequence should significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity.

Site-directed mutations and the polymorphic variant Ala160Thr in the human thromboxane receptor uncover a structural role for transmembrane helix 4.

The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala160(4.53) is highly conserved as is Gly164(4.57). Here we target Ala160(4.53) and Gly164(4.57) in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala160(4.53) and Gly164(4.57) in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions.

Hetero-oligomerization between adenosine A1 and thromboxane A2 receptors and cellular signal transduction on stimulation with high and low concentrations of agonists for both receptors.

Growing evidence indicates that G protein-coupled receptors can form homo- and hetero-oligomers to diversify signal transduction. However, the molecular mechanisms and physiological significance of G protein-coupled receptor-oligomers are not fully understood. Both ADOR1 (adenosine A(1) receptor) and TBXA2R (thromboxane A(2) receptor α; TPα receptor), members of the G protein-coupled receptor family, act on astrocytes and renal mesangial cells, suggesting certain functional correlations. In this study, we explored the possibility that adenosine A(1) and TPα receptors form hetero-oligomers with novel pharmacological profiles. We showed that these receptors hetero-oligomerize by conducting coimmunoprecipitation and bioluminescence resonance energy transfer (BRET(2)) assays in adenosine A(1) receptor and TPα receptor-cotransfected HEK293T cells. Furthermore, coexpression of the receptors affected signal transduction including the accumulation of cyclic AMP and phosphorylation of extracellular signal-regulated kinase-1 and -2 was significantly increased by high and low concentrations of adenosine A(1) receptor agonist and TPα agonists, respectively. Our study provides evidence of hetero-oligomerization between adenosine A(1) and TPα receptors for the first time, and suggests that this oligomerization affects signal transduction responding to different concentrations of receptor agonists.

Light on the structure of thromboxane A2receptor heterodimers.

The structure-based design of a mutant form of the thromboxane A(2) prostanoid receptor (TP) was instrumental in characterizing the structural determinants of the hetero-dimerization process of this G protein coupled receptor (GPCR). The results suggest that the hetero-dimeric complexes between the TPα and ß isoforms are characterized by contacts between hydrophobic residues in helix 1 from both monomers. Functional characterization confirms that TPα-TPß hetero-dimerization serves to regulate TPα function through agonist-induced internalization, with important implications in cardiovascular homeostasis. The integrated approach employed in this study can be adopted to gain structural and functional insights into the dimerization/oligomerization process of all GPCRs for which the structural model of the monomer can be achieved at reasonable atomic resolution.

[Correlation between oligohydramnios and abnormal expressions of TXA2, PGI2 and TXA2R in the umbilical arterial blood and placenta].

OBJECTIVE: To investigate the roles of thromboxane A(2) (TXA(2)) and prostaglandin I(2) (PGI(2)) in development of oligohydramnios. METHODS: The concentration of TXB(2) and 6-keto-PGF1 in umbilical cord blood collected from 30 normal parturients (control) and 30 parturients with oligohydramnios was detected by radioimmunoassay to calculate the TXA(2)/PGI(2) ratio. Immunohistochemistry was performed to detect the contents of TXA(2)R in vascular endothelial cell in the placental villi. RESULTS: Compared with the control group, the concentration of umbilical cord blood TXB(2) in oligohydramnios group was significantly increased (P<0.01), but the elevation of 6-keto-PGF(2) concentration was not statistically significant (P>0.05). The oligohydramnios group showed significantly higher positivity rates of TXB2 and 6-keto-PGF1 in than the control group (P<0.01), and the positivity rate of TXA(2)R in the vascular endothelial cells in the placental villi was also significantly higher in the oligohydramnios group (22/30, 77.3% vs 11/30, 36.7%, P<0.05). Most of the TXA(2)R-positive cases in the oligohydramnios group showed strong positivities of TXA(2)R. CONCLUSION: Abnormal elevation of TXA(2) concentration in the umbilical cord blood and the TXA(2)/PGI(2) imbalance are responsible for the development of oligohydramnios.

Thromboxane A2-induced signal transduction is negatively regulated by KIAA1005 that directly interacts with thromboxane A2 receptor.

Thromboxane A(2) (TXA(2)), a potent inducer of platelet aggregation and smooth muscle contraction, exerts its action through TXA(2) receptor (TP). There are two alternative splicing variants of TP, TP alpha and TP beta. To clarify the signal transduction of TP pathway, we searched for putative TP binding proteins using a yeast two-hybrid system with the C-terminal region of TP alpha or TP beta as bait. We found KIAA1005 as a novel interacting protein of the TP alpha and TP beta C-terminal region (TP interacting protein, TPIP). KIAA1005/TPIP was co-immunoprecipitated with TP alpha or TP beta in HEK293 cells expressing myc-KIAA1005/TPIP and FLAG-TP isoforms. Expression analysis showed a ubiquitous expression pattern of KIAA1005/TPIP mRNA, including prominent expression in the thymus. Furthermore, TP-mediated phosphoinositide hydrolysis, phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and interleukin-6 production were reduced by the expression of KIAA1005/TPIP. The expression of KIAA1005/TPIP decreased cell-surface TP alpha and TP beta levels. Thus, we show for the first time that KIAA1005/TPIP is a novel TP interacting protein that regulates TP-mediated signal transduction negatively.

Ligand-specific conformation determines agonist activation and antagonist blockade in purified human thromboxane A2 receptor.

The binding of an agonist to a G protein-coupled receptor (GPCR) causes its coupling to different G proteins, which mediate signaling. However, the binding of an antagonist to the same site of the GPCR could not induce coupling. To understand the molecular mechanism involved, the structural flexibility of the purified human thromboxane A2 receptor (TP) was characterized by spectroscopic approaches, while bound to an agonist or antagonist. Circular dichroism not only revealed that the purified TP adopted more than 50% helical conformation in solution but also showed that the antagonist, SQ29,548, could induce more of a beta-sheet structure in the TP than that of the agonist, U46619. Also, fluorescence studies showed that the antagonist induced the intrinsic Trp fluorescence signal change more than the agonist. Furthermore, three of the nine tryptophan residues involved in the different ligand-based structural changes were demonstrated by NMR spectroscopy. Low pH-induced changes in the receptor conformation and molecular interaction field dramatically increased the agonist binding but did not significantly affect the antagonist binding. Different conformational changes were also observed in the TP reconstituted into phosphatidylcholine/phosphatidylserine/phosphatydylethanolamine-formed liposomes. These studies are the first to show a possible mechanism of the ligand-specific conformation-dependent agonist activation and antagonist blockage in the GPCR.

Characterization of the prostaglandin H2 mimic: binding to the purified human thromboxane A2 receptor in solution.

For decades, the binding of prostaglandin H(2) (PGH(2)) to multiple target proteins of unrelated protein structures which mediate diverse biological functions has remained a real mystery in the field of eicosanoid biology. Here, we report that the structure of a PGH(2) mimic, U46619, bound to the purified human TP, was determined and compared with that of its conformation bound to the COX-downstream synthases, prostacyclin synthase (PGIS) and thromboxane A(2) synthase (TXAS). Active human TP protein, glycosylated and in full length, was expressed in Sf-9 cells using a baculovirus (BV) expression system and then purified to near homogeneity. The binding of U46619 to the purified receptor in a nonionic detergent-mimicked lipid environment was characterized by high-resolution NMR spectroscopy. The conformational change of U46619, upon binding to the active TP, was evidenced by the significant perturbation of the chemical shifts of its protons at H3 and H4 in a concentration-dependent manner. The detailed conformational changes and 3D structure of U46619 from the free form to the TP-bound form were further solved by 2D (1)H NMR experiments using a transferred NOE (trNOE) technique. The distances between the protons of H11 and H18, H11 and H19, H15 and H18, and H15 and H19 in U46619 were shorter following their binding to the TP in solution, down to within 5A, which were different than that of the U46619 bound to PGIS and U44069 (another PGH(2) mimic) bound to TXAS. These shorter distances led to further separation of the U46619 alpha and omega chains, forming a unique "rectangular" shape. This enabled the molecule to fit into the ligand-binding site pocket of a TP model, in which homology modeling was used for the transmembrane (TM) domain, and NMR structures were used for the extramembrane loops. The proton perturbations and 3D conformations in the TP-bound U46619 were different with that of the PGH(2) mimics bound to PGIS and TXAS. The studies indicated that PGH(2) can adopt multiple conformations in solution to satisfy the specific and unique shapes to fit the different binding pockets in the TP receptor and COX-downstream enzymes. The results also provided sufficient information for speculating the molecular basis of how PGH(2) binds to multiple target proteins even though unrelated in their protein sequences.