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Gene induction by tumor necrosis factor-alpha (TNFalpha) or interleukin-1beta (IL-1beta) is mediated in part by activation of the transcription factor nuclear factor kappaB (NF-kappaB), and requires signal adaptor molecules such as TNF receptor-associated factor (TRAFs). The latter interact with the NF-kappaB-inducing kinase (NIK), which is believed to be part of the IkappaB kinase complex. Although the precise mechanism is to be elucidated, it is well-known that antioxidant treatments inhibit the inflammatory cytokine-induced NF-kappaB activation. Thioredoxin (TRX) is a 12-kDa endogenous protein that regulates various cellular functions by modulating the redox state of proteins, overexpression of this molecule inhibits NF-kappaB activation. To elucidate the roles of TRX in the signal transduction of the cytokines, we investigated the effects of TRX on NF-kappaB activation induced by cytokine treatment or by overexpression of the signaling molecules. Our data show that TRX treatment inhibits NF-kappaB-dependent transcription at the level of downstream of TRAFs and upstream of NIK: TRX inhibited TRAF2-, TRAF5-, and TRAF6-induced NF-kappaB activation but does not inhibit NIK-, IKKalpha-, and MEKK-induced activation. In addition, we show that TRX inhibits NF-kappaB activation in a manner different from that for SAPK (stress activated protein kinase) inhibition.

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.

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.

We recently identified four isoforms of bovine prostaglandin E receptor EP3 subtype, which are coupled to different signaling pathways; EP3A is coupled to inhibition of adenylate cyclase, while EP3B and EP3C are coupled to its stimulation and EP3D is coupled to phosphatidylinositol turnover, in addition to the adenylate cyclase system (Namba, T., Sugimoto, Y., Negishi, M., Irie, A., Ushikubi, F., Kakizuka, Ito, S., A., Ichikawa, A., and Narumiya, S. (1993) Nature 365, 166-170). We examined here the identity of coupled G proteins and their regulation by one of the isoforms, EP3C, in the membranes of EP3C cDNA-transfected Chinese hamster ovary cells. M&B 28767, an EP3 agonist, stimulated the GTPase activity in the pertussis toxin (PT)-treated cell membrane, but inhibited it in the cholera toxin (CT)-treated cell membrane, while the agonist neither stimulated nor inhibited it in the both PT- and CT-treated cell membrane. In the PT- and CT-treated cell membrane reconstituted with various G proteins, M&B 28767 inhibited the GTPase activity of G(o), but stimulated that of Gs. On the other hand, M&B 28767 did not affect the GTPase activity of Gi1, Gi2, or Gi3. M&B 28767 increased the apparent affinity of G(o) for GDP without any change in that for GTP, as assessed by displacement of [35S]GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to G(o). In contrast, M&B 28767 increased the apparent affinity of Gs for GTP but decreased that for GDP. These results demonstrated that the EP3 receptor isoform is coupled to two different G proteins, and oppositely regulates their activities, inhibition of G(o), and stimulation of Gs.

Interleukin 3-dependent BNu-2c13 mast cells, mucosal type-like mast cells, exhibited a specific high-affinity binding site for [ 3 H]prostaglandin (PG) E 2 . The binding was completely displaced by M&B 28767, an EP 3 -selective agonist, but not by EP 1 - or EP 2 -selective ligands, indicating that the PGE 2 binding site is of the EP 3 subtype PGE receptor. Whereas the EP 3 subtype is presumed to be coupled to inhibition of adenylate cyclase in various tissues and cells, in BNu-2c13 cells PGE 2 had no ability to inhibit adenylate cyclase activity, while it induced concentration-dependent stimulation of phosphoinositide metabolism and caused an increase in the intracellular free Ca 2+ concentration in a pertussis toxin-sensitive manner. PGE 2 by itself did not evoke histamine release from the cells, but it markedly stimulated histamine release in concert with ionomycin, a Ca 2+ ionophore. The PGE 2 -stimulated release was also comletely blocked by pertussis toxin. Thus, the PGE receptor expressed on BNu-2c13 mast cells is of the EP 3 subtype and is linked to phosphoinositide metabolism via a pertussis toxin-sensitive G protein, and this activation leads to histamine release.

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.

A cDNA clone for the mouse thromboxane A2 receptor was isolated from mouse lung cDNA library. The cDNA has a 1,023 base pair open reading frame which encodes a protein of 341 amino acid residues. STA2 and U-46619 induced inward current in Xenopus laevis oocytes injected with the transcript of the clone. Specific binding of [3H]S-145 was found in membranes of COS-1 cells transfected with the cDNA (Kd = 3.3 nM) and was displaced with unlabeled prostaglandins and thromboxane analogues in the order of S-145 greater than STA2 greater than U-46619 greater than PGD2 greater than PGF2 alpha = PGE2. Northern blot analysis demonstrated that thromboxane A2 receptor mRNA is expressed abundantly in thymus, spleen and lung.

C3 ADP-ribosyltransferase is an exoenzyme produced by certain strains of Clostridium botulinum types C and D, which specifically ADP-ribosylates rho and rac proteins in eukaryotic cells. The enzyme was purified from a culture filtrate of C. botulinum type C strain 003-9, and the amino acid sequence from the amino-terminal Ser to Asn192 was determined by Edman degradation. Using a set of degenerate primers based on the sequence, we amplified a part of the gene for this enzyme by polymerase chain reaction. A 2.1-kilobase pair HincII fragment of C. botulinum DNA containing the whole structural gene was then identified by Southern analysis with the polymerase chain reaction product as a probe, and the complete nucleotide structure of the gene together with flanking regions was determined by cloning and DNA sequencing the HincII fragment. The gene encodes a protein of 244 amino acids with a Mr of 27,362 which begins with a putative signal peptide of 40 amino acids. Escherichia coli carrying this gene produced the active enzyme, and about 60% of it was found in the culture medium. Immunoblot analysis with antiserum against the enzyme revealed the presence of two immunoreactive proteins of 27 and 23 kDa in the cytoplasmic/membrane fraction and only the 23-kDa protein in the periplasm and the medium, suggesting that the enzyme expressed is processed in the E. coli, exported into the periplasm and released into the culture medium.

Alcohols, inhaled anesthetics, and some injectable anesthetics inhibit the function of N-methyl-d-aspartate (NMDA) receptors, but the mechanisms responsible for this inhibition are not fully understood. Recently, it was shown that ethanol inhibition of NMDA receptors was reduced by mutation of residues in the transmembrane (TM) segment 3 of the NR1 subunit (F639A) or in TM4 of the NR2A subunit (A825W), suggesting putative ethanol binding sites. We hypothesized that the actions of other anesthetics might also require these amino acids and evaluated the effects of anesthetics on the NMDA receptors expressed in Xenopus oocytes with two-electrode voltage-clamp recording. Effects of hexanol, octanol, isoflurane, halothane, chloroform, cyclopropane, 1-chloro-1,2,2-trifluorocyclobutane, and xenon were reduced or eliminated in the mutant NMDA receptors, whereas the inhibitory effects of nitrous oxide, ketamine, and benzene were not affected by these mutations. Rapid applications of glutamate and glycine by a T-tube device provided activation time constants, which suggested different properties of ketamine and isoflurane inhibition. Thus, amino acids in TM3 and TM4 are important for the actions of many anesthetics, but nitrous oxide, benzene, and ketamine seem to have distinct mechanisms for inhibition of the NMDA receptors.

The intracellular generation of reactive oxygen species (ROS) by 6-formylpterin and its effects on the human T cell functions were examined in vitro. When T cells isolated from fresh blood were incubated with 6-formylpterin for 1hr, the oxygen consumption and concomitant ROS generation were observed. The incubation of T cells with 50-500microM 6-formylpterin for 24hr brought about the elevation of intracellular ROS without inducing cell death. In contrast, the incubation of T cells with exogenously administered hydrogen peroxide (H(2)O(2)) or other pterin derivatives (6-hydroxymethylpterin, pterin-6-carboxylic acid, pterin, neopterin, biopterin and folic acid) for 24hr did not cause the intracellular ROS elevation. In the T cells stimulated with mitogenic lectin phytohemagglutinin (PHA) in conjunction with phorbol myristate acetate (PMA), 6-formylpterin suppressed the NF-kappaB-dependent transcription, the production of cytokines (IFN-gamma and IL-2) and the cell proliferation. These suppressive effects of 6-formylpterin were all reversed by N-acetyl-l-cystein (NAC). However, 6-formylpterin did not inhibit the NF-kappaB-DNA binding of the nuclear extracts obtained from the PHA/PMA-stimulated T cells. Since the NF-kappaB-DNA binding assay performed in vitro merely shows the presence or absence of NF-kappaB subunit in the nuclear extracts but not guarantees the actual binding of NF-kappaB with DNA in the nucleus, these findings suggest that intracellular ROS generated by 6-formylpterin does not affect the translocation of NF-kappaB to the nucleus but that it inhibits the NF-kappaB-dependent transcription in the nucleus, resulting in the suppression of cytokine production and cell proliferation in the activated T cells.

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.

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.

A functional cDNA clone for a third isoform of the mouse prostaglandin-E-receptor EP3 subtype, derived by alternative RNA splicing, named the EP3 gamma receptor, was obtained in addition to those for the two other isoforms, EP3 alpha and EP3 beta. The three isoforms are only different in the amino acid sequence of the putative cytoplasmic carboxy-terminal tail. When expressed, EP3 gamma shows identical ligand-binding properties to these of the other isoforms. The EP3-selective agonist, MB 28767, increased the basal cAMP level and inhibited the forskolin-induced increase in the cAMP level in EP3 gamma, while it decreased both the basal and forskolin-elevated cAMP levels in EP3 alpha and EP3 beta. The MB 28767-stimulated GTPase activity consisted of pertussis-toxin-sensitive and cholera-toxin-sensitive portions in the EP3 gamma-expressing cell membrane, suggested that EP3 gamma is coupled to both guanine nucleotide-binding inhibitory and stimulatory proteins. These results indicate that EP3 gamma is coupled to both stimulation and inhibition of adenylate cyclase, but that EP3 alpha and EP3 beta are exclusively coupled to inhibition of adenylate cyclase. Thus, alternative splicing produces a third isoform with a different carboxy-terminal tail, which differs from the other two isoforms in the specificity of coupling to a signal-transduction pathway.

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.

The functional interaction of prostaglandin E (PGE) receptor EP 3 subtype with GTP-binding proteins (G proteins) was characterized in the membranes prepared from mouse EP 3 receptor cDNA-transfected Chinese hamster ovary cells. PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in CHO cells expressing EP 3 receptor and this inhibition was abolished by pertussis toxin (PT) treatment. The PGE 2 binding to the membranes was increased by GTPγS, and PT treatment also increased the binding activity to the same level as that increased by GTPγS, but the sensitivity of GTPγS was lost. Reconstitution with PT-sensitive G proteins into the ADP-ribosylated membranes reduced the PGE 2 binding activity with the following preference: Gi 1 = Gi 2 > Gi 3 > Go , but GTPγS completely blocked the reduction by G proteins. The G-protein-induced reduction of the binding was due to the increase in K d without the change of B max , and due to suppression of association rate. [ 3 H]PGE 2 -bound EP 3 receptor solubilized from the ADP-ribosylated membranes in the presence or absence of GTPγS was eluted at the position of M r = approx . 60 kDa, similar to the relative molecular mass of EP 3 receptor deduced from its amino acid sequence. In contrast, [ 3 H]PGE 2 -bound receptor solubilized from Gi2-reconstituted membranes was eluted at the position of M r = approx . 130 kDa, corresponding to the M r of the complex of EP 3 receptor and Gi2, but GTPγS shifted the position of its elution from M r = 130 to 60 kDa. Furthermore, addition of PGE 2 stimulated the GDP release from G proteins reconstituted into the ADP-ribosylated membranes, and PGE 2 inhibited forskolin-stimulated adenylate cyclase activity in G-protein-reconstituted membranes with a selectivity order of Gi 1 = Gi 2 > Gi 3 > Go . These results indicate that EP 3 receptor can functionally couple to PT-sensitive G proteins and unusually the complex form with G proteins has low affinity for the ligand but the form not associated with G proteins has high affinity.

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.