What is the natural ligand of GPR55?

GPR55 is a seven transmembrane G protein-coupled receptor and was originally identified as a putative third cannabinoid receptor. Recently, lysophosphatidylinositol (LPI) was reported to be a GPR55 ligand. Stimulation of GPR55 by LPI activates G(12/13) and G(q/11) proteins, induces phosphorylation of the extracellular signal-regulated kinase and increases intracellular calcium concentration. Lysophospholipids are molecularly quite diverse across species and tissues. A recent report showed that the predominant fatty acyl moiety of LPI in rat brain is stearic acid followed by arachidonic acid. The biological activity of arachidonic acid-containing LPI species towards GPR55 was shown to be markedly higher than that of LPI species containing other fatty acyl groups, suggesting that 2-arachidonolyl LPI is the most likely natural ligand of GPR55.     

GPR55 and GPR35 and their relationship to cannabinoid and lysophospholipid receptors

This review presents a summary of what is known about the G-protein coupled receptors GPR35 and GPR55 and their potential characterization as lysophospholipid or cannabinoid receptors, respectively. Both GPR35 and GPR55 have been implicated as important targets in pain and cancer, and additional diseases as well. While kynurenic acid was suggested to be an endogenous ligand for GPR35, so was 2-arachidonoyl lysophosphatidic acid (LPA). Similarly, GPR55 has been suggested to be a cannabinoid receptor, but is quite clearly also a receptor for lysophosphatidylinositol. Interestingly, 2-arachidonyl glycerol (2-AG), an endogenous ligand for cannabinoid receptors, can be metabolized to 2-arachidonoyl LPA through the action of a monoacylglycerol kinase; the reverse reaction has also been demonstrated. Thus, it appears that mutual interconversion is possible between 2-arachidonoyl LPA and 2-AG within a cell, though the direction of the reaction may be site-dependent. The GPR55 natural ligand, 2-arachidonoyl LPI, can be degraded either to 2-AG by phospholipase C or to 2-arachidonoyl LPA by phospholipase D. Thus, GPR35, GPR55 and CB receptors are linked together through their natural ligand conversions. Additional agonists and antagonists have been identified for both GPR35 and GPR55, which will facilitate the future study of these receptors with respect to their physiological function. Potential therapeutic targets include pain, cancer, metabolic diseases and drug addiction.     

2-Arachidonoyl-sn-glycero-3-phosphate,an arachidonic acid-containing lysophosphatidic acid: occurrence and rapid enzymatic conversion to 2-arachidonoyl-sn-glycerol,a cannabinoid receptor ligand,in rat brain

A substantial amount of lysophosphatidic acid (LPA) (15.66 nmol/g tissue) was found to occur in the brain isolated from rats killed in liquid nitrogen. We found that a significant portion of brain LPA was accounted for by the arachidonic acid-containing species (5.4%). We obtained evidence that both 2-arachidonoyl species and 1-arachidonoyl species of LPA are present. The occurrence of 2-arachidonoyl LPA in the brain (0.53 nmol/g tissue) is a notable observation, because of its structural resemblance to 2-arachidonoyl-sn-glycerol (2-AG), an endogenous cannabinoid receptor ligand. We then examined the biological activity of 2-arachidonoyl LPA and compared it with that of 2-AG using neuroblastoma x glioma hybrid NG108-15 cells which express both the LPA receptor and cannabinoid CB1 receptor. We found that 2-arachidonoyl LPA interacts with the LPA receptor(s) to elicit the elevation of intracellular free Ca(2+) concentrations, whereas 2-AG interacts exclusively with the cannabinoid CB1 receptor. Next, we examined the possible metabolic relationship between 2-arachidonoyl LPA and 2-AG and obtained clear evidence that rapid enzymatic conversion of 2-arachidonoyl LPA to 2-AG took place in the brain homogenate. It is noteworthy that two types of endogenous ligands, that interact with different types of receptors, are closely related metabolically and rapidly interconvert.     

Lipid bilayer molecular dynamics study of lipid-derived agonists of the putative cannabinoid receptor, GPR55

Both L-α-lysophosphatidylinositol (LPI) and 2-arachidonoyl-sn-glycero-3-phosphoinositol (2-AGPI) have been reported to activate the putative cannabinoid receptor, GPR55. Recent microsecond time-scale molecular dynamics (MD) simulations and isothiocyanate covalent labeling studies have suggested that a transmembrane helix 6/7 (TMH6/7) lipid pathway for ligand entry may be necessary for interaction with cannabinoid receptors. Because LPI and 2-AGPI are lipid-derived ligands, conformations that each assumes in the lipid bilayer are therefore likely important for their interaction with GPR55. We report here the results of 70 ns NAMD molecular dynamics (MD) simulations of LPI and of 2-AGPI in a fully hydrated bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These simulations are compared with a 70 ns simulation of the cannabinoid CB1 receptor endogenous ligand, N-arachidonoylethanolamine (anandamide, AEA) in a POPC bilayer. These simulations revealed that (1) LPI and 2-AGPI sit much higher in the bilayer than AEA, with inositol headgroups that can at times be solvated completely by water; (2) the behavior of the acyl chains of AEA and 2-AGPI are similar in their flexibilities in the bilayer, while the acyl chain of LPI has reduced flexibility; and (3) both 2-AGPI and LPI can adopt a tilted headgroup orientation by hydrogen bonding to the phospholipid phosphate/glycerol groups or via intramolecular hydrogen bonding. This tilted head group conformation (which represents over 40% of the conformer population of LPI (42.2 ± 3.3%) and 2-AGPI (43.7 ± 1.4%)) may provide a low enough profile in the lipid bilayer for LPI and 2-AGPI to enter GPR55 via the putative TMH6/7 entry port.     

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a β-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.     

Lysophosphatidylinositol signalling: new wine from an old bottle

Lysophosphatidylinositol (LPI) is a bioactive lipid generated by phospholipase A2 which is believed to play an important role in several diseases. Indeed LPI can affect various functions such as cell growth, differentiation and motility, in a number of cell-types, including cancer cells, endothelial cells and nervous cells. Despite the fact that LPI-induced cellular functions had been known for more than twenty years, the recent discovery that in several cell-types the orphan G protein-coupled receptor GPR55 acts as the specific receptor for LPI has fuelled novel interest in this lysolipid. Different research groups, including our own, have recently suggested that LPI may be the specific and functional ligand for GPR55, triggering signalling cascades that are relevant to cell proliferation, migration, survival and tumourigenesis. Recently published data suggest that the LPI/GPR55 axis plays an important role in different physiological and pathological contexts. Here we review the available data supporting the role of LPI in cell signalling and the pharmacology of its putative receptor GPR55.     

Lysophosphatidylinositol causes neurite retraction via GPR55, G13 and RhoA in PC12 cells

GPR55 was recently identified as a putative receptor for certain cannabinoids, and lysophosphatidylinositol (LPI). Recently, the role of cannabinoids as GPR55 agonists has been disputed by a number of reports, in part, because studies investigating GPR55 often utilized overexpression systems, such as the GPR55-overexpressing HEK293 cells, which make it difficult to deduce the physiological role of endogenous GPR55. In the present study, we found that PC12 cells, a neural model cell line, express endogenous GPR55, and by using these cells, we were able to examine the role of endogenous GPR55. Although GPR55 mRNA and protein were expressed in PC12 cells, neither CB(1) nor CB(2) mRNA was expressed in these cells. GPR55 was predominantly localized on the plasma membrane in undifferentiated PC12 cells. However, GPR55 was also localized in the growth cones or the ruffled border in differentiated PC12 cells, suggesting a potential role for GPR55 in the regulation of neurite elongation. LPI increased intracellular Ca(2+) concentration and RhoA activity, and induced ERK1/2 phosphorylation, whereas endogenous and synthetic cannabinoids did not, thereby suggesting that cannabinoids are not GPR55 agonists. LPI also caused neurite retraction in a time-dependent manner accompanied by the loss of neurofilament light chain and redistribution of actin in PC12 cells differentiated by NGF. This LPI-induced neurite retraction was found to be G(q)-independent and G(13)-dependent. Furthermore, inactivation of RhoA function via C3 toxin and GPR55 siRNA knockdown prevented LPI-induced neurite retraction. These results suggest that LPI, and not cannabinoids, causes neurite retraction in differentiated PC12 cells via a GPR55, G(13) and RhoA signaling pathway.     

Thematic Review Series: Lysophospholipids and their Receptors: Novel lysophosphoplipid receptors: their structure and function

It is now accepted that lysophospholipids (LysoGPs) have a wide variety of functions as lipid mediators that are exerted through G protein-coupled receptors (GPCRs) specific to each lysophospholipid. While the roles of some LysoGPs, such as lysophosphatidic acid and sphingosine 1-phosphate, have been thoroughly examined, little is known about the roles of several other LysoGPs, such as lysophosphatidylserine (LysoPS), lysophosphatidylthreonine, lysophosphatidylethanolamine, lysophosphatidylinositol (LPI), and lysophosphatidylglycerol. Recently, a GPCR was found for LPI (GPR55) and three GPCRs (GPR34/LPS₁, P2Y10/LPS₂, and GPR174/LPS₃) were found for LysoPS. In this review, we focus on these newly identified GPCRs and summarize the actions of LysoPS and LPI as lipid mediators.     

Lysophosphatidylinositols in inflammation and macrophage activation: Altered levels and anti-inflammatory effects

Lysophosphatidylinositols (LPI) are bioactive lipids that are implicated in several pathophysiological processes such as cell proliferation, migration and tumorigenesis and were shown to play a role in obesity and metabolic disorders. Often, these effects of LPI were due to activation of the G protein-coupled receptor GPR55. However, the role of LPI and GPR55 in inflammation and macrophage activation remains unclear. Therefore, we thought to study the effect of macrophage activation and inflammation on LPI levels and metabolism. To do so, we used J774 and BV2 cells in culture activated with lipopolysaccharides (LPS, 100?ng/mL) as well as primary mouse alveolar and peritoneal macrophages. We also quantified LPI levels in the cerebellum, lung, liver, spleen and colon of mice with a systemic inflammation induced by LPS (300?μg/kg) and in the colon of mice with acute colitis induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) and chronic DSS-induced colitis.Our data show that LPS-induced macrophage activation leads to altered LPI levels in both the cells and culture medium. We also show that cytosolic phospholipase A2α (cPLA2α) and α/β?hydrolase domain 6 (ABHD6) are among the enzymes implicated in LPI metabolism in J774 macrophages. Indeed, ABHD6 and cPLA2α inhibition increased 20:4-LPI levels in LPS-activated macrophages. Furthermore, incubation of LPS-activated cells with LPI decreased J774 activation in a GPR55-dependent manner. In vivo, LPI levels were altered by inflammation in the liver, spleen and colon. These alterations are tissue dependent and could highlight a potential role for LPI in inflammatory processes.     

Generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in picrotoxinin-administered rat brain

The levels of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, and other molecular species of monoacylglycerols in rat brain were examined. In this study, we sacrificed the animals in liquid nitrogen to minimize postmortem changes. We found that rat brain contains 0.23 nmol/g tissue of 2-arachidonoylglycerol, which accounts for 10.5% of the total monoacylglycerol present in this tissue. We next investigated the level of 2-arachidonoylglycerol after in vivo stimulation with picrotoxinin. We found that the level of 2-arachidonoylglycerol was elevated markedly in picrotoxinin-administered rat brain (4- to 6-fold over the control level). Changes in the levels of other molecular species were relatively small or negligible. Several cannabimimetic molecules as well as Delta(9)-tetrahydrocannabinol are known to depress neurotransmission and to exert anticonvulsant activities; endogenous 2-arachidonoylglycerol produced during neural excitation may play a regulatory role in calming the enhanced synaptic transmission.     

Mechanisms of accumulation of arachidonate in phosphatidylinositol in yellowtail. A comparative study of acylation systems of phospholipids in rat and the fish species Seriola quinqueradiata.

It is known that phosphatidylinositol (PtdIns) contains abundant arachidonate and is composed mainly of 1-stearoyl-2-arachidonoyl species in mammals. We investigated if this characteristic of PtdIns applies to the PtdIns from yellowtail (Seriola quinqueradiata), a marine fish. In common with phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) from brain, heart, liver, spleen, kidney and ovary, the predominant polyunsaturated fatty acid was docosahexaenoic acid, and levels of arachidonic acid were less than 4.5% (PtdCho), 7.5% (PtdEtn) and 3.0% (PtdSer) in these tissues. In striking contrast, arachidonic acid made up 17.6%, 31.8%, 27.8%, 26.1%, 25.4% and 33.5% of the fatty acid composition of PtdIns from brain, heart, liver, spleen, kidney and ovary, respectively. The most abundant molecular species of PtdIns in all these tissues was 1-stearoyl-2-arachidonoyl. Assay of acyltransferase in liver microsomes of yellowtail showed that arachidonic acid was incorporated into PtdIns more effectively than docosahexaenoic acid and that the latter inhibited incorporation of arachidonic acid into PtdCho without inhibiting the utilization of arachidonic acid for PtdIns. This effect of docosahexaenoic acid was not observed in similar experiments using rat liver microsomes and is thought to contribute to the exclusive utilization of arachidonic acid for acylation to PtdIns in yellowtail. Inositolphospholipids and their hydrolysates are known to act as signaling molecules in cells. The conserved hydrophobic structure of PtdIns (the 1-stearoyl-2-arachidonoyl moiety) may have physiological significance not only in mammals but also in fish.     

Modulation of L-α-lysophosphatidylinositol/GPR55 mitogen-activated protein kinase (MAPK) signaling by cannabinoids

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB₁ receptor antagonist Rimonabant®, CB₂ receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB₁ receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB₂ ligand {"type":"entrez-nucleotide","attrs":{"text":"GW405833","term_id":"288331434","term_text":"GW405833"}}GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB₂ receptor ligands. The phytocannabinoids Δ⁹-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.     

Oleoyl-lysophosphatidylinositol enhances glucagon-like peptide-1 secretion from enteroendocrine L-cells through GPR119

The gastrointestinal tract is increasingly viewed as critical in controlling glucose metabolism, because of its role in secreting multiple glucoregulatory hormones, such as glucagon like peptide-1 (GLP-1). Here we investigate the molecular pathways behind the GLP-1- and insulin-secreting capabilities of a novel GPR119 agonist, Oleoyl-lysophosphatidylinositol (Oleoyl-LPI). Oleoyl-LPI is the only LPI species able to potently stimulate the release of GLP-1 in vitro, from murine and human L-cells, and ex-vivo from murine colonic primary cell preparations. Here we show that Oleoyl-LPI mediates GLP-1 secretion through GPR119 as this activity is ablated in cells lacking GPR119 and in colonic primary cell preparation from GPR119^?/? mice. Similarly, Oleoyl-LPI-mediated insulin secretion is impaired in islets isolated from GPR119^?/? mice. On the other hand, GLP-1 secretion is not impaired in cells lacking GPR55 in vitro or in colonic primary cell preparation from GPR55^?/? mice. We therefore conclude that GPR119 is the Oleoyl-LPI receptor, upstream of ERK1/2 and cAMP/PKA/CREB pathways, where primarily ERK1/2 is required for GLP-1 secretion, while CREB activation appears dispensable.     

Differential Activation of Cultured Neonatal Cardiomyocytes by Plasmalemmal Versus Intracellular G Protein-coupled Receptor 55

The L-α-lysophosphatidylinositol (LPI)-sensitive receptor GPR55 is coupled to Ca²⁺ signaling. Low levels of GPR55 expression in the heart have been reported. Similar to other G protein-coupled receptors involved in cardiac function, GPR55 may be expressed both at the sarcolemma and intracellularly. Thus, to explore the role of GPR55 in cardiomyocytes, we used calcium and voltage imaging and extracellular administration or intracellular microinjection of GPR55 ligands. We provide the first evidence that, in cultured neonatal ventricular myocytes, LPI triggers distinct signaling pathways via GPR55, depending on receptor localization. GPR55 activation at the sarcolemma elicits, on one hand, Ca²⁺ entry via L-type Ca²⁺ channels and, on the other, inositol 1,4,5-trisphosphate-dependent Ca²⁺ release. The latter signal is further amplified by Ca²⁺-induced Ca²⁺ release via ryanodine receptors. Conversely, activation of GPR55 at the membrane of intracellular organelles promotes Ca²⁺ release from acidic-like Ca²⁺ stores via the endolysosomal NAADP-sensitive two-pore channels. This response is similarly enhanced by Ca²⁺-induced Ca²⁺ release via ryanodine receptors. Extracellularly applied LPI produces Ca²⁺-independent membrane depolarization, whereas the Ca²⁺ signal induced by intracellular microinjection of LPI converges to hyperpolarization of the sarcolemma. Collectively, our findings point to GPR55 as a novel G protein-coupled receptor regulating cardiac function at two cellular sites. This work may serve as a platform for future studies exploring the potential of GPR55 as a therapeutic target in cardiac disorders.     

Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.     

Generation of lysophosphatidylinositol by DDHD domain containing 1 (DDHD1): Possible involvement of phospholipase D/phosphatidic acid in the activation of DDHD1

GPR55 is a seven-transmembrane G-protein-coupled receptor that has been proposed as a novel type of cannabinoid receptor. Previously, we identified lysophosphatidylinositol (LPI), in particular 2-arachidonoyl-LPI, as an agonist for GPR55. In the present study, we examined whether intracellular phospholipase A1 (DDHD domain containing 1, or DDHD1), previously identified as phosphatidic acid (PA)-preferring PLA1 (PA-PLA1), is involved in the formation of 2-arachidonoyl-LPI. HEK293 cells expressing DDHD1 produced [³H]arachidonic acid-containing LPI after prelabeling with [³H]arachidonic acid and subsequent activation by ionomycin; the formation of [³H]LPI was inhibited by n-butanol and the overexpression of an inactive PLD1 mutant PLD1K898R. DDHD1 was translocated from the cytosol to membranes upon ionomycin treatment. A purified recombinant DDHD1 formed [³H]LPI when incubated with [³H]PI; the V_max and apparent K_m were 190 µmol/min/mg protein and 10 mol% PI, respectively. DDHD1 binds PA, and the addition of PA to DDHD1 increased the affinity for PI (K_m ; 3 mol%) and augmented the PI-PLA1 activity. DDHD1 activated by PA was returned to a basal state by its own PA-hydrolytic activity. These results implicate DDHD1 in the formation of 2-arachidonoyl-LPI and indicate that the process is modulated by PA released by phospholipase D. Similar observations for the production of arachidonic acid-containing LPI in neuroblastoma cells suggest the DDHD1-LPI-GPR55 axis to be involved in functions in the brain.     

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

Bovine brain microsomal CDP-diacylglycerol synthetase: solubilization and properties.

CDP-diacylglycerol(DAG) synthetase (EC 2.7.7.41) has been solubilized from bovine brain microsomes by the detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate). Optimal solubilization with 1.5% CHAPS yielded 55-60% of the synthetase activity. The effect of CHAPS on the enzyme was biphasic inhibiting at 0.3% and giving maximal activity at 0.5% (the concentration used for all assays). The solubilized, but not the microsomal enzyme is activated by phosphatidylcholine (PC) and strongly inhibited by cardiolipin and lysoPC. Strong inhibition by N-ethylmaleimide, 5,5'-dithio-bis (2-nitrobenzoic acid) and p-chloromercuribenzoate supported a sulfhydryl requirement for the enzyme. Phosphatidic acid (PA) from egg lecithin and 1-stearoyl,2-arachidonoyl PA were preferred substrates for the microsomal synthetase. Solubilized synthetase showed selectivity for the latter PA which is consistent with this enzyme functioning to help form the preponderant 1-stearoyl,2-arachidonoyl species of phosphatidylinositol. Further attempts to purify the synthetase were unsuccessful. All findings suggested the enzyme exists as an unstable complex.     

The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor

Nucleotides and cysteinyl-leukotrienes (CysLTs) are unrelated signaling molecules inducing multiple effects through separate G-protein-coupled receptors: the P2Y and the CysLT receptors. Here we show that GPR17, a Gi-coupled orphan receptor at intermediate phylogenetic position between P2Y and CysLT receptors, is specifically activated by both families of endogenous ligands, leading to both adenylyl cyclase inhibition and intracellular calcium increases. Agonist-response profile, as determined by [(35)S]GTPgammaS binding, was different from that of already known CysLT and P2Y receptors, with EC(50) values in the nanomolar and micromolar range, for CysLTs and uracil nucleotides, respectively. Both rat and human receptors are highly expressed in the organs typically undergoing ischemic damage, that is, brain, heart and kidney. In vivo inhibition of GPR17 by either CysLT/P2Y receptor antagonists or antisense technology dramatically reduced ischemic damage in a rat focal ischemia model, suggesting GPR17 as the common molecular target mediating brain damage by nucleotides and CysLTs. In conclusion, the deorphanization of GPR17 revealed a dualistic receptor for two endogenous unrelated ligand families. These findings may lead to dualistic drugs of previously unexplored therapeutic potential.     

GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.