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

ABSTRACT: 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 (G?q) in response to the binding of thromboxane, while the IP signals through G?s 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.

SUBMITTER: Chakraborty R

PROVIDER: S-EPMC3554117 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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

Chakraborty Raja R Pydi Sai Prasad SP Gleim Scott S Bhullar Rajinder Pal RP Hwa John J Dakshinamurti Shyamala S Chelikani Prashen P

Molecular and cellular biology 20121029 2

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 combinat ...[more]

PMID: 23109431

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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:1. Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [(3)H]-cyclic AMP and [(3)H]-inositol phosphate production. 2. The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E(1), stimulated adenylyl cyclase activity with EC(50) values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 - 40 fold lower potency than stimulation of adenylyl cyclase. 3. The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC(50) values of 219, 166 and 398 nM, respectively, but failed to stimulate [(3)H]-inositol phosphate production. 4. Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [(3)H]-inositol phosphate production in a non-competitive manner. 5. Activation of the endogenously-expressed P(2) purinergic receptor by ATP led to an increase in [(3)H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [(3)H]-inositol phosphate production. 6. A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP(3) receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors. 7. The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant Galpha(q)RC, Galpha(14)RC and Galpha(16)QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant Galpha(s)QL. 8. These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [(3)H]-inositol phosphate production by targeting G(q/11) and/or phospholipase C in CHO cells, and that this effect is independent of IP receptors.

Dataset Information

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

Publications

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

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