GPR109a agonists. Part 2: Pyrazole-acids as agonists of the human orphan G-protein coupled receptor GPR109a

5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.     

GPR109a agonists. Part 1: 5-Alkyl and 5-aryl-pyrazole–tetrazoles as agonists of the human orphan G-protein coupled receptor GPR109a

5-Alkyl and aryl-pyrazole–tetrazoles have been identified as a new class of selective, small-molecule, agonists of the human G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.     

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.     

Identification of lysophospholipid receptors in human platelets: the relation of two agonists, lysophosphatidic acid and sphingosine 1-phosphate

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca(2+) mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets.     

Identification of a protein hydrolysate responsive G protein-coupled receptor in enterocytes

G protein-coupled receptors (GPCRs) have the potential to play a role as molecular sensors responsive to luminal dietary contents. Although such a role for GPCRs has been implicated in the intestinal response to protein hydrolysate, no GPCR directly involved in this process has been previously identified. In the present study, for the first time, we identified GPR93 expression in enterocytes and demonstrated its activation in these cells by protein hydrolysate with EC50 of 10.6 mg/ml as determined by the induction of intracellular free Ca2+. In enterocytes, GPR93 was synergistically activated by protein hydrolysate in combination with an agonist, oleoyl-l-alpha-lysophosphatidic acid (LPA), which activated the receptor in these enterocytes with EC50 of 7.9 nM. The increased intracellular Ca2+ by GPR93 activation was observed without the addition of a promiscuous Galpha protein and was pertussis toxin sensitive, which suggests Galpha(q)- and Galpha(i)-mediated pathways. Activated GPR93 also induced pertussis toxin-sensitive ERK1/2 phosphorylation. Both nuclear factor of activated T cells and 12-O-tetradecanoylphorbol 13-acetate responsive elements reporter activities were induced by protein hydrolysate in cells exogenously expressing GPR93. The peptidomimetic cefaclor by itself did not activate GPR93 but potentiated the protein hydrolysate response and further amplified the synergistic enhancement of GPR93 activation by protein hydrolysate and LPA. These data suggest that, physiologically, the composition of stimuli might determine GPR93 activity or its sensitivity toward a given activator and suggest a new mechanism of the regulation of mucosal cell proliferation and differentiation and hormonal secretion by dietary products in the lumen.     

Identification of p2y9/GPR23 as a novel G protein-coupled receptor for lysophosphatidic acid,structurally distant from the Edg family

Lysophosphatidic acid (LPA) is a bioactive lipid mediator with diverse physiological and pathological actions on many types of cells. LPA has been widely considered to elicit its biological functions through three types of G protein-coupled receptors, Edg-2 (endothelial cell differentiation gene-2)/LPA1/vzg-1 (ventricular zone gene-1), Edg-4/LPA2, and Edg-7/LPA3. We identified an orphan G protein-coupled receptor, p2y9/GPR23, as the fourth LPA receptor (LPA4). Membrane fractions of RH7777 cells transiently expressing p2y9/GPR23 displayed a specific binding for 1-oleoyl-LPA with a Kd value of around 45 nm. Competition binding and reporter gene assays showed that p2y9/GPR23 preferred structural analogs of LPA with a rank order of 1-oleoyl- > 1-stearoyl- > 1-palmitoyl- > 1-myristoyl- > 1-alkyl- > 1-alkenyl-LPA. In Chinese hamster ovary cells expressing p2y9/GPR23, 1-oleoyl-LPA induced an increase in intracellular Ca2+ concentration and stimulated adenylyl cyclase activity. Quantitative real-time PCR demonstrated that mRNA of p2y9/GPR23 was significantly abundant in ovary compared with other tissues. Interestingly, p2y9/GPR23 shares only 20-24% amino acid identities with Edg-2/LPA1, Edg-4/LPA2, and Edg-7/LPA3, and phylogenetic analysis also shows that p2y9/GPR23 is far distant from the Edg family. These facts suggest that p2y9/GPR23 has evolved from different ancestor sequences from the Edg family.     

Role of LPA4/p2y9/GPR23 in negative regulation of cell motility

Lysophosphatidic acid (LPA) is a ligand of multiple G protein–coupled receptors. The LPA_1–3 receptors are members of the endothelial cell differentiation gene (Edg) family. LPA₄/p2y9/GPR23, a member of the purinergic receptor family, and recently identified LPA₅/GPR92 and p2y5 are structurally distant from the canonical Edg LPA receptors. Here we report targeted disruption of lpa₄ in mice. Although LPA₄-deficient mice displayed no apparent abnormalities, LPA₄-deficient mouse embryonic fibroblasts (MEFs) were hypersensitive to LPA-induced cell migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase pathway by LPA₄, LPA₄ deficiency potentiated Akt and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA₄ converted LPA₄-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA₄ strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA₁ in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA₄ attenuated LPA₁-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA₄ is a suppressor of LPA-dependent cell migration and invasion in contrast to the motility-stimulating Edg LPA receptors.     

Homo- and hetero-dimerization of LPA/S1P receptors, OGR1 and GPR4

G protein coupled receptors (GPCRs) form homo- and hetero-dimers or -oligomers, which are functionally important. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophopholipids involved in diverse biological processes. We have examined homo- and hetero-dimerization among three major LPA receptors (LPA(1-3)), three major S1P receptors (S1P(1-3)), as well as OGR1 and GPR4. Using LacZ complementation assays, we have shown that LPA receptors form homo- and hetero-dimers within the LPA receptor subgroup and hetero-dimers with other receptors (S1P(1-3) and GPR4). In addition, we have found that although GPR4 and OGR1 share more than 50% homology, GPR4 forms strong homo- and hetero-dimers with LPA and S1P receptors, but OGR1 forms very weak homo-dimer and relatively weak hetero-dimers with other receptors. Using chimeric receptors between GPR4 and OGR1, we have shown that different domains of GPR4 receptor are involved in its dimerization with different GPCRs and more than one domain may be involved in some of the complex formation. Our results suggest that when studying a signal transduction induced by a stimulus, not only is the expression and activation of its own receptor(s), but also the status of the interacting receptors should be taken into consideration.     

LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor

Lysophosphatidic acid (LPA), a lipid mediator enriched in serum, stimulates cell migration, proliferation and other functions in many cell types. LPA acts on six known G protein-coupled receptors, termed LPA(1-6), showing both overlapping and distinct signaling properties. Here we show that, unexpectedly, LPA and serum almost completely inhibit the transwell migration of B16 melanoma cells, with alkyl-LPA(18:1) being 10-fold more potent than acyl-LPA(18:1). The anti-migratory response to LPA is highly polarized and dependent on protein kinase A (PKA) but not Rho kinase activity; it is associated with a rapid increase in intracellular cAMP levels and PIP3 depletion from the plasma membrane. B16 cells express LPA(2), LPA(5) and LPA(6) receptors. We show that LPA-induced chemorepulsion is mediated specifically by the alkyl-LPA-preferring LPA(5) receptor (GPR92), which raises intracellular cAMP via a noncanonical pathway. Our results define LPA(5) as an anti-migratory receptor and they implicate the cAMP-PKA pathway, along with reduced PIP3 signaling, as an effector of chemorepulsion in B16 melanoma cells.     

Lysophosphatidic acid 5 receptor induces activation of Na(+)/H(+) exchanger 3 via apical epidermal growth factor receptor in intestinal epithelial cells

Na(+) absorption is a vital process present in all living organisms. We have reported previously that lysophosphatidic acid (LPA) acutely stimulates Na(+) and fluid absorption in human intestinal epithelial cells and mouse intestine by stimulation of Na(+)/H(+) exchanger 3 (NHE3) via LPA(5) receptor. In the current study, we investigated the mechanism of NHE3 activation by LPA(5) in Caco-2bbe cells. LPA(5)-dependent activation of NHE3 was blocked by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and U0126, but not by phosphatidylinositol 3-kinase inhibitor LY294002 or phospholipase C-β inhibitor U73122. We found that LPA(5) transactivated the epidermal growth factor receptor (EGFR) and that inhibition of EGFR blocked LPA(5)-dependent activation of NHE3, suggesting an obligatory role of EGFR in the NHE3 regulation. Confocal immunofluorescence and surface biotinylation analyses showed that LPA(5) was located mostly in the apical membrane. EGFR, on the other hand, showed higher expression in the basolateral membrane. However, inhibition of apical EGFR, but not basolateral EGFR, abrogated LPA-induced regulation of MEK and NHE3, indicating that LPA(5) selectively activates apical EGFR. Furthermore, transactivation of EGFR independently activated the MEK-ERK pathway and proline-rich tyrosine kinase 2 (Pyk2). Similarly to MEK inhibition, knockdown of Pyk2 blocked activation of NHE3 by LPA. Furthermore, we showed that RhoA and Rho-associated kinase (ROCK) are involved in activation of Pyk2. Interestingly, LPA(5) did not directly activate RhoA but was required for transactivation of EGFR. Together, these results unveil a pivotal role of apical EGFR in NHE3 regulation by LPA and show that the RhoA-ROCK-Pyk2 and MEK-ERK pathways converge onto NHE3.     

Thrombogenic and atherogenic activities of lysophosphatidic acid

Lysophosphatidic acid (LPA) has been identified as a biologically active lipid in mildly-oxidized LDL, human atherosclerotic lesions, and the supernatant of activated platelets. The evidence that LPA has thrombogenic and atherogenic activities has increased substantially in recent years. Supporting the thrombogenic activity of LPA, analysis of the core region of human carotid plaques revealed recently the presence of alkyl- and acyl-molecular species from LPA with high platelet-activating potency (16:0 alkyl-LPA, 20:4 acyl-LPA). LPA, lipid extracts of atherosclerotic plaques, and the lipid-rich core elicited shape change and, in synergy with other platelet stimuli, aggregation of isolated platelets. This effect was completely abrogated by prior incubation of platelets with LPA receptor antagonists. Furthermore, LPA at concentrations approaching those found in vivo, induced platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. LPA-stimulated platelet aggregation was mediated by the ADP-stimulated activation of the P2Y(1) and P2Y(12) receptors. Supporting its atherogenic activity, LPA is a mitogen and motogen to vascular smooth muscle cells (VSMCs) and an activator of endothelial cells and macrophages. Recently, LPA has been identified as an agonist of the peroxisome proliferator activating receptor gamma (PPARgamma), which is a key regulator of atherogenesis. LPA elicits progressive neointima formation, which is fully abolished by GW9662, an antagonist of PPARgamma. We propose that LPA plays a central role in eliciting vascular remodeling and atherogenesis. Furthermore, upon rupture of lipid-rich atherosclerotic plaques, LPA may trigger platelet aggregation and intra-arterial thrombus formation. Antagonists of LPA receptors might be useful in preventing LPA-elicited thrombus formation and neointima formation in patients with cardiovascular diseases.     

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that d-erythro-SPC, but not l-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 microM, and increased the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca(2+)](i) were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca(2+)](i). However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425.These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced proliferation.     

Lysophosphatidic acid inhibits ghrelin secretion in the human gastric adenocarcinoma AGS cell line: role of mitogenic activated protein kinase signaling pathway

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor type 1a (GHS-R1a), is a 28 amino acid residue with a post-translational octanoyl modification on Ser3. Despite the biomedical interest in this hormone, the fine details of its regulation and the mechanisms controlling its secretion are largely unknown. The present study analyzes the molecular steps involved in the full lysophosphatidic acid (LPA) receptor-mediated activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and its consequent role as an inhibitor of ghrelin secretion in the gastric adenocarcinoma cell line AGS. ERK1/2 phosphorylation mediated by LPA proceeds via activation of the type 2 LPA receptor, activation of the nonreceptor tyrosine kinase c-Src, and subsequent transactivation of the epidermal growth factor receptor. Furthermore, LPA-induced ERK activation was found to be independent of matrix metalloproteinases; thus, c-Src acted as the scaffold-transactivating epidermal growth factor receptor. Finally, a correlation was observed between the mitogenic effects of LPA and ghrelin secretion in the human gastric adenocarcinoma cell line AGS. These data suggest a possible physiological role of LPA in ghrelin secretion. The relationship found between LPA and ghrelin secretion might explain the low circulating levels of ghrelin observed in obese patients, as a bona fide reflex of the energetic stores.     

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.     

Hydrolysis of phosphatidylserine-exposing red blood cells by secretory phospholipase A2 generates lysophosphatidic acid and results in vascular dysfunction

Secretory phospholipase A(2) (sPLA(2)) type IIa, elevated in inflammation, breaks down membrane phospholipids and generates arachidonic acid. We hypothesized that sPLA(2) will hydrolyze red blood cells that expose phosphatidylserine (PS) and generate lysophosphatidic acid (LPA) from phosphatidic acid that is elevated in PS-exposing red blood cells. In turn, LPA, a powerful lipid mediator, could affect vascular endothelial cell function. Although normal red blood cells were not affected by sPLA(2), at levels of sPLA(2) observed under inflammatory conditions (100 ng/ml) PS-exposing red blood cells hemolyzed and generated LPA (1.2 nM/10(8) RBC). When endothelial cell monolayers were incubated in vitro with LPA, a loss of confluence was noted. Moreover, a dose-dependent increase in hydraulic conductivity was identified in rat mesenteric venules in vivo with 5 microM LPA, and the combination of PS-exposing red blood cells with PLA(2) caused a similar increase in permeability. In the presence of N-palmitoyl L-serine phosphoric acid, a competitive inhibitor for the endothelial LPA receptor, loss of confluence in vitro and the hydraulic permeability caused by 5 microM LPA in vivo were abolished. The present study demonstrates that increased sPLA(2) activity in inflammation in the presence of cells that have lost their membrane phospholipid asymmetry can lead to LPA-mediated endothelial dysfunction and loss of vascular integrity.