G Protein-coupled Receptor 4 Research Articles

CNS distribution, signalling properties and central effects of G-protein coupled receptor 4

Information on the distribution and biology of the G-protein coupled receptor 4 (GPR4) in the brain is limited. It is currently thought that GPR4 couples to Gs proteins and may mediate central respiratory sensitivity to CO2. Using a knock-in mouse model, abundant GPR4 expression was detected in the cerebrovascular endothelium and neurones of dorsal raphe, retro-trapezoidal nucleus locus coeruleus and lateral septum. A similar distribution was confirmed using RNAscope in situ hybridisation. In HEK293 cells, overexpressing GPR4, it was highly constitutively active at neutral pH with little further increase in cAMP towards acidic pH. The GPR4 antagonist NE 52-QQ57 effectively blocked GPR4-mediated cAMP accumulation (IC50 26.8 nM in HEK293 cells). In HUVEC which natively express GPR4, physiological acidification (pH 7.4–7.0) resulted in a cAMP increase by ∼55% which was completely prevented by 1 μM NE 52-QQ57. The main extracellular organic acid, l-lactic acid (LL; 1–10 mM), suppressed pH dependent activation of GPR4 in HEK293 and HUVEC cells, suggesting allosteric negative modulation. In unanaesthetised mice and rats, NE 52-QQ57 (20 mg kg−1) reduced ventilatory response to 5 and 10% CO2. In anaesthetised rats, systemic administration of NE 52-QQ57 (up to 20 mg kg−1) had no effect on hemodynamics, cerebral blood flow and blood oxygen level dependent responses. Central administration of NE 52-QQ57 (1 mM) in vagotomised anaesthetised rats did not affect CO2-induced respiratory responses. Our results indicate that GPR4 is expressed by multiple neuronal populations and endothelium and that its pH sensitivity is affected by level of expression and LL. NE 52-QQ57 blunts hypercapnic response to CO2 but this effect is absent under anaesthesia, possibly due to the inhibitory effect of LL on GPR4.

GPR4 knockout improves renal ischemia-reperfusion injury and inhibits apoptosis via suppressing the expression of CHOP.

The aim of the present study was to investigate the effects and molecular mechanisms of GPR4 (G-protein-coupled receptor 4) in cell apoptosis and renal ischemia-reperfusion (IR) injury in vivo and in vitro GPR4-/- mice and wild-type (WT) mice underwent renal IR or sham procedures. For hypoxia/reoxygenation (HR), human umbilical vein endothelial cells (HUVECs) were subjected to 4 h of hypoxia, followed by 6 h of reoxygenation. Renal histological changes were observed by periodic acid-Schiff staining and myeloperoxidase activity. Apoptosis was detected by TUNEL staining. GPR4, C/EBP-homologous protein (CHOP) and cleaved caspase-3 protein expressions were detected by western blot. Both GPR4 and CHOP were up-regulated after renal IR in mice. GPR4-knockout mice had significantly less renal damage and decreased TUNEL-positive cells than WT controls after IR. Bone marrow chimeras demonstrated that it was due to the GPR4 inactivation in renal parenchymal cells. Moreover, GPR4 was mainly expressed in endothelial cells after renal IR. GPR4 knockdown markedly inhibited CHOP expression and cell apoptosis in the HUVECs after HR treatment. GPR4 blockade attenuated renal injury after IR and reduced the cell apoptosis through the suppression of CHOP expression.

Abstract 1993: Acidic tumor microenvironment stimulation of GPR4 alters cytoskeletal dynamics and migration of vascular endothelial cells

Abstract The acidic tumor microenvironment results from aberrant vasculature, insufficient oxygen delivery and aerobic glycolysis of cancer cells. As cancer progresses, the acidic microenvironment can lead to structural changes of endothelial cells (ECs) resulting in increased vascular permeability which may increase metastasis. Extracellular acidification can activate cellular signaling molecules, including G-protein coupled receptor 4 (GPR4), which is prominently expressed in ECs. We have previously reported that acidic activation of GPR4 induces the transcription of several pro-inflammatory and ER-stress related genes in ECs. The purpose of the study is to identify the functional response of acidosis-induced GPR4 activity using Human Umbilical Vein Endothelial Cells (HUVECs) as a model system. The expression of GPR4 was overexpressed (HUVEC/GPR4) or knocked down (GPR4 shRNA) using several genetic constructs. HUVECs were treated with media buffered to pH 6.4 or to physiological pH 7.4. Permeability of a HUVEC cell monolayer was assessed by quantifying gap formation as one indicator. Cell migration was assessed through wound-healing assays. Cytoskeletal dynamics were also investigated using Rhodamine Phalloidin to investigate actin stress fiber formation and immunocytochemistry to investigate focal adhesion dynamics. Our results showed acidic pH induced GPR4 activation changed the cytoskeletal phenotype, decreased cellular migration, and proliferation in HUVECs compared to the physiological pH treatment. There was also a further increase in gap area of a HUVEC/GPR4 monolayer and formation of actin stress fibers in response to acidic conditions. Additionally, altering expression of phosphorylated paxillin and focal adhesion kinase were observed under acidosis/GPR4 mediated stress. In conclusion, the proton sensor GPR4 induces cytoskeletal variations, reduces cellular migration, and proliferation in response to an acidic tumor microenvironment. Citation Format: Elizabeth A. Krewson, Li V. Yang, Lixue Dong. Acidic tumor microenvironment stimulation of GPR4 alters cytoskeletal dynamics and migration of vascular endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1993. doi:10.1158/1538-7445.AM2017-1993

Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the “Warburg effect”), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4-induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

Human GPR4 and the Notch signaling pathway in endothelial cell tube formation.

G protein-coupled receptor4 (GPR4) is hypothesized to function as a pH sensor and is important in the regulation of proliferation, migration and angiogenesis of vascular endothelial cells (ECs). Furthermore, the Notch signaling pathway is significant in the regulation of the angiogenic behavior of ECs. However, whether GPR4 regulates angiogenesis via the Notch signaling pathway remains unclear. The present study evaluated the effect of Notch signaling in human GPR4‑induced angiogenesis in HMEC‑1 cells. The results revealed that GPR4 increased Notch1 expression in a time‑dependent manner. In addition, the inhibition of Notch1 expression using small interfering RNA or the Notch receptor inhibitor, γ-secretase inhibitorI, significantly blocked GPR4‑induced HMEC‑1 tube formation and lymphocyte transendothelial migration. Furthermore, the inhibition of Notch1 blocked GPR4‑induced vascular endothelial growth factor and hypoxia-inducible factor1α expression. Thus, it was demonstrated that GPR4 affects ECs by regulating Notch1, a function that may be important for physiological and pathological angiogenesis.

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses.

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

Acidic pH increases cGMP accumulation through the OGR1/phospholipase C/Ca2 +/neuronal NOS pathway in N1E-115 neuronal cells

Neuronal NO synthase (nNOS)-mediated cGMP accumulation has been shown to affect a variety of neuronal cell activities, regardless of whether they are detrimental or beneficial, depending on the amount of their levels, under the physiological and pathological situations. In the present study, we examined the role of proton-sensing G protein-coupled receptors (GPCRs), which have been identified as new pH sensors, in the acidic pH-induced nNOS/cGMP activity in N1E-115 neuronal cells. In this cell line, ovarian cancer G protein-coupled receptor 1 (OGR1) and G protein-coupled receptor 4 (GPR4) mRNAs are expressed. An extracellular acidic pH increased cGMP accumulation, which was inhibited by nNOS-specific inhibitors. Acidic pH also activated phospholipase C/Ca2+ pathways and Akt-induced phosphorylation of nNOS at S1412, both of which have been shown to be critical regulatory mechanisms for nNOS activation. The acidic pH-induced activation of the phospholipase C/Ca2+ pathway, but not Akt/nNOS phosphorylation, was inhibited by small interfering RNA specific to OGR1 and YM-254890, an inhibitor of Gq/11 proteins, in association with the inhibition of cGMP accumulation. Moreover cGMP accumulation was inhibited by 2-aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate channel; however, it was not by wortmannin, a phosphatidylinositol 3-kinase inhibitor, which inhibited Akt/nNOS phosphorylation. In conclusion, acidic pH stimulates cGMP accumulation preferentially through the OGR1/Gq/11 proteins/phospholipase C/Ca2+/nNOS in N1E-115 neuronal cells. Akt-mediated phosphorylation of nNOS, however, does not appreciably contribute to the acidification-induced accumulation of cGMP.

Relations between GPR4 Expression, Microvascular Density (MVD) and Clinical Pathological Characteristics of Patients with Epithelial Ovarian Carcinoma (EOC)

G-protein coupled receptor 4 (GPR4) is a G protein-coupled receptor (GPCR) activated by sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC). Later studies indicated that GPR4 can serve as a proton sensor. GPR4 has been known to play a critical role in the tube formation of vascular endothelial cells, and GPR4 overexpression is observed in various types of malignancies, suggesting its involvement in the cancer-related angiogenesis. In this study, we examined the GPR4 expression levels in blood vessels of ovarian cancer, and analyzed the relationship between GPR4 expression and the clinical and pathological characteristics of patients with epithelial ovarian carcinomas (EOC). Results from immunohistochemistry showed that GPR4 is detectable in the endothelium of vessels of both EOC and benign ovarian tumor tissue, but the expression levels were significantly increased in EOC. Moreover the increased expression is accompanied by a higher microvascular density (MVD) in EOC compared to that in the benign ovarian tumors. We demonstrated a positive correlation between GPR4 expression density and MVD in EOC, but not benign ovarian tumor tissues. Further analyses indicated that GPR4 expression and MVD in EOC were correlated to the status of lymph node metastasis and clinical stage, but not significantly correlated to the pathological classifications, histopathological grades, the amounts of ascites, status of peritoneal cytology, tumor sizes, or patients' ages. These results suggested that GPR4 may play an important role in the development of EOC, and its overexpression might be required for the angiogenesis, tumor growth, and metastasis of EOC.

Characterization of the myometrial transcriptome in women with an arrest of dilatation during labor.

The molecular basis of failure to progress in labor is poorly understood. This study was undertaken to characterize the myometrial transcriptome of patients with an arrest of dilatation (AODIL). Human myometrium was prospectively collected from women in the following groups: (1) spontaneous term labor (TL; n=29) and (2) arrest of dilatation (AODIL; n=14). Gene expression was characterized using Illumina® HumanHT-12 microarrays. A moderated Student's t-test and false discovery rate adjustment were used for analysis. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of selected genes was performed in an independent sample set. Pathway analysis was performed on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database using Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). The MetaCore knowledge base was also searched for pathway analysis. (1) Forty-two differentially expressed genes were identified in women with an AODIL; (2) gene ontology analysis indicated enrichment of biological processes, which included regulation of angiogenesis, response to hypoxia, inflammatory response, and chemokine-mediated signaling pathway. Enriched molecular functions included transcription repressor activity, heat shock protein (Hsp) 90 binding, and nitric oxide synthase (NOS) activity; (3) MetaCore analysis identified immune response chemokine (C-C motif) ligand 2 (CCL2) signaling, muscle contraction regulation of endothelial nitric oxide synthase (eNOS) activity in endothelial cells, and triiodothyronine and thyroxine signaling as significantly overrepresented (false discovery rate <0.05); (4) qRT-PCR confirmed the overexpression of Nitric oxide synthase 3 (NOS3); hypoxic ischemic factor 1A (HIF1A); Chemokine (C-C motif) ligand 2 (CCL2); angiopoietin-like 4 (ANGPTL4); ADAM metallopeptidase with thrombospondin type 1, motif 9 (ADAMTS9); G protein-coupled receptor 4 (GPR4); metallothionein 1A (MT1A); MT2A; and selectin E (SELE) in an AODIL. The myometrium of women with AODIL has a stereotypic transcriptome profile. This disorder has been associated with a pattern of gene expression involved in muscle contraction, an inflammatory response, and hypoxia. This is the first comprehensive and unbiased examination of the molecular basis of an AODIL.

Activation of T cell death-associated gene 8 regulates the cytokine production of T cells and macrophages in vitro

An orphan G-protein-coupled receptor, T cell death-associated gene 8 (TDAG8) which has been reported to be a proton sensor, inhibits the production of pro-inflammatory cytokines induced by extracellular acidification. Recently, we have found that TDAG8 knockout mice showed significant exacerbation in various immune-mediated inflammation disease models. To elucidate the role of TDAG8, we screened an in-house library to find compounds which have a profile as a TDAG8 agonist using a cyclic adenosine 5′-monophosphate assay. Among the screening hits, we focused on (3-[(2,4-dichlorobenzyl)thio]-1,6-dimethyl-5,6-dihydro-1H-pyridazino[4,5-e][1,3,4]thiadiazin-5-one) (named BTB09089). BTB09089 did not act on other proton sensing G-protein-coupled receptors such as G-protein-coupled receptor 4 (GPR4) nor ovarian cancer G-protein-coupled receptor 1 (OGR1). Moreover, BTB09089 increased cAMP level in the splenocytes from wild-type littermates but not from TDAG8-deficient mice. Thus, BTB09089 was found to be a TDAG8 specific agonist. We then investigated the effects of BTB09089 on T cells and macrophages in vitro. In splenocytes, BTB09089 suppressed the production of IL-2 stimulated with anti-CD3 and anti-CD28 antibodies. In peritoneal exuded macrophages induced by thioglycollate, BTB09089 suppressed the production of TNF-α and IL-6 while it increased that of IL-10 when stimulated with lipopolysaccharide. These effects were observed in cells from wild type mice, but not those from TDAG8 knockout mice. These results indicate that activation of TDAG8 attenuates immune-mediated inflammation by regulating the cytokine production of T cells and macrophages.

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 ([Ca 2+] i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca 2+] 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 [Ca 2+] 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 [Ca 2+] 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.

Differential proton sensitivity of related G protein-coupled receptors T cell death-associated gene 8 and G2A expressed in immune cells

G2A, T cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) form a group of structurally related G protein-coupled receptors (GPCRs) originally proposed to bind proinflammatory lipids. More recent studies have challenged the identification of lipid agonists for these GPCRs and have suggested that they function primarily as proton sensors. We compared the ability of these four receptors to modulate pH-dependent responses by using transiently transfected cell lines. In accordance with previously published reports, OGR1 was found to evoke strong pH-dependent responses as measured by inositol phosphate accumulation. We also confirmed the pH-dependent cAMP production by GPR4 and TDAG8. However, we found the activity of the human G2A receptor and its mouse homolog to be significantly less sensitive to pH fluctuations as measured by inositol phosphate and cAMP accumulation. Sequence homology analysis indicated that, with one exception, the histidine residues that were previously shown to be important for pH sensing by OGR1, GPR4, and TDAG8 were not conserved in the G2A receptor. We further addressed the pH-sensing properties of G2A and TDAG8 in a cellular context where these receptors are coexpressed. In thymocytes and splenocytes explanted from receptor-deficient mice, TDAG8 was found to be critical for pH-dependent cAMP production. In contrast, G2A was found to be dispensable for this process. We conclude that members of this GPCR group exhibit differential sensitivity to extracellular protons, and that expression of TDAG8 by immune cells may regulate responses in acidic microenvironments.

Screening of FGF target genes in Xenopus by microarray: temporal dissection of the signalling pathway using a chemical inhibitor.

Microarray is a powerful tool for analysing gene expression patterns in genome-wide view and has greatly contributed to our understanding of spatiotemporal embryonic development at the molecular level. Members of FGF (fibroblast growth factor) family play important roles in embryogenesis, e.g. in organogenesis, proliferation, differentiation, cell migration, angiogenesis, and wound healing. To dissect spatiotemporally the versatile roles of FGF during embryogenesis, we profiled gene expression in Xenopus embryo explants treated with SU5402, a chemical inhibitor specific to FGF receptor 1 (FGFR1), by microarray. We identified 38 genes that were down-regulated and 5 that were up-regulated in response to SU5402 treatment from stage 10.5-11.5 and confirmed their FGF-dependent transcription with RT-PCR analysis and whole-mount in situ hybridization (WISH). Among the 43 genes, we identified 26 as encoding novel proteins and investigated their spatial expression pattern by WISH. Genes whose expression patterns were similar to FGFR1 were further analysed to test whether any of them represented functional FGF target molecules. Here, we report two interesting genes: one is a component of the canonical Ras-MAPK pathway, similar to mammalian mig6 (mitogen-inducible gene 6) acting in muscle differentiation; the other, similar to GPCR4 (G-protein coupled receptor 4), is a promising candidate for a gastrulation movement regulator. These results demonstrate that our approach is a promising strategy for scanning the genes that are essential for the regulation of a diverse array of developmental processes.

Feeling It in Your Bones: Sensing Acidosis

The pH of blood, which is tightly maintained at ~pH 7.4, is primarily controlled through the regulation of acid excretion by the kidneys and carbon dioxide loss through the lungs. Additionally, the bones, which store bicarbonate, act to buffer metabolic acidosis. Osteoblasts respond to physiological changes in pH; the mechanisms whereby they sense these changes, however, remain unclear. Ludwig et al. found that cell lines (CCL39 hamster fibroblasts and HEK293 cells) transfected with ovarian cancer G protein-coupled receptor 1 (OGR1) displayed strongly pH-dependent production of inositol phosphate (IP). OGR1 is a heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) that was previously identified as a receptor for sphingosylphosphorylcholine. After transfection with OGR1, IP production was half-maximal at pH 7.48 and maximal at pH 6.8. Mutational analysis indicated that pH sensitivity depended on five extracellular histidine residues. Copper and zinc, which are coordinated by histidine pairs and would be expected to stabilize them in an unprotonated state, inhibited OGR1- and pH-dependent stimulation of IP production. A closely related receptor, GPR4 (G protein-coupled receptor 4), displayed pH-dependent production of cyclic adenosine monophosphate. The authors used reverse-transcription polymerase chain reaction (RT-PCR) to show that both a human osteosarcoma cell line and primary human preosteoblasts expressed OGR1 mRNA and immunohistochemistry to demonstrate OGR1 protein in osteoblasts and osteocytes in rat bone. Both osteosarcoma cells and preosteoblasts exhibited pH-sensitive IP production. These data suggest that OGR1 and GPR4 can act as pH sensors and that OGR1 may play a role in regulating the cell-mediated responses of bone to metabolic acidosis. M.-G. Ludwig, M. Vanek, D. Guerini, J. A. Gasser, C. E. Jones, U. Junker, H. Hofstetter, R. M. Wolf, K. Seuwen, Proton-sensing G-protein-coupled receptors. Nature 425 , 93-98 (2003). [Online Journal]

Feeling It in Your Bones: Sensing Acidosis

The pH of blood, which is tightly maintained at ~pH 7.4, is primarily controlled through the regulation of acid excretion by the kidneys and carbon dioxide loss through the lungs. Additionally, the bones, which store bicarbonate, act to buffer metabolic acidosis. Osteoblasts respond to physiological changes in pH; the mechanisms whereby they sense these changes, however, remain unclear. Ludwig et al. found that cell lines (CCL39 hamster fibroblasts and HEK293 cells) transfected with ovarian cancer G protein-coupled receptor 1 (OGR1) displayed strongly pH-dependent production of inositol phosphate (IP). OGR1 is a heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) that was previously identified as a receptor for sphingosylphosphorylcholine. After transfection with OGR1, IP production was half-maximal at pH 7.48 and maximal at pH 6.8. Mutational analysis indicated that pH sensitivity depended on five extracellular histidine residues. Copper and zinc, which are coordinated by histidine pairs and would be expected to stabilize them in an unprotonated state, inhibited OGR1- and pH-dependent stimulation of IP production. A closely related receptor, GPR4 (G protein-coupled receptor 4), displayed pH-dependent production of cyclic adenosine monophosphate. The authors used reverse-transcription polymerase chain reaction (RT-PCR) to show that both a human osteosarcoma cell line and primary human preosteoblasts expressed OGR1 mRNA and immunohistochemistry to demonstrate OGR1 protein in osteoblasts and osteocytes in rat bone. Both osteosarcoma cells and preosteoblasts exhibited pH-sensitive IP production. These data suggest that OGR1 and GPR4 can act as pH sensors and that OGR1 may play a role in regulating the cell-mediated responses of bone to metabolic acidosis.M.-G. Ludwig, M. Vanek, D. Guerini, J. A. Gasser, C. E. Jones, U. Junker, H. Hofstetter, R. M. Wolf, K. Seuwen, Proton-sensing G-protein-coupled receptors. Nature 425, 93-98 (2003). [Online Journal]