| 1. |
Shingo Nojima, Yoko Fujita, Kanako Terakado Kimura, Norimichi Nomura, Ryoji Suno, Kazushi Morimoto, Masaki Yamamoto, Takeshi Noda, So Iwata, Hideki Shigematsu, Takuya Kobayashi, Cryo-EM Structure of the Prostaglandin E Receptor EP4 Coupled to G Protein., Structure (London, England : 1993), 10.1016/j.str.2020.11.007, 29, 3, 252-260, 2021.03, Prostaglandin E receptor EP4, a class A G protein-coupled receptor (GPCR), is a common drug target in various disorders, such as acute decompensated heart failure and ulcerative colitis. Here, we report the cryoelectron microscopy (cryo-EM) structure of the EP4-heterotrimeric G protein (Gs) complex with the endogenous ligand at a global resolution of 3.3 Å. In this structure, compared with that in the inactive EP4 structure, the sixth transmembrane domain is shifted outward on the intracellular side, although the shift is smaller than that in other class A GPCRs bound to Gs. Instead, the C-terminal helix of Gs is inserted toward TM2 of EP4, and the conserved C-terminal hook structure formsthe extended state. These structural features are formed by the conserved residues in prostanoid receptors (Phe542.39 and Trp3277.51). These findings may be important for the thorough understanding of the G protein-binding mechanism of EP4 and other prostanoid receptors.. |
| 2. |
Kazushi Morimoto, Ryoji Suno, Yunhong Hotta, Keitaro Yamashita, Kunio Hirata, Masaki Yamamoto, Shuh Narumiya, So Iwata, Takuya Kobayashi, Crystal structure of the endogenous agonist-bound prostanoid receptor EP3., Nature chemical biology, 10.1038/s41589-018-0171-8, 15, 1, 8-10, 2019.01, Prostanoids are a series of bioactive lipid metabolites that function in an autacoid manner via activation of cognate G-protein-coupled receptors (GPCRs). Here, we report the crystal structure of human prostaglandin (PG) E receptor subtype EP3 bound to endogenous ligand PGE2 at 2.90 Å resolution. The structure reveals important insights into the activation mechanism of prostanoid receptors and provides a molecular basis for the binding modes of endogenous ligands.. |
| 3. |
Yosuke Toyoda, Kazushi Morimoto, Ryoji Suno, Shoichiro Horita, Keitaro Yamashita, Kunio Hirata, Yusuke Sekiguchi, Satoshi Yasuda, Mitsunori Shiroishi, Tomoko Shimizu, Yuji Urushibata, Yuta Kajiwara, Tomoaki Inazumi, Yunhon Hotta, Hidetsugu Asada, Takanori Nakane, Yuki Shiimura, Tomoya Nakagita, Kyoshiro Tsuge, Suguru Yoshida, Tomoko Kuribara, Takamitsu Hosoya, Yukihiko Sugimoto, Norimichi Nomura, Miwa Sato, Takatsugu Hirokawa, Masahiro Kinoshita, Takeshi Murata, Kiyoshi Takayama, Masaki Yamamoto, Shuh Narumiya, So Iwata, Takuya Kobayashi, Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface., Nature chemical biology, 10.1038/s41589-018-0131-3, 15, 1, 18-26, 2019.01, Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.. |
| 4. |
Satoshi Yasuda, Yuta Kajiwara, Yosuke Toyoda, Kazushi Morimoto, Ryoji Suno, So Iwata, Takuya Kobayashi, Takeshi Murata, Masahiro Kinoshita, Hot-Spot Residues to be Mutated Common in G Protein-Coupled Receptors of Class A: Identification of Thermostabilizing Mutations Followed by Determination of Three-Dimensional Structures for Two Example Receptors, JOURNAL OF PHYSICAL CHEMISTRY B, 10.1021/acs.jpcb.7b02997, 121, 26, 6341-6350, 2017.07, G protein-coupled receptors (GPCRs), which are indispensable to life and also implicated in a number of diseases, construct important drug targets. For the efficient structure-guided drug design, however, their structural stabilities must be enhanced. An amino-acid mutation is known to possibly lead to the enhancement, but currently available experimental and theoretical methods for identifying stabilizing mutations suffer such drawbacks as the incapability of exploring the whole mutational space with minor effort and the unambiguous physical origin of the enhanced or lowered stability. In general, after the identification is successfully made for a GPCR, the whole procedure must be followed all over again for the identification for another GPCR. Here we report a theoretical strategy by which many different GPCRs can be considered at the same time. The strategy is illustrated for three GPCRs of Class A in the inactive state. We argue that a mutation of the residue at a position of N-BW = 3.39 (N-BW is the Ballesteros-Weinstein number), a hot-spot residue, leads to substantially higher stability for significantly many GPCRs Of Class A in the inactive state. The most stabilizing mutations of the residues with N-BW = 3.39 are then identified for two of the three GPCRs, using the improved version of our free-energy function, These identifications are experimentally corroborated, which is followed:by the determination of,new three-dimensional (3D) structures for the two GPCRs. We expect that on the basis of the strategy, the 3D structures of many GPCRs of Class A can be solved for the first time in succession.. |
| 5. |
Kazushi Morimoto, Naritoshi Shirata, Yoshitaka Taketomi, Soken Tsuchiya, Eri Segi-Nishida, Tomoaki Inazumi, Kenji Kabashima, Satoshi Tanaka, Makoto Murakami, Shuh Narumiya, Yukihiko Sugimoto, Prostaglandin E-2-EP3 Signaling Induces Inflammatory Swelling by Mast Cell Activation, JOURNAL OF IMMUNOLOGY, 10.4049/jimmunol.1300290, 192, 3, 1130-1137, 2014.02, PGE(2) has long been known as a potentiator of acute inflammation, but its mechanisms of action still remain to be defined. In this study, we employed inflammatory swelling induced in mice by arachidonate and PGE(2) as models and dissected the role and mechanisms of action of each EP receptor at the molecular level. Arachidonate- or PGE(2)-induced vascular permeability was significantly reduced in EP3-deficient mice. Intriguingly, the PGE(2)-induced response was suppressed by histamine H-1 antagonist treatment, histidine decarboxylase deficiency, and mast cell deficiency. The impaired PGE(2)-induced response in mast cell-deficient mice was rescued upon reconstitution with wild-type mast cells but not with EP3-deficient mast cells. Although the number of mast cells, protease activity, and histamine contents in ear tissues in EP3-deficient mice were comparable to those in wild-type mice, the histamine contents in ear tissues were attenuated upon PGE(2) treatment in wild-type but not in EP3-deficient mice. Consistently, PGE(2)-EP3 signaling elicited histamine release in mouse peritoneal and bone marrow-derived mast cells, and it exerted degranulation and IL-6 production in a manner sensitive to pertussis toxin and a PI3K inhibitor and dependent on extracellular Ca2+ ions. These results demonstrate that PGE(2) triggers mast cell activation via an EP3-G(i/o)-Ca2+ influx/PI3K pathway, and this mechanism underlies PGE(2)-induced vascular permeability and consequent edema formation.. |
| 6. |
Ryo Iwasaki, Kyoshiro Tsuge, Kazushi Morimoto, Tomoaki Inazumi, Osamu Kawahara, Atsuo Kawahara, Soken Tsuchiya, Yukihiko Sugimoto, Molecular and pharmacological characterization of zebrafish 'contractile' and 'inhibitory' prostanoid receptors, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2013.07.075, 438, 2, 353-358, 2013.08, Prostanoids comprising prostaglandins (PGs) and thromboxanes (TXs) have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been established. Here, we demonstrate that there exist at least five 'contractile' (Ca2+-mobilizing) and one 'inhibitory' (G(i)-coupled) prostanoid receptors in zebrafish; five 'contractile' receptors consisting of two PGE(2) receptors (EP1a and EP1b), two PGF(2 alpha) receptors (FP1 and FP2), and one TXA(2) receptor TP, and one 'inhibitory' receptor, the PGE2 receptor EP3. [H-3]PGE(2) specifically bound to the membranes of cells expressing zebrafish EP1a, EP1b and EP3 with a Kd of 4.8, 1.8 and 13.6 nM, respectively, and [H-3]PGF(2 alpha), specifically bound to the membranes of cells expressing zebrafish FP1 and FP2, with a Kd of 6.5 and 1.6 nM, respectively. U-46619, a stable agonist for human and mouse TP receptors, significantly increased the specific binding of [S-35]GTP gamma S to membranes expressing the zebrafish TP receptor. Upon agonist stimulation, all six receptors showed an increase in intracellular Ca2+ levels, although the increase was very weak in EP1b, and pertussis toxin abolished only the EP3-mediated response. Zebrafish EP3 receptor also suppressed forskolin-induced cAMP formation in a pertussis toxin-sensitive manner. In association with the low structural conservation with mammalian receptors, most agonists and antagonists specific for mammalian EP1, EP3 and TP failed to work on each corresponding zebrafish receptor. This work provides further insights into the diverse prostanoid actions mediated by their receptors in zebrafish. (C) 2013 Elsevier Inc. All rights reserved.. |
| 7. |
Kyoshiro Tsuge, Ryo Iwasaki, Kazushi Morimoto, Tomoaki Inazumi, Osamu Kawahara, Atsuo Kawahara, Soken Tsuchiya, Yukihiko Sugimoto, Molecular and pharmacological characterization of zebrafish 'relaxant' prostanoid receptors, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2013.06.017, 436, 4, 685-690, 2013.07, Prostanoids comprising prostaglandins (PGs) and thromboxanes have been shown to play physiological and pathological roles in zebrafish. However, the molecular basis of zebrafish prostanoid receptors has not been characterized to date. Here, we demonstrate that there exist at least six 'relaxant' (Gs-coupled) prostanoid receptors in zebrafish; one PGI(2) receptor IP and five PGE(2) receptors comprising two EP2 (EP2a and EP2b), and three EP4 receptors (EP4a, EP4b and EP4c). In contrast, we failed to find a zebrafish PGD(2) receptor with any structure and/or character similarities to the mammalian DP1 receptor. [H-3]iloprost, a stable IP radioligand, specifically bound to the membrane of cells expressing zebrafish IP with a Kd of 42 nM, and [H-3]PGE(2) specifically bound to the membranes of cells expressing zebrafish EP2a, EP2b, EP4a, EP4b and EP4c with a Kd of 6.9, 6.0, 1.4, 3.3 and 1.2 nM, respectively. Upon agonist stimulation, the 'relaxant' prostanoid receptors showed intracellular cAMP accumulation. The responsiveness of these zebrafish receptors to subtype-specific agonists correlated with their structural conservation to the corresponding receptor in mammals. RT-PCR analysis revealed that the six zebrafish prostanoid receptors show unique tissue distribution patterns; each receptor gene may hence be under unique transcriptional regulation. This work provides further insights into the diverse functions of prostanoids in zebrafish. (C) 2013 Elsevier Inc. All rights reserved.. |
| 8. |
Yoshitaka Taketomi, Noriko Ueno, Takumi Kojima, Hiroyasu Sato, Remi Murase, Kei Yamamoto, Satoshi Tanaka, Mariko Sakanaka, Masanori Nakamura, Yasumasa Nishito, Momoko Kawana, Naotomo Kambe, Kazutaka Ikeda, Ryo Taguchi, Satoshi Nakamizo, Kenji Kabashima, Michael H. Gelb, Makoto Arita, Takehiko Yokomizo, Motonao Nakamura, Kikuko Watanabe, Hiroyuki Hirai, Masataka Nakamura, Yoshimichi Okayama, Chisei Ra, Kosuke Aritake, Yoshihiro Urade, Kazushi Morimoto, Yukihiko Sugimoto, Takao Shimizu, Shuh Narumiya, Shuntaro Hara, Makoto Murakami, Mast cell maturation is driven via a group III phospholipase A(2)-prostaglandin D-2-DP1 receptor paracrine axis, NATURE IMMUNOLOGY, 10.1038/ni.2586, 14, 6, 554-+, 2013.06, Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A(2), regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD(2) synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.. |
| 9. |
Functions of Prostaglandin Receptors in Contact Dermatitis and Application to Drug Discovery
Contact dermatitis is an inflammatory skin disease caused by toxic factors that activate the skin innate immunity (irritant contact dermatitis) or by a T cell-mediated hypersensitivity reaction (allergic contact dermatitis). These inflammatory kin diseases are sometimes still not easy to control. Therefore, the development of new effective drugs with fewer side elects is anticipated. In the skin under pathophysiological conditions, multiple prostaglandins are produced and their receptors are expressed in time- and/or cell-dependent manners. However, the precise role of prostaglandins and their receptors in contact dermatitis has not been fully understood. Recently, studies using mice with a disruption of each prostaglandin receptor gene, as well as receptor-selective compounds revealed that prostaglandin receptors have manifold functions, sometimes resulting in opposite outcomes. Here, we review new advances in the roles of prostaglandin receptors in contact hypersensitivity as a cutaneous immune response model, and also discuss the clinical potentials of receptor-selective drugs.. |
| 10. |
Inazumi T, Shirata N, Morimoto K, Takano H, Segi-Nishida E, Sugimoto Y, Prostaglandin E₂-EP4 signaling suppresses adipocyte differentiation in mouse embryonic fibroblasts via an autocrine mechanism., Journal of lipid research, 10.1194/jlr.M013615, 52, 8, 1500-1508, 2011.08. |
| 11. |
Shigero Tamba, Rieko Yodoi, Kazushi Morimoto, Tomoaki Inazumi, Mamiko Sukeno, Eri Segi-Nishida, Yasushi Okuno, Gozoh Tsujimoto, Shuh Narumiya, Yukihiko Sugimoto, Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling, BIOCHIMIE, 10.1016/j.biochi.2010.04.006, 92, 6, 665-675, 2010.06, To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2(-/-) cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix-(ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE(2)-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions. (C) 2010 Published by Elsevier Masson SAS.. |
| 12. |
Kumiko Yamaoka, Akiko Yano, Kenji Kuroiwa, Kazushi Morimoto, Tomoaki Inazumi, Noriyuki Hatae, Hiroyuki Tabata, Eri Segi-Nishida, Satoshi Tanaka, Atsushi Ichikawa, Yukihiko Sugimoto, Prostaglandin EP3 receptor superactivates adenylyl cyclase via the G(q)/PLC/Ca2+ pathway in a lipid raft-dependent manner, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2009.09.064, 389, 4, 678-682, 2009.11, 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 Ca2+-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP3 formation and intracellular Ca2+ mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca2+ 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 Ca2+ mobilization in a lipid raft microdomain-dependent manner. (C) 2009 Elsevier Inc. All rights reserved.. |
| 13. |
Rieko Yodoi, Shigero Tamba, Kazushi Morimoto, Eri Segi-Nishida, Mika Nishihara, Atsushi Ichikawa, Shuh Narumiya, Yukihiko Sugimoto, RhoA/Rho Kinase Signaling in the Cumulus Mediates Extracellular Matrix Assembly, ENDOCRINOLOGY, 10.1210/en.2008-1449, 150, 7, 3345-3352, 2009.07, Cumulus cells surround the oocyte and regulate the production and assembly of the extracellular matrix (ECM) around the cumulus-oocyte complex for its timely interaction with sperm in the oviduct. We recently found that C-C chemokines such as CCL2, CCL7, and CCL9 are produced and stimulate integrin-mediated ECM assembly in the postovulatory cumulus to protect eggs and that prostaglandin E(2)-EP2 signaling in the cumulus cells facilitates fertilization by suppressing this chemokine signaling, which otherwise results in fertilization failure by preventing sperm penetration through the cumulus ECM. However, it remains unknown as to what mechanisms underlie chemokine-induced cumulus ECM assembly. Here we report that inhibition of EP2 signaling or addition of CCL7 augments RhoA activation and induces the surface accumulation of integrin and the contraction of cumulus cells. Enhanced surface accumulation of integrin then stimulates the formation and assembly of fibronectin fibrils as well as induces cumulus ECM resistance to hyaluronidase and sperm penetration. These changes in the cumulus ECM as well as cell contraction are relieved by the addition of Y27632 or blebbistatin. These results suggest that chemokines induce integrin engagement to the ECM and consequent ECM remodeling through the RhoA/Rho kinase/actomyosin pathway, making the cumulus ECM barrier resistant to sperm penetration. Based on these results, we propose that prostaglandin E(2)-EP2 signaling negatively regulates chemokine-induced Rho/ROCK signaling in cumulus cells for successful fertilization. (Endocrinology 150: 3345-3352, 2009). |
| 14. |
Hirotsugu Takano, Shunsuke Nakazawa, Naritoshi Shirata, Shigero Tamba, Kazuyuki Furuta, Sohken Tsuchiya, Kazushi Morimoto, Naoki Itano, Atsushi Irie, Atsushi Ichikawa, Koji Kimata, Kazuhisa Nakayama, Yukihiko Sugimoto, Satoshi Tanaka, Involvement of CD44 in mast cell proliferation during terminal differentiation, LABORATORY INVESTIGATION, 10.1038/labinvest.2008.159, 89, 4, 446-455, 2009.04, By using the recently established culture system that reproduces the terminal differentiation process of connective tissue-type mast cells, we found significant transcriptional induction of CD44. As CD44 is a primary receptor for hyaluronan (HA), which is one of the major extracellular matrix components, we investigated the role of CD44 in cutaneous mast cells. When co-cultured with fibroblasts, mouse bone marrow-derived cultured mast cells (BMMCs) were found to form clusters in an HA-dependent manner. As compared with BMMCs derived from the wild-type mice, those from the CD44(-/-) mice exhibited impaired growth during the co-cultured period. Furthermore, in the peritoneal cavities and ear tissues, mature mast cells were fewer in number in the CD44(-/-) mice than in the wild-type mice. We investigated roles of CD44 in mast cell proliferation by reconstituting BMMCs into the tissues of mast cell-deficient, Kit(W)/Kit(W-v) mice, and found that the number of metachromatic cells upon acidic toluidine blue staining in the tissues transplanted with CD44(-/-) BMMCs was not significantly changed for 10 weeks, whereas that in the tissues transplanted with the CD44(+/+) BMMCs was significantly increased. These results suggest that CD44 plays a crucial role in the regulation of the cutaneous mast cell number.. |
