Inhibition of TRPM8 function by prostacyclin receptor agonists requires coupling to Gq/11 proteins.

BACKGROUND AND PURPOSE: The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI2) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors. EXPERIMENTAL APPROACH: We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890. KEY RESULTS: Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation.

The antimalarial artemisinin is a non-electrophilic agonist of the transient receptor potential ankyrin type 1 receptor-channel.

Artemisinin and its derivatives are the main therapeutic drugs against Plasmodium protists, the causative agents of malaria. While several putative mechanisms of action have been proposed, the precise molecular targets of these compounds have not been fully elucidated. In addition to their antimalarial properties, artemisinins have been reported to act as anti-tumour agents and certain antinociceptive effects have also been proposed. We investigated the effect of the parent compound, artemisinin, on a number of temperature-gated Transient Receptor Potential ion channels (so called thermoTRPs), given their demonstrated roles in pain-sensing and cancer. We report that artemisinin acts as an agonist of the Transient Receptor Potential Ankyrin type 1 (TRPA1) receptor channel. Artemisinin was able to evoke calcium transients in HEK293T cells expressing recombinant human TRPA1, as well as in a subpopulation of mouse dorsal root ganglion (DRG) neurons which also responded to the selective TRPA1 agonist allyl isothiocyanate (AITC) and these responses were reversibly abolished by the selective TRPA1 antagonist A967079. Artemisinin also triggered whole-cell currents in HEK293T cells transiently transfected with human TRPA1, as well as in TRPA1-expressing DRG neurons, and these currents were inhibited by A967079. Interestingly, using human TRPA1 mutants, we demonstrate that artemisinin acts as a non-electrophilic agonist of TRPA1, activating the channel in a similar manner to carvacrol and menthol. These results may provide a better understanding of the biological actions of the very important antimalarial and anti-tumour agent artemisinin.

Nicotinamide Limits Replication of Mycobacterium tuberculosis and Bacille Calmette-Guérin Within Macrophages.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

A whole organism small molecule screen identifies novel regulators of pancreatic endocrine development.

An early step in pancreas development is marked by the expression of the transcription factor Pdx1 within the pancreatic endoderm, where it is required for the specification of all endocrine cell types. Subsequently, Pdx1 expression becomes restricted to the ß-cell lineage, where it plays a central role in ß-cell function. This pivotal role of Pdx1 at various stages of pancreas development makes it an attractive target to enhance pancreatic ß-cell differentiation and increase ß-cell function. In this study, we used a newly generated zebrafish reporter to screen over 8000 small molecules for modulators of pdx1 expression. We found four hit compounds and validated their efficacy at different stages of pancreas development. Notably, valproic acid treatment increased pancreatic endoderm formation, while inhibition of TGFß signaling led to α-cell to ß-cell transdifferentiation. HC toxin, another HDAC inhibitor, enhances ß-cell function in primary mouse and human islets. Thus, using a whole organism screening strategy, this study identified new pdx1 expression modulators that can be used to influence different steps in pancreas and ß-cell development.

20-HETE promotes glucose-stimulated insulin secretion in an autocrine manner through FFAR1.

The long-chain fatty acid receptor FFAR1 is highly expressed in pancreatic ß-cells. Synthetic FFAR1 agonists can be used as antidiabetic drugs to promote glucose-stimulated insulin secretion (GSIS). However, the physiological role of FFAR1 in ß-cells remains poorly understood. Here we show that 20-HETE activates FFAR1 and promotes GSIS via FFAR1 with higher potency and efficacy than dietary fatty acids such as palmitic, linoleic, and α-linolenic acid. Murine and human ß-cells produce 20-HETE, and the ω-hydroxylase-mediated formation and release of 20-HETE is strongly stimulated by glucose. Pharmacological inhibition of 20-HETE formation and blockade of FFAR1 in islets inhibits GSIS. In islets from type-2 diabetic humans and mice, glucose-stimulated 20-HETE formation and 20-HETE-dependent stimulation of GSIS are strongly reduced. We show that 20-HETE is an FFAR1 agonist, which functions as an autocrine positive feed-forward regulator of GSIS, and that a reduced glucose-induced 20-HETE formation contributes to inefficient GSIS in type-2 diabetes.

The leukotriene B4 receptors BLT1 and BLT2 form an antagonistic sensitizing system in peripheral sensory neurons.

Sensitization of the heat-activated ion channel transient receptor potential vanilloid 1 (TRPV1) through lipids is a fundamental mechanism during inflammation-induced peripheral sensitization. Leukotriene B4 is a proinflammatory lipid mediator whose role in peripheral nociceptive sensitization is not well understood to date. Two major G-protein-coupled receptors for leukotriene B4 have been identified: the high-affinity receptor BLT1 and the low-affinity receptor BLT2. Transcriptional screening for the expression G-protein-coupled receptors in murine dorsal root ganglia showed that both receptors were among the highest expressed in dorsal root ganglia. Calcium imaging revealed a sensitization of TRPV1-mediated calcium increases in a relative narrow concentration range for leukotriene B4 (100-200 nm). Selective antagonists and neurons from knock-out mice demonstrated a BLT1-dependent sensitization of TRPV1-mediated calcium increases. Accordingly, leukotriene B4-induced thermal hyperalgesia was mediated through BLT1 and TRPV1 as shown using the respective knock-out mice. Importantly, higher leukotriene B4 concentrations (>0.5 µm) and BLT2 agonists abolished sensitization of the TRPV1-mediated calcium increases. Also, BLT2 activation inhibited protein kinase C- and protein kinase A-mediated sensitization processes through the phosphatase calcineurin. Consequently, a selective BLT2-receptor agonist increased thermal and mechanical withdrawal thresholds during zymosan-induced inflammation. In accordance with these data, immunohistochemical analysis showed that both leukotriene B4 receptors were expressed in peripheral sensory neurons. Thus, the data show that the two leukotriene B4 receptors have opposing roles in the sensitization of peripheral sensory neurons forming a self-restricting system.

The G2A receptor (GPR132) contributes to oxaliplatin-induced mechanical pain hypersensitivity.

Chemotherapy-induced peripheral neuropathic pain (CIPN) is a common and severe debilitating side effect of many widely used cytostatics. However, there is no approved pharmacological treatment for CIPN available. Among other substances, oxaliplatin causes CIPN in up to 80% of treated patients. Here, we report the involvement of the G-protein coupled receptor G2A (GPR132) in oxaliplatin-induced neuropathic pain in mice. We found that mice deficient in the G2A-receptor show decreased mechanical hypersensitivity after oxaliplatin treatment. Lipid ligands of G2A were found in increased concentrations in the sciatic nerve and dorsal root ganglia of oxaliplatin treated mice. Calcium imaging and patch-clamp experiments show that G2A activation sensitizes the ligand-gated ion channel TRPV1 in sensory neurons via activation of PKC. Based on these findings, we conclude that targeting G2A may be a promising approach to reduce oxaliplatin-induced TRPV1-sensitization and the hyperexcitability of sensory neurons and thereby to reduce pain in patients treated with this chemotherapeutic agent.

Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.

19(S)-hydroxy-eicosatetraenoic acid (19(S)-HETE) belongs to a family of arachidonic acid metabolites produced by cytochrome P450 enzymes, which play critical roles in the regulation of cardiovascular, renal and pulmonary functions. Although it has been known for a long time that 19(S)-HETE has vascular effects, its mechanism of action has remained unclear. In this study we show that 19(S)-HETE induces cAMP accumulation in the human megakaryoblastic leukemia cell line MEG-01. This effect was concentration-dependent with an EC50 of 520 nM, insensitive to pharmacological inhibition of COX-1/2 and required the expression of the G-protein Gs. Systematic siRNA-mediated knock-down of each G-protein coupled receptor (GPCR) expressed in MEG-01 followed by functional analysis identified the prostacyclin receptor (IP) as the mediator of the effects of 19(S)-HETE, and the heterologously expressed IP receptor was also activated by 19(S)-HETE in a concentration-dependent manner with an EC50 of 567 nM. Pretreatment of isolated murine platelets with 19(S)-HETE blocked thrombin-induced platelets aggregation, an effect not seen in platelets from mice lacking the IP receptor. Furthermore, 19(S)-HETE was able to relax mouse mesenteric artery- and thoracic aorta-derived vessel segments. While pharmacological inhibition of COX-1/2 enzymes had no effect on the vasodilatory activity of 19(S)-HETE these effects were not observed in vessels from mice lacking the IP receptor. These results identify a novel mechanism of action for the CYP450-dependent arachidonic acid metabolite 19(S)-HETE and point to the existence of a broader spectrum of naturally occurring prostanoid receptor agonists.

Loss of FFA2 and FFA3 increases insulin secretion and improves glucose tolerance in type 2 diabetes.

Type 2 diabetes is a major health problem worldwide, and one of its key features is the inability of elevated glucose to stimulate the release of sufficient amounts of insulin from pancreatic beta cells to maintain normal blood glucose levels. New therapeutic strategies to improve beta cell function are therefore believed to be beneficial. Here we demonstrate that the short-chain fatty acid receptors FFA2 (encoded by FFAR2) and FFA3 (encoded by FFAR3) are expressed in mouse and human pancreatic beta cells and mediate an inhibition of insulin secretion by coupling to Gi-type G proteins. We also provide evidence that mice with dietary-induced obesity and type 2 diabetes, as compared to non-obese control mice, have increased local formation by pancreatic islets of acetate, an endogenous agonist of FFA2 and FFA3, as well as increased systemic levels. This elevation may contribute to the insufficient capacity of beta cells to respond to hyperglycemia in obese states. Indeed, we found that genetic deletion of both receptors, either on the whole-body level or specifically in pancreatic beta cells, leads to greater insulin secretion and a profound improvement of glucose tolerance when mice are on a high-fat diet compared to controls. On the other hand, deletion of Ffar2 and Ffar3 in intestinal cells did not alter glucose tolerance in diabetic animals, suggesting these receptors act in a cell-autonomous manner in beta cells to regulate insulin secretion. In summary, under diabetic conditions elevated acetate acts on FFA2 and FFA3 to inhibit proper glucose-stimulated insulin secretion, and we expect antagonists of FFA2 and FFA3 to improve insulin secretion in type 2 diabetes.

Conserved MIP receptor-ligand pair regulates Platynereis larval settlement.

Life-cycle transitions connecting larval and juvenile stages in metazoans are orchestrated by neuroendocrine signals including neuropeptides and hormones. In marine invertebrate life cycles, which often consist of planktonic larval and benthic adult stages, settlement of the free-swimming larva to the sea floor in response to environmental cues is a key life cycle transition. Settlement is regulated by a specialized sensory-neurosecretory system, the larval apical organ. The neuroendocrine mechanisms through which the apical organ transduces environmental cues into behavioral responses during settlement are not yet understood. Here we show that myoinhibitory peptide (MIP)/allatostatin-B, a pleiotropic neuropeptide widespread among protostomes, regulates larval settlement in the marine annelid Platynereis dumerilii. MIP is expressed in chemosensory-neurosecretory cells in the annelid larval apical organ and signals to its receptor, an orthologue of the Drosophila sex peptide receptor, expressed in neighboring apical organ cells. We demonstrate by morpholino-mediated knockdown that MIP signals via this receptor to trigger settlement. These results reveal a role for a conserved MIP receptor-ligand pair in regulating marine annelid settlement.

Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.

Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects.

Nicotinic acid (niacin): new lipid-independent mechanisms of action and therapeutic potentials.

Nicotinic acid (niacin) has been used for decades to prevent and treat atherosclerosis. The well-documented antiatherogenic activity is believed to result from its antidyslipidemic effects, which are accompanied by unwanted effects, especially a flush. There has been renewed interest in nicotinic acid owing to the need for improved prevention of atherosclerosis in patients already taking statins. In addition, the identification of a nicotinic acid receptor expressed in adipocytes and immune cells has helped to elucidate the mechanisms underlying the antiatherosclerotic as well as the unwanted effects of this drug. Nicotinic acid exerts its antiatherosclerotic effects at least in part independently of its antidyslipidemic effects through mechanisms involving its receptor on immune cells as well as through direct and indirect effects on the vascular endothelium. Here, we review recent data on the pharmacological effects of nicotinic acid and discuss how they might be harnessed to treat other inflammatory diseases such as multiple sclerosis or psoriasis.

Nicotinic acid- and monomethyl fumarate-induced flushing involves GPR109A expressed by keratinocytes and COX-2-dependent prostanoid formation in mice.

The antidyslipidemic drug nicotinic acid and the antipsoriatic drug monomethyl fumarate induce cutaneous flushing through activation of G protein-coupled receptor 109A (GPR109A). Flushing is a troublesome side effect of nicotinic acid, but may be a direct reflection of the wanted effects of monomethyl fumarate. Here we analyzed the mechanisms underlying GPR109A-mediated flushing and show that both Langerhans cells and keratinocytes express GPR109A in mice. Using cell ablation approaches and transgenic cell type-specific GPR109A expression in Gpr109a-/- mice, we have provided evidence that the early phase of flushing depends on GPR109A expressed on Langerhans cells, whereas the late phase is mediated by GPR109A expressed on keratinocytes. Interestingly, the first phase of flushing was blocked by a selective cyclooxygenase-1 (COX-1) inhibitor, and the late phase was sensitive to a selective COX-2 inhibitor. Both monomethyl fumarate and nicotinic acid induced PGE2 formation in isolated keratinocytes through activation of GPR109A and COX-2. Thus, the early and late phases of the GPR109A-mediated cutaneous flushing reaction involve different epidermal cell types and prostanoid-forming enzymes. These data will help to guide new efficient approaches to mitigate nicotinic acid-induced flushing and may help to exploit the potential antipsoriatic effects of GPR109A agonists in the skin.

An autocrine lactate loop mediates insulin-dependent inhibition of lipolysis through GPR81.

Lactate is an important metabolic intermediate released by skeletal muscle and other organs including the adipose tissue, which converts glucose into lactate under the influence of insulin. Here we show that lactate activates the G protein-coupled receptor GPR81, which is expressed in adipocytes and mediates antilipolytic effects through G(i)-dependent inhibition of adenylyl cyclase. Using GPR81-deficient mice, we demonstrate that the receptor is not involved in the regulation of lipolysis during intensive exercise. However, insulin-induced inhibition of lipolysis and insulin-induced decrease in adipocyte cAMP levels were strongly reduced in mice lacking GPR81, although insulin-dependent release of lactate by adipocytes was comparable between wild-type and GPR81-deficient mice. Thus, lactate and its receptor GPR81 unexpectedly function in an autocrine and paracrine loop to mediate insulin-induced antilipolytic effects. These data show that lactate can directly modulate metabolic processes in a hormone-like manner, and they reveal a new mechanism underlying the antilipolytic effects of insulin.

GPR109A, GPR109B and GPR81, a family of hydroxy-carboxylic acid receptors.

G-protein-coupled receptors (GPCRs) are the most versatile receptor family as they have the ability to respond to chemically diverse ligands. Despite intensive efforts during the past two decades, there are still more than 100 orphan GPCRs for which endogenous ligands are unknown. Recently, GPR109A, GPR109B and GPR81, which form a GPCR subfamily, have been deorphanized. The physiological ligands of these receptors are the ketone body 3-hydroxy-butyrate, the metabolite 2-hydroxy-propanoate (lactate) as well as the beta-oxidation intermediate 3-hydroxy-octanoate. Thus, this receptor subfamily is activated by hydroxy-carboxylic acid ligands which are intermediates of energy metabolism. All three receptors are predominantly expressed in adipocytes and mediate antilipolytic effects. In this article, we propose that the hydroxy-carboxylic acid structure of their endogenous ligands is the defining property of this receptor subfamily and that hydroxy-carboxylic acid receptors function as metabolic sensors which fine-tune the regulation of metabolic pathways.

Deorphanization of GPR109B as a receptor for the beta-oxidation intermediate 3-OH-octanoic acid and its role in the regulation of lipolysis.

The orphan G-protein-coupled receptor GPR109B is the result of a recent gene duplication of the nicotinic acid and ketone body receptor GPR109A being found in humans but not in rodents. Like GPR109A, GPR109B is predominantly expressed in adipocytes and is supposed to mediate antilipolytic effects. Here we show that GPR109B serves as a receptor for the beta-oxidation intermediate 3-OH-octanoic acid, which has antilipolytic activity on human but not on murine adipocytes. GPR109B is coupled to Gi-type G-proteins and is activated by 2- and 3-OH-octanoic acid with EC50 values of about 4 and 8 microM, respectively. Interestingly, 3-OH-octanoic acid plasma concentrations reach micromolar concentrations under conditions of increased beta-oxidation rates, like in diabetic ketoacidosis or under a ketogenic diet. These data suggest that the ligand receptor pair 3-OH-octanoic acid/GPR109B mediates in humans a negative feedback regulation of adipocyte lipolysis to counteract prolipolytic influences under conditions of physiological or pathological increases in beta-oxidation rates.

Thyrocyte-specific Gq/G11 deficiency impairs thyroid function and prevents goiter development.

The function of the adult thyroid is regulated by thyroid-stimulating hormone (TSH), which acts through a G protein-coupled receptor. Overactivation of the TSH receptor results in hyperthyroidism and goiter. The Gs-mediated stimulation of adenylyl cyclase-dependent cAMP formation has been regarded as the principal intracellular signaling mechanism mediating the action of TSH. Here we show that the Gq/G11-mediated signaling pathway plays an unexpected and essential role in the regulation of thyroid function. Mice lacking the alpha subunits of Gq and G11 specifically in thyroid epithelial cells showed severely reduced iodine organification and thyroid hormone secretion in response to TSH, and many developed hypothyroidism within months after birth. In addition, thyrocyte-specific Galphaq/Galpha11-deficient mice lacked the normal proliferative thyroid response to TSH or goitrogenic diet, indicating an essential role of this pathway in the adaptive growth of the thyroid gland. Our data suggest that Gq/G11 and their downstream effectors are promising targets to interfere with increased thyroid function and growth.

Characterization of determinants of ligand binding to the nicotinic acid receptor GPR109A (HM74A/PUMA-G).

The G-protein-coupled receptor GPR109A (HM74A/PUMA-G) has recently been shown to function as a receptor for nicotinic acid (niacin) and to mediate its antilipolytic effects. Nicotinic acid is able to strongly raise plasma levels of high-density lipoprotein cholesterol, a property that distinguishes nicotinic acid from other lipid-lowering drugs. To investigate the structural determinants of GPR109A ligand binding, we performed site-directed mutagenesis of putative ligand binding residues combined with generation of chimeric receptors consisting of GPR109A and its close relative GPR109B, which does not bind nicotinic acid. We could identify Asn86/Trp91 [transmembrane helix (TMH) 2/extracellular loop (ECL) 1], Arg111 (TMH3), Ser178 (ECL2), Phe276 (TMH7), and Tyr284 (TMH7) as amino acid residues critical for binding of nicotinic acid. Together with data from molecular modeling studies, our data suggest that the ligand binding pocket for nicotinic acid of GPR109A is distinct from that of most other group A receptors. Although Arg111 at TMH3 serves as the basic anchor point for the carboxylate ligands, the ring system of nicotinic acid is embedded between Trp91 at the junction TMH2/ECL1 and Phe276/Tyr284 at TMH7. The heterocyclic ring is also bound to Ser178 at ECL2 via an H-bond. These data will facilitate the design of new antidyslipidemic drugs acting via GPR109A.

Comparative analysis of functional assays for characterization of agonist ligands at G protein-coupled receptors.

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P(5) receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPgammaS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca(2+) via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC(50) values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPgammaS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPgammaS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays.

Sphingosine 1-phosphate and dioleoylphosphatidic acid are low affinity agonists for the orphan receptor GPR63.

Five high affinity G-protein-coupled receptors for sphingosine 1-phosphate (S1P) have been characterised so far (S1P(1,2,3,4,5) formerly referred to as edg1,5,3,6,8). In this study, we show that S1P, dihydro-sphingosine 1-phosphate (dihydro-S1P) and dioleoylphosphatidic acid (doPA) are agonists for the orphan receptor GPR63. All three phospholipids mobilise intracellular calcium in CHO cells transiently transfected with GPR63. Calcium signals required cotransfection of a chimeric Galpha(q/i) protein in a fluorometric imaging plate reader (FLIPR) assay but did not require overexpressed G proteins in an aequorin assay, using a green fluorescent protein (GFP)-aequorin fusion protein as a bioluminescent Ca(2+) reporter. GPR63 expression in CHO cells confers proliferative responses to S1P in a pertussis toxin (PTX)-insensitive manner. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) indicated highest expression in brain, especially in the thalamus and the nucleus caudatus. In peripheral tissues, highest expression was observed in thymus, stomach and small intestine; lower abundance of transcripts was detected in kidney, spleen, pancreas and heart. The discovery that S1P, dihydro-S1P and dioleoylphosphatidic acid activate GPR63 will facilitate the identification of agonists and antagonists, and help to unravel the biological function of this receptor.