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.     

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.     

Two pathways for lysophosphatidic acid production

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) is a simple phospholipid but displays an intriguing cell biology that is mediated via interactions with G protein-coupled seven transmembrane receptors (GPCRs). So far, five GPCRs, designated LPA(1-5), and, more recently, two additional GPCRs, GPR87 and P2Y5, have been identified as receptors for LPA. These LPA receptors can be classified into two families, the EDG and P2Y families, depending on their primary structures. Recent studies on gene targeting mice and family diseases of these receptors revealed that LPA is involved in both pathological and physiological states including brain development (LPA(1)), neuropathy pain (LPA(1)), lung fibrosis (LPA(1)), renal fibrosis (LPA(1)) protection against radiation-induced intestinal injury (LPA(2)), implantation (LPA(3)) and hair growth (P2Y5). LPA is produced both in cells and biological fluids, where multiple synthetic reactions occur. There are at least two pathways for LPA production. In serum or plasma, LPA is predominantly produced by a plasma enzyme called autotaxin (ATX). ATX is a multifunctional ectoenzyme and is involved in many patho-physiological conditions such as cancer, neuropathy pain, lymphocyte tracking in lymph nodes, obesity, diabetes and embryonic blood vessel formation. LPA is also produced from phosphatidic acid (PA) by its deacylation catalyzed by phospholipase A (PLA)-type enzymes. However, the physiological roles of this pathway as well as the enzymes involved remained to be solved. A number of phospholipase A(1) and A(2) isozymes could be involved in this pathway. One PA-selective PLA(1) called mPA-PLA(1)alpha/LIPH is specifically expressed in hair follicles, where it has a critical role in hair growth by producing LPA through a novel LPA receptor called P2Y5.     

Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis

G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from several studies suggesting that GPCRs can form both homo- and heterodimers. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) approaches, we established that the vasopressin V1a, V2, and the oxytocin receptors exist as homo- and hetero-dimers in transfected human embryonic kidney 293T cells. Each receptor protomer had a similar propensity to form homo- and heterodimers, indicating that their relative expression levels may determine the homo-/heterodimer ratio. The finding that immature forms of the receptor can be immunoprecipitated as homo- and heterodimers and the detection by BRET of such oligomer in endoplasmic reticulum-enriched fractions suggest that the oligomerization processes take place early during biosynthesis. Treatment with agonists or antagonists did not modify the BRET among any of the vasopressin and oxytocin receptor pairs studied, indicating that the dimerization state of the receptors is not regulated by ligand binding once they have reached the cell surface. Taken together, these results strongly support the notion that GPCR dimerization is a constitutive process.     

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.     

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.     

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.     

Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate(S1P) are potent lipid growth factors with similar abilities tostimulate cytoskeleton-based cellular functions. Their effects aremediated by a subfamily of G protein-coupled receptors (GPCRs) encoded by endothelial differentiation genes (edgs). Wehypothesize that large quantities of LPA and S1P generated by activatedplatelets may influence endothelial cell functions. Using an in vitrowound healing assay, we observed that LPA and S1P stimulated closure ofwounded monolayers of human umbilical vein endothelial cells and adultbovine aortic endothelial cells, which express LPA receptor Edg2, andS1P receptors Edg1 and Edg3. The two major components of wound healing,cell migration and proliferation, were stimulated individually by bothlipids. LPA and S1P also stimulated intracellular Ca²⁺mobilization and mitogen-activated protein kinase (MAPK)phosphorylation. Pertussis toxin partially blocked the effects of bothlipids on endothelial cell migration, MAPK phosphorylation, andCa²⁺ mobilization, implicatingG_i/_o-coupled Edg receptor signaling inendothelial cells. LPA and S1P did not cross-desensitize each other inCa²⁺ responses, suggesting involvement of distinctreceptors. Thus LPA and S1P affect endothelial cell functions throughsignaling pathways activated by distinct GPCRs and may contribute tothe healing of wounded vasculatures.

     

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 Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are blood-borne lysophospholipids with a wide spectrum of biological activities, which include stimulation of cell growth, prevention of apoptosis, regulation of actin cytoskeleton, and modulation of cell shape, cell migration, and invasion. Activated platelets appear to be a major source of both S1P and LPA in blood. Despite the diversity of their biosynthetic origins, they are considered to share substantial structural similarity. Indeed, recent investigation has revealed that S1P and LPA act via a single family of G protein-coupled receptors designated as Edg. Thus, the Edg isoforms, Edg1 (also called S1P(1)), Edg5 (S1P(2)), Edg3 (S1P(3)), Edg6 (S1P(4)), and Edg8 (S1P(5)), are specific receptors for S1P (and SPC with a lower affinity), whereas Edg2 (LPA(1)), Edg4 (LPA(2)), and Edg7 (LPA(3)) serve as receptors specific for LPA. Each receptor isoform displays a unique tissue expression pattern and coupling to a distinct set of heterotrimeric G proteins, leading to the activation of an isoform-specific panel of multiple intracellular signaling pathways. Recent studies on knockout mice have unveiled non-redundant Edg receptor functions that are essential for normal development and vascular maturation. In addition, the Edg lysophospholipid signaling system may play a role in modulating cell motility under such pathological conditions as inflammation, tumor cell dissemination and vascular remodeling.     

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.     

Expression of the lysophospholipid receptor family and investigation of lysophospholipid-mediated responses in human macrophages

Some of the biological effects of lipoproteins have been attributed to their association with lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). These lysophospholipids mediate multiple biological responses via several G protein-coupled receptors (GPR). The expression of these receptors, however, has not been systematically investigated in primary human monocytes and macrophages as major cells involved in atherosclerosis. The mRNAs for all 15 receptors described so far were detected in monocytes, macrophages, foam cells and high density lipoprotein (HDL(3))-treated cells using real time RT-PCR. Immunoblots revealed that S1P(1), S1P(2), S1P(4), LPA(1), LPA(2) and GPR65 are expressed in monocytes and macrophages, while S1P(5) and LPA(3) have not been detected. S1P(3) was induced during differentiation but down-regulated by lipid-loading and HDL(3), whereas LPA(1) was down-regulated in differentiated macrophages. The influence of S1P on macrophages was investigated and the induction of CD32 indicates an enhanced phagocytic activity. Altogether, these data give insights into the expression and regulation of lysophospholipid receptors in primary human monocytes, macrophages and foam cells.     

In vivo roles of lysophospholipid receptors revealed by gene targeting studies in mice

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are extracellular ligands for a family of G protein-coupled receptors (GPCRs), LPA1/2/3 and S1P1/2/3/4/5. Through coupling to multiple classes of G proteins and activating multiple signaling pathways, LPA/S1P receptors have been shown to be integral players for many essential cellular and physiological processes. Generation and analysis of mice deficient in each of LPA1, LPA2, S1P1, S1P2, and S1P3 have provided valuable information on the in vivo roles of these receptors. This review is focussed on expression patterns of each receptor gene in wild-type mice, targeted deletion approaches for generating mutant animals, main phenotypes of receptor-null mice, and alterations in signaling characteristics in receptor-deficient primary cells. Altogether, these data give insights to the importance of LPA/S1P receptors at the cellular and organismal level.     

A single amino acid determines lysophospholipid specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P(1)/EDG1 and LPA(1)/EDG2, respectively. We have developed a model of LPA(1) that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA(1)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P(1) study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P(1) and LPA(1) validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA(1), producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors.     

EDG receptors and hepatic pathophysiology of LPA and S1P: EDG-ology of liver injury

The biological roles of phospholipid growth factors lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been broadly investigated. The cellular effects of LPA and S1P are mediated predominantly via endothelial differentiation gene (EDG) receptors. Yet, the biological significance of LPA, S1P and their EDG receptors in cells of the liver remains unclear. Recent data demonstrate the presence of EDG2 and EDG4 mRNA for LPA receptor in a murine hepatocyte cell line transformed with human TGF-alpha, and in primary mouse hepatocytes. EDG2 receptor protein is expressed in mouse liver, where it appears to be located in nonparenchymal cells. Moreover, we have obtained data suggesting that proliferation of small hepatocyte-progenitors and stem (oval) cells during liver injury is associated with the expression of EDG2 and EDG4 receptors. LPA, and possibly S1P, appear to be essential factors that control proliferation and motility of hepatic stellate cells (HSC) and hepatoma cells. It is proposed that LPA, S1P and their respective EDG receptors play important roles in pathophysiology of chronic liver injury and fibrogenesis. The underlying mechanisms recruited by LPA and S1P in pathogenesis of liver injury remain to be investigated.     

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.     

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.