Prostaglandin E Receptors*

Prostaglandin (PG) E2 exerts its actions by acting on a group of G-protein-coupled receptors (GPCRs). There are four GPCRs responding to PGE2 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 PGE2 being the most versatile prostanoid. Studies on knock-out mice deficient in each EP subtype have defined PGE2 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.


Biochemical Properties of PGE Receptor Subtypes and Isoforms
Molecular Structures- Fig. 1 shows an alignment of the primary amino acid sequences of the mouse EP1, EP2, and EP4 and three isoforms of mouse EP3 receptors. The mouse EP1, EP2, EP3 (EP3␣), and EP4 receptors consist of 405, 362, 366, and 513 amino acids, respectively. EP4 has the longest intracellular C terminus and a relatively long intracellular third loop. The EP1 receptor also has a long third loop, whereas the EP2 and EP3 receptors have a more compact structure. A remarkable feature distinguishing the EP3 receptor from the other EP receptors is the existence of multiple variants generated by alternative splicing of the C-terminal tail. In mouse, alternative splicing creates three EP3 splice isoforms, ␣, ␤, and ␥, containing C-terminal tails of 30, 26, and 29 amino acids that do not share any structural motifs or hydrophobic features (5,6). These isoforms show similar ligand binding properties but have different signal transduction properties as described below. Multiple splice isoforms for EP3 also exist in other species including rat, rabbit, bovine, and human (3). Although all of the four EP subtypes respond to PGE 2 , the amino acid identity among the EPs is limited; the identity of EP1 to EP2, EP3, and EP4 is 30, 33, and 28%, respectively. The amino acid identity is only 31% even between the two EPs (EP2 and EP4) that couple to the activation of adenylate cyclase. The EP2 receptor is more homologous to IP (40%) and DP (44%), the other two adenylate cyclase-stimulatory prostanoid receptors, than any other EPs, and the EP1 receptor is more homologous to FP (35%) and TP (34%) than other EPs. This limited homology among EPs probably reflects the phylogenetic relationship among the prostanoid receptors (7).
Ligand Binding Properties-The EP subtypes bind most potently to PGE 2 with K d values in the range of 1-40 nM. Iloprost, an IP agonist, also binds to EP1 and EP3 with K i values of about 20 nM. The PGE analogs that have been used in conventional studies are not specific for any given EP subtype except butaprost, which is specific for EP2. Several compounds highly selective for each EP subtype have been developed using cultured cell lines stably expressing each subtype. Examples are shown in Fig. 2 (8 -11).
Signal Transduction Properties-Signal transduction pathways of EP subtypes have been studied by examining agonistinduced changes in second messengers such as cAMP, Ca 2ϩ , and inositol phosphates and agonist-induced changes in activities of downstream kinases. The EP1 receptor mediates a PGE 2 -induced elevation of the free Ca 2ϩ concentration in Chinese hamster ovary cells. This increase is dependent on extracellular Ca 2ϩ and occurs without a detectable phos-phatidylinositide response (12), suggesting that EP1 regulates Ca 2ϩ channel gating via an unidentified G protein. It was reported that EP1 expressed in Xenopus oocytes can couple to TRP5, a candidate for the receptor-activated Ca 2ϩ channel, and this coupling is inhibited by an antisense oligonucleotide for G q /G 11 but not by one for G i1 (13). The EP2 and EP4 receptors couple to G s and mediate increases in cAMP concentrations. The major signaling pathway of the EP3 receptor is inhibition of adenylate cyclase via G i . It should be noted, however, that the EP receptors do not couple exclusively to the pathways described but often to more than one G protein and signal transduction pathway (Table   1). Of interest in this respect is the presence of two EPs, EP2 and EP4, that are coupled to increases in cAMP. They apparently function redundantly in some processes. For example, both EP2 and EP4 mediate induction of RANKL through cAMP by PGE 2 in osteoclastogenesis, although the extent of the contribution by each receptor may be different (14,15). On the other hand, there are processes in which EP2 and EP4 play distinct roles. Some of these may be because of selective expression of either of them in relevant cells such as the action of EP2 during cumulus expansion in ovulation and fertilization (16) and that of EP4 in closure of the ductus arteriosus (17). However, only EP4 regulates migration of dendritic cells in the mouse although both EP2 and EP4 are expressed in these cells (18). This EP4-selective action may be related to the fact that EP4 but not EP2 couples to phosphatidylinositol 3-kinase, probably via G i , in addition to activation of adenylate cyclase (19,20). It is interesting in this respect that EP4 is also implicated in cell migration during tumor invasion (21) for ductus arteriosus closure (22) and for zebrafish gastrulation (23). As described, the EP3 receptor consists of multiple isoforms generated by alternative splicing of the C-terminal tail. Functional differences among these splice variants have been reported, including coupling to different signal transduction pathways (Table 1) (24), different sensitivities to agonist-induced desensitization (25), different extents of constitutive activity (26), different intracellular trafficking patterns (27), and different agonist-induced internalization patterns (28).
Tissue Distribution and Cellular Localization-Northern blot analysis and in situ hybridization have provided detailed information about EP receptor distribution and have shown that each receptor is specifically distributed in the body and that the expression levels are variable among tissues. The tissue distribution of the mouse EP subtypes assessed by Northern blot analyses is presented in Fig. 3A (29 -32). Among the four EPs, EP3 and EP4 receptors are the most widely distributed with their mRNAs being expressed in almost all mouse tissues examined. In contrast, the distribution of EP1 mRNA is restricted to several organs, such as  the kidney, lung, and stomach, and EP2 is the least abundant of the EP receptors. Within tissues, each EP subtype shows a distinct cellular localization. For example, in the kidney, EP3 is expressed in the tubular epithelium, the thick ascending limb, and the cortical collecting ducts in the outer medulla, EP1 in the papillary collecting ducts, and EP4 in the glomerulus (33) (Fig. 3B). This distribution pattern appears to correlate with the PGE 2 -mediated regulation of ion transport, water reabsorption, and glomerular filtration, respectively. A similar distribution of EPs in the kidney has been reported in the rabbit and human (34,35). These analyses did not detect signals for EP2 mRNA in the kidney.
Regulation of Expression-Expression of EP genes is regulated by various physiological and pathophysiological stimuli. In peritoneal resident macrophages (36) and a macrophage cell line, J774.1 (37), EP4 is expressed under basal conditions. The addition of lipopolysaccharide (LPS) induces EP2 expression markedly in both types of cells but enhances the EP4 expression only slightly in J774.1 cells and suppresses the expression of EP4 in the resident macrophages (36). Macrophages produce a large amount of PGE 2 in response to LPS, and suppression of the EP4 expression in the resident macrophages was prevented by treatment with indo-methacin and was mimicked by the addition of dibutyryl cAMP or PGE 2 but not butaprost, suggesting that EP4 expression is regulated through a negative feedback loop. The presence of EP2 and EP4 and augmentation of their expression by LPS stimulation were also seen in the RAW 264.7 murine macrophage-like cell line (38). Quantitative reverse transcription-PCR analysis indicated a 3-fold increase in EP4 mRNA 2.5 h after LPS stimulation. In thioglycolate-elicited macrophages, macrophage engagement with extracellular matrix induces expression of both EP2 and EP4 and COX-2 in a MAPK erk1/2 -dependent manner (39).
In female reproductive organs such as the ovary and uterus, hormonal exposure induces expression of the EP subtypes in specific cell types. In the ovary, the EP4 expression is found in oocytes in preantral follicles. Upon gonadotropin stimulation, this expression disappears, and EP4 is expressed first in both granulosa cells and cumulus cells and then only in granulosa cells in preovulatory follicles. EP2 expression is found in both granulosa cells and cumulus cells in preantral follicles. This expression increases upon gonadotropin stimulation and becomes confined to the cumulus cells just before ovulation. Interestingly, COX-2 expression changes in a similar pattern to EP2 upon gonadotropin stimulation in cumulus and mural granulosa cells (40). In the uterus, when mice are primed with gonadotropins and undergo pseudopregnancy, EP2 is transiently expressed on day 5 in luminal epithelial cells. EP4 expression is limited to luminal epithelial cells on day 0, sharply increases on day 3, and is then found in endometrial stromal cells as well as glandular epithelium. EP3 expression is found in longitudinal muscle layer before stimulation. After stimulation, this expression disappears and EP3 is expressed in circular smooth muscles with a further increase on days 3 and 5 (41).
Promoter analysis has been done for EP2 and EP4. Several consensus sequences relevant to inflammatory stimuli such as those for NF-IL6, NFB, and AP2 are found, and several regions responsive to progesterone have been characterized in the promoter region of the EP2 gene (42). The promoter region of the EP4 gene contains several putative cis-acting elements such as sites for AP1, AP2, Sp1, NFB, MyoD, and NF-IL6 as well as a putative glucocorticoid response element. Functional analysis detected an LPS/serum-responsive region between Ϫ554 and Ϫ116 bp (38).

Physiological Functions of EP Subtypes
Mice deficient in each EP subtype individually have been generated, and studies using these knock-out mice and subtype-specific EP agonists/antagonists have identified EP subtypes mediating various PGE 2 actions (Table 2). EP subtypes mediate many processes known to be inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). For example, the EP3 receptor mediates generation of pyrogenic fever (54), and EP1 FIGURE 3. A, tissue distribution of EP subtypes. Poly(A) ϩ RNA isolated from the indicated tissues (5 g for EP1 and EP2; 10 g for EP3 and EP4) was applied in each lane. Hybridization was carried out using the antisense RNA probes (EP1 and EP2) or cDNA fragment probes (EP3 and EP4) (29 -32). B, localization of EP subtypes in mouse kidney. In situ hybridization signals for EP1, EP3, and EP4 in renal sections are presented in a dark-field manner (33). Bar ϭ 1 mm. and EP3 signals converge at the paraventricular nucleus of the hypothalamus and mediate neuroendocrine stress response by facilitating release of corticotropin-releasing hormone (43). EP2 facilitates ovulation and fertilization by inducing expansion of the cumulus, thus clarifying the mechanism for the inhibitory effect of NSAIDs on ovulation (16). Other studies have revealed that different EP subtypes as well as the IP receptor function in hyperalgesia both at the periphery and in the CNS. For example, the acetic acid writhing test revealed the involvement of both IP and EP3 in hyperalgesia (57,61). Pain sensation that is induced by pH and heat and mediated by the capsaicin receptor TRPV1 is augmented by PGE 2 and PGI 2 acting on EP1 and IP, respectively (46). Furthermore, in the spinal cord, PGE 2 acting on EP2 in glycinergic neurons abolishes the glycine-induced tonic inhibition of pain neuron in the dorsal horn and facilitates the propagation of nociceptive signals through the spinal cord to higher areas of the CNS (48). Prostanoids, particularly PGE 2 , have been thought to play a major role in acute inflammation by acting on the peripheral circulation and inducing hyperemia and swelling. One of the lessons learned from the knock-out mouse studies, however, is that prostanoids including PGE 2 exert both pro-inflammatory and anti-inflammatory responses, and these actions are often produced through regulation of gene expression in relevant tissues. For example, consistent with the anti-inflammatory and anti-arthritic effect of NSAIDs, EP2 and EP4 (and IP) redundantly mediate development of collagen-induced arthritis (49). Intriguingly, however, the pro-inflammatory actions of these prostanoid receptors are elicited mainly by induction of arthritis-associated genes in the joint. As for inflammatory swelling, studies using the carrageenininduced paw edema model revealed involvement of IP (61), and those using carrageenin-induced pleurisy revealed participation of EP2, EP3, and IP in inflammatory exudation (62). Anti-inflammatory actions of prostanoids are seen typically in allergic or immune inflammation and are usually balanced by pro-inflammatory actions of other prostanoids. This may explain why NSAIDs are without effects on allergy and immune responses. Examples are the antagonism between the PGD 2 -DP (63) and the PGE 2 -EP3 (55) pathways in elicitation of allergic asthma. DP and EP3 are both present in the airway epithelium, and activation of the latter suppresses expression of a series of allergy-related genes and progression of allergic inflammation. Knock-out mice studies have also revealed that prostanoids work at multiple steps in hapten-induced immune responses. Interestingly, most of these actions are found in the immunization and not in the elicitation process. The PGD 2 -DP pathway suppresses (64) and the PGE 2 -EP4 pathway facilitates (18) mobilization, migration, and maturation of Langerhans cells in the skin, and the TXA 2 -TP pathway negatively modulates interaction between activated Langerhans cells and naïve T-cells, thereby suppressing T-cell proliferation and differentiation (65). PGE 2 together with other prostanoids can thus modulate various steps of inflammation in a context-dependent manner and coordinate the whole process in both pro-inflammatory and antiinflammatory directions.

Concluding Remarks
The mechanisms whereby PGE 2 exerts its pleiotropic effects, once a mystery in physiology, have been clarified through the biochemical identification and cDNA cloning of the four EP subtype receptors. Furthermore, development of highly selective agonists and antagonists to each EP subtype and information obtained by studies on mice deficient in each EP receptor now provide opportunities to apply our knowledge to manipulate various PGE 2 -mediated pathological processes.