Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M709487200 on March 17, 2008

J. Biol. Chem., Vol. 283, Issue 19, 12747-12755, May 9, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/19/12747    most recent
M709487200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hase, M.
Right arrow Articles by Nakamura, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hase, M.
Right arrow Articles by Nakamura, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins*

Momoko Hase{ddagger}, Takehiko Yokomizo{ddagger}§, Takao Shimizu{ddagger}, and Motonao Nakamura{ddagger}1

From the {ddagger}Department of Biochemistry and Molecular Biology, Faculty of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and the §Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan

Received for publication, November 19, 2007 , and in revised form, March 7, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E2-induced production of cAMP were significantly decreased. A remarkable increase in [35S]guanosine 5'-({gamma}-thio)triphosphate (GTP{gamma}S) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of Gi/o proteins. Involvement of GPR20 in the activation of Gi/o proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [35S]GTP{gamma}S incorporation and inhibition of the prostaglandin E2-induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [3H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates Gi proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian G protein-coupled receptors (GPCRs)2 are a diverse superfamily of proteins with hundreds of members, and these receptors regulate many processes in vivo via interactions with a variety of ligands, including small organic molecules, lipids, protons, hormones, short and large polypeptides, glycoproteins, and even photons. Despite the vast and longstanding efforts of academic and industrial researchers to pair GPCRs with potential ligands, more than 150 nonsensory GPCRs still remain orphan receptors, for which the cognate ligands have not yet been identified (1). Because GPCRs have proven particularly amenable to modulation by small molecules and are the targets of approximately half of currently marketed prescription drugs, the nonsensory GPCRs are clearly important therapeutic targets (2). Consequently, the orphan GPCRs constitute a vast reservoir of potential drug targets for therapeutic development. Indeed, numerous research groups, including ours, work on characterizing these receptors.

GPR20 is one of the orphan GPCRs that has been identified from human genomic DNA by PCR amplification using primers based on the sequences of the opioid/somatostatin-related receptors, GPR7 and GPR8 (3). The expression of human GPR20 has been detected in several brain regions, including the caudate nuclei, putamen, and the thalamus (3). A recently disclosed patent demonstrated that GPR20-deficient mice exhibited a hyperactivity disorder characterized by an increase in total distance traveled in an open field test (4), implying a substantial role of GPR20 in neurophysiological function. However, the physiological mechanisms of GPR20 action, including the identification of natural ligands for GPR20, have not yet been elucidated. In this study, we show that GPR20 has the potential to constitutively activate Gi-type G proteins without extracellular ligands. Furthermore, the physiological relevance of this constitutive activation of GPR20 was confirmed by RNA interference experiments and mitogenic response assays.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Prostaglandin E2 (PGE2) was purchased from Cayman Chemical (Ann Arbor, MI). 3-Isobutyl-1-methylxanthine, GDP, GTP{gamma}S, bovine serum albumin (BSA, fatty acidfree grade), and the M5 mouse monoclonal anti-FLAG antibody were purchased from Sigma. Pertussis toxin (PTX) was obtained from List Biological Laboratories (Campbell, CA). Ex-TaqDNA polymerase and restriction enzymes were purchased from Takara Bio (Tokyo, Japan). [35S]GTP{gamma}S and the AlphaScreen cAMP assay kit were obtained from PerkinElmer Life Sciences. Phycoerythrin-labeled anti-mouse IgG was obtained from Beckman Coulter (Miami, FL). Alexa Fluor 488-labeled anti-mouse IgG, Lipofectamine 2000, Opti-MEM I, and SuperScriptII reverse transcriptase were purchased from Invitrogen. G418 was purchased from Wako (Osaka, Japan). KOD Dash DNA polymerase and KOD Plus DNA polymerase were purchased from TOYOBO (Tokyo, Japan). Complete protease inhibitor mixture was purchased from Roche Diagnostics. The cAMP Biotrak EIA system was purchased from GE Healthcare.

Construction of a Phylogenetic Tree—Amino acid sequences of selected human GPCRs were obtained from GenBankTM and SwissProt. The phylogenetic tree was generated from the amino acid sequences of these GPCRs using the all-against-all matching method (available on line). The tree was constructed on the basis of point-accepted mutation distances between each pair of sequences estimated by the dynamic programming algorithm.

Construction of the Human GPR20 and BLT1-expressing Plasmids—A DNA fragment containing the human GPR20 (hGPR20) gene (GenBankTM accession number NM_005293 [GenBank] ) was obtained from human genomic DNA by PCR amplification using the sense primer, 5'-ATGGAGAAGGGGGATGCTGGGC-3', and the antisense primer, 5'-TTCAGGCCACCACATCCCATCG-3'. For construction of the hGPR20 expression plasmid, the entire open reading frame of hGPR20 was amplified from the above DNA fragment by PCR using the sense primer, 5'-CAGGATATCCCCTCTGTGTCTCCAGCGGG-3', and the antisense primer, 5'-CAGGAATTCCTAAGCCTCGGGCCCATTAG-3', and subcloned into the EcoRV and EcoRI sites of the pCXN2.1-FLAG vector, a modified version of pCXN2 (5). The resultant plasmid, designated pCXN2.1-FLAG-GPR20, was used to express an N-terminally FLAG-tagged hGPR20. For construction of the expression plasmid for human leukotriene B4 type 1 receptor (hBLT1, GenBankTM accession number NM_181657 [GenBank] ), an entire open reading frame of hBLT1 was subcloned into the pCXN2.1-FLAG vector.

Construction of Short Hairpin RNA-expressing Plasmids–Oligonucleotides for the construction of three short hairpin RNA (shRNA) plasmids directed against rat GPR20 (rGPR20) mRNA were designed using the siDirect small interfering RNA design system provided by the RNAi Co. Ltd. (Tokyo, Japan). The designed sequences are as follows: sh1 sense, 5'-GATCCCCCATCGTCTACTGTTTTATTCAAGAGATAAAACAGTAGACGATGGGGTA-3', and sh1-antisense, 5'-AGCTTACCCCATCGTCTACTGTTTTATCTCTTGAATAAAACAGTAGACGATGGGG-3'; sh2 sense, 5'-GATCCGACCCTGTCTGTATTGGGTTTCAAGAGAACCCAATACAGACAGGGTCACA-3', and sh2 antisense, 5'-AGCTTGTGACCCTGTCTGTATTGGGTTCTCTTGAAACCCAATACAGACAGGGTCG-3'; and sh3 sense, 5'-GATCCCTCAGCACCGGTTCTCACATTATTCAAGAGATGTGAGAACCGGTGCTGAGGAA-3', and sh3 antisense, 5'-AGCTTTCCTCAGCACCGGTTCTCACATCTCTTGAATAATGTGAGAACCGGTGCTGAGG-3'. After annealing the sense and antisense oligonucleotides, the resultant DNA fragments were subcloned into the BamHI and HindIII sites of the pSilencer 4.1-CMV hygro vector (purchased from Ambion (Austin, TX), abbreviated pSilencer), giving rise to three rGPR20-shRNA plasmids, pSilencer-sh1, pSilencer-sh2, and pSilencer-sh3. We used a negative control-shRNA plasmid supplied by Ambion as a control scrambled shRNA.

Construction of a Mutant hGPR20 (R148A) Plasmid—Mutagenesis of GPR20 was performed via site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions, with the exception that the KOD Plus DNA polymerase (TOYOBO) was used for PCR amplification. pCXN2.1-FLAG-GPR20 was used as a template plasmid, and the primers for mutagenesis were as follows: R148A sense, 5'-CTGCATCTGCGTGGACgccTACCTGGCCATCGTG-3', and R148A antisense, 5'-CACGATGGCCAGGTAggcGTCCACGCAGATGCAG-3'. The resultant construct was verified through complete DNA sequencing of both DNA strands.

Generation of PTX-resistant Gi-GFP Fusion Proteins—Expression plasmids for GFP-fused PTX-resistant Gi1, Gi2, and Gi3 proteins (6–10) were constructed as follows. The Cys351 residues of the Gi1 and Gi2 proteins and the Cys352 residue in the Gi3 protein were substituted with a Gly residue by PCR amplification. Next, an EcoRI site was created between Ile93 and Asp94 within the helical domain of each Gi protein by site-directed mutagenesis. A GFP sequence, bearing two Gly-rich linker sequences, Gly-Asn-Ser-Gly-Gly at the N-terminal end and Gly-Gly-Gly-Asn-Ser at the C-terminal end (9), was inserted into the EcoRI site created in each Gi protein.

Cell Culture and Transfections—Human embryonic kidney (HEK) 293 cells were maintained under 5% CO2 in air at 37 °C in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (Invitrogen). PC12h cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% fetal bovine serum. Cells were transfected with expression plasmids using Lipofectamine 2000 in serum-free Opti-MEM I according to the manufacturer's description.

Establishment of Cells Stably Expressing hGPR20—HEK293 cells were transfected with pCXN2.1-FLAG-GPR20 and cultured for 30 days in the presence of 1.0 mg/ml G418. These cells were incubated with 10 µg/ml anti-FLAG (M5) in phosphate-buffered saline (PBS) containing 2% goat serum for 30 min, followed by staining with Alexa Fluor 488-labeled anti-mouse IgG in PBS/2% goat serum for 15 min. Transformants highly expressing FLAG-hGPR20, designated HEK293-GPR20 cells, were collected as a polyclonal population by cell sorting using an EPICS ALTRA instrument (Beckman Coulter) and maintained in the presence of 0.3 mg/ml G418. Flow cytometric analyses of the FLAG-hGPR20-expressing cells were performed using an EPICS XL instrument (Beckman Coulter). cAMP Measurement—HEK293 and PC12h cells (5–8 x 104 cells) were cultured on poly-D-lysine-coated 96-well plates (for HEK293, BD Biosciences) or collagen-coated 96-well plates (for PC12h, Iwaki) for 24 h. In some experiments, cells were treated with 100 ng/ml PTX for 6 h. Cells were washed twice with buffer A (Hanks' balanced salt solution containing 25 mM HEPES-NaOH, pH 7.4, and 0.1% BSA) and preincubated in 100 µl of buffer A containing 0.5 mM 3-isobutyl-1-methylxanthine (from a 500 mM stock in dimethyl sulfoxide stored at –30 °C) for 15 min at 37 °C. Subsequently, various concentrations of PGE2 in buffer A (100 µl) were added, and cells were incubated for 30 min at 37 °C. The reactions were terminated by the addition of 10% Tween 20 (20 µl), followed by overnight storage at 4 °C. After centrifugation at 800 x g for 5 min, the cAMP concentrations in the supernatants (10 µl) were measured using an AlphaScreen cAMP assay kit (PerkinElmer Life Sciences) according to the manufacturer's instructions. For determination of the basal cAMP concentration in nonstimulated cells, a cAMP Biotrak EIA system was used. Cells were processed as described above, with the exception of the addition of 25 µl of lysis buffer supplied in the EIA kit to terminate the reactions.

Membrane Preparation—Two days after transfection, cells were harvested with PBS containing 2 mM EDTA. In some experiments, cells were pretreated with 100 ng/ml PTX for 6 h. The cells were disrupted by sonication in ice-cold sonication buffer (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mM MgCl2, 2mM EDTA) containing a complete protease inhibitor mixture and centrifuged at 10,000 x g for 10 min at 4 °C. The supernatants were collected and centrifuged at 100,000 x g for 1 h at 4 °C. The resulting membrane pellets were resuspended in sonication buffer and subjected to [35S]GTP{gamma}S binding experiments. The protein concentrations in each sample were determined by the Bradford method (11) using the protein assay kit (Bio-Rad).

[35S]GTP{gamma}S Binding Assay—Membrane preparations (10 µg of protein) were incubated in 100 µl of [35S]GTP{gamma}S binding buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 5 mM GDP, and 0.1% BSA) containing 0.5 nM [35S]GTP{gamma}S for 30 min at 30 °C. To determine nonspecific binding, unlabeled GTP{gamma}S was added to the binding mixture to a final concentration of 10 µM. Bound [35S]GTP{gamma}S was separated from free [35S]GTP{gamma}S by rapid filtration through GF/C filters and washed with 2 ml of ice-cold TMN buffer (10 mM Tris-HCl, pH 7.5, 25 mM MgCl2, and 100 mM NaCl). Radioactivity was measured using a Top Count scintillation counter (Packard Instrument Co.).

Quantitative Real Time RT-PCR Analysis—First-strand cDNAs from 16 human tissues were purchased from Clontech (human multiple tissue cDNA panel I and II). To examine the expression of hGPR20 in human tissues, the amount of the GPR20 cDNA in each sample was evaluated using a TaqMan gene expression assay (Applied Biosystems, Foster City, CA; assay code Hs00271049_s1) and the modified TaqMan Universal PCR Master Mix containing 0.01% BSA. The amount of cDNA was estimated using a LightCycler apparatus (Roche Diagnostics) and the Fit Points method in the LightCycler analysis software. For each evaluation, a specific standard curve was made using a series of dilutions from the corresponding cDNA. Mouse total RNAs were prepared from various tissues using an Absolutely RNA miniprep kit (Stratagene, La Jolla CA), and first strand cDNAs were synthesized using Superscript II. PCR amplifications were carried out in microcapillary tubes, in 20-µl reaction volumes consisting of 2 µl of cDNA solution, 1x FastStart DNA Master SYBR Green I (Roche Diagnostics), and 0.5 µM each of the forward and the reverse primers. A 194-bp fragment of the mouse GPR20 (mGPR20) cDNA was generated using following primers: forward primer, 5'-AAACCCAACCAGAAGAGTTCACC-3', and reverse primer, 5'-AAATGCCAACCAGATACCCAAGT-3'.

Evaluation of GPR20 Expression in Various Cell Lines—Total RNA was isolated from cell lines using Isogen (Nippon Gene, Toyama, Japan), followed by purification of mRNAs using a poly (A)+ isolation kit (Nippon Gene). cDNA was generated from 100 ng of the purified mRNA using Superscript II and random hexamer primers. A 187-bp fragment of hGPR20, a 194-bp fragment of mGPR20, and a 197-bp fragment of rGPR20 were generated by PCR. The primers used in these analyses were as follows: hGPR20 sense primer, 5'-GCTGGCGCTGTACGTCTTCT-3', and antisense primer, 5'-CATGTTGAGGAAGTAACCGAGGA-3'; and rGPR20, sense primer, 5'-GTCCTGGGCTACTTCCTCAACAT-3', and antisense primer, 5'-CCACCAGTCTTCACACCCAATAC-3'. The primers for mGPR20 were the same as described above. The primer sequences for β-actin were as follows: human, sense primer, 5'-CAGGATGCAGAAGGAGATCACTG-3', and antisense primer, 5'-TACTCCTGCTTGCTGATCCACAT-3'; rat, sense primer, 5'-AGACCTCTATGCCAACACAGTGC-3', and antisense primer, 5'-ATAGAGCCACCAATCCACACAGA-3'; and mouse, sense primer, 5'-CACAGGCATTGTGATGGAC-3', and antisense primer, 5'-CTTCTGCATCCTGTCAGC-3'.

Validation of RNA Interference—To validate the efficiency of the rGPR20-knockdown by pSilencer-sh1, pSilencer-sh2, or pSilencer-sh3, quantitative real time RT-PCR (qRT-PCR) was performed. Seven days after transfection of these plasmids into PC12h cells, mRNA isolation and cDNA synthesis were performed as described above. Using the resultant cDNAs, the amounts of expressed rGPR20 and β-actin mRNAs were estimated using LightCycler Fast Start DNA MasterPLUS SYBR Green I (Roche Diagnostics) according to the manufacturer's protocol. In these experiments, the primers for β-actin were the same as described above. For each evaluation, a specific standard curve was made using a series of dilutions of the corresponding cDNA.

[3H]Thymidine Incorporation—PC12h cells (1 x 104 cells) were cultured on collagen-coated 96-well plates for 48 h. Each well was pulsed with 1 µCi of [3H]methyl thymidine in the last 6 h of a 48-h culture. At the end of incubation, the cells were lysed using water, and incorporated [3H]thymidine was separated from free [3H]thymidine by rapid filtration through GF/C filters that were washed with 2 ml of water. The radioactivity was measured with a Top Count scintillation counter (Packard Instrument Co.). Incorporation of [3H]thymidine into PC12h DNA was expressed as dpm per well.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of the hGPR20 Gene—We cloned human GPR20 from human genomic DNA by PCR amplification. However, several sequences in our cloned hGPR20 were different from the reported GPR20 sequence (3). In particular, Cys53–Val54 and Ala157–Pro158–Ala159–Ala160 in the reported GPR20 were Trp53–Leu54 and Gly157–Ser158–Arg159–Arg160, respectively, in our clone. We used our hGPR20 clone in this study, because we verified the sequences of several independently isolated hGPR20 clones, and the same sequence was recently deposited in the human total genome sequence data base at NCBI (accession number NT_008046 [GenBank] ). The primary structure of hGPR20 exhibited 82.8 and 81.4% amino acid identity to mouse and rat GPR20, respectively (Fig. 1A). Phylogenetic analysis revealed that hGPR20 was closely related to functional receptors for nucleotides or lipids (e.g. TG1019, CysLT1, CysLT2, LPA4, LPA5, GPR34, and PAF receptors, Fig. 1B), raising the possibility that GPR20 may recognize nucleotides or lipids as ligands.

Tissue and Cellular Expression of GPR20 mRNA—Using cDNAs prepared from 16 human tissues, qRT-PCR was performed to evaluate the expression of hGPR20. In these tissues, hGPR20 was ubiquitously expressed, with the highest expression in small intestine (Fig. 2A). Similarly, mouse GPR20 mRNA was detected in a broad range of tissues with abundant expression in intestinal tissues, such as small intestine and colon (Fig. 2B). GPR20 mRNA was detected in various cell lines, including N1E115 (mouse neuroblastoma cells), B103 (rat neuroblastoma cells), PC12 and PC12h (rat pheochromocytoma cells), U937 (human histiocytic lymphoma cells), HL60 (human acute promyelocytic leukemia cells), and COLO320 (human colon adenocarcinoma cells), expressed detectable levels of the GPR20 mRNA (Fig. 2C). One of these cell lines, PC12h, was used for the GPR20-knockdown experiments shown below.

Reduction of cAMP Formation by the Expression of hGPR20—Because GPR20 belongs to a family of lipid/nucleotide-recognizing receptors, we carried out a ligand screening using the BioMol Screen-WellTM Bioactive Lipid Library (purchased from BioMol) and 17 nucleotides (Sigma). To facilitate this screening, we constructed an expression plasmid producing an N-terminally FLAG-tagged hGPR20 (FLAG-hGPR20) and established a HEK293 cell line stably expressing FLAG-hGPR20 (HEK293-GPR20 cells). Prior to screening, expression of FLAG-hGPR20 on the cell surface of HEK293-GPR20 cells was validated by flow cytometric analysis (Fig. 3A). Although we evaluated the agonistic activity of 198 lipids and 17 nucleotides using HEK293-GPR20 cells by monitoring intracellular calcium mobilization and cAMP formation, none of them was an effective agonist for hGPR20 (data not shown). Furthermore, FLAG-hGPR20 did not respond to changes in the extracellular pH (data not shown), even though GPR20 is closely related to the four proton-sensing GPCRs, i.e. G2A, GPR4, OGR1, and TDAG8 (12–15), in the phylogenetic tree. Additionally, we did not observe any activation of GPR20 by several steroid hormones, including hydrocortisone, corticosterone, β-estradiol, and progesterone (data not shown). During the course of these experiments, we unexpectedly detected a modest but significant decrease in basal cAMP level in HEK293-GPR20 cells, compared with that of vector-transfected cells (Fig. 3B). Moreover, we observed that the FLAG-hGPR20 expression resulted in attenuated PGE2-stimulated cAMP formation in comparison with vector-transfected cells (Fig. 3C). Similar effects were observed in HEK293 cells transiently expressing FLAG-hGPR20 (Fig. 3E), implying that these observations were not an artifact of the cloned cell lines. Because the above effects by FLAG-hGPR20 expression were abolished by treatment with PTX (Fig. 3, B, D and E), exogenous expression of hGPR20 may cause the activation of PTX-sensitive G proteins in the absence of ligand stimulation.


Figure 1
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 1.
Structural features of GPR20. A, amino acid sequence alignment of GPR20 from human, mouse, and rat. Amino acid sequences of mouse GPR20 (accession number NM_173365 [GenBank] ) and rat GPR20 (accession number NM_022216 [GenBank] ) were obtained from GenBankTM and SwissProt, respectively. Asterisks indicate amino acids conserved among all three species. Dashes represent spaces added for proper alignment. The putative transmembrane domains (TMs) of these GPR20s predicted from a Kyte-Doolittle hydrophobicity analysis are boxed and labeled as TM1–TM7. The DRY motif in the second intracellular loop is indicated by reverse type. B, phylogenetic tree of various human GPCRs including GPR20. GPR20 is shown in a black circle.

 


Figure 2
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 2.
Expression of GPR20 in various tissues and cell lines. A, expression of hGPR20 in 16 human tissues was analyzed by qRT-PCR. One arbitrary unit was defined as the relative amount of the hGPR20 mRNA expressed in U937 cells. B, expression of mGPR20 in 13 mouse tissues was analyzed by qRT-PCR. One arbitrary unit was defined as the relative amount of the mGPR20 mRNA in mouse brain. C, expression of GPR20 and β-actin in cultured cell lines was detected by RT-PCR. cDNA reverse-transcribed from purified mRNA was used as the templates for these experiments. No amplified DNA bands were obtained when nonreverse-transcribed samples were used as templates (data not shown).

 


Figure 3
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 3.
Impairment of cAMP-formation by expression of FLAG-hGPR20 in HEK293 cells. A, cell surface expression of FLAG-hGPR20 in HEK293 cells was confirmed by flow cytometric analysis. After staining with the M5 anti-FLAG and Alexa Fluor 488-labeled anti-mouse IgG as primary and secondary antibodies, respectively, the fluorescence intensity of HEK293-GPR20 cells was measured (black area). Empty vector-transfected HEK293 cells served as a negative control (gray area). B, basal cAMP content with or without PTX (100 ng/ml) pretreatment was measured in HEK293-GPR20 cells (5 x 104 cells/well in a 96-well plate, white bar) and in empty vector-transfected HEK293 cells (black bar). The cAMP Biotrak EIA system was used for the determination of these cAMP concentrations. The data are means ± S.E. of a representative experiment from two independent experiments performed in quadruplicate. n.s., not significant; **, p < 0.01 (ANOVA with Tukey's multiple comparison test). C and D, PGE2-elicited cAMP accumulation with (C) or without (D) PTX (100 ng/ml) pretreatment was measured in HEK293-GPR20 cells (5 x 104 cells/well in a 96-well plate; open triangles) and in empty vector-transfected (closed circles) HEK293 cells. The concentration of cAMP in these cells was determined using an AlphaScreen cAMP assay kit. The data are means ± S.E. of a representative experiment from two independent experiments performed in triplicate. E, PGE2 (1 µM)-stimulated cAMP accumulation in HEK293 cells transiently transfected with pCXN2.1-FLAG-GPR20 or the empty vector was measured. These examinations were performed with or without PTX (100 ng/ml) pretreatment. The data are means ± S.E. of a representative experiment from three independent experiments performed in quadruplicate. *, p < 0.05; ***, p < 0.001 (ANOVA with Bonferroni's test).

 


Figure 4
View larger version (42K):
[in this window]
[in a new window]

  		FIGURE 4.
Augmentation of [35S]GTP{gamma}S incorporation by expression of FLAG-hGPR20 in HEK293 cells. A, left, after transfection of the indicated amounts of pCXN2.1-FLAG-GPR20 (open triangles), pCXN2.1-FLAG-BLT1 (open squares), or the empty vector (closed circles) into HEK293 cells (3 x 106 cells/dish in a 100-mm collagen-coated cell culture dish), [35S]GTP{gamma}S incorporation into isolated membrane preparations from these cells was determined. The data are means ± S.E. of a representative experiment from three independent experiments performed in triplicate. Right, cell surface expression of FLAG-hGPR20 (black line) and FLAG-hBLT1 (dotted line) in HEK293 cells was confirmed by flow cytometric analysis. After staining with the M5 anti-FLAG and phycoerythrin-labeled anti-mouse IgG as primary and secondary antibodies, respectively, the fluorescence intensity of the cells was measured. Empty vector-transfected HEK293 cells served as a negative control (black area). B, effects of PTX (100 ng/ml) on FLAG-hGPR20-dependent [35S]GTP{gamma}S incorporation into the membranes. The data are means ± S.E. of a representative experiment from three independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01 (ANOVA with Tukey's multiple comparison test). C, [35S]GTP{gamma}S incorporation into membrane proteins from cells transfected with empty vector or GPR20 and a PTX-resistant Gi expression plasmid. The membrane proteins used in this experiment were prepared from cells after PTX (100 ng/ml) pretreatment for 6 h. The data are the means ± S.E. of a representative experiment from three independent experiments performed in triplicate.

 
Enhanced [35S]GTP{gamma}S Binding to Membranes Prepared from hGPR20-expressing Cells—Next we examined whether Gi-type G proteins are indeed activated in the presence of hGPR20. Using membrane preparations from HEK293 cells transiently transfected with various amounts of the FLAG-hGPR20 expression plasmid, we evaluated GDP-GTP exchange of G{alpha}-subunits by [35S]GTP{gamma}S binding assay. Because exogenously expressed GPCRs occasionally activate G protein in the absence of ligands, we compared the effect of hGPR20 with hBLT1, which is well known as a Gi-coupled receptor (16, 17). As shown in Fig. 4A, transfection of hGPR20 enhanced [35S]GTP{gamma}S incorporation into the membranes in a dose-dependent manner, even though hBLT1 did not affect the [35S]GTP{gamma}S incorporation at these doses. The augmentation of [35S]GTP{gamma}S binding was diminished by PTX treatment (Fig. 4B), which is consistent with the PTX sensitivity of PGE2-induced cAMP formation in GPR20-transfected cells (Fig. 3, B–E). To provide further evidence for the activation of Gi proteins by hGPR20, we examined whether [35S]GTP{gamma}S incorporation into exogenously expressed PTX-resistant mutants of the Gi protein (the Gi1, Gi2, and Gi3) (6–10) increased in FLAG-hGPR20 expressing cells. As shown in Fig. 4C, even in cells pretreated with PTX, increasing [35S]GTP{gamma}S binding to the PTX-resistant mutants of Gi proteins was observed in the FLAG-hGPR20-expressing cells. These results suggest that hGPR20 continuously activates Gi-type G proteins.

Involvement of the DRY Motif of GPR20 in Activation of Gi Proteins—Several reports have described the significance of a highly conserved stretch of amino acid residues located in the second intracellular loop of various GPCRs, (Glu/Asp)-Arg-Tyr, in G protein activation (18, 19). This domain is generally designated the DRY motif. To obtain more evidence for the constitutive activation of Gi proteins by GPR20, we examined whether the disruption of the DRY motif in hGPR20 (amino acids 147–149) affects Gi activation. We constructed a mutant FLAG-hGPR20, the R148A receptor, in which Asp148 is substituted by Ala. The R148A receptor was expressed on the cell surface at levels similar to the wild-type FLAG-hGPR20, as judged by flow cytometric analysis (Fig. 5A). As shown in Fig. 5B, [35S]GTP{gamma}S incorporation into membrane preparations from R148A receptor-expressing cells was remarkably reduced compared with membranes from cells expressing the wild-type receptor. Additionally, the R148A receptor was not able to inhibit PGE2-induced cAMP formation (Fig. 5C). All these results indicate that GPR20 is constitutively activated on the cell surface, leading to sustained activation of Gi proteins, and that the DRY motif in hGPR20 plays a key role in activation of Gi.


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 5.
Involvement of the DRY motif of hGPR20 in Gi activation. A, cell surface expression of FLAG-tagged hGPR20 (gray line) and R148A (black line) receptors in HEK293 cells were confirmed by flow cytometric analysis, as described under "Experimental Procedures." Empty vector-transfected cells served as a negative control (black area). B, incorporation of [35S]GTP{gamma}S into membrane preparations from hGPR20, R148A mutant, or empty vector expressing cells was measured. The data are means ± S.E. of a representative experiment from three independent experiments performed in triplicate. *, p < 0.05 (ANOVA with Tukey's multiple comparison test). C, PGE2 (1 µM) elicited cAMP accumulation in these cells was determined. The data are means ± S.E. of a representative experiment from three independent experiments performed in triplicate. **, p < 0.01 (ANOVA with Bonferroni's test).

 


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 6.
Silencing of endogenous GPR20 by rGPR20-shRNA plasmids in PC12h cells. A, three rGPR20-shRNA (pSilencer-sh1, -sh2, and -sh3) and control shRNA plasmids were transfected into PC12h cells. After culture in the presence of 300 µg/ml hygromycin for 7–9 days, the amounts of rGPR20 mRNA were measured as described under "Experimental Procedures." B, effects of rGPR20 silencing on basal cAMP levels. The data are means ± S.E. of a representative experiment from two independent experiments performed in quadruplicate. *, p < 0.05 (ANOVA with Tukey's multiple comparison test). C, effects of rGPR20 silencing on cell proliferation. After culture in the presence of 300 µg/ml hygromycin for 7 days, 1 x 104 cells transfected with control (cont) shRNA plasmid or three rGPR20-shRNA plasmids (sh1, sh2, and sh3) were grown in a 96-well plate for 48 h by adding 1 µCi of [3H]thymidine in the last 6 h of a 48-h culture. Incorporation of [3H]thymidine into PC12h DNA was expressed as disintegrations/min (dpm) per well. The data are means ± S.E. of a representative experiment from two independent experiments performed in quadruplicate. *, p < 0.05 (ANOVA with Tukey's multiple comparison test).

 
Increase in Basal cAMP Levels by Silencing of Endogenously Expressed GPR20—Although we showed that overexpression of GPR20 resulted in constitutive activation of Gi proteins, those data were obtained by using overexpression systems. Therefore, we next examined whether or not a physiological expression level of GPR20 is enough to evoke the constitutive activation of Gi proteins. For this purpose, we performed an RNAi experiment using PC12h cells that endogenously express GPR20 (Fig. 2C). We constructed three shRNA plasmids, pSilencer-sh1, pSilencer-sh2, and pSilencer-sh3, targeting the mRNA of rGPR20. Transfection of each shRNA plasmid significantly reduced the endogenous rGPR20 mRNA in PC12h cells (Fig. 6A). Introducing the most effective shRNA plasmid, pSilencer-sh3, into PC12h cells reduced the amount of rGPR20 mRNA by ~75% compared with control shRNA plasmid. In PC12h cells possessing these rGPR20-shRNA plasmids, we observed moderate but reproducible increases in the basal cAMP level compared with levels in the presence of the control shRNA plasmid (Fig. 6B). The results suggest that endogenous GPR20 also possesses constitutively or spontaneously active conformation. Furthermore, we found that RNAi-mediated knockdown of GPR20 in PC12h cells resulted in an increase in [3H]thymidine incorporation (Fig. 6C). These findings suggest that intrinsic GPR20 is involved in the regulation of cell growth. Thus, the endogenous GPR20 attenuates the mitogenic signaling of PC12h cells possibly by control cellular cAMP level through Gi activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We report here the expression pattern and characterization of an orphan GPCR, GPR20. We demonstrated that exogenously expressed hGPR20 elicits continuous activation of Gi proteins in HEK293 cells in the absence of ligand. Cell responses, such as intracellular calcium mobilization and cAMP accumulation, were not elicited by stimulation with various kinds of lipids, nucleotides, or changes in extracellular pH. Although there is still the possibility that a specific agonist for hGPR20 exists in the culture medium, we observed no effect of increasing amounts of fatty acid-free BSA, which is known to trap and decrease free lipids in the medium effectively, on the continuous activation of Gi proteins by hGPR20 (data not shown). Furthermore, mutation of the Arg148 residue in the hGPR20 DRY motif drastically abolished Gi activation (Fig. 5). Because the DRY motif is considered to maintain the receptor conformation (20, 21), and especially the central Arg residue of this motif has been reported to be directly responsible for receptor-G protein coupling (22), these results strongly support the hypothesis that GPR20 has the ability for continuous activation of Gi proteins.

There are several lines of evidence that certain GPCRs are activated in the absence of specific agonists and lead to a sustained transduction of various signals into cells (23). Here we present evidence that the exogenously expressed GPR20 activates Gi proteins without any ligand stimulation in HEK293 cells. The ligand-independent activation of GPR20 was observed not only in HEK293 cells but also in the rat hepatoma cell line RH7777 (data not shown). Although the constitutive activation of the exogenously expressed GPCR was sometimes observed because of an excess of the receptor in cultured cells (24), as shown in Fig. 4A, we demonstrated that at least GPR20 had a higher basal activity than hBLT1. Furthermore, GPR20 possibly forms a spontaneously active conformation in physiological conditions judging from the results of RNA knockdown using PC12h cells that intrinsically express GPR20 (Fig. 6). In these cells, GPR20 may have a pivotal function in maintaining the intracellular cAMP levels via regulation of transcription and/or translation. Moreover, we observed the mitogenic effect of the GPR20-silencing in PC12h cells. Several reports demonstrated that cAMP could stimulate cell proliferation by activating extracellular signal-regulated kinases (ERKs) in diverse cell types (25–27). According to previous reports, in B-Raf-expressing cells, GTP-loaded Rap1 can activate B-Raf and the mitogen-activated protein kinase cascade, and hormonal stimulation of cAMP/PKA/Rap1 in these cells activates ERKs (28). The expression of B-Raf was confirmed in PC12h cells (data not shown), and our findings that GPR20-silencing resulted in increases of cAMP production and cell proliferation are consistent with the above insight. This cAMP-mediated cell proliferation is induced by hormonal activation of GPCRs that are coupled to Gs protein. It is still unclear whether ERKs are activated in the GPR20-silencing cells, and our results provide a possibility that GPR20 expression in PC12h cells suppresses the cAMP-induced mitogenic signaling mediated by the other Gs-coupled GPCRs.

The expression of GPR20 was ubiquitous in human and mouse tissues, with the highest levels of GPR20 mRNA detected in intestinal tissues. Previously, Allander et al. (29) reported that GPR20 is abundantly expressed in gastrointestinal stromal tumors, which are well known as the most common mesenchymal tumors of the digestive tracts. The expression of GPR20 was tightly correlated with expression of the proto-oncogene KIT, whose activation is a characteristic marker for gastrointestinal stromal tumors in gene expression profiling. Thus, GPR20 may be involved in the proliferation of intestinal cells. qRT-PCR analysis also demonstrated that GPR20 is expressed in the brain and in various types of neuroblastoma cells (Fig. 2). In a recently disclosed patent (4), a crucial role for GPR20 in the central nervous system has been proposed, because GPR20-deficient mice exhibit a hyperactivity disorder characterized by an increase in total distance traveled in an open field test. Because increased cAMP production might occur in some brain regions in GPR20-deficient mice, the abnormal cAMP levels in these areas may be related to the hyperactivity disorder (4). Intriguingly, it has been demonstrated that several orphan GPCRs, including GPR3 (30, 31), GPR6, GPR12 (32), GPR26, GPR78 (33), and GPR101 (34), have the ability to augment cAMP formation by constitutive activation of Gs in a ligand-independent fashion. Specifically, GPR26, GPR78, and GPR101 are expressed in some regions of the brain, including the caudate nuclei, putamen, and the thalamus (33, 34), suggesting that they may counteract GPR20 in these areas. Because the intracellular cAMP level is essential for cellular homeostasis, including cell proliferation and differentiation, the amount of intracellular cAMP may be regulated by the balance in expression of these Gi- and Gs-coupled receptors.

At present, a large number of GPCRs are defined as orphan receptors for which the cognate ligands have not yet been identified (1). The first step in characterization of orphan GPCRs is identification of the natural agonists for these receptors, so-called deorphanization. Deorphanization of GPCRs has revolutionized many fields of biomedical research through discoveries of novel natural ligands. We have successfully identified a novel lysophosphatidic acid (LPA) receptor, LPA4 (35), and the proton-sensing behavior of G2A (13), TDAG8 (15), and 12-hydroxyheptadecatrienoic acid as a novel agonist of leukotriene B4 type 2 receptor, BLT2 (36). The most common strategy for identifying ligands for orphan receptors is to seek effective agonists among appropriate ligand libraries or tissue extracts by monitoring various cellular responses using cells overexpressing the orphan receptor. To be successful, it is necessary to know the appropriate assay format for a given orphan receptor, e.g. measurement of intracellular calcium mobilization or cAMP formation. However, it is still unclear which types of G proteins are coupled to these orphan GPCRs. Thus, multiple assays are still required for this "blinded" ligand screening. In this study, we successfully demonstrate not only that GPR20 is functionally coupled to Gi-type G proteins, but also that the coupled Gi proteins are constitutively activated by GPR20 without any ligand stimulation, resulting in a modest suppression of basal cAMP levels. However, we cannot exclude the possibility that bona fide ligands for GPR20 exist in vivo. If GPR20 possesses a potential ability for acceleration of Gi activation via the binding of specific agonists, the information obtained in this study will be helpful to develop efficient assay systems for screening specific ligands or inverse agonists.

In summary, our work demonstrates that GPR20 has the ability to activate Gi proteins without extracellular triggers, indicating the pivotal role of this receptor in determining the basal tone of cAMP in cells. Furthermore, our findings suggest that the intrinsic expression of GPR20 is involved in the regulation of cell proliferation by controlling cellular cAMP levels. In addition, if GPR20 has the potential to accelerate G protein activation by ligand binding, screening efforts to identify bona fide or surrogate ligands for GPR20 will be required to delineate the exact functions of this receptor in vivo.


   FOOTNOTES
 
* This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. S. and T. Y.) and grants from the Japan Society for the Promotion of Science (Global Center of Excellence (COE) Program), the Takeda Science Foundation, the Mitsubishi Foundation, the Naito Foundation, the Uehara Memorial Foundation, the Astellas Foundation for Research on Metabolic Disorders (to T. Y.), and the Center for NanoBio Integration at the University of Tokyo. 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. Back

1 To whom correspondence should be addressed. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail: moto-nakamura{at}umin.net.

2 The abbreviations used are: GPCR, G protein-coupled receptor; GTP{gamma}S, guanosine 5'-({gamma}-thio) triphosphate; BSA, bovine serum albumin; PGE2, prostaglandin E2; PTX, pertussis toxin; BLT1, leukotriene B4 type 1 receptor; qRT, quantitative reverse-transcription; shRNA, short hairpin RNA; LPA, lysophosphatidic acid; ERKs, extracellular signal-regulated kinases; EIA, enzyme immunoassay; ANOVA, analysis of variance; h, human; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Satoshi Ishii and Yuichiro Ihara for technical advice and useful discussions, Hideo Shindou and Daisuke Hishikawa for mouse tissue cDNA (Fig. 2), and Tokutaro Yasue and Takayoshi Ishizaki (Kyorin Pharmaceutical Co., Ltd.) for support. We also thank Junichi Miyazaki (Osaka University, Japan) for supplying pCXN2.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Civelli, O. (2005) Trends Pharmacol. Sci 26, 15–19[CrossRef][Medline] [Order article via Infotrieve]
  2. Drews, J. (2000) Science 287, 1960–1964[Abstract/Free Full Text]
  3. Odowd, B. F., Nguyen, T., Jung, B. P., Marchese, A., Cheng, R., Heng, H. H. Q., Kolakowski, L. F., Lynch, K. R., and George, S. R. (1997) Gene (Amst.) 187, 75–81[CrossRef][Medline] [Order article via Infotrieve]
  4. Brennan, T. J., Matthews, W., and Moore, M. (January 23, 2003) U. S. Patent 0018989
  5. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193–199[CrossRef][Medline] [Order article via Infotrieve]
  6. Hunt, T., Carroll, R., and Peralta, E. (1994) J. Biol. Chem. 269, 29565–29570[Abstract/Free Full Text]
  7. Senogles, S. (1994) J. Biol. Chem. 269, 23120–23127[Abstract/Free Full Text]
  8. Wise, A., Watson-Koken, M., Rees, S., Lee, M., and Milligan, G. (1997) Biochem. J. 321, 721–728[Medline] [Order article via Infotrieve]
  9. Ayoub, M., Maurel, D., Binet, V., Fink, M., Prézeau, L., Ansanay, H., and Pin, J. (2007) Mol. Pharmacol. 71, 1329–1340[Abstract/Free Full Text]
  10. Hynes, T., Mervine, S., Yost, E., Sabo, J., and Berlot, C. (2004) J. Biol. Chem. 279, 44101–44112[Abstract/Free Full Text]
  11. Bradford, M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
  12. Ludwig, M., Vanek, M., Guerini, D., Gasser, J., Jones, C., Junker, U., Hofstetter, H., Wolf, R., and Seuwen, K. (2003) Nature 425, 93–98[CrossRef][Medline] [Order article via Infotrieve]
  13. Murakami, N., Yokomizo, T., Okuno, T., and Shimizu, T. (2004) J. Biol. Chem. 279, 42484–42491[Abstract/Free Full Text]
  14. Wang, J., Kon, J., Mogi, C., Tobo, M., Damirin, A., Sato, K., Komachi, M., Malchinkhuu, E., Murata, N., Kimura, T., Kuwabara, A., Wakamatsu, K., Koizumi, H., Uede, T., Tsujimoto, G., Kurose, H., Sato, T., Harada, A., Misawa, N., Tomura, H., and Okajima, F. (2004) J. Biol. Chem. 279, 45626–45633[Abstract/Free Full Text]
  15. Ishii, S., Kihara, Y., and Shimizu, T. (2005) J. Biol. Chem. 280, 9083–9087[Abstract/Free Full Text]
  16. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620–624[CrossRef][Medline] [Order article via Infotrieve]
  17. Kuniyeda, K., Okuno, T., Terawaki, K., Miyano, M., Yokomizo, T., and Shimizu, T. (2007) J. Biol. Chem. 282, 3998–4006[Abstract/Free Full Text]
  18. Probst, W., Snyder, L., Schuster, D., Brosius, J., and Sealfon, S. (1992) DNA Cell Biol. 11, 1–20[Medline] [Order article via Infotrieve]
  19. Savarese, T., and Fraser, C. (1992) Biochem. J. 283, 1–19[Medline] [Order article via Infotrieve]
  20. Ballesteros, J., Kitanovic, S., Guarnieri, F., Davies, P., Fromme, B., Konvicka, K., Chi, L., Millar, R., Davidson, J., Weinstein, H., and Sealfon, S. (1998) J. Biol. Chem. 273, 10445–10453[Abstract/Free Full Text]
  21. Ballesteros, J., Jensen, A., Liapakis, G., Rasmussen, S., Shi, L., Gether, U., and Javitch, J. (2001) J. Biol. Chem. 276, 29171–29177[Abstract/Free Full Text]
  22. Rovati, G., Capra, V., and Neubig, R. (2007) Mol. Pharmacol. 71, 959–964[Abstract/Free Full Text]
  23. Smit, M., Vischer, H., Bakker, R., Jongejan, A., Timmerman, H., Pardo, L., and Leurs, R. (2007) Annu. Rev. Pharmacol. Toxicol. 47, 53–87[CrossRef][Medline] [Order article via Infotrieve]
  24. Chen, G., Way, J., Armour, S., Watson, C., Queen, K., Jayawickreme, C., Chen, W., and Kenakin, T. (2000) Mol. Pharmacol. 57, 125–134[Abstract/Free Full Text]
  25. van Biesen, T., Luttrell, L., Hawes, B., and Lefkowitz, R. (1996) Endocr. Rev. 17, 698–714[Abstract/Free Full Text]
  26. Dhanasekaran, N., Tsim, S., Dermott, J., and Onesime, D. (1998) Oncogene 17, 1383–1394[CrossRef][Medline] [Order article via Infotrieve]
  27. New, D., and Wong, Y. (2007) J. Mol. Signal. 2, 2[CrossRef][Medline] [Order article via Infotrieve]
  28. Stork, P., and Schmitt, J. (2002) Trends Cell Biol. 12, 258–266[CrossRef][Medline] [Order article via Infotrieve]
  29. Allander, S., Nupponen, N., Ringnér, M., Hostetter, G., Maher, G., Goldberger, N., Chen, Y., Carpten, J., Elkahloun, A., and Meltzer, P. (2001) Cancer Res. 61, 8624–8628[Abstract/Free Full Text]
  30. Eggerickx, D., Denef, J., Labbe, O., Hayashi, Y., Refetoff, S., Vassart, G., Parmentier, M., and Libert, F. (1995) Biochem. J. 309, 837–843[Medline] [Order article via Infotrieve]
  31. Mehlmann, L., Saeki, Y., Tanaka, S., Brennan, T., Evsikov, A., Pendola, F., Knowles, B., Eppig, J., and Jaffe, L. (2004) Science 306, 1947–1950[Abstract/Free Full Text]
  32. Tanaka, S., Ishii, K., Kasai, K., Yoon, S., and Saeki, Y. (2007) J. Biol. Chem. 282, 10506–10515[Abstract/Free Full Text]
  33. Jones, P., Nawoschik, S., Sreekumar, K., Uveges, A., Tseng, E., Zhang, L., Johnson, J., He, L., Paulsen, J., Bates, B., and Pausch, M. (2007) Biochim. Biophys. Acta 1770, 890–901[Medline] [Order article via Infotrieve]
  34. Bates, B., Zhang, L., Nawoschik, S., Kodangattil, S., Tseng, E., Kopsco, D., Kramer, A., Shan, Q., Taylor, N., Johnson, J., Sun, Y., Chen, H., Blatcher, M., Paulsen, J., and Pausch, M. (2006) Brain Res. 1087, 1–14[CrossRef][Medline] [Order article via Infotrieve]
  35. Noguchi, K., Ishii, S., and Shimizu, T. (2003) J. Biol. Chem. 278, 25600–25606[Abstract/Free Full Text]
  36. Okuno, T., Iizuka, Y., Okazaki, H., Yokomizo, T., Taguchi, R., and Shimizu, T. (2008) J. Exp. Med., in press

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/19/12747    most recent
M709487200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hase, M.
Right arrow Articles by Nakamura, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hase, M.
Right arrow Articles by Nakamura, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement