Human EP Research Articles

Structure-activity relationship of triaryl propionic acid analogues on the human EP3 prostanoid receptor.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Triaryl compounds bearing an ortho-substituted propionic acid moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinities of key compound on all eight human prostanoid receptors is reported.

Structure–Activity Relationship of Biaryl Acylsulfonamide Analogues on the Human EP 3 Prostanoid Receptor

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.

Phosphorylation of Glycogen Synthase Kinase-3 and Stimulation of T-cell Factor Signaling following Activation of EP2 and EP4 Prostanoid Receptors by Prostaglandin E2

Recently we have shown that the FP(B) prostanoid receptor, a G-protein-coupled receptor that couples to Galpha(q), activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 12489-12492). We now report that the EP(2) and EP(4) prostanoid receptors, which couple to Galpha(s), also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated approximately 10-fold over basal by 1 h of treatment with prostaglandin E(2) (PGE(2)) in HEK cells that were stably transfected with the human EP(2) and EP(4) receptors. This stimulation of reporter gene activity was accompanied by a PGE(2)-dependent increase in the phosphorylation of both glycogen synthase kinase-3 (GSK-3) and Akt kinase. H-89, an inhibitor of protein kinase A (PKA), completely blocked the agonist-dependent phosphorylation of GSK-3 in both EP(2)- and EP(4)-expressing cells. However, H-89 pretreatment only blocked PGE(2)-stimulated Lef/Tcf reporter gene activity by 20% in EP(4)-expressing cells compared with 65% inhibition in EP(2)-expressing cells. On the other hand wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had the opposite effect and inhibited PGE(2)-stimulated reporter gene activity to a much greater extent in EP(4)-expressing cells as compared with EP(2)-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP(2) receptors occurs primarily through a PKA-dependent pathway, whereas EP(4) receptors activate Tcf/Lef signaling mainly through a phosphatidylinositol 3-kinase-dependent pathway. This is the first indication of a fundamental difference in the signaling potential of EP(2) and EP(4) prostanoid receptors.

Structure–activity relationship of cinnamic acylsulfonamide analogues on the human EP 3 prostanoid receptor

Potent and selective antagonists of the human EP 3 receptor have been identified. The structure–activity relationship of the chemical series was conducted and we found several analogues displaying sub-nanomolar K i values at the EP 3 receptor and micromolar activities at the EP 1, EP 2 and EP 4 receptors. The effect of added human serum albumin (HSA) on the binding affinity at the EP 3 receptor was also investigated.

Key structural features of prostaglandin E(2) and prostanoid analogs involved in binding and activation of the human EP(1) prostanoid receptor.

The structure-activity relationship (SAR) of prostaglandin (PG) E(2) at the human EP(1) prostanoid receptor (designated hEP(1)) was examined via the binding and activation of this receptor by a series of 55 prostanoids and analogs. Using clonal human embryonic kidney 293 cell lines expressing recombinant hEP(1), affinity (K(i)), potency (EC(50)), and efficacy data were obtained using a radioligand competitive binding assay and an aequorin-based calcium functional assay. All compounds behaved as full agonists (90-100% of the response elicited by PGE(2)) in this assay, and the correlation between the K(i) and EC(50) values was highly significant (R(2) = 0.86). The results from the SAR analysis can be summarized as follows: 1) the existence and configuration of hydroxyl groups at the 11 and 15 positions of PGE(2) and prostanoid analog structures play a critical role in agonist activity; 2) the carboxyl group is also important for activity and modification of the carboxylic acid to various esters results in greatly reduced affinity and potency; 3) the activity of structures with moderate or weak potency can be enhanced by modification of the omega-tail; and 4) modifications to the ketone at the 9-position are better tolerated, with 9-deoxy-9-methylene-PGE(2) being the most potent agonist tested in the functional assay. The impact of other modifications on agonist potency is also discussed. The results from this study have identified, for the first time, the key structural features of PGE(2) and related prostanoids and prostanoid analogs necessary for activation of hEP(1).

Structure–activity relationship on the human EP 3 prostanoid receptor by use of solid-support chemistry

Potent and selective EP 3 receptor ligands were found by making a library using solid-support chemistry. These compounds can be obtained by a Suzuki coupling reaction of a solid-supported benzyl bromide using various boronic acids. The yields obtained for this reaction were in the range of 24–95% of arylmethyl cinnamic acid 1 after cleavage from the Wang resin.

Pharmacological characterization of [(3)H]-prostaglandin E(2) binding to the cloned human EP(4) prostanoid receptor.

Prostaglandin (PG) E(2) (PGE(2)) is a potent prostanoid derived from arachidonic which can interact with EP(1), EP(2), EP(3) and EP(4) prostanoid receptor subtypes. Recombinant human EP(4) receptors expressed in human embryonic kidney (HEK-293) cells were evaluated for their binding characteristics using [(3)H]-PGE(2) and a broad panel of natural and synthetic prostanoids in order to define their pharmacological properties. [(3)H]-PGE(2) binding was optimal in 2-[N-Morpholino]ethanesulphonic acid (MES) buffer (pH 6.0) yielding 98+/-0.7% specific binding. The receptor displayed high affinity (K(d)=0.72+/-0.12 nM; n=3) for [(3)H]-PGE(2) and interacted with a saturable number of binding sites (B(max)=6.21+/-0.84 pmol mg(-1) protein). In competition studies, PGE(2) (K(i)=0.75+/-0.03 nM; n=12) and PGE(1) (K(i)=1.45+/-0.24 nM; n=3) displayed high affinities, as did two derivatives of PGE(1), namely 11-deoxy-PGE(1) (K(i)=1.36+/-0.34 nM) and 13,14-dihydro-PGE(1) (K(i)=3.07+/-0.29 nM). Interestingly, synthetic DP receptor-specific agonists such as BW245C (K(i)=64.7+/-1.0 nM; n=3) and ZK118182 (K(i)=425+/-42 nM; n=4), and the purported EP(3) receptor-specific ligand enprostil (K(i)=43.1+/-4.4 nM), also displayed high affinity for the EP(4) receptor. Two known EP(4) receptor antagonists were weak inhibitors of [(3)H]-PGE(2) binding akin to their known functional potencies, thus: AH23848 (K(i)=2690+/-232 nM); AH22921 (K(i)=31,800+/-4090 nM). These studies have provided a detailed pharmacological characterization of the recombinant human EP(4) receptor expressed in HEK-293 cells.

New class of biphenylene dibenzazocinones as potent ligands for the human EP 1 prostanoid receptor

A new class of potent and selective ligands for the human EP 1 prostanoid receptor is described. SAR studies reported herein allowed the identification of several potent dibenzazocinones bearing an acylsulfonamide side chain. The binding affinity of these compounds on all eight human prostanoid receptors is reported.

A conserved threonine in the second extracellular loop of the human EP 2 and EP 4 receptors is required for ligand binding

G protein coupled receptors for prostaglandins are activated when agonists are bound to a binding pocket formed in part by the seven transmembrane domains. Recent studies have determined that substitution of a conserved threonine in the second extracellular loop of the prostaglandin EP 3 receptor resulted in increased affinity for ligands with a C1 methyl ester moiety. The homologous threonine in the second extracellular loop of the human prostaglandin EP 2 and EP 4 receptors was mutated to alanine. When expressed in COS1 cells, detectable radioligand binding at both of these receptors bearing the threonine to alanine substitution (EP 2T185A; EP 4T168A) was abolished, as well as the receptors' ability to stimulate intracellular [cAMP]. In contrast, EP 2 and EP 4 receptors bearing conservative threonine to serine mutations (EP 2T185S; EP 4T168S) displayed K d values for [ 3 H ]prostaglandin E 2 similar to wild type receptors: 8.8±0.7 nM for EP 2T185S compared to 12.9±1.2 nM for EP 2 wild type; 2.0±0.8 nM for EP 4T168S compared to 0.9±0.3 nM for the EP 4 wild type receptor. The EC 50 values for cAMP stimulation were 1.3±0.6 nM for EP 2 wild type; 2.7±1.3 nM for EP 2T185S; 1.1±0.3 nM for EP 4 wild type; and 1.4±0.33 nM for EP 4T168S. These studies suggest a critical role for the hydroxyl moiety on these conserved threonine residues at position 168/185 of the second extracellular loop in prostaglandin receptor–ligand interactions.

Prostanoid receptor heterogeneity through alternative mRNA splicing

Prostaglandin (PG) and thromboxane (TX) receptors are G-protein coupled receptors that mediate the physiological actions of the five principal prostanoid metabolites: PGD 2, PGE 2, PGF 2α, PGI 2 (prostacyclin) and TXA 2. Five major subdivisions of the prostanoid receptor family have been defined pharmacologically which correspond to each of the metabolites as follows: DP, EP, FP IP and TP. The EP receptors have been further classified pharmacologically into the EP 1, EP 2, EP 3 and EP 4 subtypes. Molecular biological studies have resulted in the cloning of cDNA's encoding all of these prostanoid receptors. In addition, the cloning of these receptors has revealed further heterogeneity through the use of alternative mRNA splicing. Specifically, mRNA splice variants have been identified for the EP 1, EP 3, FP and TP receptors. Interestingly, except for the EP 1 receptors, the mechanisms giving rise to these receptor isoforms involves the use of splice sites located in the cytoplasmic carboxyl termini of these receptors. Thus, the eight human EP 3 isoforms that have been identified are otherwise identical except for their carboxyl termini. Similarly, the optional use of a potential splice site encoding the carboxyl terminus gives rise to each of the two FP and TP receptor isoforms. Because the carboxyl termini of G-protein coupled receptors are generally implicated in interactions with G-proteins, it is not surprising that these receptor isoforms differ mainly with respect to their activation of second messenger pathways and not in their pharmacological characteristics. Differences also exist with respect to their levels of constitutive activity (e.g., in the absence of agonist) and in their desensitization.

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP 2 and EP 3 subtypes

We examined the contribution of specific EP receptors in regulating cell growth. By RT–PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE 2 receptor subtypes associated with cAMP signaling (EP 2, EP 3, and small amounts of EP 4). In actively growing, non-confluent primary keratinocyte cultures, the EP 2 and EP 4 selective agonists, 11-deoxy PGE 1 and 1-OH PGE 1, caused complete reversal of indomethacin-induced growth inhibition. The EP 3/EP 2 agonist (misoprostol), and the EP 1/EP 2 agonist (17-phenyl trinor PGE 2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP 2 and/or EP 4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP 3 receptors by sulprostone, which is virtually devoid of agonist activity at EP 2 or EP 4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP 3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanism, EP 3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

The C-terminal domain of the G s-coupled EP 4 receptor confers agonist-dependent coupling control to G i but no coupling to G s in a receptor hybrid with the G i-coupled EP 3 receptor

Prostaglandin E 2 receptors (EPR) belong to the family of G-protein-coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G-proteins. EP 1R are coupled to G q, EP 2 and EP 4R to G s and EP 3R to G i. Different C-terminal splice variants of the bovine EP 3R are coupled to different G-proteins. A mouse EP 3R whose C-terminal domain had been partially truncated no longer showed agonist-induced G i-protein activation and was constitutively active. In order to test the hypothesis that the C-terminal domain confers coupling specificity of the receptors on the respective G-proteins, a cDNA for a hybrid rEP 3hEP 4R, containing the N-terminal main portion of the G i-coupled rat EP 3βR including the 7th transmembrane domain and the intracellular C-terminal domain of the G s-coupled human EP 4R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP 3hEP 4R cDNA expressed a plasma membrane PGE 2 binding site with a slightly lower K d value for PGE 2 but an identical binding profile for receptor-specific ligands as cells transfected with the native rat EP 3βR. In HepG 2 cells stably transfected with the chimeric rEP 3hEP 4R cDNA PGE 2 did not increase cAMP formation characteristic of G s coupling but attenuated the forskolin-stimulated cAMP synthesis characteristic of G i coupling. This effect was inhibited by pre-treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP 3βR. Apparently, the intracellular C-terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.

Differential localization of prostaglandin E receptor subtypes in human kidney.

Four prostaglandin E2 (PGE2) receptors designated EP1, EP2, EP3, and EP4 have been pharmacologically identified, cloned, and sequenced. The present studies determined the intrarenal distribution of these EP-receptor subtypes in human kidney using in situ hybridization with riboprobes for the human EP receptors. mRNA for the phosphatidylinositol hydrolysis-coupled EP receptor was highly expressed in cortical, outer medullary, and inner medullary collecting duct. RNA for the Gi-coupled EP3 receptor was primarily expressed in the cortical and outer medullary collecting duct, as well as in the medullary thick ascending limb; however, it was absent from the inner medullary collecting duct. Expression of mRNA for EP1 and EP3 in connecting segment could not be excluded. There was no expression of the GS-coupled EP2 receptor mRNA detected in human kidney by in situ hybridization; however, mRNA for the GS-coupled EP4 receptor was highly expressed in the glomerulus. These studies demonstrate distinct regions of intrarenal expression for the different EP receptors and suggest that each receptor subtype may modulate different aspects of renal function in humans.

6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP 2 receptor antagonist

On studying the interaction of various ligands with the pharmacologically defined, recombinant human EP 2 receptor (Regan et al., Mol Pharmacol 46: 213–220, 1994), we discovered that the putative EP 1 receptor antagonist 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809) also has affinity for the human EP 2 receptor. Moreover, AH 6809 behaved as an EP 2 receptor antagonist and inhibited prostaglandin E 2 (PGE 2)-stimulated increases in cyclic AMP. These findings have significant implications for studies that employ AH 6809 to determine the pharmacological basis of PGE 2-induced responses in human cells and tissues.

Adaptive estimation of single response evoked potentials

A new adaptive filtering algorithm and structure is developed to estimate response-to-response variations in evoked responses. The evoked responses are modeled as the sum of three uncorrelated signal components: ensemble average, noise, and stochastic signal variation. A two stage time sequenced filter structure exhibiting improved convergence characteristics is developed along with a modified P-vector algorithm (mPa) which eliminates the need for a separate desired signal electrode. The mPa adaptive filter is tested with simulated and human EP data. The mPa filter is able to estimate signal variations from one response to the next.

Detection of Adenylate Cyclase-Coupled Receptors in Xenopus Oocytes by Coexpression with Cystic Fibrosis Transmembrane Conductance Regulator

To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)-a cAMP-dependent Cl − channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl − current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP 2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.

Molecular cloning and expression of human EP3 receptors: evidence of three variants with differing carboxyl termini

1. The polymerase chain reaction (PCR) was used in combination with plaque hybridization analysis to clone four variants of the EP3 prostaglandin receptor from a human small intestine cDNA library. 2. Three of these variants, i.e. the EP3A, EP3E and EP3D, share the same primary amino acid sequence except for their carboxyl termini, which diverge from one another at the same point, approximately 10 amino acids away from the end of the seventh membrane spanning domain of the receptor. The fourth variant (EP3A1) has a nucleotide coding sequence identical to EP3A but has a completely different 3' untranslated sequence. 3. The carboxyl termini of the three isoforms differ most obviously in length with the EP3A being the longest (41 amino acids) and the EP3E being the shortest (16 amino acids). They also differ in content with the EP3A containing 9 serine and threonines in its carboxyl terminus and the EP3E none. 4. Transient expression in eukaryotic cells showed that the human EP3 receptor variants had similar but not identical radioligand binding properties and differed in their functional coupling to second messenger pathways. Up to 3 pmol mg-1 protein of [3H]-prostaglandin E2 binding could be obtained with more than 95% specific binding. Using a reporter gene assay, as a measure of intracellular cyclic AMP levels, the EP3A coupled more efficiently to the inhibition of adenylyl cyclase than did the EP3E. 5. PCR was used to confirm the presence of mRNAs encoding the four human EP3 receptor variants in tissues of the human small intestine, heart and pancreas. These findings indicate that the EP3 receptor variants identified here are likely to be expressed in tissues. The differences in the carboxyl termini at the protein level, and in the 3' untranslated regions at the mRNA level, could be profound in terms of the regulation and functional coupling of these receptor isoforms.

Cloning and expression of three isoforms of the human EP 3 prostanoid receptor

Functional cDNA clones coding for three isoforms of the human prostaglandin E receptor EP 3 subtype have been isolated from kidney and uterus cDNA libraries. The three isoforms, designated hEP 3-I, hEP 3-II and hEP 3-III, have open reading frames corresponding to 390, 388 and 365 amino acids, respectively. They differ only in the length and amino acid composition of their carboxy-terminal regions, beginning at position 360. The human EP 3 receptor has seven predicted transmembrane spanning domains and therefore belongs to the G-protein-coupled receptor family. The rank order of potency for prostaglandins and related analogs in competition for [ 3H]PGE 2 specific binding to membranes prepared from transfected COS cells was comparable for all three isoforms, and as predicted for the EP 3 receptor, with PGE 2 = PGE 1 > PGF 2α = iloprost > PGD 2 ⪢ U46619. In addition, the EP 3-selective agonist MB28767 was a potent competing ligand with an IC 50 value of 0.3 nM, whereas the EP 1-selective antagonist AH6909 gave IC 50 values of 2–7 μM and the EP 2-selective agonist butaprost was inactive. In summary, we have cloned three isoforms of the human EP, receptor having comparable ligand binding properties.

In Vivo and In Vitro Erythropoietin Activities in Cultures of a Hepatocellular Carcinoma Cell Line

The present studies were undertaken to characterize erythropoietin (Ep) production in an Ep-producing hepatocellular carcinoma (Hep3B) cell line. Hep3B cells which had been maintained in culture were transplanted under the renal capsule and subcutaneously in nude mice. The Hep3B xenograft doubling time is approximately 7 days. The mean hematocrit value of the Hep3B tumor-bearing nude mice was 33.2 +/- 1.1% (n = 8), which was significantly lower than that of control nongrafted nude mice (40.8 +/- 1.7%, n = 5). The Hep3B tumor-bearing nude mice showed significantly higher Ep levels in the sera (37.5 +/- 5.5 munits/ml, n = 8) than control nude mice (13.5 +/- 2.7 munits/ml, n = 5). Ep levels in the sera were correlated (R = 0.714) with the total Ep in the tumor extracts, whereas no Ep was detectable in any of the kidney extracts. On the other hand, an inverse linear relationship (R = -0.811) between the hematocrit values and Ep levels in the sera was demonstrated in the Hep3B tumor-bearing nude mice. The Hep3B cells recultured after growing in the nude mice were capable of enhancing Ep production in response to hypoxia, very similar to the original Hep3B cells which had been maintained in culture during the same time period. In addition, 15-methyl-prostaglandin E1 at a concentration range of 4-400 ng/ml produced significant increases in Ep secretion and cAMP accumulation in Hep3B cultures under hypoxic conditions (1% O2). The Ep produced by Hep3B cells expressed 3.7 times higher in vitro bioactivity than immunoactivity. The bioactivity of Hep3B Ep was completely neutralized by an antibody to highly purified human recombinant Ep. In contrast, the in vivo bioactivity of the Hep3B Ep was less than one tenth of its immunoactivity. These results indicate that the Hep3B tumor-bearing nude mice and the in vitro Hep3B culture system may provide a reproducible model system which should be useful for studies of the mechanism of Ep production.

Fetal iron and cytochrome c status after intrauterine hypoxemia and erythropoietin administration.

Chronic fetal hypoxemia stimulates erythropoiesis and may result in a redistribution of fetal iron from plasma into erythrocytes. We studied the response of fetal plasma erythropoietin (Ep) to hypoxemia, the role of Ep in stimulating erythropoiesis in utero, and the effect of augmented erythropoiesis on fetal plasma Ep and iron and tissue cytochrome c concentrations in 19 chronically instrumented late-gestation fetal sheep. The fetuses were stimulated to produce 28 erythropoietic responses after exposure to 1) acute hypoxemia (1-5 days), 2) chronic hypoxemia (greater than 7 days), and/or 3) administration of 1,500 U recombinant human Ep concurrently during normoxemia. Plasma Ep peaked less than 12 h after the onset of hypoxemia or Ep bolus. Plasma iron decreased 24-48 h later and returned to baseline 48-96 h after normalization of Ep levels to baseline. The plasma iron response was directly related to the erythropoietin stimulus (r = 0.79, P less than 0.001) and inversely related to liver iron concentration at death (r = -0.84, P less than 0.001). Nine fetuses with depleted liver iron concentrations at autopsy had significantly lower heart and skeletal muscle iron concentrations compared with animals with 10% of control liver iron remaining. Skeletal muscle and heart iron and cytochrome c concentrations were significantly correlated. Ep has a potent biological effect on fetal erythropoiesis and iron metabolism. Augmented fetal erythropoiesis, mediated by Ep, results in decreased plasma iron, hepatic storage iron, and skeletal and cardiac muscle iron and cytochrome c. The model potentially explains the iron abnormalities found in newborn infants after fetal hypoxia.