EP3 Isoforms Research Articles

A systematic analysis of prostaglandin E2 type 3 receptor isoform signaling reveals isoform- and species-dependent L798106 Gαz-biased agonist responses

Prostaglandins are bioactive lipids involved in many physiological and pathophysiological conditions, such as pain, atherosclerosis, type II diabetes, and parturition. Prostaglandin E2 (PGE2) activates four G protein-coupled receptors (GPCRs), named the PGE2 types 1–4 receptors (EP1-4), to elicit the intracellular signaling responsible for their physiological actions. There are more than twelve EP3 isoforms in humans that differ only by the sequence of their C-termini. However, the signaling mechanisms engaged by the various isoforms have never been clearly defined. In this study, we used a recently described BRET-based biosensor technology to define the signaling profiles for each of the human isoforms on a selection of signaling pathways using the agonists, PGE2 and sulprostone, and the purportedly EP3-specific antagonist L798106. We found that L798106 is a biased agonist of the Gαz pathway for some human EP3 isoforms, an effect that is not detected in the close ortholog mouse EP3 isoform α. We also found that the presence of a threonine residue at position 107 in the binding site of human EP3, which is a serine in most other species including mice, is important for L798106-mediated Gαz efficacy. Given the reported importance of EP3-Gαz signaling on the potential therapeutic efficacy of EP3 and since many preclinical studies for these mechanisms have been performed in rodents, this finding demonstrates the importance of determining a detailed signaling profile of ligands for different species and receptor isoforms, which constitutes an important step to better understand the therapeutic potential of the EP3.

Prostaglandin E2 receptor EP3 regulates both adipogenesis and lipolysis in mouse white adipose tissue.

Among the four prostaglandin E2 receptors, EP3 receptor is the one most abundantly expressed in white adipose tissue (WAT). The mouse EP3 gene gives rise to three isoforms, namely EP3α, EP3β, and EP3γ, which differ only at their C-terminal tails. To date, functions of EP3 receptor and its isoforms in WAT remain incompletely characterized. In this study, we found that the expression of all EP3 isoforms were downregulated in WAT of both db/db and high-fat diet-induced obese mice. Genetic ablation of three EP3 receptor isoforms (EP3-/- mice) or EP3α and EP3γ isoforms with EP3β intact (EP3β mice) led to an obese phenotype with increased food intake, decreased motor activity, reduced insulin sensitivity, and elevated serum triglycerides. Since the differentiation of preadipocytes and mouse embryonic fibroblasts to adipocytes was markedly facilitated by either pharmacological blockade or genetic deletion/inhibition of EP3 receptor via the cAMP/PKA/PPARγ pathway, increased adipogenesis may contribute to obesity in EP3-/- and EP3β mice. Moreover, both EP3-/- and EP3β mice had increased lipolysis in WAT mainly due to the activated cAMP/PKA/hormone-sensitive lipase pathway. Taken together, our findings suggest that EP3 receptor and its α and γ isoforms are involved in both adipogenesis and lipolysis and influence food intake, serum lipid levels, and insulin sensitivity.

EP3 receptor isoforms are differentially expressed in subpopulations of primate granulosa cells and couple to unique G-proteins

Prostaglandin E2 (PGE2) produced within the ovarian follicle is necessary for ovulation. PGE2 is recognized by four distinct G-protein-coupled receptors. Among them, PTGER3 (also known as EP3) is unique in that mRNA splicing generates multiple isoforms. Each isoform has a distinct amino acid composition in the C-terminal region, which is involved in G-protein coupling. To determine whether monkey EP3 isoforms couple to different G-proteins, each EP3 isoform was expressed in Chinese hamster ovary cells, and intracellular signals were examined after stimulation with the EP3 agonist sulprostone. Stimulation of EP3 isoform 5 (EP3-5) reduced cAMP in a pertussis toxin (PTX)-sensitive manner, indicating involvement of Gαi. Stimulation of EP3-9 increased cAMP, which was reduced by the general G-protein inhibitor GDP-β-S, and also increased intracellular calcium, which was reduced by PTX and GDP-β-S. So, EP3-9 likely couples to both Gαs and a PTX-sensitive G-protein to regulate intracellular signals. Stimulation of EP3-14 increased cAMP, which was further increased by PTX, so EP3-14 likely regulates cAMP via multiple G-proteins. Granulosa cell expression of all EP3 isoforms increased in response to an ovulatory dose of human chorionic gonadotropin. Two EP3 isoforms were differentially expressed in functional subpopulations of granulosa cells. EP3-5 was low in granulosa cells at the follicle apex while EP3-9 was high in cumulus granulosa cells. Differential expression of EP3 isoforms may yield different intracellular responses to PGE2 in granulosa cell subpopulations, contributing to the different roles played by granulosa cell subpopulations in the process of ovulation.

EP3 Receptor Isoforms Differentially Expressed in Subpopulations of Primate Granulosa cells Couple to Unique G Proteins.

Prostaglandin (PG) E2 is necessary for ovulation of mammalian follicles. PGE2 acts through four distinct G protein coupled receptors (PTGER1-PTGER4, also called EP1-EP4). Among them, EP3 is unique in having multiple isoforms generated by alternative mRNA splicing. Each isoform has a distinct amino acid composition in the C-terminal region which couples to G proteins. Therefore, we hypothesized that each EP3 isoform couples to different G proteins to modulate intracellular signaling in primate granulosa cells. Using 3' rapid amplification of cDNA ends (3' RACE), six EP3 mRNAs were identified in cynomolgus monkey granulosa cells. Three of these isoforms shared significant sequence homology with EP3 isoform 5 (EP3-5), EP3-7, and EP3-9 as described previously in human tissues. In addition, three isoforms were identified which have not been previously reported. EP3-5, EP3-7, and EP3-9 were transiently transfected into Chinese hamster ovary (CHO) cells and were detected in a punctate pattern in the cytoplasm as well as in the plasma membrane. Stimulation of EP3-5 by sulprostone, an EP3 agonist, reduced cAMP compared to vehicle-treated cells. Pretreatment with pertussis toxin (PTX) increased sulprostone-stimulated cAMP compared to sulprostone alone, indicating that EP3-5 couples to the Gi protein. Addition of sulprostone to EP3-9 transfected cells increased cAMP, which was blocked by the general G protein inhibitor GDP-β-S. Sulprostone also increased intracellular Ca2+ in EP3-9 transfected cells, which was blocked by PTX or GDP-β-S. Therefore, EP3-9 likely couples to both Gs to increase cAMP and a PTX-sensitive G protein to regulate intracellular Ca2+. No G protein associated function was identified for EP3-7. Laser capture microdissection and qPCR were used to identify subpopulations of granulosa cells which preferentially expressed these EP3 isoforms 36 hours after an ovulatory dose of hCG, or just before ovulation. EP3-5 mRNA was significantly lower in apex granulosa cells compared to non-apex granulosa cells; EP3-9 mRNA was higher in cumulus cells when compared to mural granulosa cells. In summary, each EP3 receptor isoform may function through different G proteins. Differential expression of EP3 isoforms in subpopulations of granulosa cells may contribute to the different roles that these subpopulations play in the process of ovulation. Supported by NIH/NICHD HD054691.

Deletion of GαZ Protein Protects against Diet-induced Glucose Intolerance via Expansion of β-Cell Mass

Insufficient plasma insulin levels caused by deficits in both pancreatic β-cell function and mass contribute to the pathogenesis of type 2 diabetes. This loss of insulin-producing capacity is termed β-cell decompensation. Our work is focused on defining the role(s) of guanine nucleotide-binding protein (G protein) signaling pathways in regulating β-cell decompensation. We have previously demonstrated that the α-subunit of the heterotrimeric G(z) protein, Gα(z), impairs insulin secretion by suppressing production of cAMP. Pancreatic islets from Gα(z)-null mice also exhibit constitutively increased cAMP production and augmented glucose-stimulated insulin secretion, suggesting that Gα(z) is a tonic inhibitor of adenylate cyclase, the enzyme responsible for the conversion of ATP to cAMP. In the present study, we show that mice genetically deficient for Gα(z) are protected from developing glucose intolerance when fed a high fat (45 kcal%) diet. In these mice, a robust increase in β-cell proliferation is correlated with significantly increased β-cell mass. Further, an endogenous Gα(z) signaling pathway, through circulating prostaglandin E activating the EP3 isoform of the E prostanoid receptor, appears to be up-regulated in insulin-resistant, glucose-intolerant mice. These results, along with those of our previous work, link signaling through Gα(z) to both major aspects of β-cell decompensation: insufficient β-cell function and mass.

The prostaglandin E2 system: A toolbox for skeletal repair?

Prostaglandins, a group of lipid mediators, govern a multitude of biologic processes, including inflammation, platelet aggregation, and cell growth. Because of the key role of prostaglandins in inflammation and pain, their metabolism represents the final target of nonsteroidal antiinflammatory drugs (NSAIDs). In the US, sales volumes for NSAIDs of far more than $1 billion per year highlight the enormous economic impact of these agents and the important role of prostaglandin inhibition in clinical practice (1). Prostaglandin E2 (PGE2), the most widely produced member of the prostaglandin group, originates from arachidonic acid that is released from the cell membrane by phospholipase A2, followed by enzymatic conversion by cyclooxygenase (COX) and PGE synthase 1 (PGES-1). PGE2 not only triggers pain and inflammation but also promotes matrix degradation and tissue damage by stimulating the expression of matrixdegrading enzymes (2). Inflammatory cytokines such as interleukin-1 or tumor necrosis factor strongly increase PGE2 levels, but PGE2 secretion may also increase in response to other stimuli including growth factors, hypoxia, and a number of hormones. Apart from the well-defined role of PGE2 in tissue inflammation, there is increasing evidence that it is involved in the maintenance and repair of the skeletal system. At first glance, the effects of PGE2 on tissue homeostasis of bone and cartilage seem confusing, because they conflict with the long-known destructive potential of the prostaglandins. Nevertheless, both catabolic and anabolic effects have already been described for PGE2 in these tissues. In terms of catabolism, PGE2 can promote osteoclastic bone resorption (3,4) and enhance breakdown of the extracellular matrix by increasing the expression of matrix metalloproteinase 3 (MMP-3) or MMP-13 (2,5). Regarding its anabolic aspects, PGE2 may stimulate chondrogenesis, chondrocyte proliferation (6), cartilage matrix synthesis (7), as well as osteoblast activity (8). To shed light on the apparent ambivalent role of PGE2, a multidimensional view is necessary that addresses the developmental stage, type of tissue, disease status, concentration of PGE2, and specific receptor status of the different cell types. So far, 4 different receptors of PGE2 have been identified, designated EP1, EP2, EP3, and EP4 (9). Specific signal transduction properties characterize each receptor, as follows: EP1 stimulates intracellular Ca2 mobilization, whereas EP2 and EP4 bind GS proteins to enhance cAMP levels and activate protein kinase A. In contrast to EP2 and EP4, binding to EP3 results in decreased cAMP concentrations through Gi, Gq, or GS, depending on the EP3 isoform. The 4 receptor subtypes are expressed differentially within various types of tissues. Within articular cartilage and growth plate cartilage, all 4 receptor subtypes are detectable. Their respective expression patterns, however, vary between the different types of cartilage tissues and depend on the stage of development. Early in the process of cartilage development, PGE2 promotes proliferation and DNA synthesis of chondrocytes via binding to EP1 within the growth plate (6), while PGE2 stabilizes the phenotype of chondrocytes within articular cartilage via binding to EP2 and EP4 in later stages (10). Concentration-dependent effects of PGE2 may also contribute to its pleiotropic and sometimes even contradictory effects, as particularly shown in cartilage metabolism. In experimental settings, administration of PGE2 at low doses (slightly above the physiologic level) can promote chondrocyte proliferation (6) and suppress the expression of the degradative enzymes MMP-3 and MMP-13 (11). PGE2 may also stimulate the expression of cartilage-specific genes, including type II collagen, SOX9, and aggrecan, and inhibit the expression of Supported by the DFG (GE 1975/2-1). Kolja Gelse, MD, Christian Beyer, MD: University of Erlangen–Nuremberg and University Hospital Erlangen, Erlangen, Germany. Address correspondence to Kolja Gelse, MD, Department of Orthopaedic Trauma Surgery, University Hospital Erlangen, Krankenhausstrasse 12, 91054 Erlangen, Germany. E-mail: kolja. gelse@web.de. Submitted for publication November 1, 2010; accepted in revised form November 9, 2010.

Prostaglandin EP3 receptor superactivates adenylyl cyclase via the G q/PLC/Ca 2+ pathway in a lipid raft-dependent manner

We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G i/o-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca 2+-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP 3 formation and intracellular Ca 2+ mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca 2+ ionophore and the activation of a typical G q-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results suggest that EP3 elicits adenylyl cyclase superactivation via G q/phospholipase C activation and intracellular Ca 2+ mobilization in a lipid raft microdomain-dependent manner.

Prostanoids in health and disease

The prostanoids are a family of lipid mediators generated by the action of cyclooxygenase on a 20-carbon unsaturated fatty acid, arachidonic acid. Prostanoids are generated widely in response to diverse stimuli and, acting in a paracrine or autocrine manner, play important roles in normal physiology and disease. This review summarizes the current knowledge on prostanoid generation and the roles of individual mediators, their biosynthetic pathways, and their receptors in health and disease.

Quantitative detection of EP3-II, III and VI messenger RNA in gravid and non-gravid human myometrium using real-time RT-PCR

Objective. To examine mRNA expression of prostaglandin E2 receptor isoforms EP3-II, III and VI in lower uterine segment myometrium in the non-pregnant and pregnant state using quantitative real-time RT-PCR.Methods. Myometrial samples were obtained at the time of cesarean delivery or hysterectomy. Pregnant subjects were categorised based on the presence or absence of labour. Labour was defined as regular uterine contractions resulting in cervical change. Quantification for EP3 isoforms II, III and VI mRNA was performed using real-time RT-PCR.Results. There were no differences between non-pregnant and non-labouring pregnant subjects in mRNA expression of EP3-II, III and VI. However, when compared to pregnant subjects not in labour, labouring subjects had a 3.8-fold reduction in EP3-II expression (p < 0.001) and 5.3-fold reduction in EP3-VI expression (p = 0.022).Conclusions. In human parturition, there is decreased mRNA expression of lower-uterine segment EP3 receptor isoforms II and VI during labour. This may reflect differential relaxation of the lower segment of the uterus allowing dilatation and descent of the fetus.

PROSTAGLANDIN E2 SIGNALING IN PATHOPHYSIOLOGY OF ENDOMETRIOSIS IN HUMAN

Endometriosis is a debilitating disease affects up to 20% of reproductive age women characterized by the presence of functional endometrial glandular epithelium and stroma out side the uterine cavity. The pathophysiology of endometriosis remains as enigma in reproductive medicine. Accumulating evidence indicates that steroids, growth factors, and cytokines, and prostaglandins may promote establishment and maintenance of endometriosis. Treatment options include oral contraceptives, aromatase inhibitors, androgenic agents, and gonadotrophin releasing hormone analogues and nonsteroidal anti-inflammatory drugs and surgical removal. But the recurrence rate is up to 70% after cessation of therapy. Prostaglandin E2 (PGE2) promotes cell proliferation, migration, invasion, angiogenesis, anti-apoptosis, pain and immunomodulation. PGE2 mediated effects are primarily mediated through G-protein coupled membrane receptors designated EP that includes EP1, EP2, EP3 and EP4. EP2 and EP4 receptors are coupled to adenyl cyclase generating cAMP that in turn activates the protein kinase A signaling pathway. EP1 receptor is coupled to phospholipase C generating two second messengers, inositol triphosphate (IP3) involved in the liberation of intracellular calcium (Ca2+), and diacyl glycerol an activator of protein kinase C. There are several EP3 isoforms exhibiting a wide range of actions from inhibition of cAMP production to increases in Ca2+ and IP3. In this study, we used immortalized human endometriotic epithelial cells (11-Z, 12-Z, 49-Z, and 108-Z) and stromal cells (22-B) as in vitro models to unravel PGE2 signaling in pathophysiology of endometriosis in human. Normal endometrial epithelial and stromal cells were used as control. We investigated: (1) PGE2 production; (2) expression profiles of EP1, EP2, EP3, and EP4 receptors and (3) role of EP receptors in endometriotic cell proliferation, migration and invasion, and unraveled the underlined mechanisms. We used RT-PCR, western blot, zymography, matrigel migration and invasion assays, and ELISA. Endometriotic cells produced 8–10 fold more PGE2 than normal endometrial cells at basal condition. EP2 and EP4 receptor mRNAs and proteins were abundantly expressed whereas EP1 and EP3 receptor mRNAs and proteins are barely detectable in endometriotic epithelial and stromal cells. Inhibition of either EP2 or EP4 decreased (60%) the endometriotic epithelial and stromal cell proliferation, migration and invasion. Combined inhibition of EP2 and EP4 synergistically decreased (80–85%) endometriotic epithelial and stromal cell proliferation, migration and invasion. Inhibition of EP1 receptor did not have any effect. Inhibition of EP2 and/or EP4 decreased the expression and phosphorylation of Bcl2 protein. Inhibition of EP2 and/or EP4 decreased MMP2 and MMP9 activities. These results suggest that EP2 and/or EP4 mediated cell proliferation and invasion might be mediated respectively through Bcl2 and MMP2 and MMP9 pathways in endometriotic epithelial and stromal cells. Although endometriosis is not a malignancy its pathophysiology seems to be analogous to the mechanisms of cancer metastasis. In conclusion, these endometriotic epithelial and stromal cells can be used as ideal model to study the cellcell communications and interactions in the establishment and maintenance of endometriosis in human. Targeting PGE2 receptors and downstream signaling might be a novel therapy for endometriosis in human. (poster)

Prostaglandin E Receptors

Prostaglandin (PG) E(2) exerts its actions by acting on a group of G-protein-coupled receptors (GPCRs). There are four GPCRs responding to PGE(2) designated subtypes EP1, EP2, EP3, and EP4 and multiple splicing isoforms of the subtype EP3. The EP subtypes exhibit differences in signal transduction, tissue localization, and regulation of expression. This molecular and biochemical heterogeneity of PGE receptors leads to PGE(2) being the most versatile prostanoid. Studies on knock-out mice deficient in each EP subtype have defined PGE(2) actions mediated by each subtype and identified the role each EP subtype plays in various physiological and pathophysiological responses. Here we review recent advances in PGE receptor research.

Induction of IL-6 via the EP3 subtype of prostaglandin E receptor in rat adjuvant-arthritic synovial cells.

This study investigated the role of the prostaglandin E receptor EP3 subtype in the development of inflammatory synovitis. We examined the EP3 mRNA expression in the synovial tissue of rats with adjuvant arthritis and the effect of selective EP agonists on IL-6 production by cultured rat synovial cells. Of the two rat EP3 isoforms, rEP3B mRNA but not rEP3A mRNA was expressed in rat synovial cells. RT-PCR and ELISA were used to analyze the effects of selective EP3 agonist (ONO-AE-248) on IL-6 production by rat cultured synovial cells. This selective EP3 agonist induced IL-6 production by rat synovial cells in a time- and dose-dependent manner. Our results suggest that PGE2 upregulates IL-6 production by synovial macrophages and fibroblasts through rEP3B in rats with adjuvant arthritis.

Multiple Signal Transduction Pathways through Two Prostaglandin E Receptor EP3 Subtype Isoforms Expressed in Human Uterus

PGE2 is known to induce uterine contraction by increasing intracellular Ca2+. In the present study, to investigate other functions of PGE2 in human uterus, two EP3 isoforms were isolated by the RT-PCR method using human uterus polyadenylated ribonucleic acid (RNA). These EP3 isoforms, named EP3-V and EP3-VI, are composed of 402 and 393 amino acid residues, respectively, which are unique compared with EP3 isoforms of other species. Their N-terminal 359 amino acid residues are identical to those of previously reported human EP3 isoforms, whereas the two isoforms contained a novel amino acid sequence in their C-terminal tails. The dissociation constant values of EP3-V and EP3-VI for PGE2 were 3.9 and 1.4 nmol/L, respectively, which were consistent with those of previously reported EP3 isoforms. Signaling experiments revealed that M&B28767, an EP3 agonist, not only inhibited forskolin-induced cAMP concentrations, but also activated mitogen-activated protein kinase in Chinese hamster ovary cells stably expressing EP3-V and EP3-VI. These responses were abolished by treatment with pertussis toxin. In addition, M&B28767 increased cAMP concentrations in EP3-VI-expressing cells, whereas it did not in EP3-V-expressing cells. M&B28767 did not stimulate phosphoinositide turnover in EP3-V- or EP3-VI-expressing cells. EP3-V and EP3-VI messenger RNAs (mRNAs) were detected abundantly in human uterus, whereas weak, but substantial, bands were detected in the lung and kidney in RT-PCR specific for each mRNA. In situ hybridization revealed EP3-V and EP3-VI mRNAs in the human myometrium, but not in the endometrium. The present study suggests that EP3-V and EP3-VI are possibly involved in the proliferation of cells in human myometrium.

Different membrane targeting of prostaglandin EP3 receptor isoforms dependent on their carboxy-terminal tail structures

Mouse prostaglandin EP3 receptor consists of three isoforms, EP3α, β and γ, with different carboxy-terminal tails. To assess the role of their carboxy-terminal tails in membrane targeting, we examined subcellular localization of myc-tagged EP3 isoforms expressed in MDCK cells. Two isoforms, EP3α and EP3β, were localized in the intracellular compartment but not in the plasma membrane, while the EP3γ isoform was found in the lateral plasma membrane and in part in the intracellular compartment. Mutant EP3 receptor lacking the carboxy-terminal tail was localized in the intracellular compartment but not in the plasma membrane. Thus, EP3 isoforms differ in subcellular targeting, and the carboxy-terminal tails play an important role in determination of the membrane targeting of EP3 receptor.

Molecular biology of prostaglandin receptor and L-histidine decarboxylase

This review summarizes our studies on the molecular biology of prostaglandin (PG) receptors and L-histidine decarboxylase (HDC). Regarding PG receptors, we have cloned five basic PG receptors (DP, EP, FP, IP, TP) and four EP subtypes (EP1-EP4). The PG receptors are divided into three families related to signal transduction systems of the receptors; Gs-couple group (IP, DP, EP2 and EP4), Gq-couple group (TP, FP and EP1), and Gi-couple group (EP3 and its isoform). EP3 isoforms having different C-terminal peptides can couple to distinct G proteins (Gi, Gs, Gq). Tissue specific expression of EP subtype mRNAs was observed in various organs. The phenotypic changes of mice deficient in each receptor are; the abnormal labor in FP-deficient mice, the failure of febrile response in EP3-deficient mice, the abnormal closure of ductus arteriosus after birth in EP4-deficient mice, and the impaired inflammatory swelling and pain responses in IP-deficient mice. Regarding HDC, we have purified mouse HDC from mastocytoma cells, which is a dimer of 53 kDa subunit, and then cloned its cDNA. The size of a cDNA-deduced HDC is 74 kDa. In the rat mast cell line, the endogenous 74 kDa form of HDC was translated in the cytosol and then translocated to the ER, where it was post-translationally processed to the 53 kDa form. On the other hand, the cytosolic 74 kDa form was rapidly degraded by an ATP/ubiquitin-dependent proteasome system. The 74 kDa form without on N-terminal signal sequence is inserted into the ER membrane with a C-terminal segment.

Characterization of the prostaglandin EP3-receptor from porcine heart.

Protection of the myocardium from ischemic injury by prostacyclins and PGE1 has been repeatedly reported. Part of the antiischemic effects may be mediated by cardiac sarcolemmal EP3-receptors which have been characterized earlier in this laboratory by ligand binding studies. Of particular notice is the presence of multiple EP3 isoforms, e. g. in mouse, cattle and man which are generated by differential splicing. Here we describe the cloning and subtype characterization of the porcine EP3-receptor. We obtained two different isoforms of the receptor by screening a porcine heart cDNA library. The predicted receptor proteins consisted of 395 and 387 amino acids, respectively, with differences only in the C-terminal part of the protein. Several overlapping genomic clones were obtained from a porcine genomic library. Sequence comparison of the cDNA with the genomic DNA indicated that an intron was located in the region corresponding to the sixth transmembrane domain of the receptor protein. This position exactly matches the corresponding intron in the human TXA2-, the human PGD2- and the mouse PGE-receptor (EP1-subtype). In further experiments, we expressed the porcine EP3-receptor in COS-7 cells and performed ligand-binding-studies. In addition the expression of the porcine EP3-receptor mRNA in different tissues was demonstrated by RT-PCR.

Mouse Prostaglandin E Receptor EP3 Subtype Mediates Calcium Signals via Gi in cDNA-Transfected Chinese Hamster Ovary Cells

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified three isoforms which are produced through alternative splicing. In Chinese hamster ovary cells expressing each EP3 isoform, PGE2 induced an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i) due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. This increase was abolished by prior treatment with pertussis toxin (PT). PGE2 also stimulated an accumulation of inositol trisphosphate (IP3) in a PT-sensitive manner. Both the PGE2-induced increase in [Ca2+]i and accumulation of IP3 were blocked by the phospholipase C inhibitor U-73122. Thus, EP3 is linked to phospholipase C activation via Gi, and this activation leads to Ca2+ mobilization from internal stores and influx from the extracellular medium.

Enhancement of Adenylate Cyclase Stimulation by Prostaglandin E Receptor EP3-Subtype Isoforms with Different Efficiencies

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified two isoforms of EP3, EP3α and EP3β, which are produced through alternative splicing and differ only in the carboxyl-terminal domain. Preincubation of Chinese hamster ovary cells expressing each isoform with PGE 2 concentration-dependently enhanced both the basal and forskolin-stimulated cAMP formation, but two orders higher concentrations of PGE 2 were required for EP3β than EP3α for 50% enhancement of both formations. This enhancement by EP3 isoforms was completely blocked by pertussis toxin treatment, indicating that it is mediated through Gi activation. Thus, the two EP3 isoforms with different carboxyl-terminal tails induce enhancement of adenylate cyclase stimulation with different efficiencies.