Identification of N-arachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18

BBRC Biochemical and Biophysical Research Communications 347 (2006) 827–832 www.elsevier.com/locate/ybbrc

Identification of N-arachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18 Masashi Kohno a, Hitoshi Hasegawa a,*, Atsushi Inoue a, Masatake Muraoka a, Tatsuhiko Miyazaki b, Keizo Oka c, Masaki Yasukawa a a

Department of Bioregulatory Medicine, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan b Department of Pathogenomics, Ehime University Graduate School of Medicine, Toon, Ehime 791-0295, Japan c Integrated Center for Sciences, Shigenobu Station, Toon, Ehime 791-0295, Japan Received 29 June 2006 Available online 10 July 2006

Abstract An orphan G-protein-coupled receptor, GPR18, was cloned on the basis of degenerate-oligonucleotide PCR analysis of HUT 102 cells using primers designed from the conservative regions of the human chemokine receptor. GPR18 was expressed significantly in lymphoid cell lines, but not in non-lymphoid hematopoietic cell lines. Moreover, the expression of the GPR18 gene was higher in peripheral lymphocyte subsets (CD4+, CD4+CD45RA+, CD4+CD45RO+, CD8+, and CD19+) than in monocytes and lymphoid cell lines, and was increased after stimulation with phytohemagglutinin. By screening using a lipid library, N-arachidonylglycine (NAGly) induced an increase in intracellular Ca2+ concentration in GPR18-transfected cells, which was significantly greater than that in mock-transfected cells. NAGly also inhibited forskolin-induced cAMP production in a pertussis toxin-sensitive manner in the GPR18-transfected CHO cells. This is the first study to demonstrate that NAGly is a natural ligand for GPR18.  2006 Elsevier Inc. All rights reserved. Keywords: Lymphocytes; G-protein-coupled receptors; Lipids; Adult T-cell leukemia

The G-protein-coupled receptor (GPCR) superfamily is the largest known receptor family, characterized by seven transmembrane domains [1–3]. GPCRs transduce a variety of extracellular signals such as photons, peptides, hormone proteins, neurotransmitters, amino acids, lipids, prostanoids, and odorants through heterotrimeric G-proteins. There are about 1000 genes encoding such receptors in the human genome, and these receptors regulate numerous physiological processes, including neuronal excitability, metabolism, reproduction, development, hormonal homeostasis, and behavior. A variety of GPCRs are expressed in the immune system, including receptors for chemokines, neurotransmitters, cannabinoids, and inflammatory mediators such as *

Corresponding author. Fax: +81 89 960 5299. E-mail address: [email protected] (H. Hasegawa).

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.175

leukotrienes and prostaglandins [1,4]. Therefore, the roles of GPCRs and their ligands in the immune system have recently received attention. Previously, we have shown that increased surface expression of CCR7 and CCR4 correlates well with the involvement of lymphoid organs and skin in adult T-cell leukemia (ATL), respectively, and that chemokines and their receptors play important roles in tissue infiltration by ATL cells [5,6]. To examine chemokine receptors and GPCRs expressed in ATL cells, we have performed cloning of GPCRs by degenerateoligonucleotide PCR. While cloning, we found that an orphan receptor, GPR18, was highly expressed in HUT102 cells. In the present report, we describe the analysis of GPR18 expression in various hematopoietic cell lines and lymphocyte subsets, and reveal that N-arachidonylglycine (NAGly) is a natural ligand for GPR18.

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Materials and methods Cells. Two human T lymphoblastic cell lines, MOLT-4 and Jurkat; a human cutaneous T-cell lymphoma cell line, HUT78; a human erythroleukemia cell line, K562; two human myelomonocytoid cell lines, HL60 and U937; and a human B cell line, Raji, were obtained from the American Type Culture Collection (Rockville, MD). These cell lines were maintained in RPMI1640 medium supplemented with 10% fetal calf serum (FCS; Life Technologies, Gaithersburg, MD). HTLV-1-transformed T-cell lines, HUT102, MT-2, and MT-4, were maintained in RPMI1640 medium supplemented with 10% FCS. JPX-9 cells [7] were kindly provided by Prof. K. Sugamura, Tohoku University, Sendai, Japan. Antibodies and reagents. Human IL-2 was purchased from Roche Diagnostics GmbH, Germany. NAGly was obtained from BIOMOL, Plymouth Meeting, PA. Phytohemagglutinin (PHA) was purchased from DIFCO, Detroit, MI. Cell purification and activation. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals by Ficoll-Conray gradient centrifugation. The PBMCs were further fractionated into CD4+, CD4+CD45RA+, CD4+CD45RO+, CD8+, and CD19+ subsets and monocytes by using specific antibody-covered Micro-beads (Miltenyi Biotec GmbH, Germany), according to the manufacturer’s recommendation. The purity of the CD4+, CD4+CD45RA+, CD4+CD45RO+, CD8+, CD19+, and monocyte subsets was ascertained by FACS analysis with specific antibodies labeled with phycoerythrin (PE) or fluorescein isothiocyanate (FITC), and found to be >90%. CD4+ T cells, which had been cultured for 24 h with 10 lg/mL PHA, were used for this experiment as PHA-activated CD4+ T cells. Degenerate-oligonucleotide PCR, subcloning, and DNA sequencing. Total RNA was extracted from HUT 102 cells using TRIZOL reagent (Invitrogen, Carlsbad, CA) as described previously [8]. The cDNA was prepared with a SuperScript III CellsDirect cDNA synthesis system (Invitrogen) according to the manufacturer’s instructions. Degenerateoligonucleotide PCR was performed using the primers and conditions described by Yousefi et al. [9]. Briefly, 100 ng of single-stranded cDNA was used to seed 100 lL of reaction mixture, and then subjected to 40 cycles of PCR (95 C for 1 min, 37 C for 1 min, and 72 C for 1 min) using each degenerate-oligonucleotide primer (forward 5 0 -GAY MGI TAY YTI GCI ATH GTX CA-3 0 and reverse 5 0 -RMR TAI ADI AII GGR TTI AXR CA-3 0 ) at 3 lM in a Perkin-Elmer DNA thermal cycler (Perkin-Elmer, Norwalk, CT). Approximately 10 ng of the amplified product was ligated to 25 ng pCR2.1 vector (TA cloning kit; Invitrogen), and the contruct was introduced into 50 lL of One Shot competent cells (Invitrogen). The libraries were plated onto agar plates containing 50 lg/ mL ampicillin, 100 lM isopropylthiogalactoside (Invitrogen), and 50 lg/ mL X-Gal (Invitrogen). White colonies were picked and grown, and plasmids from selected colonies were purified using a Plasmid purification kit (Qiagen Inc., Valencia, CA). Cycle sequencing of 300 ng of plasmid DNA was performed on an automated ABI 310 sequencer (Applied Biosystems, Tokyo, Japan) with M13 reverse and forward primers. Sequence similarity searches and alignments were done using the GenBank and EMBL databases. Construction of expression vector encoding human GPR18. To amplify the fragment containing the full coding region of the human GPR18 gene [10], the following specific primers were used: forward 5 0 -AAAT GATCACCCTGAACAATCAAGA-3 0 and reverse 5 0 -ATTCATAACA TTTCACTGTTTATATTGCTTAG-3 0 . The above fragment was amplified from total RNA of HUT 102 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) using an RNA PCR kit (Takara Shuzo, Kyoto, Japan), as described previously [11]. After confirming the entire nucleotide sequence, the fragment was cloned into the EcoRI site of the pCXN2 vector [12]. Cell transfection and selection of GPR18-transfected cell lines. For transfection into the cells, the adherent cells at about 50% confluence in a 10-cm dish and the growth-phase non-adherent cells (5 · 105) were rinsed twice with Opti-MEM (Life Technologies) and transfected with 10 lg pCXN2 containing the GPR18 gene using Lipofectamine (Life

Technologies) following the instructions of the manufacturer. After incubation at 37 C for 6 h, the transfection mixture was removed. The cells were allowed to grow in fresh RPMI1640 medium supplemented with 10% FCS for 24 h and then added to G418 (Life Technologies) at a concentration of 800 lg/mL for L929, 1.5 mg/mL for K562, and 500 lg/ mL for CHO cells. After 2 weeks, the stable transformants were isolated and examined for GPR18 expression by Northern blot analysis. Total RNA preparation, cDNA synthesis, and real-time PCR for GPR18. The expression of GPR18 was quantified by real-time PCR. Briefly, for quantitative real-time PCR (qPCR) analysis, total RNAs were extracted from lymphocyte subsets and various cell lines using TRIZOL reagent (Invitrogen). The cDNA was prepared with a SuperScript III CellsDirect cDNA synthesis system (Invitrogen). The expression of the GPR18 gene was quantified using a QuantiTect SYBR Green PCR kit (Qiagen) in an ABI PRISM 7700 Sequence Detector (Applied Biosystems), and corrected with a hypoxanthine phosphoribosyl transferase 1 (HPRT1) control. Amplifications were done in a total volume of 25 lL for 45 cycles of 15 s at 95 C and 1 min at 60 C. Samples were run in triplicate and their relative expression was determined by normalizing the expression of each target to HPRT1 and then comparing this normalized value with the normalized expression in a reference control sample to calculate the fold change value. The primers for the amplicons spanned intron/exon boundaries to minimize amplification of genomic DNA. Primer sequences were as follows: GPR18, forward 5 0 -TTCTTGATCTGCTGACCATGAC AC-3 0 and reverse 5 0 -AGGGACAGGTTGATCTTGATTGTTC-3 0 ; and HPRT1, forward 5 0 -TAAGCCAGACTTTGTTGGAT-3 0 and reverse 5 0 -GAACTCTCATCTTAGGCTTT-3 0 . Assay of cytosolic Ca2+ concentration. GPR 18-expressing L929, K562, and CHO cells were seeded in 96-well plates at a density of 5 · 104 cells per well and cultured overnight. They were loaded with 4 lM Fluo-3 AM (Dojindo, Kumamoto, Japan) in Hepes–HBSS buffer (1· Hanks’ balanced salt solution containing 10 mM Hepes–NaOH (pH 7.4), 1 mM CaCl2, 0.5 mM MgCl2, and 0.1% bovine serum albumin) and 0.02% Fluronic F127 (Cosmo Bio, Tokyo, Japan) for 1 h at 37 C, washed twice, and filled with Hepes–HBSS buffer. They were then stimulated with 198 lipids contained in the Bioactive Lipid Library (BIOMOL) individually and NAGly at various concentrations, and intracellular Ca2+ mobilization was monitored with an ACAS 570 laser cytometer (MERIDIAN Inc., Okemos, Michigan) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The effect was compared with that observed with 5 lM ionomycin (Sigma–Aldrich, St. Louis, MO). Cyclic AMP assays. The cells were cultured in a 96-well plate (5 · 104 cells/well) for 12 h and then washed twice with Krebs–Ringer Hepes (KRH) buffer (1.24 mM NaCl, 5 mM KCl, 1.25 mM MgSO4, 1.45 mM CaCl2, 1.25 mM KH2PO4, 25 mM Hepes (pH 7.4), and 8 mM glucose). Then, 90 lL KRH buffer supplemented with 25 lM rolipram (Sigma–Aldrich) was added and the plates were incubated for 30 min. Then, 90 lL KRH buffer containing 1 lM forskolin (Sigma–Aldrich) and several concentrations of NAGly was added, and the plates were incubated at room temperature for 10 min. The cells were lysed by adding 20 lL lysis buffer 1A (a component of the cAMP enzyme immunoassay system, Amersham Biosciences), and 80 lL of the cell lysate was used for measurement of cAMP produced during the incubation with the kit, as recommended by the manufacturer. To examine the effect of pertussis toxin (PTX) (Sigma–Aldrich) treatment on cAMP production, the cells were cultured in medium supplemented with 100 ng/mL PTX for 4 h.

Results and discussion To analyze GPCR expression in HUT 102 cells, we performed degenerate-oligonucleotide PCR. We used primers that had been designed based on the conserved amino acid sequence found in the second intracellular loop and the seventh transmembrane domain of the known human chemokine receptors. Gel analysis of the PCR products

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generated from the HUT 102 cDNAs indicated that good amplification of products of the expected size (500– 550 bp) had been obtained. Reaction products were cloned in the pCR 2.1 vector, and approximately 200 colonies were sequenced. Comparison of their sequences with those present in the GenBank and EMBL databases indicated that eight distinct genes were represented. These eight genes included CCR2, CCR4, CCR6, CCR7, CXCR4, GPR18, and two unknown genes. Of these, one clone, GPR18, was analyzed further. The expression of the GPR18 gene in various hematopoietic cell lines was evaluated by qPCR. As shown in Fig. 1A, the GPR18 gene was significantly expressed in HTLV-1-transformed cell lines (HUT 102, MT-2, and MT-4), and lymphoid cell lines such as Jurkat, MOLT-4, HUT 78, and Raji cells, while none of the non-lymphoid hematopoietic cell lines (U937, HL60, and K562) expressed detectable levels of GPR18 mRNA. Next, we analyzed the expression of the GPR18 gene in peripheral lymphocyte subsets and monocytes. As shown in Fig. 1B, the GPR18 gene was expressed at a high level (more than 60-fold) in lymphocyte subsets such as CD4+, CD4+CD45RA+,

Fig. 1. (A) Expression of the GPR18 gene in various hematopoietic cell lines. (B) Expression of the GPR18 gene in peripheral lymphocyte subsets and monocytes. The qPCR analysis was performed on total RNA prepared from various cell lines, lymphocyte subsets, and monocytes as described in Materials and methods. Results are calculated as a ratio of GPR18 expression relative to HPRT1 expression.

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CD4+CD45RO+, CD8+, and CD19+, compared with the level seen in monocytes. Moreover, expression of the GPR18 gene was significantly increased in PHA-activated CD4+ T cells compared with the level seen in non-stimulated CD4+ T cells. To investigate whether the GPR18 gene is transactivated by Tax, we examined the kinetics of GPR18 gene expression in JPX-9 cells, a stable transformant of Jurkat with the plasmid pMAXRHneo-1 containing the metallothionein promoter-driven Tax gene. However, induction of the GPR18 gene was not observed after expression of Tax (data not shown). GPR18 is a seven-transmembrane G-protein-coupled receptor consisting of 331 amino acids [10]. The sequences of the open reading frames of the canine and human GPR18 genes are highly conserved, sharing 89% nucleotide identity and 92% amino acid similarity between the two species. The human GPR18 gene is localized in the chromosomal region 13q32.3 [10]. GPR18 has been reported to show high expression in PBMC and lymphoid tissues such as spleen and thymus, and moderate expression in brain, testis, ovary, and lung [10,13]. Taken together with our result, this suggests that GPR18 may be involved in immune system regulation. Alignment analysis [13] has revealed that GPR18 clusters with an orphan GPCR, EBI2 [14], and the lipid receptors CysL-1 [15] and -2 [16]. Among these receptors, GPR18 has closest affinity with EBI2 in view of their close chromosomal proximity and similar receptor expression patterns, suggesting that GPR18 and EBI2 may have similar biologic functions [17]. To identify the ligand for GPR18, we established a stable polyclonal population of GPR18-expressing L929 cells, since the parental L929 cells did not express detectable levels of GPR18 mRNA (data not shown), and then screened 198 lipids in the Bioactive Lipid Library by measuring intracellular Ca2+ mobilization using GPR18-expressing L929 cells. Among them, NAGly at a concentration of 10 lM induced a significant increase in intracellular Ca2+ concentration in GPR18-expressing L929 cells, compared with that in mock-transfected L929 cells (Fig. 2). To confirm that the ligand of GPR18 is NAGly, we further established two kinds (K562 and CHO cells) of stably GPR18-transfected cells, and compared their intracellular Ca2+ mobilization with that of mock-transfected cells after adding NAGly at a concentration of 10 lM. As shown in Fig. 2, the increase in intracellular Ca2+ concentration was enhanced 3-fold and 2-fold by GPR18-expressing K562 and CHO cells, compared with mock-transfected cells, respectively, although mock-transfected K562 and CHO cells displayed a slight increase in intracellular Ca2+ concentration. Similar results were obtained after adding NAGly at a concentration of 1 lM (data not shown). Next, we performed a cAMP assay to examine the effect of NAGly on cAMP accumulation in the GPR18-transfected CHO cells. As shown in Fig. 3, cAMP production in the GPR18-transfected CHO cells was clearly inhibited by NAGly, as compared with that in mock-transfected

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Fig. 2. NAGly-induced intracellular Ca2+ mobilization in GPR18-transfected cells. The three kinds of mock- (L929, K562, and CHO) or stably GPR18transfected cells (L929/GPR18, K562/GPR18, and CHO/GPR18) were loaded with 4 lM Fluo-3 AM and stimulated with 10 lM NAGly. Intracellular Ca2+ mobilization was monitored at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Results are calculated as a ratio of intensity of fluorescence induced by NAGly relative to highest intensity observed with 5 lM ionomycin.

CHO cells. Distinct inhibition (30%) was observed at 1 nM NAGly, and the inhibition increased dose-dependently to 70% at 100 nM NAGly with an IC50 value of 20 ± 8 nM. Pretreatment of the GPR18-transfected CHO cells with PTX completely abolished the inhibition of forskolin-stimulated cAMP production by NAGly. Data for intracellular Ca2+ mobilization and cAMP accumulation indicated that NAGly was a natural ligand for GPR18, and that GPR18 would be coupled with a Gai/o-protein. NAGly, the carboxylic analog of the endocannabinoid anandamide, was first identified from the bovine and rat brains [18]. Although the biological functions of NAGly are not well understood, NAGly has been reported to have analgesic properties similar to those of anandamide (N-arachidonylethanolamide) [19,20]. This is because NAGly inhibits the hydrolytic activity of fatty acid amide hydrolase (FAAH) on anandamide, causing an increase in anandamide concentration. On the other hand, NAGly

is present in a variety of tissues such as skin, small intestine, kidney, and testis, as well as brain [18]. The varying levels of NAGly in different organs likely indicate its involvement in additional physiological functions besides pain regulation. The important role of arachidonic acid-derived products (e.g., prostanoids, leukotrienes, and thromboxanes) in inflammation and pain, together with the relatively high levels of NAGly in the skin, small intestine, and brain, suggests that NAGly may serve as a modulator of inflammation [4,21]. Cannabinoids exert their effects by binding to two GPCRs, CB1 and CB2 [22–24]. The CB1 receptor is expressed predominantly in the brain, whereas cells of the immune system express high levels of CB2 receptor [24]. Cannabinoids have been shown to influence the immune system [25]. The endocannabinoid 2-arachidonoylglycerol and synthetic cannabinoids have been reported to enhance chemotaxis in human peripheral blood cells and to

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[12] Fig. 3. Effect of NAGly on forskolin-stimulated cAMP production in GPR18-transfected CHO cells. The GPR18-transfected CHO cells were subjected to stimulation of cAMP production by forskolin with (m) or without (d) pretreatment with 100 ng/mL PTX. Production of cAMP is expressed as a percentage of control in the absence of NAGly. In the absence of NAGly, mock- (h) and GPR18-transfected CHO cells accumulated 900 ± 320 fM and 1150 ± 210 fM of cAMP/5 · 104 cells, respectively. These experiments were done in triplicate.

modulate the chemokine-induced migration of T cells through the CB2 receptor [26–28]. The synthetic cannabinoids have also been shown to suppress T-cell activation and to skew cytokine production in favor of a Th2 response via a CB2 receptor-dependent pathway [29]. Although NAGly lacks affinity for the CB1 and CB2 cannabinoid receptors [18], it is likely to participate in immune regulation through its receptor GPR18, since GPR18 was expressed predominantly in T and B cells. We are now studying NAGly-GPR18-mediated immune regulation in more detail. References [1] M.D. Thompson, W.M. Burnham, D.E. Cole, The G protein-coupled receptors: pharmacogenetics and disease, Crit. Rev. Clin. Lab. Sci. 42 (2005) 311–392. [2] R.J. Lefkowitz, Historical review: a brief history and personal retrospective of seven-transmembrane receptors, Trends Pharmacol. Sci. 25 (2004) 413–422. [3] M.S. Lombardi, A. Kavelaars, C.J. Heijnen, Role of modulation of G protein-coupled receptor signaling in inflammatory processes, Crit. Rev. Immunol. 22 (2002) 141–163. [4] G.A. Cabral, Lipids as bioeffectors in the immune system, Life Sci. 77 (2005) 1699–1710. [5] H. Hasegawa, T. Nomura, M. Kohno, N. Tateishi, Y. Suzuki, N. Maeda, R. Fujisawa, O. Yoshie, S. Fujita, Increased chemokine receptor CCR7/EBI1 expression enhances the infiltration of lymphoid organs by adult T-cell leukemia cells, Blood 95 (2000) 30–38. [6] O. Yoshie, R. Fujisawa, T. Nakayama, H. Harasawa, H. Tago, D. Izawa, K. Hieshima, Y. Tatsumi, K. Matsushima, H. Hasegawa, A. Kanamaru, S. Kamihira, Y. Yamada, Frequent expression of CCR4 in adult T-cell leukemia and human T-cell leukemia virus type 1transformed T cells, Blood 99 (2002) 1505–1511. [7] S. Miura, K. Ohtani, N. Numata, M. Niki, K. Ohbo, Y. Ina, T. Gojobori, Y. Tanaka, H. Tozawa, M. Nakamura, K. Sugamura, Molecular cloning and characterization of a novel glycoprotein, gp34,

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