Poster

Poster 2 Cell Signaling 1 ポスター 2 細胞内外のシグナル1

P2-1 Analysis of regulatory factors of G-protein-coupled receptors localization at the primary cilium Gタンパク質共役型受容体の1次繊毛局在を制御する因子の解析 Shikada Sho（鹿田星），三好耕，吉村武，韓薩日娜，天野元揮，高村明孝，片山泰一 Dept Child Development & Mol Brain Sci, United Grad Sch Child Development, Osaka Univ On the surface of nearly every neurons in vertebrates there are protrusions of the cell membrane called primary cilia. In recent years, primary cilia have been studied as having a role as a non-synaptic sensor surveying various signals, and transmitting those signals into the cell. Defective formation or function of primary cilia is implicated in the pathogenesis of many human developmental disorders and diseases, termed ciliopathies. There are multiple G protein-coupled receptors (GPCRs) as signaling factors in primary cilia, and it has been clarified that many of them are important for various nerve functions. Serotonin receptor 6 (HTR6) selectively localizes to neuronal primary cilia, and it has been suggested that this GPCR is implicated in social cognition. Meanwhile, since serotonin receptor 7 (HTR7), highly homologous to HTR6, does not localize to primary cilia, localization of GPCR in primary cilia is likely to be specifically regulated by other factors. Therefore, in this study, in order to clarify regulatory mechanisms of GPCR localization in primary cilia, we performed immunoprecipitation or immunofluorescence analysis, and attempted to identify factors that specifically bind to and regulate HTR6. We found that HTR6 interacts with β-arrestin while HTR7 does not, and HTR6-β-arrestin interaction is likely to important for the cilia localization of HTR6.		P2-2 TULP3 regulates the formation of primary cilia and the localization of ciliary membrane proteins in hTERT-RPE1 hTERT-RPE1細胞においてTULP3は一次繊毛の形成と膜タンパクの局在を制御する Han Sarina（韓薩日娜），三好耕，鹿田星，天野元揮，高村明孝，吉村武，片山泰一 Molecular Bra Sci, United Grad Sch of Child Develo p, Osaka Univ, Osaka, Japan The primary cilia are known as biosensors that receive extracellular signals and transduce them to the cell body in vertebrate cells. In humans, impairment of cilia-mediated signaling causes ciliopathies characterized by cognitive deficits, obesity, renal abnormalities, retinal degeneration, and so on. Similar to phenotype of ciliopathy, spontaneous “tubby” mice, in which the Tub gene is mutated, also show obesity, retinal dystrophy. Tub and Tubby-like protein 1~4 (Tulp1~4) share the carboxyl-terminal tubby domain and are collectively referred to as Tubby family proteins, in which Tub and Tulp3 are expressed in the brain. In humans, a mutation in TUB is associated with learning difficulty, obesity and retinal dystrophy. Disruption of mouse Tulp3 is embryonically lethal, displaying neural tube closure defect, while the biological role of TULP3 in development is poorly understood. In this study, we generated hTERT-RPE1 cell lines with biallelic disruption of the TULP3 gene by the genome editing CRISPR/Cas9 system. Absence of the TULP3 proteins in the established TULP3-KO hTERT-RPE1 cells were confirmed. Formation of cilia and localization of ciliary membrane proteins in the TULP3-KO cell lines and parental hTERT-RPE1 cells were observed by immunofluorescent study. TULP3 ablation affected the cilium number and the cilium length. Moreover, ADP-ribosylation factor-like protein 13B (ARL13B) and 72 kDa inositol polyphosphate 5-phosphatase (INPP5E), ciliopathy-related ciliary membrane proteins, were undetectable at the cilia of TULP3-KO cells. TULP3 overexpression rescued the ciliary formation and the ciliary membrane protein localization in TULP3-KO cells. Our findings suggest that TULP3 is involved in formation and membrane protein localization of cilia in hTERT-RPE1 cells.

P2-3 Primary cilia length in energy balance signaling involved in melanin-concentrating hormone receptor エネルギー代謝状態と一次繊毛長：一次繊毛局在型Gタンパク質共役型受容体MCHR1の関与 Okada Tomoya（岡田智哉），三木大輔，河渕省吾，小林勇喜，斎藤祐見子 Grad Schl of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan Energy homeostasis is maintained by balancing the intake and expenditure of energy. Excessive accumulation of fat by disrupting balance system causes overweight and obesity. Intriguing subset of genes associated with obesity cause a dysfunction of primary cilia, organelles that function as hubs for select signaling pathways. Many neurons in the mammalian brain possess primary cilia that are enriched for certain G protein-coupled receptors (GPCRs), including melanin-concentrating hormone (MCH) receptor 1 (MCHR1). The MCH-MCHR1 system has been intensively studied for its involvement in the regulation of feeding behavior and body weight. Recently, we reported that MCH causes an effective reduction in cilia length via ciliary MCHR1-expressing epithelial RPE1 cells. Although short cilia have been observed in genetically obese mice, a possible correlation between MCHR1-positive neuronal cilia length and energy metabolism has not been characterized. Here, we report that 48 h-starved mice displayed shorter MCHR1 positive cilia than did normal diet-fed mice in the hippocampal CA1 region. Although many cilia carrying MCHR1 were detected in the CA3 region, a significant difference in the average length of the cilia was not observed as compared with fed mice. Further, MCH-treated hippocampal slice cultures exhibited cilia length shortening of MCHR1 positive cilia in the CA1 region, which did not occur in the CA3 region. The present results provide the first evidence that MCHR1-positive neuronal cilia length is actively changed by metabolic alteration in vivo, and this appears to selectively occur in hippocampal CA1 neurons.		P2-4 Ciliary G-protein-coupled receptor MCHR1 and SSTR3 affects cilia length control via different mechanisms 中枢性Gタンパク質共役型受容体MCHR1とSSTR3による一次繊毛縮退機構の解析 Tomoshige Sakura（友重桜子）¹，今門宏輔¹，宮本達夫²，小林勇喜¹，斎藤祐見子¹ ¹Graduate School of Integrated Arts and Sciences, Hiroshima University ²Research Institute for Radiation Biology and Medicine, Hiroshima University Primary cilia are specialized sensory organelle protruding from the surface of most vertebrate cell types. Primary cilium coordinates multiple cellular signaling pathways through the myriad of transmembrane receptors embedded in the cilium membrane. Among various receptors, the G-protein-coupled receptors (GPCRs) are essential for neuronal primary cilia function, and neuronal cilia integrity is essential for normal brain development and neuronal interactions in the adult brain. During postnatal development, the cilium membrane becomes equipped with a set of GPCR such as melanin-concentrating hormone (MCH) receptor 1 (MCHR1) and somatostatin (SST) receptor 3 (SSTR3). We have previously shown that the MCH-ciliary MCHR1 axis causes cilia shortening via the Gi/o-dependent pathway with an EC50 value of 0.54 nM. Here we report that SST concentrations in the nanomolar range also causes cilia shortening in clonal ciliary SSTR3-expressing RPE1 cells but via different mechanisms from those of MCHR1-expressing cells. First, on the time course, the MCH-ciliary MCHR1 system acts as a more efficient effector for cilia shortening than the SST-SSTR3 system. Second, while the MCHR1-expressing cells display a transient increase of ligand-dependent Akt and GSK3b signaling, the SSTR3-expressing cells can be kept in a state of continuous activation of two components for up to 60 min. Moreover, by using cells with fewer cilia by Kif3A siRNA treatment, we observed several different signaling characteristics between ligand-treated MCHR1- and SSTR3-expressing cells. Collectively, our results indicate that MCHR1 and SSTR3 affect ciliary length control and Akt/GSKb signaling via different mechanisms.

P2-5 Rho-kinase regulates the Ras activity through phosphorylation of SynGAP1 RhoキナーゼはSynGAP1のリン酸化を介してRasの活性化を制御する Wu Mengya（呉夢雅），船橋靖弘，高野哲也，坪井大輔，許伊凡，アハマドリズゥアン，天野睦紀　，貝淵弘三 Dept. of Cell Pharmacol. Med., Univ. of Nagoya The small GTPase RhoA and its downstream effector Rho-kinase are considered as one of the key regulators in dendritic spine formation and synaptic plasticity. However, how RhoA/Rho-kinase signaling involved in modulating synaptic plasticity still remains unknown. We have recently developed a phosphoproteomic analysis method that uses affinity beads coated with 14-3-3 proteins to enrich phosphorylated proteins and established the kinase-associated neural phosphosignaling (KANPHOS) database that provides the phosphorylated sites identified by our phosphoproteomic approaches. Using KANPHOS database, we identified SynGAP1, which is a synaptic Ras-GTPase activating protein, as a novel Rho-kinase substrate. In this study, we found that phosphorylation of SynGAP1 by Rho-Kinase increased its interaction with 14-3-3 but decreased with PSD-95, which is a major scaffolding protein in the postsynaptic densities of dendritic spines. SynGAP1 was dispersed from spines upon LTP induction in cultured neurons, and this dispersion depends on phosphorylation of SynGAP1 by Rho-kinase. Moreover, we found that Rho-kinase increased Ras and ERK activity through phosphorylation of SynGAP1. These results suggest, the synaptic dispersion of SynGAP1 which phosphorylated by Rho-kinase during LTP represents supposed to be a curtail signaling element. Also make RhoA/Rho-kinase activity transduce Ras-ERK signaling-mediated spine enlargement, and synaptic potentiation.