Prostaglandin EP4 Receptor Research Articles

A Cluster of Aromatic Amino Acids in the i2 Loop Plays a Key Role for Gs Coupling in Prostaglandin EP2 and EP3 Receptors

To assess the structural requirements for G(s) coupling by prostaglandin E receptors (EPs), the G(s)-coupled EP2 and G(i)-coupled EP3beta receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3beta left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr(143) into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His(140) enabled EP3beta to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His(140) into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent Trp(141) to Ala but not at the following Tyr(142) weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe(144) to Ala but not at Tyr(145) reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing G(s)-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for G(s) coupling.

Prostaglandin EP receptor agonists differentially modulate aspirin-induced generation of 15-HETE in aspirin-sensitive asthmatics

Rationale Aspirin triggers generation of 15-hydroxyeicosaterraenoic acid (15-HETE) in peripheral blood leukocytes from aspirin-sensitive (AS) but not aspirin-tolerant (AT) patients with asthma/rhinosinusitis. The aim of this study was to determine the effect of prostaglandin EP receptor agonists (misoprostol and sulprostone) on aspirin-induced 15-HETE generation. Methods Peripheral blood leukocytes (PBLs) were isolated from AS and AT patients with asthma, and the 15-HETE release into the cell supernatant was measured with a specific immunoassay (Assay Design, MA). Results Incubation of PBLs from AS asthmatics with 200μM of lysine-aspirin significantly increased generation of 15-HETE (mean increase +260%) but lysine-aspirin did not affect 15-HETE generation in PBLs from AT asthmatics. Preincubation of cells with misoprostol or sulprostone did not affect basal 15-HETE in AS or AT patients. However, both misoprostol and sulprostone inhibited in a dose-dependent manner the ASA-induced increase in 15-HETE generation in AS asthmatics (maximal inhibition being 89% and 80% after misoprostol and sulprostone respectively). In contrast in AT patients preincubation of PBLs with misoprostol or sulprostone resulted in a significant increase in generation of 15-HETE after addition of 200μM lysine-aspirin. The maximal increase of 15-HETE generation was observed with 10 μg/ml of misoprostol or sulprostone (+73% and +86% respectively). Conclusions Our data suggest that aspirin-triggered 15-HETE release is differentially regulated by prostaglandin EP receptors in PBLs from AS and AT asthmatics.

Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma.

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the progression of human lung adenocarcinoma. However, the mechanism underlying COX-2's effect on tumor progression remains largely unknown. Lymphangiogenesis, the formation of new lymphatic vessels, has recently received considerable attention and become a new frontier of tumor metastasis research. Here, we study the interaction between COX-2 and the lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, in human lung cancer cells and their implication in patient outcomes. We developed an isopropyl-beta-D-thiogalactopyranoside-inducible COX-2 gene expression system in human lung adenocarcinoma CL1.0 cells. We found that VEGF-C gene expression but not VEGF-D was significantly elevated in cells overexpressing COX-2. COX-2-mediated VEGF-C up-regulation was commonly observed in a broad array of non-small cell lung cancer cell lines. The use of pharmacological inhibitors or activators and genetic inhibition by EP receptor-antisense oligonucleotides revealed that prostaglandin EP(1) receptor but not other prostaglandin receptors is involved in COX-2-mediated VEGF-C up-regulation. At the mechanistic level, we found that COX-2 expression or prostaglandin E(2) (PGE(2)) treatment could activate the HER-2/Neu tyrosine kinase receptor through the EP(1) receptor-dependent pathway and that this activation was essential for VEGF-C induction. The transactivation of HER-2/Neu by PGE(2) was inhibited by way of blocking the Src kinase signaling using the specific Src family inhibitor, PP1, or transfection with the mutant dominant negative src plasmid. Src kinase was involved in not only the HER-2/Neu transactivation but also the following VEGF-C up-regulation by PGE(2) treatment. In addition, immunohistochemical staining of 59 lung adenocarcinoma specimens showed that COX-2 level was highly correlated with VEGF-C, lymphatic vessels density, and other clinicopathological parameters. Taken together, our results provided evidence that COX-2 up-regulated VEGF-C and promotes lymphangiogenesis in human lung adenocarcinoma via the EP(1)/Src/HER-2/Neu signaling pathway.

Cyclooxygenase-2 Induction by Bradykinin in Human Pulmonary Artery Smooth Muscle Cells Is Mediated by the Cyclic AMP Response Element through a Novel Autocrine Loop Involving Endogenous Prostaglandin E2, E-prostanoid 2 (EP2), and EP4 Receptors

Bradykinin (BK) is an important mediator in several inflammatory and vascular diseases that acts in part via induction of cyclooxygenase-2 (COX-2). The mechanisms involved in BK-mediated COX-2 induction are unclear. Here we characterized the transcriptional mechanisms involved in human pulmonary artery smooth muscle cells. BK stimulated the activity of a transiently transfected 966-bp (-917 to + 49) COX-2 promoter luciferase reporter construct. There was no reduction in BK-induced luciferase activity in cells transfected with COX-2 promoter constructs of 674, 407, 239, and 135 bp or constructs with mutated CCAAT/enhancer-binding protein- or NF-kappaB-binding sites. In contrast luciferase activity was reduced in cells transfected with a 407-bp COX-2 promoter fragment containing a mutated cAMP response element (CRE)-binding site, suggesting that the CRE binding site is critical. Electrophoretic mobility shift assays using oligonucleotides specific for the CRE-binding region of the COX-2 promoter and consensus oligonucleotides showed strong specific binding. Furthermore BK increased consensus cAMP-responsive luciferase reporter (p6CRE/luc)-mediated luciferase expression. CRE activation occurred by BK inducing cytosolic phospholipase A2-mediated arachidonic acid release and rapid prostaglandin E2 (PGE2) production, thereby increasing cAMP. Indomethacin inhibited BK-induced PGE2 production, cAMP accumulation, and CRE/luc reporter and COX-2 promoter luciferase activity. Exogenous PGE2 and EP2 (ONO-AE1 259) and EP4 (ONO-AE1 329) PGE2 receptor agonists mimicked the effect of BK. Collectively these studies indicate that COX-2 induction by BK in human pulmonary artery smooth muscle cells is mediated by the CRE through a novel autocrine loop involving endogenous PGE2.

Functional domains essential for Gs activity in prostaglandin EP2 and EP3 receptors

The interaction of cell surface hormone receptors with heterotrimeric G proteins is crucial for hormonal actions. The domains of the receptor, which interact with and activate G protein, have been extensively studied. However, precise molecular mechanisms underlying regulation of the receptor-induced G protein activation are still poorly understood. Prostaglandin E 2 (PGE 2) receptors comprise of four subtypes, EP1, EP2, EP3 and EP4. Among them, EP2 and EP4 couple to Gs and EP3 to Gi. To assess the functional domains essential for Gs activation in prostanoid receptors, EP2, EP3β and each intracellular loop- (IC-) interchanged EP2/EP3 chimeras were tested for agonist binding and functional responses. In EP2 receptor, substitution of IC1 or IC3 resulted in loss of binding activity, while substitution of IC2, N- (IC2N) or C-terminal half region of IC2 (IC2C) had no effects on the binding activity. Wild-type EP2 and IC2C-substituted EP2 showed agonist-induced Gs activity, but IC2- and IC2N-substituted EP2 failed to elicit Gs activity upon agonist stimulation. On the other hand, in EP3 receptor substitution of IC1 resulted in loss of PGE 2 binding, while substitution of IC2, IC3, IC2N or IC2C had no effects on binding activity. Wild-type EP3β, IC3- or IC2C-substituted EP3 failed to show Gs activity upon agonist stimulation, but IC2- or IC2N-substituted EP3 chimera showed agonist-dependent Gs activity. These results indicated that the second intracellular loop of the EP2 plays an essential role in activation of Gs.

Effects of Mechanical Strain on the Function of Gap Junctions in Osteocytes Are Mediated through the Prostaglandin EP2 Receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.

Temporal and tissue-specific expression of prostaglandin receptors EP2, EP3, EP4, FP, and cyclooxygenases 1 and 2 in uterus and fetal membranes during bovine pregnancy.

Uteroplacental prostaglandins (PGs) play pivotal roles in maintenance and /or termination of pregnancy in mammals. Regulation of PG biosynthetic and signaling mechanisms in uteroplacental tissues during maintenance of pregnancy is largely unknown. In the present study, we have characterized the expression of PGE2 receptors (EP2, EP3, EP4), PGF2alpha receptor (FP), and cyclooxygenase (COX) types 1 and 2 in placentome caruncle (CAR), intercaruncle, and fetal membrane tissues during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. The levels of expression of EP2, EP3, and FP mRNAs differ depending on tissues and days of gestation (days <50 to >250). EP4 mRNA was undetectable in all the tissues studied. The expression levels of PG receptor mRNAs were as follows: placentome CAR FP>EP2>P3, intercaruncle EP2>EP3> or =FP, and fetal membranes EP3> or =EP2 >>FP. EP2 and EP3 expressions were modulated in uteroplacental tissues, depending on days of pregnancy, whereas FP was uniformly expressed. COX-1 mRNA and protein were constitutively expressed, whereas COX-2 was highly modulated in uteroplacental tissues throughout pregnancy. Immunohistochemistry showed that EP2 and COX-2 proteins were colocalized in most cell types of placentome CAR, endometrium, and myometrium. Our study indicates that EP2 is the primary cAMP-generating PGE2 receptor expressed in uteroplacental tissues during bovine pregnancy. Temporal and tissue-specific expression of PGE2 and PGF2alpha receptors and COX-1 and -2 at the maternal-fetal interface suggests a selective and distinctive role for PGE2 and PGF2alpha in uterine activities during pregnancy in bovine.

Transactivation of steroidogenic acute regulatory protein in human endometriotic stromalcells is mediated by the prostaglandin EP2 receptor.

Steroidogenic acute regulatory protein (StAR) regulates the first committed step in the biosynthesis of steroids, and thus aberrant expression of StAR in endometriotic implants plays a critical role in the etiology of endometriosis. However, the mechanism responsible for abnormal expression of StAR in ectopic endometriotic tissues remains unknown. In the present study, we demonstrate that prostaglandin (PG) E(2) stimulates StAR protein expression at the cellular and molecular levels. PGE(2) caused a rapid increase in StAR expression that involves activation of the EP2 receptor-coupled protein kinase A pathway. Activation of EP2 receptor-induced phosphorylation of ERK and cAMP response element binding protein (CREB). However, activation of ERK did not involve in CREB phosphorylation or concomitantly StAR expression. Phosphorylation of CREB induced by PGE(2) increased the recruitment of CREB binding protein and thus histone H3 acetylation. Chromatin immunoprecipitation experiments showed that acetylated histone H3 bound to the proximal region of the StAR promoter was increased after 30 min treatment with PGE(2), and this was mirrored by an increase in nascent StAR RNA transcription. Treatment with the histone deacetylase inhibitor, tricostatin A, enhanced PGE(2)-induced nascent StAR RNA transcription. We conclude that increased histone H3 acetylation involving the EP2 receptor, protein kinase A, CREB, and CREB binding protein is responsible for PGE(2)-induced StAR gene activation in endometriotic stromal cells. Our current report may provide new insights in understanding mechanism of abnormally local production of estrogen and the etiology of endometriosis.

Characteristics of thermoregulatory and febrile responses in mice deficient in prostaglandin EP1 and EP3 receptors

Characteristics of thermoregulatory and febrile responses in mice deficient in prostaglandin EP1 and EP3 receptors

The effect of deletion of cyclooxygenase-2, prostaglandin receptor EP2, or EP4 in bone marrow cells on osteoclasts induced by mouse mammary cancer cell lines

The inducible prostaglandin (PG) synthesis enzyme, cyclooxygenase-2 (COX-2), is involved in osteoclast (OC) formation in cocultures of mouse mammary cancer cell lines (MMT060562 or BALB/c-MC) and bone marrow cells through production of PGE 2. There are four PGE 2 receptors but only the EP2 and EP4 receptors are reported to be important for OC formation. We have investigated the role of COX-2, EP2 receptor, and EP4 receptor in marrow cells for osteoclastogenesis in cocultures of cancer cells and bone marrow cells. We cocultured cancer cell lines with bone marrow cells from COX-2 knockout (−/−), EP2 −/− or EP4 −/− mice compared to wild-type mice. In addition, an EP4 receptor antagonist (EP4 RA) was added in some cocultures. Disruption of COX-2 gene in bone marrow cells had no effect on PGE 2 production and OC formation in cocultures with MMT060562, while it abrogated PGE 2 production and OC formation in cocultures with BALB/c-MC. Disruption of the EP2 gene in bone marrow cells had no effect on OC formation in the cocultures, while disruption of the EP4 gene in bone marrow cells abrogated OC formation in the cocultures. Furthermore, EP4 RA suppressed OC formation and prevented the increase in receptor activator of nuclear factor κB ligand (RANKL) mRNA levels in the cocultures. We conclude that COX-2 in cancer cells is responsible for PGE 2 and OC production in cocultures with MMT060562, while COX-2 in bone marrow cells, not cancer cells, is responsible for PGE 2 and OC production in cocultures with BALB/c-MC, and EP4 receptors are essential for OC formation in both cocultures.

Mice deficient in prostaglandin EP3 receptors do not induce fever but produce psychological stress-induced rise in core temperature

Mice deficient in prostaglandin EP3 receptors do not induce fever but produce psychological stress-induced rise in core temperature

Shear-induced Cyclooxygenase-2 via a JNK2/c-Jun-dependent Pathway Regulates Prostaglandin Receptor Expression in Chondrocytic Cells

Using cDNA microarrays coupled with bioinformatics tools, we elucidated a signaling cascade regulating cyclooxygenase-2 (COX-2), a pivotal pro-inflammatory enzyme expressed in rheumatic and osteoarthritic, but not normal, cartilage. Exposure of T/C-28a2 chondrocytic cells to fluid shear results in co-regulation of c-Jun N-terminal kinase2 (JNK2), c-Jun, and COX-2 as well as concomitant downstream expression of prostaglandin receptors EP2 and EP3a1. JNK2 transcript inhibition abrogated shear-induced COX-2, EP2, and EP3a1 mRNA up-regulation as well as c-Jun phosphorylation. Functional knock-out experiments using an antisense c-Jun oligonucleotide revealed the abolition of shear-induced COX-2, EP2, and EP3a1, but not JNK2, transcripts. Moreover, inhibition of COX-2 activity eliminated mRNA upregulation of EP2 and EP3a1 induced by shear. Hence, a biochemical pathway exists wherein fluid shear activates COX-2, via a JNK2/c-Jun-dependent pathway, which in turn elicits downstream EP2 and EP3a1 mRNA synthesis.

Characteristics of thermoregulatory and febrile responses in mice deficient in prostaglandin EP1 and EP3 receptors.

Previous studies have disagreed about whether prostaglandin EP1 or EP3 receptors are critical for producing febrile responses. We therefore injected lipopolysaccharide (LPS) at a variety doses (1 microg kg(-1)-1 mg kg(-1)) intraperitoneally (i.p.) into wild-type (WT) mice and mice lacking the EP1 or the EP3 receptors and measured changes in core temperature (Tc) by using telemetry. In WT mice, i.p. injection of LPS at 10 microg kg(-1) increased Tc about 1 degrees C, peaking 2 h after injection. At 100 microg kg(-1), LPS increased Tc, peaking 5-8 h after injection. LPS at 1 mg kg(-1) decreased Tc, reaching a nadir at 5-8 h after injection. In EP1 receptor knockout (KO) mice injected with 10 microg kg(-1) LPS, only the initial (< 40 min) increase in Tc was lacking; with 100 microg kg(-1) LPS the mice showed no febrile response. In EP3 receptor KO mice, LPS decreased Tc in a dose- and time-dependent manner. Furthermore, in EP3 receptor KO mice subcutaneous injection of turpentine did not induce fever. Both EP1 and EP3 receptor KO mice showed a normal circadian cycle of Tc and brief hyperthermia following psychological stress (cage-exchange stress and buddy-removal stress). The present study suggests that both the EP1 and the EP3 receptors play a role in fever induced by systemic inflammation but neither EP receptor is involved in the circadian rise in Tc or psychological stress-induced hyperthermia in mice.

Correction to “Prostaglandin receptor EP4 mediates the bone anabolic effects of PGE2”: Fig. 1.

Correction to “Prostaglandin receptor EP₄ mediates the bone anabolic effects of PGE₂”: Fig. 1.

Comparison of Prostaglandin F2α, Bimatoprost (Prostamide), and Butaprost (EP2 Agonist) on Cyr61 and Connective Tissue Growth Factor Gene Expression

Connective tissue growth factor (CTGF) and Cyr61 (cysteine-rich angiogenic protein 61) are members of the CCN gene family that encode multifunctional, extracellular matrix-associated signaling proteins. Because the mechanism of action of certain anti-glaucoma drugs involves extracellular matrix remodeling of ocular ciliary muscle, with a resultant increase in drainage of aqueous humor from the eye, we compared the effects of three pharmacologically distinct ocular hypotensive agents on Cyr61 and CTGF gene expression. Thus, prostaglandin F2alpha (PGF2alpha) (FP receptor agonist), Butaprost (EP2 receptor agonist), and Bimatoprost (a prostamide) were compared. Using Affymetrix gene chip technology, we first identified that PGF2alpha dramatically up-regulated Cyr61 and CTGF mRNA expression in HEK 293/EBNA cells (hFP-HEK 293/EBNA). Northern blot further confirmed the Cyr61 and CTGF up-regulation is in a dose- and time-dependent manner. PGF2alpha-induced up-regulation of Cyr61 appeared to exclusively involve the Rho pathway, and up-regulation of CTGF was via multiple intracellular pathways. Because prostamide receptors are, to date, defined only at the pharmacological level, Bimatoprost effects on Cyr61 and CTGF were studied in the isolated feline iris sphincter preparation, a tissue highly responsive to prostamides. Both PGF2alpha and Bimatoprost up-regulated Cyr61 mRNA expression in the cat iris tissue. Only PGF2alpha up-regulated CTGF mRNA expression in the cat iris. Therefore, PGF2alpha and Bimatoprost appear to interact with different receptors populations in the cat iris, according to their markedly different effects on CTGF. Activation of prostaglandin EP2 receptors (Gs-coupled) also up-regulated Cyr61 but not CTGF mRNA expression in the isolated cat iris. Similar data were observed in human primary ciliary smooth muscle cells. Thus, despite quite different signal transduction pathways, FP receptor stimulation up-regulates CTGF and Cyr61. The prostamide analog Bimatoprost and an EP2-selective agonist affects only Cyr61.

The prostaglandin E2 analogue sulprostone antagonizes vasopressin-induced antidiuresis through activation of Rho.

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells by activation of vasopressin V2 receptors and the subsequent translocation of water channels (aquaporin-2, AQP2) from intracellular vesicles into the plasma membrane. Prostaglandin E2 (PGE2) antagonizes AVP-induced water reabsorption; the signaling pathway underlying the diuretic response is not known. Using primary rat inner medullary collecting duct (IMCD) cells, we show that stimulation of prostaglandin EP3 receptors induced Rho activation and actin polymerization in resting IMCD cells, but did not modify the intracellular localization of AQP2. However, AVP-, dibutyryl cAMP- and forskolin-induced AQP2 translocation was strongly inhibited. This inhibitory effect was independent of increases in cAMP and cytosolic Ca2+. In addition, stimulation of EP3 receptors inhibited the AVP-induced Rho inactivation and the AVP-induced F-actin depolymerization. The data suggest that the signaling pathway underlying the diuretic effects of PGE2 and probably those of other diuretic agents include cAMP- and Ca2+-independent Rho activation and F-actin formation.

Functional evidence for interaction between prostaglandin EP3 and κ-opioid receptor pathways in tactile pain induced by human immunodeficiency virus type-1 (HIV-1) glycoprotein gp120

HIV-1 glycoprotein gp120 administered intrathecally induces tactile pain (allodynia) in animals. In the present study, we investigated the mechanism of gp120-induced allodynia and possible functional connections with factors modulating pain transmission at the spinal level. Gp120 evoked allodynia in a dose-dependent manner with the maximum effect at 1 pg/mouse, and stimulated a rapid increase in intracellular free Ca 2+ concentration ([Ca 2+]i) in the dorsal horn cells of the spinal cord. These responses evoked by gp120 were blocked by galactocerebroside. The gp120-induced allodynia was also attenuated by the non-steroidal anti-inflammatory drug indomethacin, which inhibits prostaglandin synthesis, and did not develop in mice lacking the EP3 prostaglandin E receptor subtype (EP3 –/–). Pretreatment of spinal slices with indomethacin dose-dependently decreased the percentage of the cells that showed increased [Ca 2+]i in response to gp120, and the decrease was reversed by addition of the selective EP3 agonist ONO-AE-248. The κ-opioid agonist U-50,488 significantly enhanced the gp120-stimulated increase in [Ca 2+]i in spinal slices prepared from EP3 –/– mice, and the simultaneous addition of U-50,488 with gp120 reproduced the gp120-induced allodynia in EP3 –/– mice. These results suggest that gp120 induced allodynia by increasing [Ca 2+]i, concomitant with activation of prostanoid EP3 and κ-opioid receptors in the spinal cord.

Chimeric exchanges within the bradykinin B2 receptor intracellular face with the prostaglandin EP2 receptor as the donor: importance of the second intracellular loop for cAMP synthesis

The prostaglandin E2 (PGE 2) EP2 receptor (EP2R) type is G protein coupled (GPCR) and links to Gαs. Through this receptor PGE 2 activates cAMP production. The bradykinin (BK) B2 receptor (BKB2R) is also a GPCR but links to G αq and Gαi and does not activate cAMP production in response to bradykinin. In an attempt to convert the BKB2R into a Gαs-linked adenylate cyclase-activating receptor we proceeded to make global and discrete motif replacements of the intracellular (IC) face of the BKB2R with the corresponding regions of the human EP2R. With this approach we produced hybrid receptors which, when stably transfected into wild type (WT) Rat-1 cells, bound BK but produced cAMP. Replacement of the second loop (IC2), third loop (IC3), the entire C terminus, and the distal C terminus resulted in receptors which bound BK. However, only the IC2 and IC3 exchanges resulted in cAMP-producing receptors. Of these two regions, the IC2 exchange was by far the better cAMP-generating receptor, producing cAMP at approximately 6.6-fold above WT BKB2R or approximately one fourth the amount produced by WT EP2R-transfected Rat-1 cells. Both human and rat EP2R and human β2-adrenergic receptor exchanges of the IC2 produced equal quantities of cAMP. Focusing on the rBKB2R/hEP2R IC2 chimeras, the region consisting of residues 136–147 (BKB2R residue numbering) proved to contain a cAMP-generating motif. Within this region, the proximal six amino acids from the EP2R (HPYFYQ) at position 136–141 proved crucial for cAMP production (10-fold over WT BKB2R). The distal part of this region, the six residues at 142–147, played no role in cAMP production. On the other hand, the ALV motif of the BKB2R IC2, residues 133–135, proved important with respect to phosphatydilinositol (PI) turnover. Replacing the entire IC2 of BKB2R resulted in poor PI turnover, while including the AVL of BKB2R retained approximately half of the WT PI turnover. With respect to receptor uptake, all the IC2 mutants endocytosed as WT BKB2R (60% in 1 h). However, the exchange of the distal and the whole C termini resulted in a marked drop in endocytosis (30% in 1 h). These results demonstrate that the construction of a cAMP-producing BKB2/EP2 receptor hybrid is possible, with the IC2 region distal to DRYLALV proving important to Gαs linkage and the LALV motif within the IC2 of BKB2R and the region proximal to it proving important for Gαq and Gαi linkage. Additionally, our results confirm the importance of the distal C terminus in determining receptor uptake.

Cancer-associated immunodeficiency and dendritic cell abnormalities mediated by the prostaglandin EP2 receptor.

Prostaglandin E(2) (PGE(2)), a major COX metabolite, plays important roles in several facets of tumor biology. We characterized the contribution of the PGE(2) EP2 receptor to cancer-associated immune deficiency using EP2(-/-) mice. EP2(-/-) mice exhibited significantly attenuated tumor growth and longer survival times when challenged with MC26 or Lewis lung carcinoma cell lines as compared with their wild-type littermates. While no differences in T cell function were observed, PGE(2) suppressed differentiation of DCs from wild-type bone marrow progenitors, whereas EP2-null cells were refractory to this effect. Stimulation of cells in mixed lymphocyte reactions by wild-type DCs was suppressed by treatment with PGE(2), while EP2(-/-)-derived DCs were resistant to this effect. In vivo, DCs, CD4(+), and CD8(+) T cells were significantly more abundant in draining lymph nodes of tumor-bearing EP2(-/-) mice than in tumor-bearing wild-type mice, and a significant antitumor cytotoxic T lymphocyte response could be observed only in the EP2(-/-) animals. Our data demonstrate an important role for the EP2 receptor in PGE(2)-induced inhibition of DC differentiation and function and the diminished antitumor cellular immune responses in vivo.

Cancer-associated immunodeficiency and dendritic cell abnormalities mediated by the prostaglandin EP2 receptor

Prostaglandin E2 (PGE2), a major COX metabolite, plays important roles in several facets of tumor biology. We characterized the contribution of the PGE2 EP2 receptor to cancer-associated immune deficiency using EP2–/– mice. EP2–/– mice exhibited significantly attenuated tumor growth and longer survival times when challenged with MC26 or Lewis lung carcinoma cell lines as compared with their wild-type littermates. While no differences in T cell function were observed, PGE2 suppressed differentiation of DCs from wild-type bone marrow progenitors, whereas EP2-null cells were refractory to this effect. Stimulation of cells in mixed lymphocyte reactions by wild-type DCs was suppressed by treatment with PGE2, while EP2–/–-derived DCs were resistant to this effect. In vivo, DCs, CD4+, and CD8+ T cells were significantly more abundant in draining lymph nodes of tumor-bearing EP2–/– mice than in tumor-bearing wild-type mice, and a significant antitumor cytotoxic T lymphocyte response could be observed only in the EP2–/– animals. Our data demonstrate an important role for the EP2 receptor in PGE2-induced inhibition of DC differentiation and function and the diminished antitumor cellular immune responses in vivo.