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GPR65

Family: Class A Orphans

Contents:
Gene and Protein Information
Previous and Unofficial Names
Database Links
Agonists
Antagonists
Transduction Mechanisms
Tissue Distribution
Expression Datasets
Functional Assays
Physiological Functions
Physiological Consequences of Altering Gene Expression
Phenotypes, Alleles and Disease Models
Gene Expression and Pathophysiology
Biologically Significant Variants
General Comments
References
Gene and Protein Information
class A G protein-coupled receptor
Species TM AA Chromosomal Location Gene Symbol Gene Name Reference
Human 7 337 14q31-q32.1 GPR65 G protein-coupled receptor 65 9
Mouse 7 337 12 E Gpr65 G-protein coupled receptor 65
Rat 7 337 6q32 Gpr65 G-protein coupled receptor 65
Previous and Unofficial Names
TDAG8
Psychosine receptor
T cell death-associated protein 8
Dig1
Gpcr25
GPR65
G-protein coupled receptor 65
hTDAG8
Gpr65_predicted
LOC299242
G-protein coupled receptor 65 (predicted)
T cell death associated protein 8
rCG_20955
Database Links
Ensembl ENSG00000140030 (Hs), ENSMUSG00000021886 (Mm), ENSRNOG00000003806 (Rn)
Entrez Gene 8477 (Hs), 14744 (Mm), 299242 (Rn)
GeneCards GPR65 (Hs)
GenitoUrinary Development Molecular Anatomy Project Gpr65 (Mm)
HomoloGene 2675 (Hs)
Human Protein Reference Database 07057 (Hs)
InterPro Q8IYL9 (Hs), Q61038 (Mm)
KEGG Gene hsa:8477 (Hs), mmu:14744 (Mm), rno:299242 (Rn)
OMIM 604620 (Hs)
PharmGKB Gene PA28909 (Hs)
PhosphoSitePlus Q8IYL9 (Hs), Q61038 (Mm)
Protein Ontology (PRO) PRO:000001665 (Hs)
RefSeq Nucleotide NM_003608 (Hs), NM_008152 (Mm), NM_001106751 (Rn)
RefSeq Protein NP_003599 (Hs), NP_032178 (Mm), NP_001100221 (Rn)
TreeFam ENSG00000140030 (Hs), ENSMUSG00000021886 (Mm), ENSRNOG00000003806 (Rn)
UniGene Hs. 513440 (Hs)
UniProt Q8IYL9 (Hs), Q61038 (Mm)
Wikipedia GPR65
Orphan and other 7TM receptors
Natural/Endogenous Ligand(s)
(lyso)phospholipid mediators
Agonist Comments
GPR65 is a proton-sensing GPCR coupling to adenylyl cyclase. A study has found proton sensitivity to be inhibited by coupling to psychosine [22]. A separate study found the receptor to be internalised upon acid stimulation, with cAMP accumulation, RhoA activation and actin rearrangement. However this was not affected by psychosine [6]. It was initially believed that psychosine and other lysolipids acted at human GPR65 to mediate drug-induced inhibition of forskolin driven cAMP rise in a concentration-dependent manner (D-glucosyl-b-1,19 sphingosine (GlcPSY), LacPSY, and lysosulfatide, but not by N-acetyl PSY, sphingosine 1-phosphate, lysophosphatidic acid, ceramide 1-phosphate, or lysophosphatidylcholine [5]). It has since been reported that these drugs may actually be antagonists at GPR65 [20,22].
Antagonist Comments
Psychosine related lysosphingolipids behave as antagonists against proton-sensing GPR65 [22].
Primary Transduction Mechanisms
Transducer Effector/Response
Gs family Adenylate cyclase stimulation
Comments:  GPR65 response to psychosine was not blocked by pretreatment of RH7777 cultures with pertussis toxin, suggesting the involvement of PTX-insensitive G proteins, perhaps Gαs. Proton stimulation of the receptor causes cAMP accumulation [6,17]. This proton-receptor interaction may be mediated by Gs proteins [22]. TDAG8, at least partly, mediates the extracellular acidification-induced inhibition of proinflammatorycytokine production through the Gs protein/cAMP/PKA
References:  6,14,17,22
Tissue Distribution
Natural killer cells, monocytic cell lines (U937,THP-1)
Species:  Human
Technique:  RT-PCR
References:  11
Peripheral blood leukocytes, spleen, lymph nodes, thymus
Species:  Human
Technique:  Northern blot
References:  9
Differentiated HL-60 cells, neutrophils
Species:  Human
Technique:  qRT-PCR
References:  15
Monocytes, macrophages
Species:  Human
Technique:  RT-PCR, immunoblotting
References:  2
Spleen, lymph node, peripheral blood leukocytes (low level expression in all tissue extracts)
Species:  Human
Technique:  Microarray analysis
References:  5
Peritoneal macrophages
Species:  Mouse
Technique:  RT-PCR
References:  14
Brain, spinal cord, dorsal root ganglion, and trigeminal ganglion
Species:  Mouse
Technique:  RT-PCR
References:  4
Eosinophils
Species:  Mouse
Technique:  Northern blot
References:  8
Hypothalamus, amygdala, hippocampus, frontal cortex, striatum, medulla
Species:  Rat
Technique:  RT-PCR
References:  13
Tissue Distribution Comments
GPR65 is overexpressed by more than 5x in a range of human cancer tissues (kidney, ovarian, colon, breast) screened using quantitative fluorescence-based real-time PCR [21]. Enhanced green fluorescent protein reporter has been knocked into the disrupted GPR65 locus to allow the analysis of TDAG8 expression in living cells [16]. GPR65 is expressed in nociceptors of the lumbar dorsal root ganglion in mice (RT-PCR) [4]. Receptor expression is increased in eosinophils in murine models of allergic asthma and human populations during acute asthma exacerbations [8].
Expression Datasets

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Log average relative transcript abundance in mouse tissues measured by qPCR from Regard, J.B., Sato, I.T., and Coughlin, S.R. (2008). Anatomical profiling of G protein-coupled receptor expression. Cell, 135(3): 561-71. [PMID:18984166] [Raw data: website]

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Functional Assays
Site directed mutagenesis of conserved H17 and H20 indicates that these residues are required for the proton-sensing ability of GPR65
Species:  Human
Tissue:  COS7 cells
Response measured:  Proton-insensitivity
References:  22
Psychosine induces multinuclear cells in GPR65-expressing RH7777 cultures
Species:  Human
Tissue:  RH777 hepatoma cells
Response measured:  Globoid cell formation
References:  2
Murine GPR65 is upregulated during apoptosis of immature thymocytes as a result of activation with either anti-CD3 plus anti-CD28 or with glucocorticoids
Species:  Mouse
Tissue:  Lymphoid tissues
Response measured:  Receptor upregulation
References:  1
GalSph, GlcSph, and LacSph significantly induced NK cell chemotaxis, apoptosis, and inhibit IFN-γ secretion and cytotoxicity
Species:  Human
Tissue:  NK cells
Response measured:  Chemotaxis and reduced cytotoxicity
References:  11
GalSph and GlcSph induced multinuclear formation in NK cells
Species:  Human
Tissue:  NK cell culture
Response measured:  Multinucleation and globoid-like formation
References:  11
Expression of GPR65 in WHEI7.2 cells is sufficient to induce apoptosis
Species:  Mouse
Tissue:  WHEI7.2 lymphoma cell culture
Response measured:  Apoptosis
References:  12
Glucocorticoids induce GPR65 expression in multiple models of glucocorticoid-mediated apoptosis
Species:  Mouse
Tissue:  WEHI7.2 cells, S49.A2 cells, primary thermocytes
Response measured:  Receptor activation by psychosine enhances dexamethasone-mediated apoptosis
References:  12
Increased cAMP and phosphorylated CREB concentrations are observed in GPR65 expressing cells. Acidosis-induced toxicity is also attenuated
Species:  Rat
Tissue:  Transfected CHO cells
Response measured:  Increased intracellular cAMP and phosphorylated CREB
References:  13
Functional Assay Comments
Cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPR65 [3].
Physiological Functions
Enhances glucocorticoid-mediated apoptosis
Species:  Mouse
Tissue:  Leukocyte cell lines
References:  12
GPR65 regulates viability of allergen-elicited BALF eosinophils, most likely through a caspase-dependent mechanism
Species:  Mouse
Tissue:  Eosinophils
References:  8
Physiological Functions Comments
Receptor mediated increased intracellular cAMP concentration may mediate the acidic pH-induced inhibition of superoxide anion (O(2)(-)) production [15].
Physiological Consequences of Altering Gene Expression
Pro-survival phenotype. Repression of GPR65 inhibits dexamethasone mediated apoptosis
Species:  Mouse
Tissue:  WHEI7.2 lymphoma cell line
Technique:  RNA interference (RNAi)
References:  12
Overexpression of TDAG8 in HEK293 cells leads to transcriptional activation from SRE- and CRE-driven promoters, independent of exogenously added ligand. This leads to a tumour-formation phenotype
Species:  Human
Tissue:  HEK293 cell line
Technique:  Gene over-expression
References:  21
Ectopic overexpression of GPR65 causes up to 10x increase in cAMP production, even in alkaline conditions. This increase in cAMP was amplified in acidic conditions.
Species:  Human
Tissue:  293T cells
Technique:  Gene over-expression
References:  17
GPR65 is required for pH-dependent cAMP production in immune cells
Species:  Mouse
Tissue:  Thermocytes and splenocytes
Technique:  Genomic DNA fragment containing exon 2-derived coding sequences was replaced by a construct encoding promoterless IRES-EGFP sequences and the neomycin resistance cassette flanked by loxP sites. Mice were back-crossed for 6 generations.
References:  17
Knockout of GPR65 has no effect on thermocyte maturation, selection, or on major immune functions. Thermocytes also displayed normal apoptosis following glucocorticoid treatment
Species:  Mouse
Tissue:  Thermocytes
Technique:  Gene knock-outs
References:  16
GPR65 homozygous knockout ablates the acid-dependent upregulation of cAMP production, and inhibits the acid-induced increase in eosinophil viability.
Species:  Mouse
Tissue:  Eosinophils
Technique:  Gene knockouts
References:  8
siRNA specific to GPR65 (TDAG8), but not to GPR132 (G2A), clearly attenuated the acidification-induced inhibition of TNF-alpha, but not isoproterenol or prostaglandin-E2, in mouse peritoneal macrophages
Species:  Mouse
Tissue:  Peritoneal macrophages
Technique:  RNA interference (RNAi)
References:  14
Knockdown of GPR65 prevented the elevation of Bcl-2 and Bcl-xL in response to acidic conditions, due to inhibition of MEK/ERK signalling
Species:  Human
Tissue:  WEHI7.2 cells
Technique:  RNA interference (RNAi)
References:  19
Physiological Consequences of Altering Gene Expression Comments
Gene profiling of GPR65 indicates that mRNA is down regulated in leukemic cells overexpressing growth factor independence 1B gene [7].
Phenotypes, Alleles and Disease Models Mouse data from MGI

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Allele Composition & genetic background Accession Phenotype Id Phenotype Reference
Gpr65tm1Witt Gpr65tm1Witt/Gpr65tm1Witt
B6.Cg-Gpr65		 MGI:108031  MP:0001819 abnormal immune cell physiology PMID: 15665078 
Gpr65tm1Witt Gpr65tm1Witt/Gpr65tm1Witt
B6.Cg-Gpr65		 MGI:108031  MP:0002442 abnormal leukocyte physiology PMID: 15665078 
Gpr65tm1Witt Gpr65tm1Witt/Gpr65tm1Witt
C.Cg-Gpr65		 MGI:108031  MP:0002442 abnormal leukocyte physiology PMID: 15665078 
Gpr65Tn(sb-lacZ,GFP)T1.88Jtak Gpr65Tn(sb-lacZ,GFP)T1.88Jtak/Gpr65Tn(sb-lacZ,GFP)T1.88Jtak
B6.Cg-Gpr65		 MGI:108031  MP:0002451 abnormal macrophage physiology PMID: 19234222 
Gpr65Tn(sb-lacZ,GFP)T1.88Jtak Gpr65Tn(sb-lacZ,GFP)T1.88Jtak/Gpr65Tn(sb-lacZ,GFP)T1.88Jtak
B6.Cg-Gpr65		 MGI:108031  MP:0008561 decreased tumor necrosis factor secretion PMID: 19234222 
Gene Expression and Pathophysiology Comments
Chromosomal location and tissue distribution implicates GPR65 in T-cell-associated diseases [9]. Association with the ligand psychosine indicates a role for the receptor in globoid cell leukodystrophy [5]. Receptor overexpression in a range of human cancer tissues suggest contribution to tumor development [21]. Genome-wide association and fine mapping of genetic loci indicates Gpr65 in predisposition to colon carcinogenesis in mice [10].
Biologically Significant Variants
I231L
SNP accession:  rs3742704 
Type:  Naturally occurring SNPs.
Species:  Human
References: 
General Comments
GPR65 may be a Stat3alpha gene target [18].
Available Assays
DiscoveRx PathHunter® CHO-K1GPR65 (Orphan) High Expression β-Arrestin Cell Line Human Cat No. 93-0428C2A
DiscoveRx PathHunter® eXpress GPR65 CHO-K1 β-Arrestin (Orphan) GPCR Assay Human Cat No. 93-0428E2ACP1

REFERENCES

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1. Choi, JW; Lee, SY; Choi, Y. (1996) Identification of a putative G protein-coupled receptor induced during activation-induced apoptosis of T cells. Cell. Immunol., 168 (1): 78-84. [PMID:8599842]

2. Duong, CQ; Bared, SM; Abu-Khader, A; Buechler, C; Schmitz, A; Schmitz, G. (2004) Expression of the lysophospholipid receptor family and investigation of lysophospholipid-mediated responses in human macrophages. Biochim. Biophys. Acta, 1682 (1-3): 112-9. [PMID:15158762]

3. Fukunaga, S; Setoguchi, S; Hirasawa, A; Tsujimoto, G. (2006) Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach. Life Sci., 80 (1): 17-23. [PMID:16978657]

4. Huang, CW; Tzeng, JN; Chen, YJ; Tsai, WF; Chen, CC; Sun, WH. (2007) Nociceptors of dorsal root ganglion express proton-sensing G-protein-coupled receptors. Mol. Cell. Neurosci., 36 (2): 195-210. [PMID:17720533]

5. Im, DS; Heise, CE; Nguyen, T; O'Dowd, BF; Lynch, KR. (2001) Identification of a molecular target of psychosine and its role in globoid cell formation. J. Cell Biol., 153 (2): 429-34. [PMID:11309421]

6. Ishii, S; Kihara, Y; Shimizu, T. (2005) Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor. J. Biol. Chem., 280 (10): 9083-7. [PMID:15618224]

7. Koldehoff, M; Zakrzewski, JL; Klein-Hitpass, L; Beelen, DW; Elmaagacli, AH. (2008) Gene profiling of growth factor independence 1B gene (Gfi-1B) in leukemic cells. Int. J. Hematol., 87 (1): 39-47. [PMID:18224412]

8. Kottyan, LC; Collier, AR; Cao, KH; Niese, KA; Hedgebeth, M; Radu, CG; Witte, ON; Khurana Hershey, GK; Rothenberg, ME; Zimmermann, N. (2009) Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner. Blood, 114 (13): 2774-82. [PMID:19641187]

9. Kyaw, H; Zeng, Z; Su, K; Fan, P; Shell, BK; Carter, KC; Li, Y. (1998) Cloning, characterization, and mapping of human homolog of mouse T-cell death-associated gene. DNA Cell Biol., 17 (6): 493-500. [PMID:9655242]

10. Liu, P; Lu, Y; Liu, H; Wen, W; Jia, D; Wang, Y; You, M. (2012) Genome-wide association and fine mapping of genetic loci predisposing to colon carcinogenesis in mice. Mol. Cancer Res., 10 (1): 66-74. [PMID:22127497]

11. Maghazachi, AA; Knudsen, E; Jin, Y; Jenstad, M; Chaudhry, FA. (2004) D-galactosyl-beta1-1'-sphingosine and D-glucosyl-beta1-1'-sphingosine induce human natural killer cell apoptosis. Biochem. Biophys. Res. Commun., 320 (3): 810-5. [PMID:15240120]

12. Malone, MH; Wang, Z; Distelhorst, CW. (2004) The glucocorticoid-induced gene tdag8 encodes a pro-apoptotic G protein-coupled receptor whose activation promotes glucocorticoid-induced apoptosis. J. Biol. Chem., 279 (51): 52850-9. [PMID:15485889]

13. McGuire, J; Herman, JP; Ghosal, S; Eaton, K; Sallee, FR; Sah, R. (2009) Acid-sensing by the T cell death-associated gene 8 (TDAG8) receptor cloned from rat brain. Biochem. Biophys. Res. Commun., 386 (3): 420-5. [PMID:19501050]

14. Mogi, C; Tobo, M; Tomura, H; Murata, N; He, XD; Sato, K; Kimura, T; Ishizuka, T; Sasaki, T; Sato, T; Kihara, Y; Ishii, S; Harada, A; Okajima, F. (2009) Involvement of proton-sensing TDAG8 in extracellular acidification-induced inhibition of proinflammatory cytokine production in peritoneal macrophages. J. Immunol., 182 (5): 3243-51. [PMID:19234222]

15. Murata, N; Mogi, C; Tobo, M; Nakakura, T; Sato, K; Tomura, H; Okajima, F. (2009) Inhibition of superoxide anion production by extracellular acidification in neutrophils. Cell. Immunol., 259 (1): 21-6. [PMID:19539899]

16. Radu, CG; Cheng, D; Nijagal, A; Riedinger, M; McLaughlin, J; Yang, LV; Johnson, J; Witte, ON. (2006) Normal immune development and glucocorticoid-induced thymocyte apoptosis in mice deficient for the T-cell death-associated gene 8 receptor. Mol. Cell. Biol., 26 (2): 668-77. [PMID:16382156]

17. Radu, CG; Nijagal, A; McLaughlin, J; Wang, L; Witte, ON. (2005) Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells. Proc. Natl. Acad. Sci. U.S.A., 102 (5): 1632-7. [PMID:15665078]

18. Redell, MS; Tsimelzon, A; Hilsenbeck, SG; Tweardy, DJ. (2007) Conditional overexpression of Stat3alpha in differentiating myeloid cells results in neutrophil expansion and induces a distinct, antiapoptotic and pro-oncogenic gene expression pattern. J. Leukoc. Biol., 82 (4): 975-85. [PMID:17634277]

19. Ryder, C; McColl, K; Zhong, F; Distelhorst, CW. (2012) Acidosis Promotes Bcl-2 Family-mediated Evasion of Apoptosis: INVOLVEMENT OF ACID-SENSING G PROTEIN-COUPLED RECEPTOR GPR65 SIGNALING TO MEK/ERK. J. Biol. Chem., 287 (33): 27863-75. [PMID:22685289]

20. Seuwen, K; Ludwig, MG; Wolf, RM. (2006) Receptors for protons or lipid messengers or both?. J. Recept. Signal Transduct. Res., 26 (5-6): 599-610. [PMID:17118800]

21. Sin, WC; Zhang, Y; Zhong, W; Adhikarakunnathu, S; Powers, S; Hoey, T; An, S; Yang, J. (2004) G protein-coupled receptors GPR4 and TDAG8 are oncogenic and overexpressed in human cancers. Oncogene, 23 (37): 6299-303. [PMID:15221007]

22. Wang, JQ; 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; Okajima, F. (2004) TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor. J. Biol. Chem., 279 (44): 45626-33. [PMID:15326175]

To cite this database page, please use the following:

Amy E. Monaghan.
Class A Orphans: GPR65. Last modified on 02/11/2012. Accessed on 18/06/2013. IUPHAR database (IUPHAR-DB), http://iuphar-db.org/DATABASE/ObjectDisplayForward?objectId=113.


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