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A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

ABSTRACT: We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A(2) receptor (TxA(2)R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA(2)R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA(2)R, U46619 did not increase cytosolic free Ca(2+) concentration, indicating loss of receptor function. The TxA(2)R antagonist [(3)H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA(2)R. This is the second naturally occurring TxA(2)R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA(2)R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA(2)R function in vivo.

SUBMITTER: Mumford AD

PROVIDER: S-EPMC4405764 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

Mumford Andrew D AD Dawood Ban B BB Daly Martina E ME Murden Sherina L SL Williams Michael D MD Protty Majd B MB Spalton Jennifer C JC Wheatley Mark M Mundell Stuart J SJ Watson Steve P SP

Blood 20091014 2

We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A(2) receptor (TxA(2)R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA(2)R. In Chinese hamster ovary-K1 cells expressing the variant D304 ...[more]

PMID: 19828703

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Project description:BackgroundThromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostaglandin H(2), into thromboxanes. Some of the thromboxanes are known to be biologically active on cancer cells. The aim of the study was to investigate the expression of thromboxane synthases, TBXAS1 and the thromboxane A2 receptor, TBXA2R in a cohort of human breast cancer patients and also to assess their potential clinical relevance.MethodsHuman breast tumour tissues (n = 120) and non-neoplastic mammary tissues (n = 32) were studied. Levels of TBXA2R and TBXAS1 transcripts were quantified using quantitative real-time RT-PCR analysis and correlated with clinical/pathological information including nodal status, grade, prognosis and long term survival (median follow-up period 120 months).ResultsBreast tumour tissue expressed higher levels of TBXA2R compared with normal mammary tissues, although the difference was not statistically significant (p = 0.09). There was no difference between tumour and normal tissues for TBXAS1. However, TBXA2R expression was significantly increased in grade 3 tumours(p = 0.006 vs grade 1), while TBXAS1 was significantly reduced in grade 3 tumours (p = 0.026 vs grade 1 tumours). A similar differential expression pattern was seen in tumours from patients with different prognosis, in that patients with predicted poor prognosis had higher, but not statistically different, levels of TBXA2R, and significantly lower levels of TBXAS1 (p = 0.008). Finally, Kaplan-Meier survival analysis has shown that patients with high levels of TBXA2R had significantly shorter disease free survival (103.8 (79.1-128.5) months) compared with those with low levels (123.7 (112.0-135.3)) months, p = 0.043.ConclusionThromboxane synthases are differentially expressed in human breast cancer. While TBXA2R is highly expressed in aggressive tumours and linked with poor prognosis, TBXAS1 is expressed at significantly low levels in high grade tumours and tumour patients with poor prognosis. TBXA2R thus has a significant prognostic value in clinical breast cancer.

Project description: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.

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A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

Publications

A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

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