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J. Biol. Chem., Vol. 280, Issue 10, 9083-9087, March 11, 2005

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Identification of T Cell Death-associated Gene 8 (TDAG8) as a Novel Acid Sensing G-protein-coupled Receptor^*

Satoshi Ishii, Yasuyuki Kihara, and Takao Shimizu

From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, the University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, July 12, 2004 , and in revised form, November 30, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
T cell death-associated gene 8 (TDAG8) is a G-protein-coupled receptor mainly expressed in lymphoid organs and cancer tissues. TDAG8 shares high amino acid sequence homologies with recently reported proton-sensing G-protein-coupled receptors, G2A, OGR1, and GPR4. Here we have identified TDAG8 as a novel proton-sensing receptor. Upon acid stimulation, stably expressed TDAG8 was internalized from the plasma membrane. As a signaling pathway downstream of TDAG8, accumulation of cyclic AMP was observed in response to solutions with a pH value lower than 7.2. Furthermore, RhoA activation and actin rearrangement were elicited by acid-stimulated TDAG8. These results suggest that TDAG8 may play biological roles in immune response and cellular transformation under conditions accompanying tissue acidosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Immature thymocytes undergo apoptosis in vitro by cross-linking T cell receptor or treatment with glucocorticoids (1). During activation-induced cell death, expression of T cell death-associated gene 8 (TDAG8,¹ also known as GPR65) was significantly increased (2). The primary structure of TDAG8 suggested that the gene encoded a putative G-protein-coupled receptor (GPCR). Subsequently, Im et al. (3) identified TDAG8 as a receptor for 1--D-galactosylsphingosine (psychosine); TDAG8 mediated calcium mobilization and inhibition of forskolin-evoked cAMP accumulation in response to psychosine, yet specific binding of psychosine to TDAG8 was not reported (3, 4). They also showed that TDAG8 mRNA expression was rich in peripheral blood leukocytes, lymph nodes, and spleen in humans (3). Whereas psychosine is barely detectable in vivo, the inherited deficiency of the catabolic enzyme galactosylceramidase (Krabbe disease, also known as globoid cell leukodystrophy) results in accumulation of psychosine in brain (5).

TDAG8 shares high amino acid sequence homologies with three GPCRs, i.e. ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) (6), GPR4 (7, 8), and G2A (9). Although all these receptors were initially defined as orphan receptors, cognate ligands were identified several years later. OGR1 and G2A were reported to be receptors for sphingosylphosphorylcholine (10) and lysophosphatidylcholine (11), respectively. Meanwhile, GPR4 was shown to bind both sphingosylphosphorylcholine and lysophosphatidylcholine (12). However, OGR1, GPR4, and G2A were also identified as proton-sensing receptors (13, 14). The relatively high level of sequence homology between TDAG8 and these three receptors therefore suggested the possibility that TDAG8 is also activated in a pH-dependent manner. In this study, we show that TDAG8, heterologously expressed in mammalian cells, is a proton-sensing GPCR that stimulates cAMP formation, activates Rho, and induces stress fiber formation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents—HEPES and MES were purchased from Dojindo (Kumamoto, Japan). EPPS, psychosine, and 3-isobutyl-1-methylxanthine (IBMX) were from Sigma-Aldrich (St. Louis, MO). Forskolin was from Wako (Osaka, Japan). Psychosine, IBMX, and forskolin were dissolved in dimethylsulfoxide (Sigma-Aldrich) at 5, 50, and 10 mM, respectively. These solutions were stocked frozen at –30 °C. Amidinophenylmethanesulfonyl fluoride and proteinase inhibitor mixture were from Sigma-Aldrich and Roche Applied Science, respectively. Bovine serum albumin (BSA), fatty acid-free and very low endotoxin grade, was from Serologicals Proteins (Kankakee, IL). The Bioactive Lipid Library and 1-oleoyl lysophosphatidic acid (LPA) was from Biomol%20Research%20Laboratories">Biomol Research Laboratories (Plymouth Meeting, PA) and Avanti (Alabaster, AL), respectively. Other chemicals were of analytical grade.

Cell Culture—CHO-S cells were purchased from Invitrogen. This cell line was established from adult Chinese hamster ovary but has been distinguished as a separate subclone from the common CHO-K1 cell line. They were cultured on type I collagen-coated dishes in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, catalog no. D-5796) supplemented with 10% fetal bovine serum (Equitech-Bio, Inc., Kerrville, TX) and 0.1 mM minimum Eagle's medium non-essential amino acids (Invitrogen) in a 37 °C humidified incubator at 5% CO₂. For serum-starvation, the cells were washed three times and incubated for 18 h with DMEM at pH 7.9 containing 0.1% BSA, 100 IU/ml penicillin and 100 µg/ml streptomycin (Roche Applied Science).

Stable Expression of TDAG8—A DNA fragment containing the entire open reading frame of TDAG8 (NCBI accession number BC035633 [GenBank] ) was first amplified from human genomic DNA by PCR using Pfu turbo DNA polymerase (Stratagene, La Jolla, CA) and oligonucleotides (sense primer, 5'-TTCGATGTTCAAAACAAACTACAAA-3'; antisense primer, 5'-CTCACATGCTAGATTTCCTTTTCA-3'). The entire open reading frame of TDAG8 with an additional sequence encoding a hemagglutinin (HA) epitope (YPYDVPDYA) at the 5'-end was subsequently amplified from the resultant PCR products using KOD-Plus DNA polymerase (Toyobo, Osaka, Japan) and oligonucleotides (sense primer containing KpnI and HA-tag sequence, 5'-GGGGTACCGCCATGTACCCCTACGACGTGCCCGACTACGCCAACAGCACATGTATTGAA-3' and antisense primer containing XbaI sequence, 5'-GCTCTAGACTACTCAAGGACCTCTAA-3'). The resultant DNA fragment was digested with KpnI and XbaI and subsequently cloned into mammalian expression vector pCXN2.1 (15), a slightly modified version of pCXN2 (16) with multiple cloning sites, between the KpnI and NheI sites. The HA epitope-tagged human platelet-activating factor (PAF) receptor cDNA (17) was also cloned into a KpnI/NheI-digested pCXN2.1. CHO-S cells were transfected using Lipofectamine 2000 reagent (Invitrogen). After 48 h, the transient expression of the HA epitope on the cell surface was confirmed by flow cytometric analysis (EPICS XL, Beckman Coulter, Fullerton, CA) with 3F10 rat monoclonal anti-HA antibody (Roche Applied Science) and phycoerythrin-labeled anti-rat IgG (Beckman Coulter) as the second antibody. Then stable transfectants were selected with 2 mg/ml G418 (Wako) for 17 days. After staining the drug-resistant cells as described above, HA-positive cells were sorted by flow cytometry (EPICS ALTRA, Beckman Coulter). The expression levels of the HA epitope on the sorted cells, which were maintained with 0.3 mg/ml G418, were reanalyzed by flow cytometry.

Western Blotting—CHO-S cells were harvested and homogenized by sonication in 20 mM Tris-HCl (pH 7.5), 0.3 M sucrose, 5 mM 2-mercaptoethanol, 20 µM amidinophenylmethanesulfonyl fluoride, and proteinase inhibitor mixture. The cell debris was removed by centrifugation at 2,000 x g for 10 min. The supernatant was then centrifuged at 100,000 x g for 60 min, and the resulting pellet was homogenized in a buffer containing 180 mM sodium phosphate (pH 7.3) and 20 mM EDTA. Protein concentration of the homogenate was determined by Bradford assay (Bio-Rad). 10 µg of protein was digested with 2 units of N-glycosidase F (Roche Applied Science) to remove asparagine-bound N-glycans according to the manufacturer's instructions. The protein sample (with 5% 2-mercaptoethanol) was analyzed by 10% SDS-polyacrylamide gel electrophoresis without heat denaturation followed by transfer to a Hybond ECL nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% skim milk (Difco) and then probed with 3F10 rat anti-HA antibody. The bands were visualized with an ECL chemiluminescence detection system (Amersham Biosciences) using horseradish peroxidase-conjugated anti-rat IgG (Santa Cruz Biotechnology, Santa Cruz, CA).

Buffers—Physiological salt solution (PSS) contained 130 mM NaCl, 0.9 mM NaH₂PO₄, 5.4 mM KCl, 0.8 mM MgSO₄, 1.0 mM CaCl₂, 25 mM glucose, and 0.1% BSA (13). Unless otherwise stated, PSS was buffered with HEPES/EPPS/MES (8.3 mM each, HEM-PSS). A series of HEM-PSS were prepared by the addition of 40-fold concentrated buffers at pH 6.0–8.0 adjusted at room temperature. In experiments with serum-starved cells, bicarbonate-buffered DMEM (Sigma-Aldrich, catalog no. D-5648) containing 0.1% BSA was used for pH stimulation in a 37 °C humidified incubator at 5% CO₂. The actual pH values of buffers were measured with a pH meter (HM30V, Toa Electronics, Tokyo, Japan) under the experimental conditions.

cAMP Measurement—Subconfluent CHO-S cells were detached from a collagen-coated 10-cm dish with PBS containing 2 mM EDTA, washed, and then suspended in HEM-PSS (pH 7.2) containing 0.5 mM IBMX for 15 min at room temperature. After application of cells (1.5 x 10⁵/well) to 96-well V-bottom plates, the plates were centrifuged at 210 x g for 3 min. Supernatants were decanted, and stimulation was initiated by suspending the cells in 40 µl of HEM-PSS at various pH values. After 30 min of incubation at room temperature, the reaction was terminated by adding 4 µl of 10% Tween 20 followed by cooling on ice. Following overnight storage at 4 °C, cAMP concentrations in the reaction mixture were measured in quadruplicate by a Fusion system (PerkinElmer Life Sciences) using the AlphaScreen cAMP assay kit (PerkinElmer Life Sciences) following the manufacturer's instructions. Because cAMP concentrations were apparently underestimated at lower pH values, a correction for the pH effect was made. Because no saturation in the dose-response curve was reached, the data were not fitted by the logistic function to calculate half-maximal activation pH values of the receptors. Instead, they were calculated from the plots of cAMP concentration versus pH value by linear interpolation from the two adjacent data points.

Receptor Internalization Assay—Subconfluent CHO-S cells were detached from a collagen-coated 10-cm dish with PBS containing 2 mM EDTA, washed, and then suspended in HEM-PSS (pH 7.7). After centrifugation, aliquots of 7.5 x 10⁵ cells were stimulated with varying pH of HEM/PSS buffer in Eppendorf tubes. Following incubation at 37 °C for 30 min, the cells were fixed with PBS containing 1% paraformaldehyde for 10 min at room temperature and blocked with PBS containing 2% goat serum (Invitrogen). The expression level of HA-epitope on the cell surface was examined by flow cytometric analysis as described above.

Stress Fiber Observation—Cells were seeded onto a collagen-coated glass-bottomed 3.5-cm dish (MatTek Corp., Ashland, MA) and serum-starved for 18 h. Following stimulation with DMEM at pH 6.4 and 8.1 containing 0.1% BSA for 15 min at 37 °C, the cells were fixed with PBS containing 1.85% paraformaldehyde for 30 min at 37 °C. As a positive control, 1 µM 1-oleoyl LPA dissolved in DMEM at pH 8.1 containing 0.1% BSA was used. The cells were incubated with 1 unit/ml rhodamine-phalloidin (Molecular Probes, Eugene, OR) in PBS containing 0.1% Triton X-100 for 30 min at room temperature. Following washing three times with PBS, images were obtained using an LSM510 laser-scanning confocal microscope (Carl Zeiss, Tokyo, Japan) equipped with an argon laser as the light source.

Rho Activity Assay—Subconfluent CHO-S cells in a collagen-coated 10-cm dish were starved for 18 h and stimulated with DMEM at pH 6.4 and 8.1 containing 0.1% BSA at 37 °C for the indicated time points. As a positive control, 1 µM 1-oleoyl LPA dissolved in DMEM at pH 8.1 containing 0.1% BSA was used. Following washing with ice-cold Tris-buffered saline once, the cells were lysed with 0.5 ml of ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 500 mM NaCl, 1% Triton X-100, and proteinase inhibitor mixture). The cell debris was removed by centrifugation at 300 x g for 10 min. Half of the supernatant was incubated with 100 µg of GST-rhotekin immobilized on glutathione-Sepharose beads (Cytoskeleton, Inc., Denver, CO) at 4 °C for 60 min. After the beads were washed twice with ice-cold wash buffer (25 mM Tris-HCl (pH 7.5), 30 mM MgCl₂, and 40 mM NaCl), the bound proteins were dissolved in wash buffer and separated in 12% SDS-polyacrylamide gel. The proteins were transferred onto a Hybond ECL nitrocellulose membrane. After blocking with 5% skim milk, the membrane was probed with 26C4 mouse monoclonal anti-RhoA antibody (Santa Cruz Biotechnology). The GTP-bound form of RhoA was visualized with an ECL chemiluminescence detection system using horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences). Total RhoA in the original cell lysate was detected by Western blotting as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
pH-dependent Activation of cAMP Formation by TDAG8—To test whether TDAG8, which shares high homologies with G2A, OGR1, and GPR4, is also a proton sensor, CHO-S cells were stably transfected with the expression vector for TDAG8. The receptor was tagged with an HA epitope at the N terminus to detect the level of expression on the cell surface (Fig. 1A). With the use of the epitope and fluorescence-activated cell sorting, the drug-resistant cells were enriched giving a polyclonal population of TDAG8-expressing cells. This population is almost free of the variations of stable clones that may cause functional deviation. The molecular size of the transfected protein was measured by Western analysis using the membrane fraction of the cell homogenate. Consequently, we detected an 35-kDa protein in the sorted CHO-S cells expressing HA-tagged TDAG8 and HA-tagged PAF receptor but not cells transfected with empty vector (Fig. 1B). As was previously observed in leukotriene B4 receptor (18), the detected proteins ran faster than the predicted molecular masses (40 kDa for both TDAG8 and PAF receptor). This may be caused by conformational and charge effects on the receptors.

View larger version (31K): [in this window] [in a new window]   FIG. 1. Cell surface expression of TDAG8. A, flow cytometry analysis. CHO-S cells were stably transfected with the expression vector for TDAG8 tagged with HA epitope at the N terminus. After staining with anti-HA antibody and phycoerythrin-conjugated secondary antibody, HA-positive cells were sorted with a cell sorter and then subcultured. Data shown are the surface expression level of HA epitope in subcultured polyclonal cells analyzed by flow cytometry. Empty vector-transfected polyclonal cells served as a negative control. Numbers above the bars depict the percentages of positively stained cells out of 2.0 x 104 cells. Means of fluorescence intensity (arbitrary units) are 20.1 in TDAG8 and 0.5 in vector. B, Western blotting of HA-tagged receptors. Membrane fractions of CHO-S cells stably transfected with empty vector, TDAG8, or PAF receptor were examined for receptor expression by anti-HA antibody. 10 µg of total protein was applied in each lane.

View larger version (13K): [in this window] [in a new window]   FIG. 2. pH-dependent cAMP formation by TDAG8. A, pH dependence of cAMP formation. TDAG8-transfected (closed squares) and vector-transfected (open squares) CHO-S cells were incubated in HEM-PSS at the indicated pH values in the presence of 0.5 mM IBMX. After a 30-min incubation at room temperature, the cells were solubilized, and cAMP concentrations in the cell lysates were measured. Plots of cAMP concentration versus pH value are shown. Data are representative of seven independent experiments with similar results. B, failure of psychosine to affect cAMP formation. TDAG8-transfected (closed symbols) and vector-transfected (open symbols) CHO-S cells were incubated with various concentrations of psychosine in 25 mM HEPES-PSS at pH 7.2 in the presence of 0.5 mM IBMX either with (squares) or without (circles) 50 µM forskolin. After a 30-min incubation at room temperature, the cells were solubilized, and cAMP concentrations in the cell lysates were measured. Plots of cAMP concentration versus logarithm of psychosine concentration are shown. Data are representative of three independent experiments with similar results.

Receptor Internalization—Following activation by ligands, many GPCRs undergo internalization, which plays an important role in the temporal regulation of receptor function and can be a major determinant in the regulation of signaling specificity and coupling to downstream effector molecules. To characterize TDAG8 as a GPCR, we observed the internalization of TDAG8 by acidic HEM-PSS buffers using a fluorescence-activated cell sorter-based assay. Acid treatment for 30 min at pH 5.7 and 6.0 resulted in the loss of 58 and 45%, respectively, of TDAG8 on the cell surface at pH 7.7 (Fig. 3). Meanwhile, acid treatment had little or no effect on the fluorescence intensity of vector- or PAF receptor-transfected cells. Again, 10 µM psychosine did not elicit the internalization of TDAG8 (data not shown). Immunocytochemical analysis for subcellular localization of TDAG8 consistently showed that acid treatment with DMEM at pH 6.4 induced the internalization of TDAG8 within 30 min; whereas the fluorescence intensity of the plasma membrane was significantly reduced, that of the cytosol was relatively increased (data not shown).

View larger version (41K): [in this window] [in a new window]   FIG. 3. pH-dependent internalization of TDAG8. TDAG8-transfected, PAF receptor-transfected, and vector-transfected CHO-S cells were treated with HEM-PSS at pH 5.7 (shown in red), 6.0 (blue), and 7.7 (green) for 30 min at 37 °C. After fixation with paraformaldehyde, the cells were stained with anti-HA antibody and phycoerythrin-conjugated secondary antibody. Fluorescence intensity was measured by flow cytometry as in Fig. 1A. Data are representative of five independent experiments with similar results.

View larger version (54K): [in this window] [in a new window]   FIG. 4. Stress fiber formation and Rho activation by acid-stimulated TDAG8. A, organization of filamentous actin observed by confocal fluorescence microscopy. Serum-starved CHO-S cells were treated with DMEM-based stimuli, i.e. acid at pH 6.4 and 1 µM 1-oleoyl LPA, for 15 min at 37 °C and then stained with rhodamine-phalloidin. In DMEM at pH 8.1, stress fibers are distributed only at low levels in the cytoplasm of both TDAG8- and vector-transfected cells (top). Acid treatment with DMEM at pH 6.4 significantly induced stress fiber formation in TDAG8-transfected cells but not in vector-transfected cells (middle). LPA dissolved in DMEM at pH 8.1 also stimulated stress fiber formation (bottom). Scale bar, 10 µm. Data are representative of three independent experiments with similar results. B, RhoA activation demonstrated by GST pull-down assay. Serum-starved CHO-S cells were treated with DMEM-based stimuli, i.e. acid at pH 6.4 (Acid) and 1 µM 1-oleoyl LPA (LPA), for the indicated periods of time at 37 °C. The cell lysates were incubated with GST-rhotekin immobilized on glutathione-Sepharose beads. The amount of GTP-bound RhoA was determined by Western blotting. Total amounts of RhoA in the cell lysates are also shown. Data are representative of three independent experiments with similar results.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the current study, we demonstrated the proton-sensing properties of a GPCR, TDAG8. Activation of TDAG8 promoted cAMP formation. Furthermore, TDAG8 mediated actin-based cytoskeletal changes under acidic conditions concomitant with RhoA activation. Many reports have shown that G_12/13-mediated pathways regulate the activation of Rho (23). Thus, our results suggest that TDAG8 couples with multiple G-proteins, probably G_s and G_12/13. No Ca²⁺-mobilizing potency was observed for TDAG8.

The identification of TDAG8 (in addition to GPR4, OGR1, and G2A) as a proton-sensing GPCR will improve our understanding of the versatile roles of extracellular pH (13, 14). OGR1 and G2A stimulated inositol phosphate formation, whereas GPR4 (like TDAG8) elicited cAMP formation. It was reported that half-maximal cAMP formation by GPR4 was achieved with pH 7.55 in transiently transfected HEK293 cells (13). Meanwhile, we observed in polyclonal stable transfectants of TDAG8 that the half-maximal activation pH value of TDAG8 was 6.74 (Fig. 3). Although the difference between the two experimental conditions should be taken into account, TDAG8 seems to be activated at a lower pH than GPR4. Dot blotting of human tissue mRNAs showed expression of both TDAG8 and GPR4 transcripts in lymph nodes and spleen (3, 12). In lymphoid organs, each proton sensor may control the distinct responsiveness to the pH of the expressing cells according to its own dynamic range.

Certain subsets of lymphoid cells, such as thymocytes or peripheral B cells, undergo apoptosis after treatment with agents that elevate intracellular cAMP (24, 25). Because strong induction of TDAG8 mRNA is associated with activation-induced cell death of thymocytes (2), TDAG8 might play a role in negative selection of thymocytes in a pH-dependent manner. Indeed, TDAG8-overexpressing mice suggested that the receptor was critical for glucocorticoid-induced apoptosis in CD4⁺ CD8⁺ double-positive thymocytes (26). It is intriguing to note that the thymic microenvironment is composed of a network of epithelial and reticular cells, which form discrete cortical and medullary compartments (27). The compartmentalized thymocytes might be exposed to acidic fluids because of their own production of acid metabolites.

TDAG8 mRNA is distributed not only in thymus but also in lymph nodes, peripheral blood leukocytes (3), natural killer cells (28), T cell lymphoma lines (29), and bone marrow-derived cells (2). In addition, Sin et al. (30) recently reported that TDAG8 mRNA was overexpressed in a range of human cancer tissues. Therefore, it is plausible that TDAG8 has other unknown biological functions in peripheral tissues under acidotic conditions. Of note, the extracellular pH can lower to values less than 6.0 in inflammation foci and tumor interstitial fluids (31). The protons are released by lysed cells along with degranulation of different mediators, or they come from hypoxic metabolism. Activation of TDAG8 may account for the enhancing or suppressive effect of extracellular acidosis on immunocompetent cells, including neutrophils, macrophages, lymphocytes, natural killer cells, and dendritic cells (31, 32). Alternatively, TDAG8 may mediate cellular transformation (30). Indeed, recent studies have uncovered the oncogenic and metastatic potential of Rho proteins (33). Thus, further characterization of TDAG8 functions will pave the way for developing novel pharmaceutical approaches to treat cancer, inflammation, and immune diseases.

While this article was under review, Wang et al. (34) published their independent discovery of TDAG8 as a proton-sensing GPCR. They reported cAMP formation by acid treatment, which agrees with our data. However, they described that pH-dependent cAMP formation of TDAG8, when expressed in CHO cells, was inhibited by psychosine. Previously, Im et al. showed that psychosine inhibited forskolin-evoked cAMP accumulation in RH7777 cells through TDAG8 in a dose-dependent manner (3). Under the present assay conditions, we observed no effect of psychosine on TDAG8. The differences in the cAMP assay method, parental cell lines, and sublines, or intracellular localization of the expressed receptor may explain the discrepancy between our results and the previous reports (3, 34). In addition, high concentrations of psychosine have been reported to possess a detergent-like hemolytic activity in vitro (35). Concerning calcium mobilization, Wang et al. (34) and also we were unable to see the stimulatory effect of psychosine, whereas Im et al. (3) described a psychosine-induced Ca²⁺ response. Further studies are needed to explain these differences and to determine whether or not TDAG8 works as a proton sensor in vivo.

	FOOTNOTES

 
^* This work was supported in part by grants-in-aid from the Ministry of Education, Science, Culture, Sports and Technology of Japan (to T. S. and S. I.) and a grant-in-aid for Comprehensive Research on Aging and Health from the Ministry of Health, Labor and Welfare, Japan (to S. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-3-5802-2925; Fax: 81-3-3813-3732; E-mail: mame{at}m.u-tokyo.ac.jp.

¹ The abbreviations used are: TDAG8, T cell death-associated gene 8; GPCR, G-protein-coupled receptor; psychosine, 1--D-galactosylsphingosine; MES, 4-morpholineethanesulfonic acid; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; IBMX, isobutylmethylxanthine; BSA, bovine serum albumin; LPA, lysophosphatidic acid; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; PSS, physiological salt solution; HEM, HEPES/EPPS/MES; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PAF, platelet-activating factor.

	ACKNOWLEDGMENTS

 
We thank F. Hamano for excellent technical assistance and Drs. T. Yokomizo, N. Murakami, and K. Noguchi (University of Tokyo) for vital discussions and critical suggestions. We also thank Dr. J.-i. Miyazaki (Osaka University, Japan) for supplying pCXN2 and J. H. Jennings for critical reading of the manuscript.

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