Biochem. J. (1999) 337, 67–75 (Printed in Great Britain) 67 The novel sphingosine 1-phosphate receptor AGR16 is coupled via pertussis toxin-sensitive and -insensitive G-proteins to multiple signalling pathways Koichi GONDA*†, Hiroyuki OKAMOTO*‡, Noriko TAKUWA*, Yutaka YATOMI§, Hiroshi OKAZAKIR, Takeshi SAKURAI¶, Sadao KIMURA**, Rannar SILLARD††, Kiyonori HARII† and Yoh TAKUWA*R‡‡1 *Department of Molecular and Cellular Physiology, University of Tokyo Graduate School of Medicine, Tokyo 113–0033, Japan, †Department of Plastic and Reconstructive Surgery, University of Tokyo Graduate School of Medicine, Tokyo 113–0033, Japan, ‡Department of Vascular Surgery, University of Tokyo Graduate School of Medicine, Tokyo 113–0033, Japan, §Department of Laboratory Medicine, Yamanashi Medical University, Tamaho-cho, Yamanashi 408–3898, Japan, RDepartment of Cardiovascular Biology, University of Tokyo Graduate School of Medicine, Tokyo 113–0033, Japan, ¶Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–0006, Japan, **Division of Cardiovascular Biology, Center for Biomedical Sciences, Chiba University Medical School, Chiba 260–0856, Japan, ††Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden, and ‡‡Foundation for Advancement of International Science, Tsukuba, Ibaraki 305–0005, Japan In the present study, we determined the agonist specificity and (PTX), different from the case of the recently identified S1P the signalling mechanisms of a putative sphingosine 1-phosphate receptor EDG1. Also in CHO-AGR16 cells, but not in CHO (S1P) receptor, AGR16. In CHO cells transiently transfected cells, S1P at higher concentrations activated mitogen-activated with an AGR16 expression vector, but not in cells transfected protein kinase (MAPK) in a PTX-sensitive and Ras-dependent with an empty vector, the addition of a low concentration of S1P manner. S1P also induced the activation of two stress-activated (1 nM) caused an increase in the intracellular free Ca#+ con- MAPKs, c-Jun N-terminal kinase and p38, in a manner that #+ #+ centration ([Ca ]i) by mobilization of Ca from both intra- and was totally insensitive to PTX. In CHO-AGR16 cells, S1P extra-cellular pools. To determine the spectrum of agonists for induced stress-fibre formation, with an increase in myosin AGR16, we employed K562 cells, which in the naive state do not light chain phosphorylation, in a PTX-insensitive and Rho- respond at all to either S1P or structurally related lipids with an dependent manner. S1P also induced an increase in the #+ increase in [Ca ]i. In K562 cells stably expressing AGR16, cellular cAMP content in CHO-AGR16 cells, which contrasts S1P and sphingosylphosphorylcholine (SPC) dose-dependently sharply with the case of EDG1. These results establish #+ increased [Ca ]i with half-maximal values of 3 nM and 100 nM that the S1P receptor AGR16 is coupled via both PTX- respectively. In CHO cells stably expressing AGR16 (CHO- sensitive and -insensitive G-proteins to multiple effector AGR16), but not in parental CHO cells, we observed specific pathways. binding of [$#P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P Key words: adenylate cyclase, G-protein-coupled receptor, stimulated the production of inositol phosphates and Ca#+ lysophospholipid, mitogen-activated protein kinase, mobilization which was only 30% inhibited by pertussis toxin phospholipase C. INTRODUCTION and SPC activate multiple intracellular signalling molecules, including phospholipase C [8,16,17], phospholipase D [6,16], Accumulating evidence now indicates that the lysosphingolipids mitogen-activated protein kinase (MAPK)\extracellular-signal- sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine regulated kinase (ERK) [8,18], protein kinase C (PKC) [19] and + (SPC) constitute a group of important extracellular lipid the K [IK(Ach)] channel [20]. Also, many of the lysosphingolipid- mediators [1–4]. Both S1P and SPC have been shown to regulate induced cellular responses were shown to be inhibited by pertussis such diverse cellular functions as proliferation [5,6], migration toxin (PTX) pretreatment [8,16,21]. Moreover, it was demon- [7,8] and cell shape change [9,10] in a variety of cell types. It was strated that the addition of S1P to culture medium, but not the demonstrated previously [11–13] that S1P at high concentrations intracellular injection of S1P, induced morphological changes in could act directly on intracellular Ca#+ stores to induce Ca#+ NIE-115 neuronal cells [9]. It was also reported that immobilized release. This observation, together with the finding that growth S1P covalently linked to glass particles was able to activate factors and other extracellular stimuli induce activation of platelets [22]. These observations favour the view that lyso- sphingolipid metabolism leading to the production of S1P sphingolipids exert many of their biological activities via cell- [8,14,15], suggested that S1P might act as an intracellular second surface receptors [4]. Importantly, it was recently demonstrated messenger. However, subsequent studies have revealed that S1P [23] that S1P is present in the plasma at a readily detectable level, 2+ 2+ Abbreviations used: ATF, activating transcription factor; [Ca ]i, intracellular free Ca concentration; EDG, endothelial differentiation gene; ERK, extracellular-signal-regulated kinase; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; LPA, lysophosphatidic acid; MAPK, mitogen- activated protein kinase; MLC, 20 kDa myosin light chain; PKC, protein kinase C; PTX, pertussis toxin; SPC, sphingosylphosphorylcholine; S1P, sphingosine 1-phosphate. 1 To whom correspondence should be addressed: Department of Molecular and Cellular Physiology, University of Tokyo School of Medicine, 7-3- 1 Hongo, Bunkyo-ku, Tokyo 113, Japan (e-mail yohtakwa!m.u-tokyo.ac.jp). 68 K. Gonda and others implying physiological and pathophysiological roles for S1P as a presence of 0.2 mM 3-isobutyl-1-methylxanthine, and then stimu- first messenger. lated with S1P for 5 min. The reaction was terminated by adding The present study demonstrates that AGR16\H218\EDG5 HCl (final concentration 0.1 M), and the amount of cAMP in the (endothelial differentiation gene 5) [24,25], a member of EDG acid extracts was measured by RIA using a Yamasa cAMP kit receptor family with a seven-transmembrane structure [26–28], is (Choshi, Japan) [34]. a functional receptor for S1P, with signalling characteristics clearly distinct from those of EDG1, the first S1P receptor identified [29–31]. We originally cloned AGR16 from rat aortic Plasmids and transfections smooth muscle [24]. We have defined the intracellular signalling pathways activated by AGR16, and determined the ligand Rat full-length AGR16 cDNA (DDBJ\GenBank\EMBL ac- specificity of AGR16, by studying responses in mammalian cells cession no. AB016931) was cloned by hybridization screening of (CHO and K562) transiently or stably transfected with an AGR16 a λgt10 cDNA library made from rat aortic smooth muscle cells expression vector. While this paper was in preparation, it was [24]. AGR16 cDNA was ligated into the mammalian expression reported [32] that the overexpression of AGR16 in Jurkat T-cells vector pME18S (obtained from Dr. K. Maruyama, Tokyo and Xenopus oocytes conferred the ability to respond to S1P and Medical and Dental College, Tokyo, Japan) at the EcoRI site SPC, with stimulation of serum-response-element-driven trans- downstream of the SRα promoter. Transfections of cells were criptional activity and Ca#+ efflux respectively. However, other carried out by using Lipofectamine (GIBCO-BRL) as described functional aspects of AGR16 as the receptor for S1P, as well as [36]. Stable transfectants were selected with G418 as the agonist specificity of AGR16, have not been elucidated. described [35]. It was demonstrated recently that another S1P receptor, EDG1, is coupled to MAPK in a PTX-sensitive manner in mammalian cells [29,30] and to inhibition of adenylate cyclase in Assays of MAPK, JNK and p38 activities both mammalian cells [33] and Sf9 insect cells [30]. In addition to these observations, we independently found that EDG1 is coupled For measurement of the MAPK activity in transiently transfected exclusively via Gi to multiple signalling pathways, including cells, CHO cells in 35 mm-diam. dishes were co-transfected with activation of phospholipase C, Ca#+ mobilization and inhibition pME18S-Myc-MAPK [36] and either pME18S-[Asn"(]Ras of adenylate cyclase, in mammalian expression systems [31]. ([Asn"(]Ras cDNA was obtained from Dr. G. Cooper, Harvard Unlike EDG1, we report that AGR16 is coupled largely Medical School, Cambridge, MA, U.S.A.) or the empty vector via PTX-insensitive G-proteins to phospholipase C, Ca#+ pME18S. The cells were lysed in lysis buffer containing 50 mM mobilization, and c-Jun N-terminal kinase (JNK) and p38 Tris (pH 8.0), 1 mM EDTA, 150 mM NaCl, 1 mM Na$VO%, MAPKs. When activated with higher concentrations of S1P, 1 mM PMSF, 10 µg\ml each of leupeptin and aprotinin, 0.5% AGR16 is coupled via a PTX-sensitive G-protein to the Ras\ sodium deoxycholate, 0.1% SDS and 1% Nonidet P-40. Myc- MAPK pathway. tagged MAPK was immunoprecipitated by using a mouse monoclonal anti-(Myc epitope) antibody (clone 9E10). The MAPK activity associated with the immune complex was assayed MATERIALS AND METHODS in itro using myelin basic protein (Sigma) as a substrate, as Cells described [36].