Characterization of the first insect prostaglandin (PGE2) receptor: MansePGE2R is expressed in oenocytoids and lipoteichoic acid (LTA) increases transcript expression.

In arthropods, eicosanoids derived from the oxygenated metabolism of arachidonic acid are significant in mediating immune responses. However, the lack of information about insect eicosanoid receptors is an obstacle to completely decipher immune mechanisms underlying both eicosanoid downstream signal cascades and their relationship to immune pathogen-associated molecular patterns (PAMPs). Here, we cloned and sequenced a G protein-coupled receptor (MW 46.16 kDa) from the model lepidopteran, Manduca sexta (Sphingidae). The receptor shares similarity of amino acid motifs to human prostaglandin E2 (PGE2) receptors, and phylogenetic analysis supports its classification as a prostaglandin receptor. In agreement, the recombinant receptor was activated by PGE2 resulting in intracellular cAMP increase, and therefore designated MansePGE2R. Expression of MansePGE2R in Sf9 cells in which the endogenous orthologous receptor had been silenced showed similar cAMP increase upon PGE2 challenge. Receptor transcript expression was identified in various tissues in larvae and female adults, including Malpighian tubules, fat body, gut and hemocytes, and in female ovaries. In addition to the cDNA cloned that encodes the functional receptor, an mRNA was found featuring the poly-A tail but lacking the predicted transmembrane (TM) regions 2 and 3, suggesting the possibility that internally deleted receptor proteins exist in insects. Immunocytochemistry and in situ hybridization revealed that among hemocytes, the receptor was exclusively localized in the oenocytoids. Larval immune challenges injecting bacterial components showed that lipoteichoic acid (LTA) increased MansePGE2R expression in hemocytes. In contrast, injection of LPS or peptidoglycan did not increase MansePGE2R transcript levels in hemocytes, suggesting the LTA-associated increase in receptor transcript is regulated through a distinct pathway. This study provides the first characterization of an eicosanoid receptor in insects, and paves the way for establishing the hierarchy in signaling steps required for establishing insect immune responses to infections.

PTGER2 activation induces PTGS-2 and growth factor gene expression in endometrial epithelial cells of cattle.

The prostaglandin E2 receptor 2 (PTGER2) is present in the endometrium and its gene expression is accompanied with endometrial growth, however, it is unknown whether there is endometrial repair through stimulation of growth factor gene expression that is promoted by PTGER2 activation in cattle. The aim of this study was to investigate whether PTGER2 activation can induce prostaglandin-endoperoxide synthase-2 (PTGS-2) and growth factor gene expression by activating PKA and ERK signaling pathways in endometrial epithelial cells of cattle. Results demonstrated that the PTGER2 agonist, butaprost, induced cAMP/PKA and ERK activation and up-regulated PTGS-2, VEGF, CTGF, TGF-ß1 and IL-8 gene expression. These activations were less after PTGER2 antagonist, AH6809, treatment. Data suggested that PTGS-2 gene expression was induced by PTGER2 activation through the PKA and ERK pathways. Furthermore, PTGER2 activation promoted several growth factor gene expressions in endometrial epithelial cells. One potential implication of this finding is that PTGER2 activation in the endometrium of cattle could induce endometrial repair by stimulating VEGF, CTGF, TGF-ß1 and IL-8 gene expression.

An aromatic amino acid within intracellular loop 2 of the prostaglandin EP2 receptor is a prerequisite for selective association and activation of Gαs.

We previously demonstrated that the aromatic moiety of Tyr143 within the intracellular loop 2 (ICL2) region of the prostaglandin EP2 receptor plays a crucial role in Gs coupling. Here we investigated whether the ICL2 of the EP2 receptor directly binds to Gαs and whether an aromatic moiety affects this interaction. In Chinese hamster ovary cells, mutations of Tyr143 reduced the ability of the EP2 receptor to interact with G proteins as demonstrated by GTPγS sensitivity, as well as the ability of agonist-induced cAMP formation, with the rank order of Phe>Tyr (wild-type)=Trp>Leu>Ala (=0). We found that the wild-type ICL2 peptide (i2Y) and its mutant with Phe at Tyr143 (i2F) inhibited receptor-G protein complex formation of wild-type EP2 in membranes, whereas the Ala-substituted mutant (i2A) did not. Specific interactions between these peptides and the Gαs protein were detected by surface plasmon resonance, but Gαs showed different association rates, with a rank order of i2F>i2Y≫i2A, with similar dissociation rates. Moreover, i2F and i2Y, but not i2A activated membrane adenylyl cyclase. These results indicate that the ICL2 region of the EP2 receptor is its potential interaction site with Gαs, and that the aromatic side chain moiety at position 143 is a determinant for the accessibility of the ICL2 to the Gαs protein.

Human DP and EP2 prostanoid receptors take on distinct forms depending on the diverse binding of different ligands.

Human D-type prostanoid (DP) and E-type prostanoid 2 (EP2) receptors are G protein-coupled receptors and are regarded as the most closely related receptors among prostanoid receptors because they are generated by tandem duplication. The DP receptor-cognate ligand, prostaglandin D2 (PGD2 ) has the ability to activate not only DP receptors but also EP2 receptors. Likewise, the EP2 receptor-cognate ligand, prostaglandin E2 (PGE2 ) has the ability to activate DP receptors in addition to EP receptors in order to stimulate cAMP formation. However, since PGD2 and/or PGE2 activate DP and EP2 receptors to similar maximal levels, that is, their similar efficacies, differences between the ligands in each receptor have not yet been determined in detail except for their different affinities. Herein we demonstrated, using an in silico simulation to predict binding patterns among DP or EP2 receptors and PGD2 , PGE2 , or prostaglandin F2α as the reference prostanoid, that DP and EP2 receptors plausibly take on distinct forms depending on the diverse binding of different ligands. Since these ligands have the potential to make these receptors form distinct conformations with discrete signaling pathways, they are consequently regarded as endogenous biased ligands. Moreover, by using functional assays, the susceptibilities of the DP receptors to the noncognate ligands were approximately 10 times lower than those of EP2 receptors. Thus, EP2 receptors seem to be able to distinguish endogenous ligands better than DP receptors, thereby both receptors are plausibly gaining role-sharing functions with respect to one another as the copies of duplicated gene.

A facile and dynamic assay for the detection of peptide aggregation.

We report on a facile method to detect the aggregation and co-aggregation of peptides by tryptophan fluorescence spectroscopy. Peptide aggregates (PAs) play a pivotal role in neurodegenerative diseases, such as Alzheimer's and Parkinson's. The detection of the formation of aggregates, especially in the early stage, will facilitate the diagnosis and treatment of the associated disease. In this study, by choosing a tryptophan-containing peptide of EP2, we investigated its fluorescence spectroscopic characteristics in the process of PAs. The results showed that the intensity of emission spectra was significantly enhanced with the formation of PAs within 48 h. In addition, by employing EP2 as a fluorescence probe, we found that EP2 was able to effectively monitor the aggregation of other peptides/proteins that are otherwise difficult to detect with conventional approach. Thus, these preliminary data provide a promising diagnostic tool to detect the formation of PAs.

Molecular cloning and expression analysis of prostaglandin E receptor 2 gene in cashmere goat (Capra hircus) skin during hair follicle development.

As a member of the four subtypes of receptors for prostaglandin E2 (PGE2), prostaglandin E receptor 2 (PTGER2) is in the family of G-protein coupled receptors and has been characterized to be involved in the development and growth of hair follicles. In this study, we cloned and characterized the full-length coding sequence (CDS) of PTGER2 gene from cashmere goat skin. The entire open reading frame (ORF) of PTGER2 gene was 1047 bp and encoded 348 amino acid residues. The deduced protein contained one G-protein coupled receptors family 1 signature, seven transmembrane domains, and other potential sites. Tissue expression analysis showed that PTGER2 gene was expressed strongly in the skin. The general expression tendency of PTGER2 gene at different hair follicle developmental stages in the skin was gradually decreased from anagen to catagen to telogen. After comparing with the expression of BMP4 gene and related reports, we further presume that it seems to have a relationship between the hair follicle cycle and the expression level of PTGER2 gene in cashmere goat skin.

Discovery of a novel EP2/EP4 dual agonist with high subtype-selectivity.

A series of γ-lactam prostaglandin E(1) analogs bearing a 16-phenyl moiety in the ω-chain and aryl moiety in the α-chain were synthesized and biologically evaluated. Among the tested compounds, γ-lactam PGE analog 3 designed as a structural hybrid of 1 and 2 was discovered as the most optimized EP2/EP4 dual agonist with excellent subtype-selectivity (K(i) values: mEP2=9.3 nM, mEP4=0.41 nM). A structure-activity relationship study is presented.

The soluble EP2 receptor FuEP2/Ex2 suppresses endometrial cancer cell growth in an orthotopic xenograft model in nude mice.

Endometrial cancer is one of the most common gynecologic malignancies and many factors influence in its growth and development. As in many other types of cancer, prostaglandin E(2) (PGE(2)) is thought to be an accelerator of cell proliferation and endometrial cancer progression. In this study, we examined the effect of FuEP2/Ex2, a soluble decoy receptor for PGE(2) on growth of endometrial cancer cells. A stable transfectant expressing FuEP2/Ex2 was established from human endometrial cancer Ishikawa cells (Ish-FuEP2/Ex2). Ish-FuEP2/Ex2 cells expressed FuEP2/Ex2 mRNA and protein. Expression levels of E-prostanoid receptor 1 (EP1), EP2, EP3, EP4, and F-prostanoid receptor (FP) were almost the same in Ish-FuEP2/Ex2 and vector control cells. Growth rates of Ish-FuEP2/Ex2 under normal culture conditions were also similar to vector control cells, although PGE(2)-induced growth stimulation was completely inhibited in Ish-FuEP2/Ex2 or by Ish-FuEP2/Ex2 culture medium. Moreover, phosphorylation of extracellular signal-regulated kinase (ERK) and induction of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), cyclin D1, and c-fos mRNA by PGE(2) were not observed in Ish-FuEP2/Ex2 and Ish-FuEP2/Ex2 culture medium-treated vector control cells, although they were found when treated with prostaglandin F(2α). An orthotopic xenograft model in athymic nude mice revealed that Ish-FuEP2/Ex2-injected mice had significantly decreased mean tumor area. The proportion of Ki-67-positive cells in the tumor lesion was also significantly lower in Ish-FuEP2/Ex2-injected mice. These findings suggest that an EP-targeting strategy using FuEP2/Ex2 may be of use in the treatment of endometrial cancer.