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Prostaglandin (PG) E receptor EP3D is coupled to both Gi and Gs. To examine the roles of the interaction of alpha-carboxylic acid of PGE2 and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine residue to the noncharged glutamine. PGE2 with a negatively charged alpha-carboxylic acid and sulprostone, an EP3 agonist with a noncharged modified alpha-carboxylic acid, inhibited the forskolin-stimulated adenylate cyclase activity via Gi activation in the EP3D receptor in the same concentration-dependent manner. In contrast, the adenylate cyclase stimulation via Gs activation by sulprostone was much lower than that by PGE2. On the other hand, both PGE2 and sulprostone showed potent Gi activity but failed to show Gs activity in the mutant receptor. EP3D receptor showed a high affinity binding for PGE2 in the form coupled to either Gi or Gs. Although the mutant receptor showed high affinity binding when coupled to Gi, it lost high affinity binding in the condition of Gs coupling. Furthermore, sulprostone bound to the Gi-coupled EP3D receptor with higher affinity than the Gs-coupled receptor. Among various EP3 agonists, alpha-carboxylic acid-unmodified agonists showed both Gi and Gs activities, but the modified agonists showed only Gi activity. These findings suggest that the interaction between the alpha-carboxylic acid of PGE2 and the arginine residue of the receptor regulates the selectivity of the G protein coupling.

A cDNA clone for rat prostacyclin receptor was isolated. The cDNA encodes a protein of 416 amino acid residues (M(r) 44,662) with putative seven transmembrane domains, and belongs to the G protein-coupled receptor superfamily. Specific binding of [3H]iloprost was found in membrane of COS-7 cells transfected with the cDNA (Kd = 1.3 nM) and was displaced with unlabeled prostaglandins in the order of iloprost = cicaprostPGE1STA2 = PGE2 = PGD2PGF2 alpha. Northern blot analysis demonstrated that rat prostacyclin receptor mRNA is expressed in the lung, spleen, heart, pancreas, thymus, stomach and aorta.

BACKGROUND Prostacyclin elicits a potent vasodilation and inhibition of platelet aggregation through binding to its membrane receptor. The impairment of prostacyclin receptor activity is implicated in various human cardiovascular diseases. In the present study, we succeeded in the isolation and characterization of human prostacyclin receptor cDNA and elucidated its gene expression in human tissues. METHODS AND RESULTS We isolated a cDNA clone encoding the human prostacyclin receptor from a human lung cDNA library. The isolated cDNA clone encodes a 386-amino acid protein with seven putative transmembrane domains, which belongs to the G protein-coupled receptor superfamily. [3H]iloprost, a prostacyclin receptor agonist, specifically bound to the receptor transiently expressed in COS-7 cells. The binding was inhibited in the rank order of iloprost = cicaprost, another prostacyclin receptor agonist, > prostaglandin E1 (PGE1) > PGE2, PGF2 alpha, PGD2, STA2. In addition, iloprost dose-dependently stimulated cAMP generation in these COS-7 cells. These results are consistent with the characteristics of the human prostacyclin receptor. Northern blotting analysis on human tissues revealed that prostacyclin receptor mRNA is abundantly expressed in the aorta, lung, atrium, ventricle, and kidney. CONCLUSIONS We cloned human prostacyclin receptor cDNA and elucidated its abundant gene expression in the human cardiovascular system. The present study will lead to better understanding of the significance of prostacyclin in humans and further facilitate the clinical application of prostacyclin.

Three isoforms of the mouse prostaglandin-E-receptor EP3 subtype (EP3), EP3 alpha, EP3 beta and EP3 gamma, with different C-termini, which are produced through alternative splicing, showed different efficiencies with respect to heterotrimeric GTP-binding protein activation and adenylate cyclase inhibition [Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S.Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718; Irie, A., Sugimoto, Y., Namba, T., Harazono, A., Honda, A., Watabe, A., Negishi, M., Narumiya, S.Ichikawa, A. (1993) Eur. J. Biochem. 217, 313-318]. To assess the role of the C-terminus in GTP-binding protein coupling, we truncated the C-terminus of EP3 at an alternative splicing site and expressed the mutant receptor. The truncated receptor retained the ability to physically associate with Gi2, forming an agonist/receptor/Gi2 ternary complex, and to undergo the characteristic conversion of its agonist-binding affinity, mediated by a guanine nucleotide from a low-affinity state to a high-affinity state. However, sulprostone, an EP3 agonist, failed not only to inhibit the forskolin-induced cAMP accumulation in the mutant receptor-expressing cells but also to stimulate the GTPase activity in the mutant receptor-expressing cell membrane. These results indicated that the C-terminus of EP3 is essential for the activation of GTP-binding protein.

Distribution of the mRNAs for three subtypes of prostaglandin E (PGE) receptors in the mouse kidney was investigated by in situ hybridization. The mRNA for EP1 subtype, which is coupled to Ca2+ mobilization, was specifically localized to the collecting ducts from the cortex to the papilla. The mRNA for EP2 subtype, which is linked to stimulation of adenylate cyclase, was localized to the glomeruli. The mRNA for EP3 subtype, which is coupled to inhibition of adenylate cyclase, was located densely in the tubules in the outer medulla and in the distal tubules in the cortex. These results exhibit distinct cellular localization of three subtypes of PGE receptor in the kidney and suggest that PGE2 exerts multiple functions via these subtypes expressed in different segments of the nephron.

We have recently revealed that the thymus is the organ showing the highest expression of thromboxane (TX) A2 receptor in mice. In this study, thymic cell populations expressing the receptor were identified, and the effects of a TXA2 agonist on these cells were examined. Radioligand binding using a TXA2 receptor-specific radioligand revealed a single class of binding sites in the thymocytes with an affinity and specificity identical to those reported for the TXA2 receptor. The receptor density in these cells was comparable to that seen in blood platelets. This receptor is most highly expressed in CD4-8- and CD4+8+ immature thymocytes, followed by CD4+8- and CD4-8+ cells. The receptor density in splenic T cells was less than one fifth of that in CD4+8+ cells and no binding activity was detectable in splenic B cells. The addition of a TXA2 agonist, STA2, to thymocytes induced the disappearance of the CD4+8+ cells in a time- and concentration-dependent manner and caused DNA fragmentation. These changes were blocked by a specific TXA2 antagonist, S-145. These results demonstrate that TXA2 induces apoptotic cell death in immature thymocytes by acting on the TXA2 receptor on their cell surface and suggest a role for the TXA2/TXA2 receptor system in the thymic micro-environment.

1. Expression of prostacyclin receptor (IP receptor) mRNA was examined in various mouse organs, and the cells expressing IP receptor mRNA were identified by in situ hybridization studies. Co-localization of mRNA for the IP receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively. 2. IP receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes. 3. IP receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe. 4. IP receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small- and large-sized neurones were labelled but no labelling was found in the glia. Expression of PPTA mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP receptor mRNA, and about half of the IP receptor-positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP receptor-positive neurons co-expressed the EP1, EP3 and EP4 receptor, respectively. 5. These results not only verified IP receptor expression in various cells and tissues known to be sensitive to prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of prostacyclin in these tissues. They also indicated that the actions of prostacyclin on blood vessels and platelets are mediated by the same type of receptor. Absence of IP receptor mRNA in the JG cells suggests that the action of prostacyclin on renin release may be indirect.

We previously cloned a cDNA for a mouse PGE receptor positively coupled to adenylate cyclase from mouse mastocytoma cells, and reported it as EP2 subtype of PGE receptor [Honda, A., Sugimoto, Y., Namba, T., Watabe, A., Irie, A., Negishi, M., Narumiya, S. and Ichikawa, A. (1993) J. Biol. Chem. 268, 7759–7762]. However, it is not sensitive to one of the EP2 agonists, butaprost. Recently another subtype of PGE receptor coupled to adenylate cyclase has been identified pharmacologically and named EP4. These findings have led us to examine whether the cloned receptor is the EP4 subtype. AH23848B, a selective EP4 antagonist, not only displaced the [3H]PGE2 binding to the cloned receptor but antagonized the PGE2-stimulated cAMP formation in the receptor. In contrast, EP2 specific agonists, butaprost and 19(R)OH-PGE2 neither bound to the receptor nor stimulated the cAMP formation. These results suggest that this receptor previously reported as ‘EP2’ subtype is identical to the pharmacologically defined EP4 subtype and not of EP2 subtype.

Distribution of the messenger RNA for the prostaglandin E receptor subtype EP3 was investigated by in situ hybridization in the nervous system of the mouse. The hybridization signals for EP3 were widely distributed in the brain and sensory ganglia and specifically localized to neurons. In the dorsal root and trigeminal ganglia, about half of the neurons were labeled intensely. In the brain, intensely labeled neurons were found in Ammon's horn, the preoptic nuclei, lateral hypothalamic area, dorsomedial hypothalamic nucleus, lateral mammillary nucleus, entopeduncular nucleus, substantia nigra pars compacta, locus coeruleus and raphe nuclei. Moderately labeled neurons were seen in the mitral cell layer of the main olfactory bulb, layer V of the entorhinal and parasubicular cortices, layers V and VI of the cerebral neocortex, nuclei of the diagonal band, magnocellular preoptic nucleus, globus pallidus and lateral parabrachial nucleus. In the thalamus, moderately labeled neurons were distributed in the anterior, ventromedial, laterodorsal, paraventricular and central medial nuclei. Based on these distributions, we suggest that EP3 not only mediates prostaglandin E2 signals evoked by blood-borne cytokines in the areas poor in the blood-brain barrier, but also responds to those formed intrinsically within the brain to modulate various neuronal activities. Possible EP3 actions are discussed in relation to the reported neuronal activities of prostaglandin E2 in the brain.

A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost ⪢ carbacyclin ⪢ prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF2α did not inhibit it. Iloprost in a concentration-dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.

A functional cDNA for a mouse prostacyclin receptor was isolated from a mouse cDNA library by reverse transcription polymerase chain reaction and hybridization screening. The cDNA encodes a polypeptide of 417 amino acid residues with putative seven transmembrane domains and an calculated molecular weight of 44,722. The amino acid sequence is 30-40% identical in the transmembrane domains to those of the mouse prostaglandin (PG) E receptor subtypes and thromboxane A2 receptor. [3H]Iloprost, a specific prostacyclin receptor radioligand, specifically bound to the membrane of Chinese hamster ovary cells permanently expressing the cDNA with Kd of 4.6 nM. This binding was displaced with unlabeled prostanoids in the order of cicaprost > iloprost, both prostacyclin agonists > PGE1 > carbacyclin >> PGD2 approximately STA2, a thromboxane A2 agonist approximately PGE2 > PGF2 alpha. Iloprost in a concentration-dependent fashion increased cAMP level and generated inositol phosphates in these cells, indicating that the receptor couples to multiple signal transduction pathways. Northern blot analysis revealed that the mRNA is expressed most abundantly in thymus, followed by spleen, heart, and lung. In situ hybridization of thymus showed that it is expressed exclusively in medulla and not in cortex.

Thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) are two of the most representative eicosanoids that are formed from arachidonic acid. They produce a broad spectrum of biological effects mediated through specific cell surface receptors. We have mapped genetic loci for TXA2 receptor, Tbxa2r, and for PGE2 receptor subtypes EP2 and EP3, Ptgerep2 and Ptgerep3, respectively, using restriction fragment length variants in interspecific backcross mice. None of the three loci cosegregated with each other. Tbxa2r mapped to Chr 10, Ptgerep2 mapped to the centromeric region of Chr 15, and Ptgerep3 mapped to the distal end of Chr 3. Possible human loci for these receptors are predicted based on the homology between mouse and human chromosomes.

A functional cDNA clone encoding a mouse EP1 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library by cross-hybridization with the mouse thromboxane A2 receptor cDNA. The clone isolated encodes a protein consisting of 405 amino acid residues with putative seven-transmembrane domains. [3H]PGE2 specifically bound to the membrane of Chinese hamster ovary cells expressing this clone. The binding to the membrane was displaced with unlabeled PGs in the order of PGE2 > iloprost (a prostacyclin analogue) > PGE1 > PGF2 alpha > U-46619 (a thromboxane A2 analogue) > PGD2. The binding was also inhibited by 17-phenyl trinor PGE2 (an EP1 agonist) and sulprostone (an EP1 and EP3 agonist) but not by 11-deoxy PGE1 (an EP2 and EP3 agonist) and butaprost (an EP2 agonist). PGE2 induced a rapid increase in intracellular Ca2+ concentration in Chinese hamster ovary cells expressing the receptor. These results suggest that this receptor belongs to EP1 subtype of PGE receptor. Northern blot analysis demonstrated that the mRNA of this receptor is expressed abundantly in kidney and in a lessor amount in lung.

PEPTIDE hormones, neurotransmitters, and autacoids activate a family of seven-transmembrane-domain receptors1. Each of these receptors specifically couples to one of several G proteins, Gs, Gi, Go and Gp, to activate a specific second messenger system2. Cell surface receptors for prostanoids have been characterized pharmacologically3 and the complementary DNAs for thrombox-ane A2 receptor4,5 and the EP3 subtype of the prostaglandin (PG)E receptor6 reveal that they belong to the seven-transmembrane-domain receptor family. The EP3 receptor mediates the diverse physiological actions of PGE2 (ref. 3). Although most of them occur through coupling of the EP3 receptor to Gi and inhibition of adenylyl cyclase, the EP3-mediated contraction of uterine muscle can only occur by activation of another second messenger pathway7. In chromaffin cells, two different second messenger pathways are activated by PGE2 binding to an apparently single EP3 receptor class8. Here we show that at least four isoforms of the EP3 receptor, which differ only at their C-terminal tails and are produced by alternative splicing, couple to different G proteins to activate different second messenger systems.

A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening. The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains. The sequence revealed significant homology to the human thromboxane A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding. The binding was displaced with unlabeled PGs in the order of PGE2 = PGE1 greater than iloprost greater than PGF2 alpha greater than PGD2. The EP3-selective agonists, M&B 28,767 or GR 63799X, potently competed for the [3H]PGE2 binding, but no competition was found with EP1- or EP2-selective ligands. PGE2 and M&B 28,767 decreased forskolin-induced cAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA. Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.

Functional cDNA clones for two isoforms of the mouse prostaglandin E receptor EP3 subtype derived from alternative RNA splicing were obtained. The two isoforms are only different in the sequence of the putative cytoplasmic carboxyl-terminal tail and their hydrophobicity; one isoform, named EP3 alpha, has a hydrophilic tail, and the other, named EP3 beta, has a hydrophobic tail. When expressed, the two receptors displayed identical ligand binding properties but different responses to guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). Without a change in the Bmax value, GTP gamma S increased Kd for prostaglandin E2 of EP3 beta and decreased that of EP3 alpha. These effects were abolished by the treatment of membranes with pertussis toxin and restored by the addition of Gi2. Although both isoforms exerted inhibition of forskolin-induced cAMP accumulation, three orders lower concentrations of agonists were required for EP3 alpha than EP3 beta for 50% inhibition of cAMP formation. A similar difference in agonist potency was observed also for agonist-induced stimulation of GTPase activity in membranes. Thus, the two receptors with different carboxyl-terminal tails show different coupling to the Gi protein, leading to the opposite responses to GTP in the ligand binding affinity and to different affinities of the agonist-occupied receptors to the G proteins.

A functional cDNA clone for mouse prostaglandin (PG) F receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of cloned prostanoid receptors, and cross-hybridization screening. The mouse PGF receptor consists of 366 amino acid residues with putative seven transmembrane domains. The sequence revealed the highest homology to the EP1 subtype of PGE receptor and thromboxane (TX) A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGF2 alpha binding. The binding was displaced with unlabeled PGs in the order of PGF2 alpha = 9 alpha, 11 beta PGF2 > PGF 1 alpha > PGD2 > STA2 (a stable TXA2 agonist) > PGE2 > iloprost (a stable PGI2 agonist). PGF2 alpha increased inositol trisphosphate formation in a concentration-dependent manner in COS cells expressing PGF receptor. RNA blot and in situ hybridization analyses demonstrated that the PGF receptor transcripts are abundantly expressed in luteal cells of corpus luteum and in a lesser amount in kidney, heart, stomach, and lung.