Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4

Sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) are bioactive lipid molecules involved in numerous biological processes. We have recently identified ovarian cancer G protein-coupled receptor 1 (OGR1) as a specific and high affinity receptor for SPC, and G2A as a receptor with high affinity for LPC, but low affinity for SPC. Among G protein-coupled receptors, GPR4 shares highest sequence homology with OGR1 (51%). In this work, we have identified GPR4 as not only another high affinity receptor for SPC, but also a receptor for LPC, albeit of lower affinity. Both SPC and LPC induce increases in intracellular calcium concentration in GPR4-, but not vector-transfected MCF10A cells. These effects are insensitive to treatment with BN52021, WEB-2170, and WEB-2086 (specific platelet activating factor (PAF) receptor antagonists), suggesting that they are not mediated through an endogenous PAF receptor. SPC and LPC bind to GPR4 in GPR4-transfected CHO cells with K(d)/SPC = 36 nm, and K(d)/LPC = 159 nm, respectively. Competitive binding is elicited only by SPC and LPC. Both SPC and LPC activate GPR4-dependent activation of serum response element reporter and receptor internalization. Swiss 3T3 cells expressing GPR4 respond to both SPC and LPC, but not sphingosine 1-phosphate (S1P), PAF, psychosine (Psy), glucosyl-beta1'1-sphingosine (Glu-Sph), galactosyl-beta1'1-ceramide (Gal-Cer), or lactosyl-beta1'1-ceramide (Lac-Cer) to activate extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration- and time-dependent manner. SPC and LPC stimulate DNA synthesis in GPR4-expressing Swiss 3T3 cells. Both extracellular signal-regulated kinase activation and DNA synthesis stimulated by SPC and LPC are pertussis toxin-sensitive, suggesting the involvement of a G(i)-heterotrimeric G protein. In addition, GPR4 expression confers chemotactic responses to both SPC and LPC in Swiss 3T3 cells. Taken together, our data indicate that GPR4 is a receptor with high affinity to SPC and low affinity to LPC, and that multiple cellular functions can be transduced via this receptor.     

Manganese and cobalt activate zebrafish ovarian cancer G-protein-coupled receptor 1 but not GPR4

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) is activated by some metals in addition to extracellular protons and coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebrafish OGR1, zebrafish GPR4, and human GPR4 (zOGR1, zGPR4, and hGPR4, respectively) could sense the metals and activate the intracellular signaling pathways. On one hand, we found that only manganese and cobalt of the tested metals stimulated SRE-promoter activities in zOGR1-overexpressed HEK293T cells. On the other hand, none of the metals tested stimulated the promoter activities in zGPR4- and hGPR4-overexpressed cells. The OGR1 mutant (H4F), which is lost to activation by extracellular protons, did not stimulate metal-induced SRE-promoter activities. These results suggest that zOGR1, but not GPR4, is also a metal-sensing G-protein-coupled receptor in addition to a proton-sensing G-protein-coupled receptor, although not all metals that activate hOGR1 activated zOGR1.     

Prostaglandin I(2) production and cAMP accumulation in response to acidic extracellular pH through OGR1 in human aortic smooth muscle cells

Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4 have recently been identified as proton-sensing or extracellular pH-responsive G-protein-coupled receptors stimulating inositol phosphate production and cAMP accumulation, respectively. In the present study, we found that OGR1 and GPR4 mRNAs were expressed in human aortic smooth muscle cells (AoSMCs). Acidic extracellular pH induced inositol phosphate production, a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), and cAMP accumulation in these cells. When small interfering RNAs (siRNAs) targeted for OGR1 and GPR4 were transfected to the cells, the acid-induced inositol phosphate production and [Ca(2+)](i) increase were markedly inhibited by the OGR1 siRNA but not by the GPR4 siRNA. Unexpectedly, the acid-induced cAMP accumulation was also largely inhibited by OGR1 siRNA but only slightly by GPR4 siRNA. Acidic extracellular pH also stimulated prostaglandin I2 (PGI(2)) production, which was again inhibited by OGR1 siRNA. The specific inhibitors for extracellular signal-regulated kinase kinase and cyclooxygenase attenuated the acid-induced PGI(2) production and cAMP accumulation without changes in the inositol phosphate production. A specific inhibitor of phospholipase C also inhibited the acid-induced cAMP accumulation. In conclusion, OGR1 is a major receptor involved in the extracellular acid-induced stimulation of PGI(2) production and cAMP accumulation in AoSMCs. The cAMP accumulation may occur through OGR1-mediated stimulation of the phospholipase C/cyclooxygenase/PGI(2) pathway.     

Lysophospholipid receptors: signalling, pharmacology and regulation by lysophospholipid metabolism

The lysophospholipids, sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), activate diverse groups of G-protein-coupled receptors that are widely expressed and regulate decisive cellular functions. Receptors of the endothelial differentiation gene family are activated by S1P (S1P(1-5)) or LPA (LPA(1-3)); two more distantly related receptors are activated by LPA (LPA(4/5)); the GPR(3/6/12) receptors have a high constitutive activity but are further activated by S1P and/or SPC; and receptors of the OGR1 cluster (OGR1, GPR4, G2A, TDAG8) appear to be activated by SPC, LPC, psychosine and/or protons. G-protein-coupled lysophospholipid receptors regulate cellular Ca(2+) homoeostasis and the cytoskeleton, proliferation and survival, migration and adhesion. They have been implicated in development, regulation of the cardiovascular, immune and nervous systems, inflammation, arteriosclerosis and cancer. The availability of S1P and LPA at their G-protein-coupled receptors is regulated by enzymes that generate or metabolize these lysophospholipids, and localization plays an important role in this process. Besides FTY720, which is phosphorylated by sphingosine kinase-2 and then acts on four of the five S1P receptors of the endothelial differentiation gene family, other compounds have been identified that interact with more ore less selectivity with lysophospholipid receptors.     

Lysophospholipid receptors: Signalling, pharmacology and regulation by lysophospholipid metabolism

The lysophospholipids, sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), activate diverse groups of G-protein-coupled receptors that are widely expressed and regulate decisive cellular functions. Receptors of the endothelial differentiation gene family are activated by S1P (S1P_1-5) or LPA (LPA_1-3); two more distantly related receptors are activated by LPA (LPA_4/5); the GPR_3/6/12 receptors have a high constitutive activity but are further activated by S1P and/or SPC; and receptors of the OGR1 cluster (OGR1, GPR4, G2A, TDAG8) appear to be activated by SPC, LPC, psychosine and/or protons. G-protein-coupled lysophospholipid receptors regulate cellular Ca²⁺ homoeostasis and the cytoskeleton, proliferation and survival, migration and adhesion. They have been implicated in development, regulation of the cardiovascular, immune and nervous systems, inflammation, arteriosclerosis and cancer. The availability of S1P and LPA at their G-protein-coupled receptors is regulated by enzymes that generate or metabolize these lysophospholipids, and localization plays an important role in this process. Besides FTY720, which is phosphorylated by sphingosine kinase-2 and then acts on four of the five S1P receptors of the endothelial differentiation gene family, other compounds have been identified that interact with more ore less selectivity with lysophospholipid receptors.     

G2A is a proton-sensing G-protein-coupled receptor antagonized by lysophosphatidylcholine

G2A (from G2 accumulation) is a G-protein-coupled receptor (GPCR) that regulates the cell cycle, proliferation, oncogenesis, and immunity. G2A shares significant homology with three GPCRs including ovarian cancer GPCR (OGR1/GPR68), GPR4, and T cell death-associated gene 8 (TDAG8). Lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC) were reported as ligands for G2A and GPR4 and for OGR1 (SPC only), and a glycosphingolipid psychosine was reported as ligand for TDAG8. As OGR1 and GPR4 were reported as proton-sensing GPCRs (Ludwig, M. G., Vanek, M., Guerini, D., Gasser, J. A., Jones, C. E., Junker, U., Hofstetter, H., Wolf, R. M., and Seuwen, K. (2003) Nature 425, 93-98), we evaluated the proton-sensing function of G2A. Transient expression of G2A caused significant activation of the zif 268 promoter and inositol phosphate (IP) accumulation at pH 7.6, and lowering extracellular pH augmented the activation only in G2A-expressing cells. LPC inhibited the pH-dependent activation of G2A in a dose-dependent manner in these assays. Thus, G2A is another proton-sensing GPCR, and LPC functions as an antagonist, not as an agonist, and regulates the proton-dependent activation of G2A.     

Receptors for protons or lipid messengers or both?

The subfamily of G protein-coupled receptors comprising GPR4, OGR1, TDAG8, and G2A was originally characterized as a group of proteins mediating biological responses to the lipid messengers sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine. We challenged this view by reporting that OGR1 and GPR4 sense acidic pH and that this process is not affected by concentrations of SPC or LPC previously reported as agonistic. The original publications describing GPR4, OGR1, and G2A as receptors for LPC or SPC have now been retracted, and the first studies exploring receptors of this family as pH sensors in physiology have appeared. Here we review the status of this field and we confirm that GPR4, OGR1, and TDAG8 should be considered as proton-sensing receptors. Negative regulation of these receptors by high micromolar concentrations of lipids appears not specific in our experiments.     

Heteromerization of G2A and OGR1 enhances proton sensitivity and proton-induced calcium signals

Proton-sensing G-protein-coupled receptors (GPCRs; OGR1, GPR4, G2A, TDAG8), with full activation at pH 6.4 ～ 6.8, are important to pH homeostasis, immune responses and acid-induced pain. Although G2A mediates the G13-Rho pathway in response to acid, whether G2A activates Gs, Gi or Gq proteins remains debated. In this study, we examined the response of this fluorescence protein-tagged OGR1 family to acid stimulation in HEK293T cells. G2A did not generate detectable intracellular calcium or cAMP signals or show apparent receptor redistribution with moderate acid (pH?≥?6.0) stimulation but reduced cAMP accumulation under strong acid stimulation (pH?≤?5.5). Surprisingly, coexpression of OGR1- and G2A-enhanced proton sensitivity and proton-induced calcium signals. This alteration is attributed to oligomerization of OGR1 and G2A. The oligomeric potential locates receptors at a specific site, which leads to enhanced proton-induced calcium signals through channels.     

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.     

Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled receptors and receptor-mediated signal transduction

In recent years, certain lysophospholipids (lyso-PLs) have been recognized as important cell signaling molecules. Among them, two phosphorylcholine-containing lyso-PLs, sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), have been shown to be involved in many cellular processes and are produced under physiological and pathological conditions. Although signaling properties of SPC and LPC have been studied in a variety of cellular systems, specific cell membrane receptors for SPC and LPC have not been identified previously. Recently, ovarian cancer G protein-coupled receptor 1 (OGR1, also known as GPR68), G protein-coupled receptor 4 (GPR4), and G2A have been identified as receptors for SPC and LPC. The signaling and ligand-binding properties of these receptors are reviewed here. These discoveries provide an intriguing opportunity and a novel approach in studying the pathophysiological roles of SPC and LPC and their receptors.     

Receptors for Protons or Lipid Messengers or Both?

The subfamily of G protein-coupled receptors comprising GPR4, OGR1, TDAG8, and G2A was originally characterized as a group of proteins mediating biological responses to the lipid messengers sphingosylphosphorylcholine (SPC), lysophosphatidylcholine (LPC), and psychosine. We challenged this view by reporting that OGR1 and GPR4 sense acidic pH and that this process is not affected by concentrations of SPC or LPC previously reported as agonistic. The original publications describing GPR4, OGR1, and G2A as receptors for LPC or SPC have now been retracted, and the first studies exploring receptors of this family as pH sensors in physiology have appeared. Here we review the status of this field and we confirm that GPR4, OGR1, and TDAG8 should be considered as proton-sensing receptors. Negative regulation of these receptors by high micromolar concentrations of lipids appears not specific in our experiments.     

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.     

Sphingosylphosphorylcholine-biological functions and mechanisms of action

Compared to the lysophospholipid mediators, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), little information is available regarding the molecular mechanisms of action, metabolism and physiological significance of the related sphingosylphosphorylcholine (SPC). S1P and LPA have recently been established as agonists at several G-protein-coupled receptors of the EDG family, S1P additionally serves an intracellular second messenger function. Several cellular effects of SPC can be explained by low-affinity binding to and activation of S1P-EDG receptors. However, certain cellular and subcellular actions of SPC are not shared by S1P, suggesting that SPC, which has been identified in normal blood plasma, ascites and various tissues, is a lipid mediator in its own right. This concept was corroborated by the recent discovery of specific high-affinity G-protein-coupled SPC receptors. In this article, our present knowledge on cellular actions and biological functions of SPC will be reviewed.     

Novel clusters of receptors for sphingosine-1-phosphate, sphingosylphosphorylcholine, and (lyso)-phosphatidic acid: new receptors for "old" ligands

The (lyso)phospholipid mediators sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC), and phosphatidic acid (PA) regulate diverse cellular responses such as proliferation, survival and death, cytoskeletal rearrangements, cell motility, and differentiation among many others. Signaling is complex and many signaling events are mediated through the activation of cell surface seven transmembrane (7TM) G protein coupled receptors. Five high affinity receptors for S1P have been identified so far and named S1P(1, 2,3,4,5) (formerly referred to as endothelial differentiation gene (edg)1, 5, 3, 6, 8). Recently, the orphan receptor GPR63 was identified a low affinity S1P receptor structurally distant from the S1P(1-5) family. The orphan GPR3, 6, 12 cluster, phylogenetically related to the edg and melanocortin receptors appears to be subject to modulation by S1P and SPC although all three receptors are strong constitutive stimulators of the Galphas-adenylyl cyclase (AC) pathway and would not require additional ligand stimulation but rather inverse agonism to control activity. Ovarian cancer G protein coupled receptor 1 (OGR1) and GPR4, two structurally closely related receptors were assigned in functional and binding studies as high affinity molecular targets for SPC. Very recently, however, both OGR1 and GPR4 were described as receptors endowed with the ability to signal cells in response to protons. LPA exerts its biological effects through the activation of G protein coupled LPA(1-3) receptors (formerly referred to as edg2, 4, 7). A fourth high affinity LPA receptor has been identified: P2Y9 (GPR23) structurally related to nucleotide receptors and phylogenetically quite distant from the high affinity LPA(1-3) cluster. This review attempts to give an overview about the existing families of lysophosholipid receptors and the spectrum of lipid agonists they use as high or low affinity ligands to relay extracellular signals into intracellular responses. Recently deorphaned lipid receptors, within and outside the known lipid receptor clusters will receive particular attention.     

Abnormalities in Osteoclastogenesis and Decreased Tumorigenesis in Mice Deficient for Ovarian Cancer G Protein-Coupled Receptor 1

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK) activation and nitric oxide (NO) production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1''s role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases.     

Sphingosylphosphorylcholine as a novel calmodulin inhibitor

S1P (sphingosine 1-phosphate) and SPC (sphingosylphosphorylcholine) have been recently recognized as important mediators of cell signalling, regulating basic cellular processes such as growth,differentiation, apoptosis, motility and Ca2+ homoeostasis.Interestingly, they can also act as first and second messengers. Although their activation of cell-surface G-protein-coupled receptors has been studied extensively, not much is known about heir intracellular mechanism of action, and their target proteins are yet to be identified. We hypothesized that these sphingolipids might bind to CaM (calmodulin), the ubiquitous intracellular Ca2+sensor. Binding assays utilizing intrinsic tyrosine fluorescence of the protein, dansyl-labelled CaM and surface plasmon resonance revealed that SPC binds to both apo- and Ca2+-saturated CaM selectively, when compared with the related lysophospholipid mediators S1P, LPA (lysophosphatidic acid) and LPC (lysophosphatidylcholine). Experiments carried out with the model CaM-binding domain melittin showed that SPC dissociates the CaM-target peptide complex, suggesting an inhibitory role. The functional effect of the interaction was examined on two target enzymes, phosphodiesterase and calcineurin, and SPC inhibited the Ca2+/CaM-dependent activity of both. Thus we propose that CaM might be an intracellular receptor for SPC, and raise the possibility of a novel endogenous regulation of CaM.     

The proton-activated G protein coupled receptor OGR1 acutely regulates the activity of epithelial proton transport proteins

The Ovarian cancer G protein-coupled Receptor 1 (OGR1; GPR68) is proton-sensitive in the pH range of 6.8 - 7.8. However, its physiological function is not defined to date. OGR1 signals via inositol trisphosphate and intracellular calcium, albeit downstream events are unclear. To elucidate OGR1 function further, we transfected HEK293 cells with active OGR1 receptor or a mutant lacking 5 histidine residues (H5Phe-OGR1). An acute switch of extracellular pH from 8 to 7.1 (10 nmol/l vs 90 nmol/l protons) stimulated NHE and H(+)-ATPase activity in OGR1-transfected cells, but not in H5Phe-OGR1-transfected cells. ZnCl(2) and CuCl(2) that both inhibit OGR1 reduced the stimulatory effect. The activity was blocked by chelerythrine, whereas the ERK1/2 inhibitor PD 098059 had no inhibitory effect. OGR1 activation increased intracellular calcium in transfected HEK293 cells. We next isolated proximal tubules from kidneys of wild-type and OGR1-deficient mice and measured the effect of extracellular pH on NHE activity in vitro. Deletion of OGR1 affected the pH-dependent proton extrusion, however, in the opposite direction as expected from cell culture experiments. Upregulated expression of the pH-sensitive kinase Pyk2 in OGR1 KO mouse proximal tubule cells may compensate for the loss of OGR1. Thus, we present the first evidence that OGR1 modulates the activity of two major plasma membrane proton transport systems. OGR1 may be involved in the regulation of plasma membrane transport proteins and intra- and/or extracellular pH.     

TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1- and GPR4-expressing cells but not for prostaglandin E(2)- and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR1.     

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G_q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP₃) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G_q/11 protein and inositol-1,4,5-trisphosphate-induced Ca²⁺ mobilization in human ASMCs.     

Identification of T cell death-associated gene 8 (TDAG8) as a novel acid sensing G-protein-coupled receptor

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.