International GPCR Symposium

June 29 (Fri) -30 (Sat) Siran Kaikan, Kyoto University

Programme International GPCR Symposium

Date and time: Friday, June 29th - Saturday, June 30th, 2018

Venue: Shiran kaikan, Kyoto University, Kyoto University medical school area, Yoshida Konoe-chou, Sakyou-ku, Kyoto, 606-8501, Japan

Purpose of the Session: G protein-coupled receptors (GPCRs), also called as seven-transmembrane receptors, are the largest family of membrane receptors in the human genome and consist of approximately 800 receptors. Importantly, about half of which are GPCRs that interact with endogenous ligands and are the targets for many therapeutic drugs currently in use. In addition, because a substantial number of GPCRs still remain orphan, their biological roles and therapeutic potential are of special interests. This WCP2018 satellite symposium is aimed to exchange the newest insights as well as up-to-date overview of GPCR research and discuss the therapeutic potentials. This satellite symposium is organized as an international meeting of the 15th GPCR Conference, which has been held annually in Japan since 2004. We sincerely look forward to seeing you in Kyoto.

Chair: Prof. Atsuro Miyata Kagoshima University Vice chair: Prof. Hitoshi Hashimoto Osaka University Auditor: Prof. Seiji Shioda Hoshi University Prof. Kenji Kangawa National Cerebral and Cardiovascular Center Research Institute Committee: Prof. So Iwata Kyoto University Prof. Masayasu Kojima Kurume University Prof. Takeshi Sakurai Tsukuba University Dr. Yutaka Takahashi Kobe University Prof. Masaki Tanaka Kyoto Prefectual University of Medicine Prof. Kazuwa Nakao Kyoto University Dr. Mikiya Miyazato National Cerebral and Cardiovascular Center Research Institute Dr. Mayumi Furuya Kyoto University Dr. Makiko Suwa Aoyama Gakuin University Prof. Toshihiko Yada Jichi Medical University Prof. Masamitsu Nakazato University of Miyazaki Secretary General: Dr. Yuki Kambe Kagoshima University

1 Venue

Shiran Kaikan

Stage

Oral presentation Inamori Hall

Poster Registration desk Gala dinner Yamauchi Hall

From Kansai International Airport to Kyoto Kyoto city map

Local map

For chairpersons and speakers

For Chairpersons Please take the next chairperson’s seat at least 15 minutes before the beginning of your session.

For Symposists and Oral Presenters Presenters are asked to bring your own laptop PC. The types of connecters available are a D-sub 15 pin (mini) and an HDMI (type A) only. Please bring your own connecter in case your PC does not accept the D-sub 15 pin (mini) or HDMI (type A) connecter. The resolution of the projection screen is XGA (1024 ×768 pixels). Be sure to disable a screensaver and a power-saving mode prior to your presentation. For presenters who want to use PC in the presentation room (Windows PC only), save your presentation data on a USB flash drive, register it at the Operation Desk no later than 60 minutes prior to your presentation. Please visit the Operation Desk located inside the Inamori Hall, no later than 30 minutes prior to your presentation in order to check the connection and operation. Please take the next speaker’s seat during the presentation up to 15 minutes prior to your own presentation.

For Poster Presenters Posting and removal times are scheduled as shown below. Note that any posters remaining past the removal time will be disposed of by the Secretariat. The size of panel is 160 * 90 cm. The Presentation number will be provided in advance. Posting June 29 (Fri) 12:30 - 14:30 Presentation June 29 (Fri) 16:30 - 17:30 Removal June 30 (Sat) 12:00 - 13:30

Programme

Day 1, Jun/29/2018 (Fri)

12:00 - 12:50 Registration

12:50 - 13:00 Opening remark

13:00 - 14:30 Session 1. Historical comments on GPCR by Japanese pioneers Chair: Seiji Shioda (Hoshi University) Chair: Mayumi Furuya (Kyoto University) 1. Dynamic control of glutamatergic transmission via distinct neurotransmitter-receptor interactions Shigetada Nakanishi SUNTORY Foundation for Life Sciences, Bioorganic Research Institute 2. Personal Memories of Biochemical Studies on G Protein-Coupled Receptors Tatsuya Haga Tokyo University Medical School 3. GPCR biology of lipid ligands Takao Shimizu Lipid Signaling Project, National Center for Global Health and Medicine, Department of Lipidomics, Faculty of Medicine, The University of Tokyo, Tokyo, Japan

14:30 - 14:50 Coffee break

14:50 - 16:20 Session 2. New technologies for GPCRs Chair: Masayasu Kojima (Kurume University) Chair: Takanori Ida (University of Miyazaki) 1. The G protein-coupled receptor database, GPCRdb David E. Gloriam Department of Drug Design and Pharmacology, University of Copenhagen Denmark 2. Fluorescence and bioluminescence approaches to study ligand binding to GPCRs and RTKs Stephen J Hill School of Life Sciences, University of Nottingham, Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK 3. Split-luciferase-based G protein biosensors Francois Marie Ngako Kadji Graduate School of Pharmaceutical Sciences, Tohoku University

Programme

16:30 - 17:30 Poster session

17:40 - 18:40 Plenary Lecture 1 Chair: Yumiko Saito (Hiroshima University) Orphan GPCRs history Olivier Civelli Department of Pharmacology, University of California, Irvine, USA

19:00 - Gala dinner

Programme

Day 2, Jun/30/2018 (Sat)

9:00 - 10:30 Session 3. 3D Structure of GPCRs Chair: Hitoshi Hashimoto (Osaka University) Chair: Yosuke Toyoda (Tsinghua University) 1. Crystal structure of the human prostaglandin E receptor EP4 Yosuke Toyoda Department of Medical Chemistry and Cell Biology, Kyoto University Graduate School of Medicine; School of Medicine, Tsinghua University

2. A crystal structure of the lysophospholipid receptor LPA6and insights into lipid ligand entry and a hereditary hair disease Asuka Inoue Graduate School of Pharmaceutical Sciences, Tohoku University, PRIME, Japan Agency for Medical Research and Development (AMED) 3. Endothelin Receptor – Ligand binding and conformational changes Tomoko Doi Graduate School of Science, Department of Biophysics, Kyoto University, Japan

10:30 - 10:50 Coffee break

11:00 - 12:00 Plenary Lecture 2 Chair: Takao Shimizu (The University of Tokyo) BLT1 and BLT2, two GPCRs for lipid mediators Takehiko Yokomizo Department of Biochemistry, Graduate School of Medicine, Juntendo University, Japan

12:00 - 13:00 Break for lunch (by your own)

Programme

13:00 - 14:30 Session 4. Drug discovery based on GPCRs Chair: Kenji Kangawa (National Cerebral and Cardiovascular Center), Chair: Hiroyuki Kaiya (National Cerebral and Cardiovascular Center) 1. The Discovery of Histamine H4 Receptor Antagonists for the treatment of Inflammation Robin L. Thurmond Janssen Research & Development, LLC San Diego, CA 92121 2. Employing novel chemogenetic approaches to defining GPCR-biology Andrew B. Tobin Institute for Molecular and cell and systems biology, University of Glasgow, Glasgow, G12 8QQ, UK 3. Prediction of cancer-associated hotspot mutations that affect GPCR oligomerization Wataru Nemoto Department of Science and Technology, Tokyo Denki University, Saitama, Japan

14:30 - 15:00 Coffee break

15:00 - 16:30 Session 5. GPCR and Diseases Chair: Kazuwa Nakao (Kyoto University) Chair: Masaki Tanaka (Kyoto Prefectural University of Medicine) 1. Structure-based Discovery of Non-metabolite Synthetic Ligands for Metabolite Receptors - exploding Orthosteric versus Allosteric Locks Thue W.Schwartz Center for Basic metabolic Research, University of Copenhagen, Copenhagen Denmark 2. Autocrine Regulation of Metabolic Functions Through Metabolite GPCRs Stefan Offermanns Dept. of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany, Medical Faculty, J. W. Goethe University, Frankfurt, Germany 3. Functional analysis of dry syndrome by PACAP and its prevention and possible use for therapeutic application Seiji Shioda Peptide Drug Innovation, Global Research Center for Innovative Life Science, Hoshi University

16:30 - 16:40 Closing remark

Programme

Poster presentations (16:30 - 17:30, Day 1, Jun/29/2018 (Fri))

1. Allosteric transmission with rigidity propagation across GPCR networks Adnan Sljoka Kwansei Gakuin University, Japan 2. Illuminating G12-coupled GPCRs Asuka Inoue Graduate School of Pharmaceutical Sciences, Tohoku University, PRIME, Japan Agency for Medical Research and Development (AMED) 3. Identification of ghrelin and its receptor in Schlegel's Japanese gecko, Gekko japonicus Hiroyuki Kaiya Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Japan 4. Effect of MRS2578, a P2Y6 receptor inhibitor, on IgE-dependent activation of human basophils Manabu Nakano Graduate School of Health Sciences, Department of Bioscience and Laboratory Medicine, Hirosaki University, Japan, 2 Graduate School of Health Sciences, Research Center for Biomedical Sciences, Hirosaki University Japan 5. RNA editing of 5-HT2C in the nucleus accumbens participates in alcohol drinking behavior Masaki Tanaka Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, Japan 6. The Endogenous Pituitary Adenylate Cyclase-Activating Polypeptide increases food intake by modulating the expression of neuropeptides in the mouse hypothalamus Nguyen Thanh Trung Department of Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, Japan 7. Recent progress on screening for novel GPCR ligands using GPCR-G fusion proteins Shigeki Takeda Faculty of Science and Technology, Division of Molecular Science, Gunma University

Programme

8. Functional analysis of promotion of sweat secretion by PACAP Takahiro Hirabayashi Peptide Drug Innovation, Global Research Center for Innovative Life Science, Hoshi University, Tokyo, Japan 9. Identification of novel bioactivepeptide, LURY-1 Takanori Ida Center for Animal Disease Control, University of Miyazaki 10. Distribution, characterization, and regulatory effect on feeding behavior of PACAP/ PAC1 receptors system in zebrafish Tomoya Nakamachi Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama, Toyama 930-8555, Japan

11. Crystal structure of leukotriene B4 receptor 1 bound with an inverse agonist Toshiaki Okuno Department of Biochemistry, Juntendo University School of Medicine, 12. Regulator of G protein signaling 8 (RGS8) modulates depression-like behavior via melanin-concentrating hormone receptor 1 activity Yuki Kobayashi Graduate School of Medical and Dental Sciences, Department of Pharmacology, Hiroshima University, Japan

Programme

Matsuo Award (16:30 - 17:30, Day 1, Jun/29/2018 (Fri))

1. Characterization of orexigenic neuropeptide receptor in neuronal primary cilia in vitro and in vitro. Daisuke Miki Graduate School of Integrated Arts and Sciences, Department of Behavioral Neuroscience, Hiroshima University, Japan 2. Calcitonin receptor modulates body temperature rhythm in mammals Hiroyuki Shimatani Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan 3. Structure-guided development of subtype-selective muscarinic acetylcholine receptor antagonists Hongtao Liu Beijing Advanced Innovation Center for Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China 4. PACAP induces differentiation of neural progenitor cells into glial lineage via radial glia Jun Watanabe Department of Biochemistry, Center for Laboratory Animal Science, School of Medicine, Showa University, Tokyo, Japan 5. The orphan receptor GPRL mediates light-induced activation of clock gene expression in the suprachiasmatic nucleus. Kaoru Goto Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Japan 6. Comprehensive analysis of Gα-protein-coupling ability among orphan GPCRs Kouki Kawakami Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan. 7. Agonist-independent cAMP-repressing activity of the orphan receptor Gpr176 requires Gz. Shumpei Nakagawa Development of Systems Biology, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University

Programme

8. Development of high throughput screening assay method for Gz-linked orphan receptor Gpr176 Tianyu Wang Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan 9. Generation of HEK293 cells devoid of Gi proteins. Yuki Ono Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University 10. PACAP-provoked PAC1 receptor signaling and internalization through two isoform of β-arrestins dependent mechanisms Yusuke Shintani Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Japan

Plenary Lecture 1

Orphan GPCRs history

Olivier Civelli1, Yan Zhang2 and Yumiko Saito1 1 Department of Pharmacology, University of California, Irvine, USA, 2 School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China, 3 Graduate School of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan

The majority of the G protein-coupled receptors (GPCRs) began as orphan GPCRs, genes found on the basis of their sequences to belong to the GPCR family but which lack endogenous ligands. Finding these ligands,”deorphanizing” the putative GPCRs, began by matching known transmitters or hormones to particular orphan GPCRs. Deorphanization of the dopamine D2 and 5-HT1A receptors were the first to be reported. These successes were rapidly followed by a large number of deorphanizations and ultimately most known transmitters matched to particular orphan GPCRs.

However at the same time, mining of the genome pointed at the existence of GPCRs still orphan. We designed a strategy that use orphan GPCRs as targets to identify novel endogenous transmitters. We were successful in discovering OFQ/N the first new orphan GPCR neuropeptide. This strategy has since led to the discovery of several neuropeptides. However, the discovery of novel neurotransmitters or neuropeptides is only the first step in this strategy. The second one is to find the functions of these new transmitters in the organism. This is a difficult yet exciting aim as it leads to the possibility of pharmacological intervention. Moreover, GPCRs are known to modulate higher brain functions. When higher brain functions are disrupted, psychiatric disorders arise. Therefore studying these deorphanized GPCR systems can have a direct impact in psychiatry. In this presentation, I will discuss one such “deorphanized” system, that may have neuropsychiatric implications..

The discovery of novel endogenous transmitters via orphan GPCR strategy while very successful earlier, it has slowed down over the last decade. Yet there still exist at least 100 orphan GPCRs in the human genome. The difficulties at finding the natural ligand of an orphan GPCR will be discussed in the second part of my presentation by following our attempt at deorphanizing a GPCR that has all the hallmarks of a neuropeptide receptor.

Plenary Lecture 2

BLT1 and BLT2, two GPCRs for lipid mediators

Takehiko Yokomizo Department of Biochemistry, Graduate School of Medicine, Juntendo University, Japan

BLT1 is a high-affinity GPCR for leukotriene B4 (LTB4), a potent lipid chemoattractant for phagocytes (1). Although numerous study using BLT1-KO mice revealed that BLT1 is involved in various inflammatory diseases including arthritis, asthma, psoriasis, atherosclerosis and cancer, no BLT1 antagonists are currently available for clinical treatments for patients (2). Recently, we succeeded in the crystallization of BLT1 with an antagonist BIIL 260, and showed that the benzamidine moiety of this compound could be applied as a common structure for inverse agonists against various GPCRs (3). We identified BLT2 as a low-affinity LTB4 receptor (4), and a cyclooxygenase (COX) product 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) as a high-affinity ligand for BLT2 (5). Analyses using BLT2-KO mice showed that 12-HHT/BLT2 axis is important in acceleration of wound healing in skin (6) and cornea (7), and maintenance of epithelial barrier function (8). Some of the adverse effects of non-steroidal anti-inflammatory drugs are caused by the reduced production of 12-HHT in vivo (6). In my talk, I will briefly review our works on BLT1 and BLT2, and also discuss the novel roles of BLT1 and BLT2 over some unpublished data on BLT1- or BLT2-KO mice.

References 1) Yokomizo, T.et al. Nature, 387: 620-624 (1997) 2) Yokomizo, T.et al. J Clin Invest: (2018) In press. 3) Hori, T.et al. Nat Chem Biol, 14: 262-269 (2018) 4) Yokomizo, T.et al. J Exp Med, 192: 421-432 (2000) 5) Okuno, T.et al. J Exp Med, 205: 759-766 (2008) 6) Liu, M.et al. J Exp Med, 211: 1063-1078 (2014) 7) Iwamoto, S.et al. Sci Rep, 7: 13267 (2017) 8) Ishii, Y.et al. FASEB J, 30: 933-947 (2016)

Session 1. Historical comments on GPCR by Japanese pioneers 1

Dynamic control of glutamatergic transmission via distinct neurotransmitter-receptor interactions

Shigetada Nakanishi SUNTORY Foundation for Life Sciences Bioorganic Research Institute

Glutamate receptors are essential for learning, memory and other brain functions and classified as ion-permeating AMPA and NMDA receptors and G protein-coupled metabotropic receptors (mGluRs). When we started investigations of glutamate receptors at late 1980, the molecular entity of glutamate receptors was poorly understood, due to their tight embedding into cell membranes. We therefore developed a new cloning strategy for these membrane proteins by combining electrophysiology and the Xenopus oocyte expression system. This strategy allowed us to molecularly elucidate the family of mGluRs and NMDA receptors. The diverse members of glutamate receptors are widely but distinctly distributed in various neuronal cell types and play specialized roles in a variety of brain functions. Of particular interest in our findings is that inhibitory mGluR6 and excitatory AMPA receptor serve to discriminate light and dark signals at the On and Off parallel pathways in the retinal network. Analogous mechanism underlying neural information processing takes place in the basal ganglia circuit, in which the stimulatory D1 and inhibitory D2 dopamine receptors are separately expressed in parallel striatal neural pathways and play a key role for evoking opposite behavioral responses, namely, reward-seeking and aversive actions. Hence, the dynamic control of neurotransmitter-receptor interactions is crucial in rapid discrimination and processing of neural information in response to environmental changes.

Session 1. Historical comments on GPCR by Japanese pioneers 2

Personal Memories of Biochemical Studies on G Protein-Coupled Receptors

Tatsuya Haga Tokyo University Medical School

In 1975-1977, Tatsuya and Kazuko Haga had a chance to study on the signal transduction from adrenaline to cAMP in Gilman’s Laboratory in Virginia University. He had shown that the agonist binding is affected by GTP. We determined hydrodynamic properties of the ligand binding component (adrenergic receptor) and the catalytic component as distinct entities. Ross and Gilman demonstrated that the catalytic component is composed of two units a GTP-related one (later termed as G protein Gs) and a catalytic one (adenylate cyclase). In 1977-1980, ample evidence indicated that GTP is involved in the inhibition as well as in the activation of adenylate cyclase. We studied on GTP-dependent, high-affinity agonist binding of  adrenergic receptors and muscarinic acetylcholine receptors (mAChRs) in Ichiyama’s Labo. in Hamamatsu U. Sch. Med. Nukada et al. cloned cDNAs for the relevant GTP-binding proteins (transducin, Gi etc.) in collaboration with Numa’s Labo. in Kyoto U. In 1981, we studied on ligands and physical properties of mAChRs in Burgen’s Labo. in London. In 1983-1985, K. Haga developed an affinity chromatography system for mAChRs and purified them to homogeneity. In 1986, we showed that purified mAChRs interact with purified G proteins Gi or Go in collaboration with Ui’s Labo. in Hokkaido U., providing direct evidence for the function of mAChRs, and purification of mAChRs enabled us to determine their partial sequences in collaboration with Matsuo’s Labo. in Miyazaki U. Med. Sch., which were used to clone cDNAs for mAChRs (M1 and M2 receptors) in collaboration with Numa’s Labo. In the same year a cDNA for  adrenergic receptors was also cloned. Thus mAChRs and  adrenergic receptors as well as rhodopsin, which are typical receptors for a transmitter, a hormone, and an outer signal respectively, were shown to have the same structural characteristics with seven-transmembrane segments and the same functional activity that is to interact with and activate G proteins. These three proteins served as prototypes for the concept of G protein-coupled receptors. In 1988-2011, we studied on Gq and Gq-mAChR interaction with Nakamura and others, and on the regulation of mAChRs, particularly on the agonist-dependent phosphorylation of M2 receptors by G protein-coupled receptor kinase 2 (GRK2) and their internalization with Kameyama and others in Tokyo U. and Gakushuin U. We also attempted to crystalize mAChRs more than 10 years with little success. In 2011 we sent purified mAChRs, which were prepared with help of Kobayashi et al. in Kyoto U., to Kobilka’s Labo. in Stanford U., where the crystal structure of M2 receptors was determined.

Ref. T. Haga: Molecular properties of muscarinic acetylcholine receptors. Proc. Jpn. Acad. 89, 226-256 (2013).

Session 1. Historical comments on GPCR by Japanese pioneers 3

GPCR biology of lipid ligands

Takao Shimizu Lipid Signaling Project, National Center for Global Health and Medicine, Department of Lipidomics, Faculty of Medicine, The University of Tokyo, Tokyo, Japan

Among several hundred GPCRs, about 40 lipid GPCRs have been identified, which include prostaglandins, leukotrienes, platelet-activating factor (PAF), sphingosine 1-phosphate, lysophosphatidic acids (LPAs), endocannabinoids, bile acids, fatty acids and sterol derivatives. I will summarize three lipid GPCRs with different categories; their different cloning strategies, intracellular signaling, and biological roles as revealed by gene targeting or antagonist studies. 1. PAF receptor By Xenopus oocyte expression system combined with electrophysiology, we identified a GPCR for PAF in 1991 (Honda et al., Nature 349, 342-346, 1991). This is the first example of lipid GPCRs. PAFR couples with a variety of G-proteins activating multiple intracellular signaling. Gene knockout studies showed that PAF is involved in sepsis, bronchial hypersensitivity, allergic encephalomyelopathy, collagen-induced arthritis etc (Ishii and Shimizu, Prog. Lipid Res., 39, 41-82, 2001). Recently, a PAF antagonist with a histamine blocker is on the market to treat allergic disorders. 2. BLT1 and 2, two types of leukotriene B4 receptors By subtraction strategy between differentiated and undifferentiated HL-60 cells, we identified BLT1, a high-affinity receptor for leukotriene B4 (LTB4), a potent chemoattractant (Yokomizo et al., Nature 387, 620-624, 1997). BLT2, originally thought as the low-affinity receptor for LTB4 is later identified as a 12-HHT high affinity receptor (Okuno et al., J. Exp. Med, 205, 759-766, 2008). They are involved various inflammatory disorders and wound healing, respectively (Nakamura and Shimizu, Chem. Rev. 111, 6231-6298, 2011). 3. LPA4, a Non-Edg family of LPA receptor. Three distinct LPA receptors were identified in late 90’s, which are all classified as Edg (endothelial differential gene) family, close to 5 different S1P receptors and endocannabinoid receptors. By the ordinal deorphaning strategy, we have identified a receptor in purinergic family, as the novel class of receptor for LPA (LPA4) (Noguchi et al., J. Biol. Chem., 278, 25600-25606, 2003). Eventually, 2 additional receptors (LPA5 and LPA6) have been discovered with structural similarities with LPA4. LPA-LPA4 axis is important in vascular and lymphatic development (Sumida et al., Blood, 116, 5060-5070), and adipose tissue remodeling and fatty liver generation, possibly independent of vascular abnormalities.

Session 2. New technologies for GPCRs 1

The G protein-coupled receptor database, GPCRdb

David E. Gloriam1 1Department of Drug Design and Pharmacology, University of Copenhagen Denmark

The G protein-coupled receptor database, GPCRdb G protein-coupled receptors (GPCRs) are the most abundant mediators of both human signalling processes and therapeutic effects. The GPCR database, GPCRdb comprises reference data, online analysis tools and interactive visualisation (www.gpcrdb.org) 1.

Recent GPCRdb sections that will be presented: 1. Drugs: drug statistics, target mapping and browsing of drugs, targets, indications, clinical progression. Based on our analysis of the new trends for GPCR drugs, targets and indications covering FDA-approved drugs and agents in clinical trials 2. 2. Signal Proteins: facilitates investigation of GPCR-G protein coupling profiles, interfaces and mutations; and was added as part of a landmark study on GPCR-G protein selectivity 3. 3. Genetic variation: features variation statistics, browsing and an estimated economic burden; and was added as part of a publication on pharmacogenomics of GPCR drug targets 4. 4. GPCRome-wide homology models: models of unprecedented quality are provided for inactive, intermediate and active states 1. 5. Ligand database with biological activities and supplier info for 150,000 GPCR ligands 1. 6. Crystallisation construct design tool based on all available GPCR structures 5 7. Biased agonist database. Preliminary results will be shared for a forthcoming resource of ligands with a proposed signal pathway-bias annotated from publications and patents.

References 1 Pandy-Szekeres, G. et al. GPCRdb in 2018: adding GPCR structure models and ligands. Nucleic Acids Res 46, D440-D446, (2018). 2 Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schioth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nature reviews. Drug discovery 16, 829-842, (2017). 3 Flock, T. et al. Selectivity determinants of GPCR-G-protein binding. Nature 545, 317-322, (2017). 4 Hauser, A. S. et al. Pharmacogenomics of GPCR Drug Targets. Cell 172, 41-54 e19, (2018). 5 Munk, C. et al. GPCR crystallisation constructs and conditions - In-depth analysis and public resource. Manuscript.

Session 2. New technologies for GPCRs 2

Fluorescence and bioluminescence approaches to study ligand binding to GPCRs and RTKs

Stephen J Hill1, Leigh A Stoddart1, Chloe J Peach1, Diana C Alcobia1, Laura E Kilpatrick1 1School of Life Sciences, University of Nottingham, Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, UK

Previous work in our lab, using fluorescent agonists and antagonists, has provided novel insights into the allosteric regulation of adenosine A3 (A3AR), A1 (A1AR) receptors and 1-adrenoceptors by allosteric ligands and receptor dimerization in single living cells (May et al 2011 FASEB J 25, 3465; Gherbi et al 2015 FASEB J 29, 2859). More recently, we have developed novel ligand binding assays for both G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (particularly VEGFR2) using cell surface receptors tagged with a novel bright luciferase (NanoLuc; Promega) and bioluminescence resonance energy transfer (BRET) to a fluorescent ligand (Stoddart et al 2015 Nature Methods, 12, 661: Kilpatrick et al, 2017 Biochem Pharmacol136, 62; Stoddart et al 2018 Trend Pharmacol Sci 39:136). My presentation will summarize some of our recent developments with fluorescent ligands and NanoBRET technologies to study the kinetics of ligand binding to GPCRs, VEGFR2 and Neuropilin-1.

Session 2. New technologies for GPCRs 3

Split-luciferase-based G protein biosensors

Francois Marie Ngako Kadji1, Asuka Inoue1,2, Kouki Kawakami1, Junken Aoki1,3 1Graduate School of Pharmaceutical Sciences, Tohoku University, 2PRIME, Japan Agency for Medical Research and Development (AMED), 3LEAP, AMED

Background The heterotrimeric G proteins are classified into four subfamily including Gs, Gi, Gq and G12 based on downstream signaling in GPCR activation. A classical method to measure G protein activation is GTPS binding assay. A more recent study describes BRET-based G protein sensors. However, both assays have limitations: the former assay shows high background, and require a filtration step and detecting only Gi activation by reason of sensitivity, whereas the latter demonstrates weak signals and overlap between donor and acceptor emission peaks. Split-Luciferase assay overcomes these disadvantages using genetically-encoded enzyme (non-chemically modified) and evaluating all subfamily of G proteins activation. Methods Taking advantage of NanoBiT (NanoLuc binary technology, developed by Promega) system, it has become possible to measure with accuracy protein interaction. We aim at developing and applying a new GPCR activation assay namely NanoBiT-G protein assay. We can measure NanoBiT-G protein dissociation signal in living cells and in membrane preparation in real time. The transfection of parental or knockout (G protein or -arrestin) HEK293 cells is done with plasmid constructs which includes an insertion of LgBiT (luciferase large fragment) into -helical domain of G subunit and SmBiT (luciferase small fragment) attached to N-terminus of G or G subunit. Results From several potential combinations of the three heterotrimeric G proteins including all subfamilies of sensors, their activations via known receptors including V2, D2, TP and DREADD were effective. Interestingly, G proteins activation was also observed in homogenate preparation and membrane fraction with no negative effect on freeze-and-thaw membrane fraction. Conclusion Using the Split-luciferase-based G protein biosensors, we get larger rang of signals with all G subfamily and lower luminescence count variation as compare to previous assays for G proteins activation. Moreover, it requires less in term of economy and applicable for GPCR membrane fractions.

Session 3. 3D Structure of GPCRs 1

Crystal structure of the human prostaglandin E receptor EP4

Yosuke Toyoda1,13; Kazushi Morimoto1; Ryoji Suno1; Shoichiro Horita1; Keitaro Yamashita2; Kunio Hirata2; Yusuke Sekiguchi1; Satoshi Yasuda3,6; Mitsunori Shiroishi4; Tomoko Shimizu8; Yuji Urushibata8; Yuta Kajiwara7; Tomoaki Inazumi5; Yunhon Hotta1; Hidetsugu Asada1; Takanori Nakane1; Yuki Shiimura1; Tomoya Nakagita1; Kyoshiro Tsuge5; Suguru Yoshida9; Tomoko Kuribara9; Takamitsu Hosoya9; Yukihiko Sugimoto5; Norimichi Nomura1; Miwa Sato10; Takatsugu Hirokawa11; Masahiro Kinoshita6; Takeshi Murata3; Kiyoshi Takayama8; Masaki Yamamoto2; Shuh Narumiya12; So Iwata1,2; Takuya Kobayashi1 1 Department of Medical Chemistry and Cell Biology, Kyoto University Graduate School of Medicine; 2 RIKEN SPring-8 Center; 3 Department of Chemistry, Graduate School of Science, Chiba University; 4 Graduate School of Pharmaceutical Sciences, Kyushu University, 5 Department of Pharmaceutical Biochemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University; 6 Institute of Advanced Energy, Kyoto University; 7 Graduate School of Energy Science, Kyoto University; 8 NB Health Laboratory Co. Ltd.; 9 Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University; 10 Biomedical Department, Mitsui Knowledge Industry Co., LTD.; 11 Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST); 12 Medical Innovation Center, Kyoto University Graduate School of Medicine; 13 School of Medicine, Tsinghua University

Prostaglandins (PGs) are a family of bioactive lipids that control physiological processes such as cardiovascular homeostasis, fertilization and parturition and mediate inflammatory responses including pain, fever, and swelling. PGE 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 and a functional 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.

Session 3. 3D Structure of GPCRs 2

A crystal structure of the lysophospholipid receptor LPA6 and insights into lipid ligand entry and a hereditary hair disease

Asuka Inoue1,2, Reiya Taniguchi3, Osamu Nureki3, Junken Aoki1,4 1Graduate School of Pharmaceutical Sciences, Tohoku University, 2PRIME, Japan Agency for Medical Research and Development (AMED), 3Graduate School of Sciences, Tokyo University, 4LEAP, AMED

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid consisting of a glycerol backbone, a phosphate group and a fatty acid chain. The human genome encodes six genes for LPA receptors (gene symbols, LPAR1-6; protein names, LPA1-6). Genetic studies in human have identified mutations in the LPAR6 gene as a cause of a hereditary hair disease called autosomal recessive wooly hair/hypotrichosis (ARWH) (Shimomura et al. Nat Genet 2008, Pasternack et al. Nat Genet 2008). Individuals carrying hypomorphic LPAR6 alleles show a hair disorder characterized by sparse and fragile hair. A secretory LPA-producing enzyme, membrane-associated phosphatidic acid-selective phospholipase A1 (PA-PLA1) encoded by the LIPH gene, has been identified as another causative gene for the ARWH (Kazantseva et al.

Science 2006). In hair follicles, PA-PLA1 produces 2-acyl-LPA and activates LPA6, inducing proper proliferation/differentiation of an epithelial layer. Mechanistically, downstream of LPA6 involves a G12/13-ADAM17-TGF-EGFR pathway (Inoue et al. EMBO J 2011). Despite a need for potential drugs for individuals with LIPH mutations, development of LPA6 agonists is challenging owing to difficulty in detecting G12/13 signaling as well as lack of structural information on LPA6.

The six LPA receptors are classified into two distant families, namely the EDG family (LPA1-3) and the non-EDG family (LPA4-6). The EDG family and its neighboring members include structurally determined lipid GPCRs (LPA1, a sphingosine 1-phosphate receptor (S1P1) and a cannabinoid receptor (CB1)). By contrast, structural information on the non-EDG family and its related lipid GPCRs is limited. We have recently solved the crystal structure of LPA6 (Taniguchi et al. Nature 2017). Strikingly, the othosteric ligand pocket is laterally open toward the lipid bilayer and an acyl chain is bound in the lateral cavity consisting of TM4 and TM5. The unique pocket structure indicates a lateral entry mechanism of a lipid ligand. A docking study and a mutagenesis experiment reveal binding mode of LPA and ligand-induced conformational change of LPA6. The model also explains how mutations found in the ARWH individuals affect receptor functions. In this presentation, we will discuss a shared ligand entry mechanism for the non-EDG family and related lipid GPCRs and comparison with P2Y nucleotide receptors.

Session 3. 3D Structure of GPCRs 3

Endothelin Receptor – Ligand binding and conformational changes

Tomoko Doi Graduate School of Science, Department of Biophysics, Kyoto University, Japan

Endothelin-1 (ET-1), a 21-amino acid long peptide hormone, underlies important physiological processes such as regulation of vascular tone, humoral homeostasis, neural crest cell development, and cell growth. It also participates in the development of various diseases and pathological conditions via G-protein-coupled endothelin receptor activation. ET-1 adopts a unique cyclic structure, comprising the N-terminal region crosslinked by two disulphide bonds to the central -helical segment, and the following flexible C-terminal region located at the position 17-21. ET receptors bind ET-1 in a quasi-irreversible manner with sub-nanomolar affinities. To understand how ET-1 forms such tight interactions and subsequently drive the receptor conformation to the active state, we have studied the crystal structures of endothelin type B (ETB) receptor in a ligand-free form and in a complex with ET-1 (Shihoya et al., Nature, 2016). The structure revealed wide-range interactions between ETB and ET-1, in which the -helical region of ET-1 interacts with the outer vestibule of the ETB binding pocket, whereas the C-terminal region penetrates deeply into the transmembrane core, forming an extensive charge network. Further, we have also studied the structure of non-peptide antagonist, bosentan-bound ETB in an inactive state (Shihoya et al., Nat. Mol. Struct. Biol., 2017). The comparison of these structures suggests helical movements of ETB by the ligand binding. The conformational changes induced by ligands as well as ligand binding properties of endothelin receptors will be discussed. Details of the interactions between ETB and ligands will improve our understanding of endothelin signal transduction and facilitate the development of improved ligands.

Session 4. Drug discovery based on GPCRs 1

The Discovery of Histamine H4 Receptor Antagonists for the Treatment of Inflammation

Robin L. Thurmond Janssen Research & Development, LLC San Diego, CA 92121

The histamine H4 receptor (H4R) is a high affinity GPCR for histamine and was discovered via a genomics method about 17 years ago. The discovery of the H4R provided a new avenue for the exploration of the physiological role of histamine, as well as providing a new drug target for the development of novel antihistamines. Since its discovery numerous ligands, both antagonists and agonists, have been described for the receptor. The identification of these selective ligands has been crucial in uncovering the functions of the receptor. Most of this work has been in the preclinical space, but over the past few years clinical data have been reported furthering our understanding for the role of the receptor in human physiology and pathophysiology. One of the first selective H4R antagonists discovered was JNJ 7777120, which has become one of the standard ligands for the H4R. The use of this ligand, along with studies in H4R –deficient mice, has pointed to a role for the receptor in a number of human diseases including pruritus, asthma and atopic dermatitis. The use of antagonist/agonist pairs, along with studies in receptor-deficient mice, has been crucial in separating H4R-specific pharmacology from off-target pharmacology inherent in any compound. While JNJ 7777120 has been very successful as a tool for understanding the function of the receptor, it has drawbacks, including a short in vivo half-life and hypoadrenocorticism toxicity in rats and dogs, that prevented advancing it into clinical studies. Further research let to the discovery of JNJ 39758979, which, similar to JNJ 7777120, was a potent and selective H4R antagonist and showed anti-inflammatory and anti-pruritic activity preclinically. JNJ 39758979 advanced into human clinical studies and showed efficacy in reducing experimental pruritus and in patients with atopic dermatitis. However, development of this compound was terminated due to the occurrence of drug-induced agranulocytosis. This was overcome by developing another H4R antagonist with a different chemical structure, toreforant, that does not appear to have this side effect. Toreforant has been tested in clinical studies in patients with rheumatoid arthritis, asthma or psoriasis. The experience with the development of H4R antagonists demonstrates the difficulties in finding receptor ligands with the right properties needed to become a successful human therapeutic. Despite some of the failures, the clinical efficacy shown to date with H4R antagonists point to their potential as future therapeutics for the treatment of atopic dermatitis, pruritus and perhaps other indications.

Session 4. Drug discovery based on GPCRs 2

Employing novel chemogenetic approaches to defining GPCR-biology

Andrew B. Tobin Institute for Molecular and cell and systems biology, University of Glasgow, Glasgow, G12 8QQ, UK

Defining the in vivo function of G protein coupled receptors (GPCRs) and validating receptors as targets in drug discovery has been a major challenge. Although genetic knockout studies have been extremely valuable in addressing this issue there are serious limitations, particularly in regard to defining the action of selective drug targeting. Using the muscarinic and free fatty acid receptors as exemplar GPCR classes this talk will describe how we have combined novel genetic models with unique pharmacological tools to define the modes of in vivo action of GPCRs. I will describe how these approaches have been used to determine the importance of M1-muscarinic receptor phosphorylation in mediating key neurological responses of this receptor subtype. Further, using mutant free fatty acid receptors expressed in engineered mice I will describe how we are revealing the physiological role of this “hard to target” class of receptors in a manner that might be generally applied to other receptor subtypes. In this way, I will describe our view on GPCR target validation and the methods we use to define the correct pharmacology that will result in clinical efficacy with reduced toxicity.

Session 4. Drug discovery based on GPCRs 3

Prediction of cancer-associated hotspot mutations that affect GPCR oligomerization

Wataru Nemoto1, Shunsuke Fujishiro, Vachiranee Limviphuvadh2, Sebastian Maurer-Stroh2, Yoshihiro Yamanishi3, Hiroyuki Toh4 1Department of Science and Technology, Tokyo Denki University, Saitama, Japan, 2Bioinformatics Institute, Agency for Science, Technology and Research, Singapore, 3Medical Institute of Bioregulation, Kyushu University, Japan, 4School of Science and Technology, Kwansei Gakuin University, Japan

We developed a high-performance method to predict interacting pairs for G-Protein Coupled Receptors (GPCRs) oligomerization, GPCR-GPCR Interaction Pair predictor (GGIP), by integrating the structure and sequence information [Nemoto et al. Proteins. 2016;84:1224-33]. In addition, we launched a prediction server, which is available at http://protein.b.dendai.ac.jp/GGIP/. A recent study reported that somatic mutations in GPCR-encoding genes are frequently found in various types of cancer. Among the somatic mutations, hotspot mutations are defined as recurrent amino acid changes occurring in coding sequences. It has been revealed that many GPCRs have hotspot mutations in the same types of cancer tissues. However, most of the hotspot mutations have not been characterized yet, and their effects on the cancer pathways remain unknown. Some of the hotspot mutations may be related to cancers through modifying GPCR oligomerization, since they are considered to be present on the surface of transmembrane helices. Hence, we examined the predicted interacting pairs including the GPCRs with hotspot mutations. We will discuss the characteristics of these mutations and introduce several examples.

Session 5. GPCR and Diseases 1

Structure-based Discovery of Non-metabolite Synthetic Ligands for Metabolite Receptors - exploding Orthosteric versus Allosteric Locks

Mette Trauelsen, Michael Lückman, Thomas M.Frimurer and Thue W.Schwartz Center for Basic metabolic Research, University of Copenhagen, Copenhagen Denmark

A number of key metabolites function not only as fuel and metabolic building blocks but also as extracellular signaling molecules, which in analogy to transmitters and hormones are recognized by specific selective GPCRs (1). These signaling metabolites are both dietary nutrient metabolites, gut microbial metabolites and intermediary metabolism metabolites being sensed as ‘fuels’ and as metabolic ‘stress signals’ (1). However, we badly need synthetic non-metabolite agonists and antagonists in order to characterize the physiological importance and pharmacological potential of these metabolite GPCRs, as the metabolites themselves obviously also have confounding functions as basic metabolites. Succinate, accumulates during hypoxia and functions as a metabolic stress signaling through activation of GPR91 expressed on for example stellate cells in the liver and neurons secreting VGEF in the retina. Based on the identification of an empty pocket next to the orthosteric binding site for succinate in GPR91 and the fact that certain backbone modifications of succinate were allowed we virtually screened the ZINC database by use of a substructure-based query combined with molecular docking to identify two serial libraries of a total of 245 compounds from which we identified back-bone modified succinate analogs in which amide linked hydrophobic moieties exploited the side-pocket in GPR91 as demonstrated by both gain and loss of function mutagenesis (2). These novel ligands functioned as potent selective GPR91 agonists devoid of any succinate metabolic effects. In the long chain fatty acid receptor GPR40/FFAR1 synthetic agonists are known to bind in both an inter-helical site between TM-III and –IV and a lipid-exposed groove between the intracellular segments of these helices. Molecular dynamics simulations with agonist removed demonstrated closure of a solvent-exposed pocket between the extracellular poles of TM-I, -II and -VII. A synthetic compound designed to bind in this pocket and thereby prevent its closure was identified through structure-based virtual screening and shown to function both as an agonist and allosteric modulator of receptor activation. This demonstrates both the power of including molecular dynamics in the drug discovery process and that this specific, clinically proven, but difficult anti-diabetes target can be addressed by completely novel chemotypes. 1. Husted A.S. et al. Cell Metabol (2017) 25: 777-7962 2. Trauelsen M et al Mol.Metabol. (2017) 6: 1585-96

Session 5. GPCR and Diseases 2

Autocrine Regulation of Metabolic Functions Through Metabolite GPCRs

Stefan Offermanns1, 2 1Dept. of Pharmacology, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany 2Medical Faculty, J. W. Goethe University, Frankfurt, Germany

G-protein-coupled receptors (GPCRs) have traditionally been regarded as receptors for hormones, neurotransmitters, and other mediators which are produced solely for the purpose of carrying a signal and to serve cell-cell communication. This view has changed during the last decade, as a growing number of GPCRs are being identified, for which the ligands are energy substrates or metabolic intermediates. Among these ligands are saturated and unsaturated free fatty acids (FFAs) as well as hydroxy carboxylic aids (HCAs), such as lactate and ketone bodies, which exert cellular effects through GPCRs named FFA1-FFA4 and HCA1-HCA3, respectively. These receptors are widely expressed in the human body and regulate the metabolic, endocrine or immune system to maintain homeostasis under changing dietary conditions. Data will be presented on the role of FFA1, FFA2 and FFA3 in the regulation of β-cell function by natural ligands acting in part in an autocrine manner.

Session 5. GPCR and Diseases 3

Functional analysis of dry syndrome by PACAP and its prevention and possible use for therapeutic application

Seiji Shioda1, Tomoya Nakamachi2, Takahiro Hirabayashi1, Fumiko Takenoya1, Nobuhiro Wada1, Naoko Nonaka3, and Junko Shibato1 1Peptide Drug Innovation, Global Research Center for Innovative Life Science, Hoshi University, Japan, 2Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, Japan, 3Department of Anatomy and Developmental Biology, Showa University School of Dentistry, Japan

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide/glucagon/secretin family of peptides. PACAP and its three receptor subtypes (PAC1R, VPAC1R, VPAC2R) are expressed in neural tissues. PAC1R is dominantly expressed in the eye, including the retina, cornea and lacrimal gland, and the eccrine sweat gland in the skin, however in the salivary glands VPAC1R is mainly expressed. PACAP is known to exert pleiotropic effects on the central nervous system and in eye tissues where it plays important roles in protecting against dry eye. There are many dry disease patients, the number of these patients is increasing year by year. In our study using PACAP gene-deficient mice, these animals cause keratinization of the corneal epithelium especially with elderly females, a dry eye-like symptom was observed, and a remarkable decrease in lacrimal fluid. In addition, the instillation of PACAP promoted the transfer of aquaporin 5 (AQP 5), a water channel protein present in the lacrimal gland, from the cytoplasm to the cell membrane. PACAP promotes phosphorylation of AQP5 and is found to promote transport of water molecules from the basal to apical part of the acinar cells by AQP5. In addition, dry animals called dry syndrome, dry skin and dry mouth etc. we first found PACAP as a key role in protecting dry symptoms via AQP5. Thus, PACAP is considered an important peptide that regulates the water metabolism regulation function in the body. I will make an overview of current knowledge regarding dry symptoms in aged animals and humans and the protective effects, mechanisms of action. I also refer to the development of a new preventive/therapeutic method by PACAP for dry syndrome patients.

References 1) Nakamachi T, Shioda S et al., Nat Commun 7: 12034 (2016) 2) Sasaki S, Shioda S et al., Br J Dermatol 176: 413-22 (2017) 3) Shioda S, Takenoya F et al., J Mol Neurosci (in press)

Poster session 1

Allosteric transmission with rigidity propagation across GPCR networks

Adnan Sljoka Kwansei Gakuin University, Japan

Allostery can be viewed as an effect of binding at one site of the protein to a second, often significantly distant functional site, enabling regulation of the protein function. Allostery is of particular interest for GPCRs because GPCR dynamics and activation is often dependent on long range signaling via transmembrane region. We have recently developed a rigidity-transmission allostery (RTA) algorithm [1,2], a computational method based on mathematical rigidity theory and applied it on several GPCR structures. Given a crystal structure, we model the protein as a constraint network consisting of nodes (atoms) and edges (i.e., constraints including covalent bonds, electrostatic bonds, hydrogen bonds, and hydrophobic contacts). RTA algorithm provides a mechanical interpretation of allosteric signaling and is designed to predict if perturbation of rigidity (mimicking ligand binding) at one site of the protein can transmit and propagate across a protein network and in turn cause a transmission and change in conformational degrees of freedom at a second distant site, resulting in allosteric transmission. Starting with a set of known GPCR 3-dimensional crystal structures (A2A-adenosine receptor, CC chemokine receptor 2, beta1 and beta2 AR) we have applied the RTA method to detect if ligand binding at either orthosteric site or extrcellular loops can trigger small rigidity changes which propagate to the critical G-protein binding regions, and in turn cause a rearrangement and alteration of the shape of the cytoplasmic binding region. As our method is computationally very fast, we can rapidly scan many unknown sites, identifying potential new allosteric sites and quantify their allosteric response. We will illustrate our method and demonstrate the key quantifiable differences observed in rigidity based communication in number of active-like (agonist-bound) GPCRs structures versus the inactive structures. We will also present the prediction of the allosteric pathways crucial for signal transmission.

[1] Kim T., Mehrabi P., Sljoka A. et al Ing C., Bezginov A., Pomes R., Prosser S. and Pai E., The Role of Dimer Asymmetry and Protomer Dynamics in Enzyme Catalysis, Science 355, 262, 2017.

[2] Ye L., C. Neale, Sljoka A., Pichugin D., Tsuchimura N., Sunahara R., Prosser S. et al, Bidirectional Regulation of the A2A Adenosine G Protein-Coupled Receptor by Physiological Cations, Nature Communication, 1 9:1372, 2018.

Poster session 2

Illuminating G12-coupled GPCRs

Asuka Inoue1,2, Francois Marie Ngako Kadji1, Kouki Kawakami1, Yuji Shinjo1, Junken Aoki1,3 1Graduate School of Pharmaceutical Sciences, Tohoku University, 2PRIME, Japan Agency for Medical Research and Development (AMED), 3LEAP, AMED

Among the four classes of the heterotrimeric G proteins (Gs, Gi, Gq and G12), the G12 subfamily, as well as G12-coupled GPCRs, remains poorly understood. This is mainly due to limitation of existing assays to detect downstream of G12 signaling. Since importance of the G12 subfamily, which includes G12 and G13 subunits, is known from gene-knockout mice studies and mutational analysis of human diseases such as lymphoma, new G12-signaling assays are desired. In this presentation, we will describe development and application of our recent G12-signaling assays/tools. The first assay originates from a previously established method named as the TGF shedding assay (Inoue et al., Nat Methods, 2012), which measures activation of G12- and/or Gq-coupled receptors. Although the TGF shedding assay was powerful for analyzing G12-coupled receptors (e.g., a successful structural determination of a lysophosphatidic acid receptor LPA6 (Taniguchi, Inoue et al., Nature, 2017)), the crosstalk between Gq signaling and G12 signaling disturbed selective measurement of G12 activation. By using a library of G protein-KO HEK293 cells (Milligan, Inoue, Trends Pharmacol Sci, 2018), we have set up a system to selectively measure G12 signaling in which G12 activation is monitored as TGF shedding response in the Gq subfamily-KO cells and this is further confirmed by silenced response in the Gq and G12 subfamily-KO cells. Interestingly, the G12-selective TGF shedding assay could identify many GPCRs as being coupled with G12. The second method is a RhoA biosensor based on a NanoBiT system (Promega), in which interaction between GTP-bound, activated RhoA and its effector (PKN1) is measured as luminescent signal in real time in living cells. Using the G protein-KO cells, we found that, in HEK293 cells, the RhoA sensor detects G12 signaling and Gq signaling, and Gi signaling to a lesser extent. The third method is a NanoBiT-based G protein biosensor, which will be described in an accompanying presentation (Kadji et al.). In this assay, we monitor dissociation of LgBiT-inserted G12 or G13 subunit from heterotrimeric G protein containing SmBiT-fused G or G subunit. Assay optimization found SmBiT-fused Gt1 and co-expression of RIC8A to achieve robust dissociation signal. The NanoBiT-G protein assay showed that some GPCRs clearly distinguish between G12 and G13. Using our sensitive G12-signaling assays, we uncover previously unrecognized G12-coupled receptors. This information will help to develop a G12-coupled designer GPCR (DREADD) and also potential drug targets for G12-related diseases.

Poster session 3

Identification of ghrelin and its receptor in Schlegel's Japanese gecko, Gekko japonicus

Hiroyuki Kaiya1, MK Park2, Kenji Kangawa1, Mikiya Miyazato1 1 Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Japan, 2 Department of Biological Science, Graduate School of Science, The University of Tokyo, Japan.

Ghrelin has known as a growth hormone-releasing and appetite-stimulating hormone in mammals. However, little is known about the structure and function of ghrelin in reptiles. In this study, we identified ghrelin and its receptor cDNAs in a squamata, Japanese gecko, Gekko japonicus, and conducted functional characterization of the receptor using mammalian HEK293 cell. Complementary DNA of ghrelin in Japanese gecko (geckoGHRL) was amplified from the stomach. It was 501 bps in length, containing a 61 bps of 5’-untranslated region (UTR), a 345 bps of coding region, and a 95 bps of 3’-UTR. The geckoGHRL cDNA encoded a 114-amino acid (aa) preproghrelin precursor, including 26-aa mature ghrelin peptide. Identify of mature geckoGHRL was the highest in emu (bird) and green anole (reptile) : human (57%), chicken (54%), emu (73%), green anole (73%), red-eared slider turtle (65%), bullfrog (25%), tree frog (40%), and zebrafish (46%). Quantitative PCR (qPCR) revealed that ghrelin mRNA predominantly expressed in the stomach, and followed by the eshophagus. Lower levels of expression were detected in the brain, lung, and middle intestine. The ghrelin receptor cDNA of Japanese gecko (geckoGHS-R1a) could be amplified in the brain, stomach, intestine and testis. Complementary DNA of geckoGHS-R1a was 1457 bps in length, containing a 317 bps of 5’- UTR, a 1089 bps of coding region, and a 51 bps of 3’-UTR. The geckoGHS-R1a cDNA encoded a 362-aa G-protein coupled receptor. Identify of geckoGHS-R1a was the highest in green anole (reptile) : human (74%), chicken (80%), green anole (85%), Chinese soft-shell turtle (73%), painted turtle (83%), bullfrog (74%), tree frog (73%), zebrafish-1a (71%) and zebrafish-2a (68%). qPCR revealed that GHS-R1a mRNA mainly expressed in the brain, and pituitary. GeckoGHSR-1a expressed in HEK293cell was functional, and responded to ghrelin of rat, chicken, bullfrog, newt, tilapia, and GHRP-6 and hexarelin. In summary, we identified ghrelin and its receptor in Japanese gecko, indicating the presence of the ghrelin system in this species. Ghrelin receptor was expressed in the brain and pituitary, suggesting involvement of feeding behavior and regulation of pituitary hormone secretion as seen in other vertebrates.

Poster session 4

Effect of MRS2578, a P2Y6 receptor inhibitor, on IgE-dependent activation of human basophils

Manabu Nakano1, 2, Marina Kikuchi3, Syun Aburakawa3, Naofumi Tokita3, Kyoko Ito1, Hideki Takami1, Koichi Ito1, 2 1 Graduate School of Health Sciences, Department of Bioscience and Laboratory Medicine, Hirosaki University, Japan, 2 Graduate School of Health Sciences, Research Center for Biomedical Sciences, Hirosaki University Japan, 3School of Health Sciences, Department of Medical Technology, Hirosaki University, Japan

Nucleotides are released from damaged cells or secreted spontaneously or by activated cells. Extracellular nucleotides regulate a variety of biological functions through P2 purinergic receptors expressed on cell surfaces. Among the P2Y receptor subtypes, P2Y6 is known to be a proinflammatory receptor, which is specifically stimulated by Uridine diphosphate (UDP). Our previous studies showed that UDP stimulation via the P2Y6 receptor enhanced IgE-dependent degranulation of basophils. In addition, our data suggested that basophils secreted UDP, and had stimulated P2Y6 receptor. In the present study, we confirmed that basophils secrete UDP. Furthermore, we examined the effect of MRS2578, a P2Y6 receptor inhibitor, on IgE-dependent degranulation of human basophils. HPLC analysis indicated basophils spontaneously secrete UTP. Basophils were also found to express E-NTPDase2 and E-NTPDase3, which hydrolyzed UTP to UDP, suggesting that basophils stimulated P2Y6 receptor by regulating the UDP concentration. Next, we cultured human basophils with MRS2578 and then stimulated them with anti-IgE antibody. MRS2578 inhibited expression of CD203c and CD63 on basophils and production of IL-4 induced by anti-IgE antibody stimulation. Activated basophils release histamine and leukotriene and produce cytokines such as IL-4 and IL-13; this has an important role in allergic reactions. Inhibition of IgE-dependent activation of basophils by MRS2578 suggests a possible treatment for allergic diseases.

Poster session 5

RNA editing of 5-HT2C in the nucleus accumbens participates in alcohol drinking behavior

Masaki Tanaka1, Takahira Shirahase2, Shin Kwak3, Yoshihisa Watanabe4 1 Department of Anatomy and Neurobiology, 2 Department of Dental Medicine, and 4 Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan 3 Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan

The nucleus accumbens (NAc) in the forebrain is known to have an important role in the addiction and reward system in primate and rodents. Messenger RNA editing, particularly RNA editing of neurotransmitter receptors such as serotonin 2C receptor (5-HT2CR) and glutamate receptor A2 is closely related to various neuronal functions. We previously found that chronic ethanol exposure elevated the RNA editing frequency of 5-HT2CR in the NAc of alcohol-preferring C57BL/6J mice (Watanabe Y, Int J Neuropsychopharmacol, 2014). This finding led us to speculate that RNA editing in the NAc may control alcohol-drinking behavior. To investigate this possibility, we generated NAc-specific ADAR2 knockout mice using AAV-GFP/Cre, and performed various behavioral analyses including assessment of alcohol-drinking behavior. Unlike control mice injected with AAV-GFP, NAc-specific ADAR2 knockout mice did not show enhanced alcohol consumption and alcohol preference after chronic ethanol vapor exposure. This result suggests that alcohol-drinking behavior and alcohol preference are regulated by RNA editing in the NAc.

Poster session 6

The Endogenous Pituitary Adenylate Cyclase-Activating Polypeptide increases food intake by modulating the expression of neuropeptides in the mouse hypothalamus

Nguyen Thanh Trung, Yuki Kambe, Takashi Kurihara and Atsuro Miyata

Department of Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima University, Japan

It is well known that pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, which are strongly expressed in some nuclei of brain, are responsible for appetite regulation in hypothalamus. However, the mechanism by which food intake is decreased in PACAP-null mice is still under discussion. To address this issue, we attempted to elucidate if PACAP might involve in the manipulation of energy homeostasis. We found that nocturnal and daily food intake of PACAP-null mice was significantly lower than those of wild-type (WT) mice, but both diurnal food intake was unchanged. In addition, food consumption at 4, 8, or 24 hours of refeeding after fasting for 2 days showed that the food intake at 8 hours of the refeeding was significantly lower in PACAP-null mice than in the WT mice. We also observed that the expression level of agouti-related peptide (AgRP) was decreased in PACAP-null mice but that of proopiomelanocortin (POMC) was increased in PACAP-null mice after refeeding for

4 hours. The intracerebroventricular (i.c.v.) administration of a PACAP antagonist PACAP6-38 (1 nmol) reduced the diurnal food intake of WT mice after 1, 2, 4 and, 24 hours of injection. Furthermore, the expression levels of AgRP and neuropeptide Y (NPY) were down-regulated by the injection of PACAP6-38 at 4 hours after refeeding in WT mice. The gene expression of c-Fos increased in the PVH and ARC region of mouse brain after 2 days fasting. Taken together, our study demonstrates that food intake in mice was enhanced by the endogenous PACAP in not only night-time/daytime but also fasting/refeeding paradigm via the modulation of neuropeptides AgRP and/or POMC in the mouse hypothalamus, indicating PACAP inhibition as a potential strategy for the development of anti-obesity drug.

Poster session 7

Recent progress on screening for novel GPCR ligands using GPCR-G fusion proteins

Shigeki Takeda Faculty of Science and Technology, Division of Molecular Science Gunma University

Various kinds of methods have been proposed for high-throughput screening of GPCR ligands. Most common methods are the cell-based assay which monitors downstream events of signal cascades, although the cell-based assay system has drawbacks such as false–positive responses due to endogenous receptors on host cells. Since 2004, we have tried to use the fusion protein as a tool for ligand screening (S. Takeda, et al., J. Biochem., 135, 597 (2004)). The binding of [35S]GTPS to cell membrane fractions expressing a -opioid receptor-Gi fusion protein was measured to screen a chemical library. We successfully found a few structurally unique compounds that showed agonist activity for a -opioid receptor. We also tried to screen structurally similar chemicals with them and reached to the improved compound. Subtype specificity of the compound (GUM1) was examined by using -, -, and -opioid receptor-Gi fusion proteins and revealed to be similar with morphine. Hargreaves method was employed to investigate in vivo activity of our new opioid ligand and resulted that 3 mg/kg of subcutaneous injection for wild-type rats showed significant extension of withdrawal latency. This extension was inhibited by pre-treatment of naloxone. Moreover, this analgesic activity was also observed for morphine tolerance rat (Y. Nikaido et al., Eur. J. Pharmacol., 767, 193-200 (2015)). The membrane bound bile acid receptor (GPBA) mediates bile acids signaling and regulates energy homeostasis. Thus, GPBA ligands have attractive potential for the development of anti-diabetic drugs. We performed screening for novel human GPBA ligands by using a GPBA-Gs fusion protein. Finally, a unique agonist, GUM2, was successfully identified. We then tested the effects of GUM2 intraperitoneal administration on the plasma glucose levels in mice during glucose tolerance test. We observed a statistically significant blood glucose reduction in GUM2 treated mice (2 mg/kg) compared with the vehicle treated mice at 15 min and 30 min after glucose load, suggesting a glucose lowering effect of GUM2 in vivo (R. Enomoto, A. et al., Eur. J. Pharmacol., 814, 130-137 (2017)).

Poster session 8

Functional analysis of promotion of sweat secretion by PACAP

Takahiro Hirabayashi1, Sayaka Endo1, Mifuyu Shioda1, Tomoya Nakamachi2, Shun Sasaki3 Fumiko Takenoya1, Brian J.P. Harvey4, and Seiji Shioda1 1Peptide Drug Innovation, Global Research Center for Innovative Life Science, Hoshi University, Tokyo, Japan, 2Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, Japan, 3Department of Dermatology, Showa University School of Medicine, Tokyo, Japan, 4 The Royal College of Surgeons in Ireland, Ireland

Pituitary adenylate-cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which shows particularly high homology to vasoactive intestinal peptide (VIP) and these peptides share three common G protein-coupled receptors (GPCRs), PAC1-R, VPAC1-R and VPAC2-R. PACAP exhibits pleiotropic functions in the central nervous system, including neurotransmission, neuroprotection, and vasodilatation. Recently, we have reported that PACAP gene-deficient mice develop dry eye-like symptoms such as corneal keratinization and tear reduction, and we have shown that PACAP eye drops stimulate tear secretion [1, 2]. In this study, we investigated the sweat secretion promoting by PACAP. Subcutaneous administration of PACAP into the mouse footpad was found to promote sweat secretion by the starch iodine test [3]. Further, to elucidate the molecular mechanism of sweat secretion by PACAP, we performed using the human eccrine sweat gland cell line NCL-SG3 cells established by Simian virus 40 infection of primary cultures from human eccrine sweat glands [3]. Strong expression of PAC1-R mRNA was found in the cells, while VPAC1-R and VPAC2-R mRNA expressions were significantly lower. PACAP increased cytosolic Ca2+ concentration in these cells, and this could be blocked by PAC1-R antagonist PACAP6-38. These results indicate that PACAP stimulates eccrine sweat gland to promote sweat secretion via PAC1-R. These findings suggest that PACAP may provide new therapeutic options to combat sweating disorders.

References [1] Nakamachi T, Shioda S et al., Nat Commun 7: 12034 (2016) [2] Shioda S, Hirabayashi T et al., J Mol Neurosci (in press) [3] Sasaki S, Shioda S et al., Br J Dermatol 176: 413-22 (2017) [4] Lee CM, Dessi J., J Cell Sci 92: 241-9 (1989)

Poster session 9

Identification of novel bioactivepeptide, LURY-1

Takanori Ida Center for Animal Disease Control, University of Miyazaki

In both vertebrates and invertebrates, there are many orphan G protein-coupled receptors (GPCRs), for which ligands have not yet been identified. Identification of their endogenous ligands is very important for understanding the function and regulation of such GPCRs. Indeed, That has enhanced our understanding of many physiological processes including feeding behavior, sleep-awake system, stress reaction, immunological system and reproduction. Here, we identified five Drosophila endogenous ligands, CCHamide-1, CCHamide-2, dRYamide-1, dRYamide-2 and trissin of the Drosophila orphan GPCRs and LURY-1 of the C.elegans orphan GPCRs using functional assays with the reverse pharmacological technique. And, in various invertebrates, we identified orthologous peptides to Drosophila peptides. Some of these peptides modulate feeding behavior and metabolism. These results suggest that identification of novel invertebrates bioactive peptide might facilitate the elucidation of various physiological function and have a useful possibility for the ligand searching of mammal orphan GPCRs.

Figure

Poster session 10

Distribution, characterization, and regulatory effect on feeding behavior of PACAP/ PAC1 receptors system in zebrafish

Tomoya Nakamachi1, Ayano Tanigawa1, Hiroyuki Kaiya2, Norifumi Konno1, and Kouhei Matsuda1 1 Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama, Toyama 930-8555, Japan. 2 Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Suita 565-8565, Japan

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide with the amino acid sequence that is well conserved among vertebrates. In teleosts, including zebrafish, the PACAP gene (adcyap1) and PACAP receptor (PAC1R) gene (adcyap1r1) has been duplicated at teleost specific whole-genome duplication to adcyap1a (coding PACAP1) and adcyap1b (coding PACAP2), and to adcyap1r1a (coding PAC1Ra) and adcyap1r1b (coding PAC1Rb), respectively. However differences of the duplicated PACAPs and PAC1Rs in teleost has not been clarified yet. In this study, we first examined expression pattern of PACAP and PAC1R mRNA in zebrafish by real-time PCR methods. All PACAP and PAC1R mRNA were dominantly expressed in the brain. adcyap1b mRNA level was almost 15 times greater than adcyap1b mRNA level, and adcyap1r1a level was almost 2 times greater than adcyap1r1b level. Meanwhile, adcyap1a mRNA was detected in peripheral tissues such as gut and testis. To characterize the zebrafish PACAPs and PAC1Rs, zebrafish (zf) PAC1Ra or zfPAC1Rb were transiently expressed in CHO cells. The cells were treated with zfPACAP1 or zfPACAP2, and measured changes in intracellular Ca2+ and cAMP levels. zfPACAP1 or zfPACAP2 treatment increased the Ca2+ and cAMP level with no difference responsibility in both zfPAC1Rs, suggesting that zfPAC1Rs were functional PACAP receptors. When compared the responsiveness between zfPAC1Rs, cAMP response of zfPACAPs was almost similar, but Ca2+ response of zfPAC1Ra was greater than that of zfPAC1Rb. Next, we examined the effect of ICV administration of zfPACAPs on the feeding behavior in zebrafish and feeding-induced changes in the expression of zfPACAPs and zfPAC1R mRNA in the brain. ICV administration of 2 pmol/g BW of zfPACAPs significantly suppressed food consumption in zebrafish. adcyap1a mRNA level significantly elevated 2 hs after feeding, whereas adcyap1b and zfPAC1Rs mRNA levels did not change significantly. These results suggest that duplicated zfPACAPs and zfPAC1Rs have almost similar character, but partially differentiated the distribution, intracellular signaling, and expression change by feeding status.

Poster session 11

Crystal structure of leukotriene B4 receptor 1 bound with an inverse agonist

Toshiaki Okuno1, Tetsuya Hori2, Shigeyuki Yokoyama2, Takehiko Yokomizo1 1Department of Biochemistry, Juntendo University School of Medicine, 2Riken Structural Biology Laboratory

The leukotriene (LT) B4 receptor 1, BLT1 is a high affinity G-protein-coupled receptor (GPCR) for LTB4, a chemotactic and inflammatory lipid mediator. In this study, we determined the crystal structure of BLT1 bound with an inverse agonist, BIIL260 which is a chemical bearing a benzamidine moiety. Most GPCRs are commonly stabilized in the inactive state by the formation of the sodium ion-centered water cluster with the conserved Asp2.50 inside the seven-transmembrane domain. We found that the amidine group of BIIL260 occupies the sodium ion and water locations, interacts with D662.50, and mimics the entire sodium ion-centered water cluster. Thus, the amidine group of BIIL260 fixes BLT1 in the inactive state, and the transmembrane helices of BLT1 cannot change their conformations into the active state. Importantly, the benzamidine molecule alone serves as a negative allosteric modulator for BLT1. Considering that the residues involved in the benzamidine binding are widely conserved among various GPCRs, the unprecedented inverse-agonist mechanism by the benzamidine moiety could be adapted to other GPCRs, allowing the rational development of inverse agonists specific for each GPCR.

Poster session 12

Regulator of G protein signaling 8 (RGS8) modulates depression-like behavior via melanin-concentrating hormone receptor 1 activity

Yuki Kobayashi1, Tomoya Okada1, Risa Takemoto1, Shogo Yamato1, Michihiko Iijima2, Yoshikatsu Uematsu2, Shigeyuki Chaki2 and Yumiko Saito1 1 Graduate School of Medical and Dental Sciences, Department of Pharmacology, Hiroshima University, Japan, 2Research Headquarter, Taisho Pharmaceutical Co. Ltd., Japan

Regulator of G protein signaling (RGS) proteins modulate Gα-directed signals through their GTPase-activating protein activity and terminate G protein-coupled receptor (GPCR)-associated signaling. Among the RGS proteins, RGS8 mRNA is highly expressed in several brain regions involved in movement and mood. As previously reported, mice with global RGS8 knockout appeared healthy but have not been examined in behavioral tests. Here, we report that RGS8 transgenic mice (RGS8tg) overexpressing RGS8 protein in the hippocampal CA1 region exhibit decreased depression-like behavior. Some RGS proteins interact directly and selectively with GPCR to modulate their activity. RGS8 was previously found to be a potent negative regulator for melanin-concentrating hormone receptor 1 (MCHR1) activity in heterologous cell expression systems (Matsubara-Miyamoto et al, 2008). Interestingly, treatment with MCHR1 antagonist SNAP94847 did not have an antidepressant effect in RGS8tg, but did affect wild-type mice (WT). Meanwhile, a significant effect of selective noradrenergic reuptake inhibitor desipramine was observed in RGS8tg. Importantly, the data suggest that the mechanism for the antidepressant-like phenotype observed in RGS8tg may be associated with a change in the MCHR1 active state, but be distinct from that of conventional antidepressants acting on the monoaminergic system. Next, we observed that MCHR1 is localized in neuronal primary cilia, antenna-like sensory organelles that act as a signaling platform. We further analyzed the average length of MCHR1-positive cilia, finding that the cilia were significantly longer in the CA1 region of RGS8tg compared with that of WT. Although MCHR1-positive cilia were also detected in the CA3 region, a significant difference in the average length of MCHR1-positive cilia was not observed in RGS8tg compared with WT. Thus, it can be speculated that a significant change in cilia length may be associated with the behavioral consequences observed in RGS8tg. Overall, our in vivo study provides the first meaningful insights that alteration in the RGS8-ciliary MCHR1 interaction in the CA1 region may be an important factor in the pathway leading to depression-like behavior. In addition, these findings implicate RGS8 as a potential target for the development of novel drugs to treat depressive disorders wherein modulation of MCHR1 activity has therapeutic potential.

Matsuo Award 1

Characterization of orexigenic neuropeptide receptor in neuronal primary cilia in vitro and in vitro.

Daisuke Miki1, Tomoya Okada1, Shogo Kobuchi2, Yuki Kobayashi and YumikoSaito1, 2 1Graduate School of Integrated Arts and Sciences, Department of Behavioral Neuroscience, Hiroshima University, Japan, 2School of Integrated Arts and Sciences, Department of Behavioral Neuroscience, Hiroshima University, Japan

A non-motile primary cilium is detected on most mammalian cells where it provides sensory and signaling function as a cellular antenna. Their importance is highlighted by the studies of a group of diseases, known as ciliopathies, which include cystic kidney disease, neurodevelopmental abnormalities, blindness, obesity, and perhaps even psychiatric disorders. Different functions of primary cilia are reflected by their diverse morphology and unique signaling components localized in the ciliary membrane. Indeed, many neurons in the mammalian brain possess primary cilia that are enriched for a set of G protein-coupled receptors (GPCRs) such as melanin-concentrating hormone (MCH) receptor 1 (MCHR1) and somatostatin receptor 3 (Hilgendorf et al. 2016). The MCH-MCHR1 system is known to mediate distinct aspects of feeding, energy homeostasis, sleep and emotional processing. We previously found that MCH signaling through a Gi/o-dependent Akt pathway induces cilia length shortening in ciliary MCHR1-expressing RPE1 cells (Hamamoto et al. 2015; Tomoshige et al. 2016). Although short cilia have been observed in genetic obese mice, a possible correlation between MCHR1-positive neuronal cilia length and energy metabolism has not be characterized. For this purpose, first we established a novel protocol to detect MCHR1 colabeled with the neuronal ciliary marker (AC3; adenylyl cyclase 3) in frozen section of mice brain. Quantitative analysis revealed that neuronal primary cilia carrying AC3/MCHR1 (MCHR1-positive cilia) were abundant in the hippocampal CA1 and CA3 region, while many MCHR1-negative cilia were observed in the dentate gyrus of the hippocampus. Next, brains were collected from mice subjected to a 48-h fast. Starved mice displayed shorter MCHR1-positive cilia than did normal diet-fed mice in their hippocampal CA1 but not in CA3. Further, we have developed a novel hippocampal slice culture model derived from P10 rat. As well as from a frozen section of mouse brain, many cilia carrying MCHR1 were detected in the CA1 and CA3 region. Importantly, exposure of slice culture to MCH at nanomolar order selectively caused MCHR1-positive cilia shortening in CA1. The present results provide the first evidence that MCHR1-positive cilium length is actively reduced by metabolic alteration, and this appears to occur in region-specific manner, possibly via discrete MCHR1 signaling pathways.

Matsuo Award 2

Calcitonin receptor modulates body temperature rhythm in mammals

Hiroyuki Shimatani, Iori Murai, and Masao Doi Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan

The body temperature rhythm (BTR) is one of the most conspicuous outputs of the circadian clock and is crucial for maintaining homeostasis in metabolism and sleep as well as entraining the peripheral clock in mammals. In humans, body temperature increases during wakefulness and decreases during sleep. As daily variations in BTR are robust and parallel fluctuations in locomotor activity rhythms, BTR is widely used to monitor circadian rhythms in mammals. The molecular mechanisms that regulate BTR remain largely uncharacterized, although a study in which subsets of neurons in the brains of rats were surgically ablated suggests that locomotor activity rhythms and BTR are controlled by different output pathways that originate from the suprachiasmatic nucleus (SCN). In humans, body temperature fluctuates even when locomotor activity is restricted, and BTR and locomotor activity rhythms can be experimentally dissociated, a phenomenon known as spontaneous internal desynchronization. These accumulating data suggest that BTR is likely controlled separately from locomotor activity rhythms. However, no molecular evidence supporting this possibility has been reported. We recently show that calcitonin receptor (Calcr) is expressed in the SCN and mediates BTR during the night (active phase for mice). Since Calcr is not involved in locomotor activity rhythmicity, these findings provide the first molecular evidence that BTR is regulated separately from locomotor activity rhythms (Genes Dev 32, 140-155, 2018).

Matsuo Award 3

Structure-guided development of subtype-selective muscarinic acetylcholine receptor antagonists

Hongtao Liu1, Josefa Hofmann2, Inbar Fish3,4, Benjamin Schaake2, Katrin Eitel2, Amelie Bartuschat2, Jonas Kaindl2, Hannelore Rampp2, Ashutosh Banerjee2, Harald Hübner2, Mary J. Clark5, Sandra G. Vincent6, John T. Fisher6, Markus R. Heinrich2, Kunio Hirata7, Xiangyu Liu1, Roger K. Sunahara5, Brian K. Shoichet3, Brian K. Kobilka1,8, Peter Gmeiner2 1 Beijing Advanced Innovation Center for Structural Biology, School of Medicine, Tsinghua University, Beijing 100084, China, 2 Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich Alexander University, Nikolaus-Fiebiger-Straße 10, 91058 Erlangen, German, 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, USA, 4 Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, Israel, 5 Department of Pharmacology, University of California San Diego School of Medicine, 9500 Gilman Drive, La Jolla, California 92093, USA, 6 Department of Biomedical & Molecular Sciences and Division of Respirology, Department of Medicine, Queen's University, Kingston, Ontario K7L 3N6, Canada, 7 Advanced Photon Technology Division, Research Infrastructure Group, SR Life Science Instrumentation Unit, RIKEN/SPring-8 Center, 1-1-1 Kouto Sayo-cho Sayo-gun, Hyogo 679-5148 Japan, 8 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a profound effect on health, but can suffer from their lack of selectivity against the M2 muscarinic receptor (M2R) subtype, which modulates heart rate. Beginning with the crystal structures of the M2R and M3R, we exploited a single amino-acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over the M2R in affinity and over 1000-fold selectivity in vivo. The crystal structure of a new picomolar M3 receptor antagonist in complex with the M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

Matsuo Award 4

PACAP induces differentiation of neural progenitor cells into glial lineage via radial glia

Jun Watanabe1,2, Hirokazu Ohtaki3, Satoru Arata1,2, Seiji Shioda4 1Department of Biochemistry, 2Center for Laboratory Animal Science, 3 Department of Anatomy, School of Medicine, Showa University, Tokyo, Japan 4Department of Neuropeptide Drug Discovery, Hoshi University School of Pharmacy and Pharmaceutical Sciences,

Neural development is controlled by region-specific factors that regulate cell proliferation, migration and differentiation. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts a wide range of effects on different cell types in the brain as early as the fetal stage. Previously, we reported that PACAP induced differentiation of cultured mouse embryonic neural progenitor cells (NPCs) into astrocytes. Here, we report that the effect of PACAP in vivo on the glial differentiation. We investigated the localization of PACAP specific receptor (PAC1-R) during neural development by use of immunohistochemistry. The immunereactivity of PAC1-R and proliferation marker, Ki67, showed similar distribution in E14 mouse embryo. Double-immunostaining showed that immunoreactivity for PAC1-R was co-localized with radial glia marker, vimentin. These data suggest that endogenous PACAP leads differentiation of NPCs into astrocytes via radial glia differentiation. Addition of PACAP into primary cultured-NPCs increased the expression of another radial grail marker, GLAST after 4 days culture. Furthermore, intracerebroventricular injection of PACAP into the ventricle of telencephalon of E13 embryos in utero increased the number of GLAST immunopositive cells. These data suggest that PACAP plays crucial roles in the differentiation of NPCs into astrocytes via radial grail lineage.

Matsuo Award 5

The orphan receptor GPRL mediates light-induced activation of clock gene expression in the suprachiasmatic nucleus.

Kaoru Goto, Masao Doi and Hitoshi Okamura Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Japan

G-protein-coupled receptors (GPCRs) participate in a broad range of physiological functions. There still exist a considerable number of orphan GPCRs whose cognate ligand and physiological functions are not known. The suprachiasmatic nucleus (SCN) is the principal central oscillator that drives circadian rhythms in behavior and physiology. Therefore, clarifying how the SCN entrains to the solar light/dark cycle is critical to better understand the mechanism of daily adaptation to environmental change. In this study, we report that the orphan receptor GPRL mediates light-induced activation of Per1 mRNA and c-Fos expression in the SCN. A significant attenuation was observed for light-responsive expression of Per1 and c-Fos in the GPRL-deficient SCN. When mice were exposed to light at subjective night, GPRL transcripts were acutely increased in the ventral part of the SCN. Our data suggest that the orphan receptor GPRL may contribute to photic entrainment of the central clock in the brain.

Matsuo Award 6

Comprehensive analysis of Gα-protein-coupling ability among orphan GPCRs

Kouki Kawakami1, Asuka Inoue1,2, Takeaki Shibata1, Junken Aoki1,3 1Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan. 2AMED-PRIME, Japan Agency for Medical Research and 3 Development, Japan. AMED-LEAP, Japan Agency for Medical Research and Development, Japan.

G protein-coupled receptors (GPCRs) form the largest membrane and druggable protein families accounting for about 900 genes and 40% of approved drug targets. Although rhodopsin-type (class A) GPCRs are most studied, one-third to one-fourth of these GPCRs are still classified in “orphan GPCRs”, whose ligand are unknown or understudied.

GPCR signaling is primarily mediated by four types of G proteins (Gs, Gi, Gq and

G12/13) and β-arrestins. To identify ligands for orphan GPCRs, we should use proper assays corresponding to downstream signaling pathways. Some groups reported Gs,

Gi and β-arrestins coupling abilities for orphan GPCRs based on ligand-independent constitutive activity1,2. However, it is still unknown which orphan GPCRs can be coupled to Gq and G12/13. Therefore, we evaluated Gq and G12/13 coupling potencies for orphan GPCRs based on their constitutive activity. We focused on 68 orphan GPCRs listed in the IUPHAR database3 and constructed the GPCR expression vectors containing FLAG-epitope tag at N-terminus. And then we measured the constitutive Gq and G12/13 activities using TGFα shedding assay, and found that about 25 GPCRs activated Gq and G12/13 without their ligands and the rest of the GPCRs had weak or no responses.

In addition, We used each G proteins depleted HEK293 cell lines to dissect Gq and

G12/13 signals and found that most of the activating GPCRs can be coupled to both Gq and G12/13, but some GPCRs, especially GPR35 and GPR132/G2A, can only be coupled to G12/13. Moreover, by introducing a particular activating mutation4 to inactive GPCRs, we identified some GPCRs including GPR68 can be coupled to Gq and G12/13. This information provides advantages of selecting suitable GPCR assays and also speculating these orphan GPCRs functions.

1PLOS ONE 10, e0138463 (2015), 2 Nat. Struct. Mol. Biol. 22, 362–369 (2015), 3IUPHAR/BPS Guide to PHARMACOLOGY, 4Nat. Genet. 48, 675–680 (2016)

Matsuo Award 7

Agonist-independent cAMP-repressing activity of the orphan receptor Gpr176 requires Gz.

Shumpei Nakagawa, Sumihiro Kunisue, Masao Doi Development of Systems Biology, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University

There are still more than 140 orphan GPCRs whose cognate ligands are not known, and deciphering their function remains a priority for fundamental and clinical research. Here we report the identification of ligand-independent (constitutive) basal activity of the orphan receptor Gpr176. To analyze this activity, we generated doxycycline-inducible stable cell lines expressing the untagged wild-type receptor. Out of the assay formats for measuring cAMP and IP1 levels, we only detected a significant reduction of cAMP in induced cells. On the other hand, no reduction was observed when a mutant receptor was induced, indicating that this receptor bears an agonist-independent intrinsic activity to reduce cAMP synthesis. Pertussis toxin (PTX) has been widely used to test the involvement of Gi signaling. Yet, interestingly, PTX treatment did not affect this activity, suggesting a potential participation of PTX-insensitive G-protein. Gz is a unique Gi/o subfamily member that can repress adenylyl cyclases in a PTX-insensitive manner. To test the potential involvement of Gz, Gz pathway was inactivated by siRNA-mediated knockdown of the endogenous Gz protein expression, lentiviral transduction of the regulator of G protein signaling (RGS) protein family member RGSZ1, which selectively inhibits Gz, and overexpression of a dominant negative Gz (DN-Gz) in the cells. We observed that all treatments abrogate the agonist-independent cAMP repressing activity of Gpr176. These results suggest that Gpr176 requires Gz for its activity.

Matsuo Award 8

Development of high throughput screening assay method for Gz-linked orphan receptor Gpr176

Tianyu Wang and Masao Doi Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan

The suprachiasmatic nucleus (SCN), the brain’s circadian pacemaker, governs daily rhythms in behaviour and physiology. Gpr176 is an SCN-enriched orphan G-protein-coupled receptor that sets the pace of circadian behaviour. Gpr176 is expressed in a circadian manner by SCN neurons, and molecular characterization reveals that it represses cAMP signalling via a unique G protein, Gz, in an agonist-independent manner (Nat Commun 7: 10583, 2016). While Gpr176 and other orphan GPRCs have no known natural ligands, small molecules that act as ligands could be developed as potential drug candidates. A key impediment to GPCR deorphanization is uncertainty about the coupling partner via which they signal, making functional assays problematic. In this sense, the evidence that Gpr176 couples to Gz is advantageous. By coupling with Gz, Gpr176 displays a relatively strong basal activity to repress cAMP. This would provide a merkmal activity for the purpose of assaying inverse agonists. In this study, we established a cAMP luminescence detection method to determine the constitutive activity of Gpr176. To monitor the intracellular cAMP concentration change caused by Gpr176, we constructed a doxycycline (DOX)-induced Gpr176 cell line with stable expression of a luciferase-based cAMP biosensor (GloSensor). Cells were cultured in 96 well plate or 384 well plate for 6 hours at 37°C with luciferin, followed by incubation with either DOX or vehicle solution for 15 hours. Before luminescence detection, the cell plate was acclimatized to 27°C room temperature for 1 hour. We used a HTP luminescence detector system FDSS/µCELL (Hamamatsu Photonics) to measure the luminescence in real time. After determining the baseline for 5 minutes, forskolin (FSK) was added to stimulate cAMP synthesis and we traced the luminescence for 25 minutes. Compared to the vehicle cells, DOX-treated cells showed a 50% reduction of luminescence, indicating cAMP-repressing activity of Gpr176. Importantly, these results are congruent with the activity of Gpr176 determined by cAMP-ELISA, attesting to the versatility of the GloSensor cAMP monitoring system. The GloSensor-based cell line that we developed in this study may be applicable for HTP ligand screening for Gpr176.

Matsuo Award 9

Generation of HEK293 cells devoid of Gi proteins.

Yuki Ono1, Aska Inoue1,2, Satoru Ishida1, Kouki kawakami1, Junken Aoki11,3 1 Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 2AMED-PRIME, 3AMED-LEAP

[Background and Aim] There are 16 G genes in the human genome. These gene products can be divided into four classes (Gs, i, q, 12) based on sequence similarity. Gi family has 8 members (i1/ i2/ i3/ o, t1, t2, t3, z) and is the largest family in the G genes. Gi family members have important roles in chemotaxis and cell proliferation, but the functional difference among Gi members still remain unknown. In this study, we generated HEK293 cells devoid of Gi family proteins by CRISPR-Cas9 system. [Method] The expression levels of Gi members in HEK293 cells were asesessed by qRT-PCR. 2-3 sgRNAs were designed against each gene expressed in HEK293 cells. sgRNAs which have high mutagenetic activity were selected and transfected to HEK293 cells. The cells were cloned by limiting dilution. Cells with mutations in target genes were screened by using restriction enzyme and the mutated DNA sequences were determined. Then, the selected cells were transfected with Gi coupled receptors and the response against its ligand were measured by Glosensor cAMP assay. [Results] We generated multiple clones of HEK293 cells devoid of Gi proteins expressed in HEK293 cells (except Gt3). When the Gi KO cells were transfected with Gi coupled receptors (D2R, MOR) and stimulated with its ligand, cAMP level did not decrease. When the cells were co-transfected with Gi members, cAMP decrease were observed and each Gi member showed different response. [Discussion] Gi KO cells will be powerful tools to dessect the difference among Gi members. For example, Gi coupled GPCR and Gi members coupling specificity can be determined. Many mutations in Gi genes have been reported in some diseases, such as, Auriculocondylar syndrome (Gi3) and Neurological development disorder (Go) and these mutants’ function could be analyzed by the Gi KO cells. Together with already established Gs, Gq, G12 family KO cells, these G-protein KO cell library should offer great advantages to dissect GPCR and downstream signaling.

Matsuo Award 10

PACAP-provoked PAC1 receptor signaling and internalization through two isoform of β-arrestins dependent mechanisms

Yusuke Shintani1, Atsuko Hayata-Takano1, 2, Keita Moriguchi1 and Hitoshi Hashimoto1, 2, 3, 4 1 Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Japan, 2 Center for Child Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Japan, 3 Division of Bioscience, Institute for Datability Science, Osaka University, Japan, 4 Department of Transdimensional Life Imaging, Open and Transdisciplinary Research Initiatives, Osaka University, Japan

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide widely expressed in the nervous systems and acts as a neurotransmitter and neurotrophic factor via three subtypes of G protein-coupled receptors (GPCRs). Among them, PAC1 receptor is highly expressed in the brain and involved in the regulation of, e.g., emotion, stress response, and pain transmission. Two nonvisual β-arrestins, β-arrestin1 and β-arrestin2 act as functional adaptor proteins that interact with agonist-occupied GPCRs and mediate activation of G-protein-independent several signaling pathways such as ERK1/2, Akt and Src. Recent studies have proposed that the β-arrestin-mediated signaling can be therapeutic targets for several disorders with improved therapeutic potential. However, the detailed roles of the two β-arrestin isoforms in PACAP–PAC1R signaling remain unclear. In this study, we examined PACAP-induced interaction between PAC1R and the two β-arrestin isoforms, β-arrestin-dependent PAC1R subcellular trafficking and ERK1/2 activation using the NanoBiT system, HaloTag technology and siRNA-mediated silencing in HEK293T cells and primary cultured cortical neurons. PAC1R interacted with both β-arrestins with similar PACAP dose- and time-dependency. Upon PACAP stimulation, the complex of PAC1R and β-arrestin2 was translocated from the cell surface into cytosol, but that of β-arrestin1 remained at the cell surface regions. siRNA-mediated silencing of β-arrestin2 significantly reduced PACAP-induced PAC1R internalization and prolonged ERK1/2 activation, but silencing of β-arrestin1 showed no significant effect on PAC1R internalization and rather increased ERK1/2 phosphorylation. Taken together, these results suggest that the fine and precise tuning of the PAC1R signaling pathways is mediated by the differential actions of β-arrestin1 and β-arrestin2. The present observations are expected to contribute to therapeutic drug development.

Acknowledgement

Supported by:

Daiichi Sankyo Company, LTD

Shin Nippon Biomedical Laboratories, LTD

The Uehara Memorial Foundation