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Purpose G-protein coupled receptor (GPR 34) has been found to play important roles in some cancers and regulates the proliferation, apoptosis, and migration of these cancer cells. However, the mechanisms underlying how GPR34 functions to regulate growth and proliferation of colorectal cancer cells remains to be clarified. Methods We employed stable GPR34 knockdown LS174T cell models, GPR34 Mab blocking, a CCK-8 kit, and a colony formation assay to characterize the effect of GPR34 on the proliferation of LS174T in vitro and xenograft tumor growth in vivo. The mRNA level of GPR34 was detected by RT-PCR in tumor tissues and adjacent normal tissues from 34 CRC patients. Results Based on RT-PCR results, GPR34 exhibited high level in tumor samples compared with adjacent normal samples. Increased expression of GPR34 is more associated with poor prognosis of CRC as shown in The Cancer Genome Atlas (TCGA) dataset by Kaplan–Meier survival analysis. Furthermore, we showed that GPR34 knockdown inhibited the proliferation of LS174T colon cancer cells and related xenograft tumor growth. Searching for the distinct molecular mechanism, we identified several contributors to proliferation of LS174T colon cancer cells: PI3K subunits/PTEN, PDK1/AKT, and Src/Raf/Ras/ERK. GPR34 knockdown inhibited the proliferation of LS174T cells by upregulating expression of PTEN, and downregulating expression of PI3K subunits p110-beta. Conclusion Our findings provide direct evidence that GPR34 regulates the proliferation of LS174T cells and the growth of LS174T tumor xenografts by regulating different pathways. High expression of GPR34 mRNA could then be used to predict poor prognosis of CRC.

Membrane phospholipid metabolism forms lysophospholipids, which possess unique biochemical and biophysical properties that influence membrane structure and dynamics. However, lysophospholipids also function as ligands for G-protein–coupled receptors that influence embryonic development, postnatal physiology, and disease. The 2 most well-studied species—lysophosphatidic acid and S1P (sphingosine 1-phosphate)—are particularly relevant to vascular development, physiology, and cardiovascular diseases. This review summarizes the role of lysophosphatidic acid and S1P in vascular developmental processes, endothelial cell biology, and their roles in cardiovascular disease processes. In addition, we also point out the apparent connections between lysophospholipid biology and the Wnt (int/wingless family) pathway, an evolutionarily conserved fundamental developmental signaling system. The discovery that components of the lysophospholipid signaling system are key genetic determinants of cardiovascular disease has warranted current and future research in this field. As pharmacological approaches to modulate lysophospholipid signaling have entered the clinical sphere, new findings in this field promise to influence novel therapeutic strategies in cardiovascular diseases.

GPR34 translocation and mutation are specifically associated with salivary gland MALT lymphoma (SG-MALT-lymphoma). The majority of GPR34 mutations are clustered in its C-terminus, resulting in truncated proteins lacking the phosphorylation motif important for receptor desensitization. It is unclear why GPR34 genetic changes associate with SG-MALT-lymphoma and how these mutations contribute to the development of lymphoma. We generated isogenic Flp-InTRex293 cell lines that stably expressed a single copy of GPR34 or its various mutants and performed a range of in vitro assays. We found that the GPR34 Q340X truncation, but not the R84H and D151A mutants, conferred a significantly increased resistance to apoptosis and greater transforming potential than the GPR34 wild type. The GPR34 truncation mutant had a significantly delayed internalization compared with the wild type after ligand (lysophosphatidylserine) stimulation. Among the 9 signaling pathways examined, the GPR34 Q340X truncation, and to a lesser extent the D151A mutant, significantly activated CRE, NF-κB, and AP1 reporter activities, particularly in the presence of ligand stimulation. We further described the enhanced activities of phospholipase-A1/2 in the culture supernatant of Flp-InTRex293 cells that expressed the GPR34 Q340X mutant, as well as their potential to catalyze the synthesis of lysophosphatidylserine from phosphatidylserine. Importantly, phospholipase-A1 was abundantly expressed in the duct epithelium of salivary glands and those involved in lymphoepithelial lesions (LELs). Our findings advocate a model of paracrine stimulation of malignant B cells via GPR34, in which phospholipase A is released by LELs and hydrolyzes the phosphatidylserine exposed on apoptotic cells, generating lysophosphatidylserine, the ligand for GPR34. Thus, GPR34 activation potentially bridges LELs to genesis of SG-MALT-lymphoma.

Lysophosphatidylserine (LysoPS), an endogenous ligand of G protein-coupled receptors, consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages, respectively. An ester linkage of phosphatidylserine can be hydrolyzed at the 1-position or at the 2-position to give 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. 2-Acyl lysophospholipid is in nonenzymatic equilibrium with 1-acyl lysophospholipid in vivo. On the other hand, 3-acyl lysophospholipid is not found, at least in mammals, raising the question of whether the reason for this might be that the 3-acyl isomer lacks the biological activities of the other isomers. Here, to test this idea, we designed and synthesized a series of new 3-acyl lysophospholipids. Structure-activity relationship studies of more than 100 "glycol surrogate" derivatives led to the identification of potent and selective agonists for LysoPS receptors GPR34 and P2Y10. Thus, the non-natural 3-acyl compounds are indeed active and appear to be biologically orthogonal with respect to the physiologically relevant 1- and 2-acyl lysophospholipids.

When lipid mediators bind to G-protein-coupled receptors (GPCRs), the ligand first enters the lipid bilayer, then diffuses laterally in the cell membrane to make hydrophobic contact with the receptor protein, and finally enters the receptor's binding pocket. In this process, the location of the hydrophobic contact point on the surface of the receptor has been little discussed even in cases in which the crystal structure has been determined, because the ligand binding pocket is buried inside the transmembrane (TM) domains. Here, we coupled an activator ligand to a series of membrane phospholipid surrogates, which constrain the depth of entry of the ligand into the lipid bilayer. Consequently, via measurement of the receptor-activating activity as a function of the depth of entry into the membrane, these surrogates can be used as molecular rulers to estimate the location of the hydrophobic contact point on the surface of GPCR. We focused on lysophosphatidylserine (LysoPS) receptor GPR34 and prepared a series of simplified membrane-lipid-surrogate-conjugated lysophospholipid analogues by attaching alkoxy amine chains of varying lengths to the hydrophobic tail of a potent GPR34 agonist. As expected, the activity of these lipid-conjugated LysoPS analogues was dependent on chain length. The predicted contact position matches the position of the terminal benzene ring of a nonlipidic ligand that protrudes between TMs 4 and 5 of the receptor. We further found that the nature of the terminal hydrophilic functional group of the conjugated membrane lipid surrogate strongly influences the activity, suggesting that lateral hydrophilic contact of LysoPS analogues with the receptor's surface is also crucial for ligand-GPCR binding.

Lysophospholipids (LPLs), particularly sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA), are bioactive lipid modulators of cellular homeostasis and pathology. The discovery and characterization of five S1P- and six LPA-specific G protein-coupled receptors (GPCRs), S1P(1–5) and LPA(1–6), has expanded their known involvement in all mammalian physiological systems. Resolution of the S1P(1), LPA(1), and LPA(6) crystal structures has fueled the growing interest in these receptors and their ligands as targets for pharmacological manipulation. In this review, we have attempted to provide an integrated overview of the three crystallized LPL GPCRs with biochemical and physiological structure-function data. Finally, we provide a novel discussion of how chaperones for LPLs may be considered when extrapolating crystallographic and computational data towards understanding actual biological interactions and phenotypes.

Abundance of G protein-coupled receptor 34 (GPR34) mRNA is greater in granulosa cells (GC) of cystic vs. normal follicles of cattle. The present experiments were designed to determine if GPR34 mRNA in GC changes during selection and growth of dominant follicles in cattle as well as to investigate the hormonal regulation of GPR34 mRNA in bovine GC in vitro. In Exp. 1, estrous cycles of non-lactating cows were synchronized and then ovariectomized on either day 3-4 or 5-6 after ovulation. GPR34 mRNA abundance in GC was 2.8- to 3.8-fold greater (P < 0.05) in small (1–5 mm) and large (≥ 8 mm) estrogen-inactive dominant follicles than in large estrogen-active follicles. Also, GPR34 mRNA tended to be greater (P < 0.10) in F2 than F1 follicles on day 3-4 post-ovulation. In Exp. 2–7, ovaries were collected at an abattoir and GC were isolated and treated in vitro. Expression of GPR34 was increased (P < 0.05) 2.2-fold by IGF1. Tumor necrosis factor (TNF)-α decreased (P < 0.05) the IGF1-induced GPR34 mRNA abundance in small-follicle GC, whereas IGF1 decreased (P < 0.05) GPR34 expression by 45% in large-follicle GC. Treatment of small-follicle GC with either IL-2, prostaglandin E2 or angiogenin decreased (P < 0.05) GPR34 expression whereas FSH, cortisol, wingless 3A or hedgehog proteins did not affect (P > 0.10) GPR34 expression. In Exp. 6 and 7, two presumed ligands of GPR34, L-a-lysophosphatidylserine (LPPS) and LPP-ethanolamine, increased (P < 0.05) GC numbers and estradiol production by twofold or more in small-follicle GC, and this response was only observed in IGF1-treated GC. In conclusion, GPR34 is a developmentally and hormonally regulated gene in GC, and its presumed ligands enhance IGF1-induced proliferation and steroidogenesis of bovine GC.

Background Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. Methods mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography–tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. Results GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. Conclusions Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain. Electronic supplementary material The online version of this article (10.1186/s12974-019-1458-8) contains supplementary material, which is available to authorized users.

Summary Neutrophils migrate rapidly to damaged tissue and play critical roles in host defense and tissue homeostasis. Here we investigated the mechanisms whereby neutrophils participate in tissue repair. In an intestinal epithelia injury model, neutrophil depletion exacerbated colitis and associated with reduced interleukin (IL)-22 and limited activation of type 3 innate lymphoid cells (ILC3s). Co-culture with neutrophils activated ILC3s in a manner dependent on neutrophil apoptosis. Metabolomic analyses revealed that lysophosphatidylserine (LysoPS) from apoptotic neutrophils directly stimulated ILC3 activation. ILC3-specific deletion of Gpr34, encoding the LysoPS receptor GPR34, or inhibition of downstream PI3K-AKT or ERK suppressed IL-22 production in response to apoptotic neutrophils. Gpr34−/− mice exhibited compromised ILC3 activation and tissue repair during colon injury, and neutrophil depletion abrogated these defects. GPR34 deficiency in ILC3s limited IL-22 production and tissue repair in vivo in settings of colon and skin injury. Thus, GPR34 is an ILC3-expressed damage-sensing receptor that triggers tissue repair upon recognition of dying neutrophils.

Dendritic cells (DCs) are specifically equipped with the G protein–coupled receptor 34 (GPR34). Tight regulation of GPR34 gene expression seems highly important for proper immunological functions, because the absence of this receptor leads to an alteration of the immune response, whereas overexpression was reported to be involved in neuroinflammation. However, the regulatory mechanism of GPR34 expression has not yet been investigated. Whole-transcriptome RNA sequencing analysis from spleens and DCs of GPR34 knockout and wild-type mice, combined with protein–protein interaction data, revealed functional modules affected by the absence of this receptor. Among these, NF-κB, MAPK, and apoptosis-signaling pathways showed high significance. Using murine DCs we experimentally show that NF-κB and MAPK pathways are involved in the downregulation of GPR34. DCs lacking GPR34 have a higher caspase-3/7 activity and increased apoptosis levels. Our study reveals a novel role of GPR34 in the fate of DCs and identifies a regulatory mechanism that could be relevant for treatment of GPR34-overexpressing pathologies, such as neuroinflammatory or cancer conditions.

MicroRNAs (miRNAs) have emerged as the vital post-transcriptional regulators and control the growth and progression of different cancers types. The current study aimed at exploration of the role of microRNA-381 (miRNA-381) in human cervical cancer with emphasis on the evaluation of the underlying molecular mechanism. The results revealed a significant (P < 0.05) downregulation of miRNA-381 was found in cervical cancer tissues and cancer cell lines. Overexpression of miRNA-381 in cervical cancer cells significantly (P < 0.05) inhibited their proliferation through the induction of cell apoptosis which was accompanied by depletion of Bcl-2 and increase in Bax expression. Additionally, the cleavage of caspase-3 and 9 was also activated upon miRNA-381 overexpression. The Overexpression of miRNA-381 further inhibited the migration and invasion of cervical cancer cells. In silico analysis together with dual luciferase assay revealed G protein-Coupled receptor 34 (GPR34) to be the target of miRNA-381. The expression of GPR34 was significantly (P < 0.05) upregulated in the cervical cancer tissues and cell lines. Nonetheless, miRNA-381 overexpression caused a remarkable decrease in the expression of GPR34. The GPR34 knockdown and overexpression proved that the tumor-suppressive effects of miRNA-381 are mediated via GPR34. The study elucidated the essence of miRNA-381/GPR34 molecular regulatory axis in cervical cancer and unraveled the possibility of targeting this molecular axis as an important therapeutic approach against human cervical cancer.

MALT lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterised. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (GPR34 and CCR6) not previously reported in human malignancies, 3 genes (PIK3CD, TET2, TNFRSF14) not previously implicated in MALT lymphoma, and a further 2 genes (TBL1XR1, NOTCH1) recently described in MALT lymphoma. The majority of mutations in GPR34 and CCR6 were nonsense and frameshift changes clustered in the C-terminal cytoplasmic tail, and would result in truncated proteins that lack the phosphorylation motif important for β-arrestin mediated receptor desensitization and internalisation. Screening of these newly identified mutations, together with previously identified genetic changes, identified distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterised by frequent TBL1XR1 and GPR34 mutations, thyroid by frequent TET2, TNFRSF14 and PIK3CD mutations, and ocular adnexa by frequent TNFAIP3 mutation. Interestingly, in MALT lymphoma of the salivary gland, there was a significant positive association between TBL1XR1 mutation and GPR34 mutation/translocation (P=0.0002). In those of ocular adnexa, TBL1XR1 mutation was mutually exclusive from TNFAIP3 mutation (P=0.049), but significantly associated with IGHV3-23 usage (P=0.03) and PIK3CD mutation (P=0.009). These findings unravel novel insights into the molecular mechanisms of MALT lymphoma and provide further evidence for potential oncogenic cooperation between receptor signalling and genetic changes. ispartof: Haematologica vol:103 issue:8 pages:1329-1336 ispartof: location:Italy status: published

Research on GPR34, which was discovered in 1999 as an orphan G protein-coupled receptor of the rhodopsin-like class, disclosed its physiologic relevance only piece by piece. Being present in all recent vertebrate genomes analyzed so far it seems to improve the fitness of species although it is not essential for life and reproduction as GPR34-deficient mice demonstrate. However, closer inspection of macrophages and microglia, where it is mainly expressed, revealed its relevance in immune cell function. Recent data clearly demonstrate that GPR34 function is required to arrest microglia in the M0 homeostatic non-phagocytic phenotype. Herein, we summarize the current knowledge on its evolution, genomic and structural organization, physiology, pharmacology and relevance in human diseases including neurodegenerative diseases and cancer, which accumulated over the last 20 years.

Mucosa-associated lymphoid tissue (MALT) lymphoma originates from a background of diverse chronic inflammatory disorders at various anatomic sites. The genetics underlying its development, particularly in those associated with autoimmune disorders, is poorly characterized. By whole exome sequencing of 21 cases of MALT lymphomas of the salivary gland and thyroid, we have identified recurrent somatic mutations in 2 G-protein coupled receptors (

Lysophosphatidylserine (LysoPS) has been shown to have lipid mediator-like actions to induce mast cell degranulation and suppress T lymphocyte proliferation. Recently, three G protein-coupled receptors (GPCRs), LPS

The ligands of certain G-protein-coupled receptors (GPCRs) have been identified as endogenous lipids, such as lysophosphatidylserine (LysoPS). Here, we analyzed the molecular basis of the structure-activity relationship of ligands of GPR34, one of the LysoPS receptor subtypes, focusing on recognition of the long-chain fatty acid moiety by the hydrophobic pocket. By introducing benzene ring(s) into the fatty acid moiety of 2-deoxy-LysoPS, we explored the binding site's preference for the hydrophobic shape. A tribenzene-containing fatty acid surrogate with modifications of the terminal aromatic moiety showed potent agonistic activity toward GPR34. Computational docking of these derivatives with a homology modeling/molecular dynamics-based virtual binding site of GPR34 indicated that a kink in the benzene-based lipid surrogates matches the L-shaped hydrophobic pocket of GPR34. A tetrabenzene-based lipid analogue bearing a bulky tert-butyl group at the 4-position of the terminal benzene ring exhibited potent GPR34 agonistic activity, validating the present hydrophobic binding pocket model.

Tyrosine 177 and the Src homology 2 (SH2) domain play important roles in linking p185Bcr-Abl to downstream pathways critical for cell growth and survival. However, a mutant p185(Y177FR552L) (p185(YR)), in which tyrosine 177 and arginine 552 in the SH2 domain are mutated, is still capable of transforming hematopoietic cells in vitro. Transplant of these cells into syngeneic mice also leads to leukemogenesis, albeit with a phenotype distinct from that produced by wild-type p185Bcr-Abl (p185(wt))-transformed cells. Here we show that G-protein coupled receptor 34 (Gpr34) expression is markedly up-regulated in p185(YR)-transformed cells compared to those transformed by p185(wt). Knockdown of Gpr34 in p185(YR) cells is sufficient to suppress growth factor-independent proliferation and survival in vitro and attenuate leukemogenesis in vivo. The Erk and phosphatidylinositol 3-kinase/Akt pathways are activated in p185(YR) cells and the activation is dependent on Gpr34 expression. These studies identify Gpr34 as an alternative pathway that may mediate p185Bcr-Abl-induced transformation and leukemogenesis.

G-protein-coupled receptors (GPCRs) that recognize the lysophospholipids (LPLs) are grouped into two phylogenetically distinct families: the endothelial differentiation gene (Edg) and non-Edg GPCRs. Owing to their more recent identification, and hindered by a lack of selective pharmacological tools, our understanding of the functions and signaling pathways of the non-Edg GPCRs is still in its infancy. Targeting the non-conserved allosteric binding sites of the LPL GPCRs shows particular promise for the development of selective modulators by structure-based drug design. However, only one Edg GPCR (S1PR1) structure has been determined to date, and it has low sequence identity with the non-Edg GPCRs (

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) signal through G-protein coupled receptors (GPCRs) which couple to multiple G-proteins and their effectors. These GPCRs are quite efficacious in coupling to the Gα12/13 family of G-proteins, which stimulate guanine nucleotide exchange factors (GEFs) for RhoA. Activated RhoA subsequently regulates downstream enzymes that transduce signals which affect the actin cytoskeleton, gene expression, cell proliferation and cell survival. Remarkably many of the enzymes regulated downstream of RhoA either use phospholipids as substrates (e.g. phospholipase D, phospholipase C-epsilon, PTEN, PI3 kinase) or are regulated by phospholipid products (e.g. protein kinase D, Akt). Thus lysophospholipids signal from outside of the cell and control phospholipid signaling processes within the cell that they target. Here we review evidence suggesting an integrative role for RhoA in responding to lysophospholipids upregulated in the pathophysiological environment, and in transducing this signal to cellular responses through effects on phospholipid regulatory or phospholipid regulated enzymes. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Oxidized phospholipids (OxPLs) are increasingly recognized to play a role in a variety of normal and pathological states. OxPLs were implicated in regulation of inflammation, thrombosis, angiogenesis, endothelial barrier function, immune tolerance and other important processes. Rapidly accumulating evidence suggests that OxPLs are biomarkers of atherosclerosis and other pathologies. In addition, successful application of experimental drugs based on structural scaffold of OxPLs in animal models of inflammation was recently reported. This review briefly summarizes current knowledge on generation, methods of quantification and biological activities of OxPLs. Furthermore, receptor and cellular mechanisms of these effects are discussed. The goal of the review is to give a broad overview of this class of lipid mediators inducing pleiotropic biological effects.

There are limitations in the current classification of danger-associated molecular patterns (DAMP) receptors. To overcome these limitations, we propose a new paradigm by using endogenous metabolites lysophospholipids (LPLs) as a prototype. By utilizing a data mining method we pioneered, we made the following findings: (1) endogenous metabolites such as LPLs at basal level have physiological functions; (2) under sterile inflammation, expression of some LPLs is elevated. These LPLs act as conditional DAMPs or anti-inflammatory homeostasis-associated molecular pattern molecules (HAMPs) for regulating the progression of inflammation or inhibition of inflammation, respectively; (3) receptors for conditional DAMPs and HAMPs are differentially expressed in human and mouse tissues; and (4) complex signaling mechanism exists between pro-inflammatory mediators and classical DAMPs that regulate the expression of conditional DAMPs and HAMPs. This novel insight will facilitate identification of novel conditional DAMPs and HAMPs, thus promote development of new therapeutic targets to treat inflammatory disorders.

Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34.

Genetic aberrations, including trisomies 3 and 18, and well-defined IGH translocations, have been described in marginal zone lymphomas (MZLs); however, these known genetic events are present in only a subset of cases. Here, we report the cloning of an IGH translocation partner on chromosome X, t(X;14)(p11.4;q32) that deregulates expression of an poorly characterized orphan G-protein–coupled receptor, GPR34. Elevated GPR34 gene expression was detected independent of the translocation in multiple subtypes of non-Hodgkin lymphoma and distinguished a unique molecular subtype of MZL. Increased expression of GPR34 was also detected in tissue from brain tumors and surface expression of GPR34 was detected on human MZL tumor cells and normal immune cells. Overexpression of GPR34 in lymphoma and HeLa cells resulted in phosphorylation of ERK, PKC, and CREB; induced CRE, AP1, and NF-κB–mediated gene transcription; and increased cell proliferation. In summary, these results are the first to identify a role for a GPR34 in lymphoma cell growth, provide insight into GPR34-mediated signaling, identify a genetically unique subset of MZLs that express high levels of GPR34, and suggest that MEK inhibitors may be useful for treatment of GPR34-expressing tumors.

Lyso-PS (lyso-phosphatidylserine) has been shown to activate the Gi/o-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca(2+) ion levels ([Ca(2+)](i)). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) that deacylates fatty acid at the sn-1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA(1), stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA(1) but not with the mutant PS-PLA(1). PS treated with PLA(1) was much more effective at stimulating AP-TGFα release than PS treated with PLA(2). In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position.

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.

Sphingosine 1-phosphate (S1P) signaling in the treatment of multiple sclerosis (MS) has been highlighted by the efficacy of FTY720 (fingolimod), which upon phosphorylation can modulate S1P receptor activities. FTY720 has become the first oral treatment for relapsing MS that was approved by the FDA in September 2010. Phosphorylated FTY720 modulates four of the five known S1P receptors (S1P(1), S1P(3), S1P(4), and S1P(5)) at high affinity. Studies in human MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have revealed that FTY720 exposure alters lymphocyte trafficking via sequestration of auto-aggressive lymphocytes within lymphoid organs, representing the current understanding of its mechanism of action. These effects primarily involve S1P(1), which is thought to attenuate inflammatory insults in the central nervous system (CNS). In addition, FTY720's actions may involve direct effects on S1P receptor-mediated signaling in CNS cells, based upon the known expression of S1P receptors in CNS cell types relevant to MS, access to the CNS through the blood-brain barrier (BBB), and in vitro studies. These data implicate lysophospholipid signaling--via S1P(1) and perhaps other lysophospholipid receptors--in therapeutic approaches to MS and potentially other diseases with immunological and/or neurological components.

Previous studies on lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) using various approaches have shown that both the molecules can act as intercellular signaling molecules. The discovery of the Edg subfamily of G-protein-coupled receptors (GPCRs) (later renamed LPA(1-3) and S1P(1-5)) for these molecules has opened up a new avenue for pathophysiological research on lysophospholipids. Genetic and molecular studies on lysophospholipid GPCRs have elucidated pathophysiological impacts and roles in cellular signaling pathways. Recently, lysophospholipid GPCR genes have been used to develop receptor subtype-selective agonists and antagonists. The discovery of FTY720, a novel immune modulator, along with other chemical tools, has provided a means of elucidating the functions of each lysophospholipid GPCR on an organ and the whole body level. This communication attempts to retrospectively review the development of agonists and antagonists for lysophospholipid GPCRs, provide integrated information on pharmacological tools for lysophospholipid GPCR signaling, and speculate on future drug development.

Emerging data indicates the existence of novel molecular targets for cannabinoid ligands and recently it has been suggested that the orphan G-protein coupled receptor, GPR55 can be activated by a range of endogenous, plant and synthetic cannabinoids. However, to date, the most potent ligand identified for GPR55 is the endogenous phospholipid, lysophosphatidylinositol (LPI). GPR55 is thought to link predominantly G-protein alpha(13), where it promotes Rho-dependent signalling. Additional events downstream of GPR55 include activation of ERK-MAP kinase and Ca(2+) release from stores, as well as the induction of a number of transcription factors. Although GPR55 has only a low sequence identity with the CB1 or CB2 cannabinoid receptors, it clearly interacts with certain cannabinoid ligands. However, the nature and scope of these effects are presently unclear and they may be influenced by the assay and cellular background used for their study. This article reviews the current status of GPR55 pharmacology and its putative endogenous ligand, lysophosphatidylinositol LPI.

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

It is now widely accepted that lysophospholipids (LPLs), a product of the phospholipase A reaction, function as mediators through G-protein-coupled receptors. Notably, recent studies of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) have revealed their essential roles in vivo. On the other hand, other LPLs such as lysophosphatidylserine (LPS), lysophosphatidylthreonine (LPT), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI) and lysophosphatidylglycerol (LPG) have been reported to show lipid mediator-like responses both in vivo (LPS and LPT) and in vitro (LPS, LPT, LPE and LPG), while very little is known about their receptor, synthetic enzyme and patho-physiological roles. In this review, we summarize the actions of these LPLs as lipid mediators including LPS, LPT, LPE and LPG.

Intercellular lipid mediators such as prostaglandins and lysophosphatidic acid (LPA) interact with their G-protein-coupled receptors (GPCR) in the plasma membrane to modulate functions of target cells or tissues. Discovery of new members of intercellular lipid mediators and their GPCRs have been milestones in lipid biology and the foundation for drug development. Recent advances in intercellular lipid mediators are very interesting. New lipid molecules have been recognized as intercellular signaling mediators acting on GPCRs including resolvin E1, eoxin, acylethanolamides (arachidnonylethanolamide and oleoylethanolamide), fatty acids, bile acids, lipoamino aicd (N-palmitoyl glycine and N-arachidonyl glycine), estrogen, 5-oxo-ETE and 9-hydroxyoctadecadienoic acid, among others. Also new GPCRs for LPA have been identified. New intercellular lipid mediators and their GPCRs are reviewed.

Absrtract—G protein–coupled receptors (GPCRs) are widely expressed cell surface receptors that have been successfully exploited for the treatment of a variety of human diseases. Recent studies in genetically engineered mouse models have led to the identification of several GPCRs important for lymphatic vascular development and function. The adrenomedullin receptor, which consists of an oligomer between calcitonin receptor-like receptor and receptor activity modifying protein 2, is required for normal lymphatic vascular development and regulates lymphatic capillary permeability in mice. Numerous studies also suggest that lysophospholipid receptors are involved in the development of lymphatic vessels and lymphatic endothelial cell permeability. Given our current lack of pharmacological targets for the treatment of lymphatic vascular diseases like lymphedema, the continued identification and study of GPCRs in lymphatic endothelial cells may eventually lead to major breakthroughs and new pharmacological strategies for the treatment of lymphedema.

The lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), stimulate chemotaxis and induce differentiation of human keratinocytes. As Ca(2+) plays an important role in keratinocyte differentiation, we studied Ca(2+) signaling by S1P and LPA in these cells, known to express mRNA transcripts of the S1P(1-5) and LPA(1-3) receptors, and the receptor subtypes involved in this process. S1P and LPA caused transient increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)), with pEC(50) values of 8.5+/-0.11 and 7.5+/-0.23, respectively. The [Ca(2+)](i) increases are apparently mediated by stimulation of phospholipase C and involve Ca(2+) mobilization from thapsigargin-sensitive stores and subsequent Ca(2+) influx. The LPA-induced [Ca(2+)](i) increases were not inhibited by the LPA(1/3) receptor antagonist, dioctanoylglycerol pyrophosphate. The S1P-induced [Ca(2+)](i) increases were largely inhibited by the putative S1P(3) antagonist, BML-241, and the S1P(1/3) antagonist, VPC23019. The S1P(1)-specific agonist, SEW2871, did not increase [Ca(2+)](i) but stimulated chemotaxis of keratinocytes, which was fully blocked by S1P(1) antisense oligonucleotides. The data indicate that LPA and S1P potently increase [Ca(2+)](i) in human keratinocytes and that the effect of LPA is mediated by LPA(2), whereas that of S1P is mediated at least to a large part by S1P(3). The S1P(1) receptor, without stimulating [Ca(2+)](i) increases, mediates chemotaxis of keratinocytes.

BACKGROUND Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are two prominent signaling lysophospholipids (LPs) exerting their functions through a group of G protein-coupled receptors (GPCRs). This review covers current knowledge of the LP signaling in the function and pathology of the reproductive system. METHODS PubMed was searched up to May 2008 for papers on lysophospholipids/LPA/S1P/LPC/SPC in combination with each part of the reproductive system, such as testis/ovary/uterus. RESULTS LPA and SIP are found in significant amounts in serum and other biological fluids. To date, 10 LP receptors have been identified, including LPA(1-5) and S1P(1-5). In vitro and in vivo studies from the past three decades have demonstrated or suggested the physiological functions of LP signaling in reproduction, such as spermatogenesis, male sexual function, ovarian function, fertilization, early embryo development, embryo spacing, implantation, decidualization, pregnancy maintenance and parturition, as well as pathological roles in ovary, cervix, mammary gland and prostate cancers. CONCLUSIONS Receptor knock-out and other studies indicate tissue-specific and receptor-specific functions of LP signaling in reproduction. More comprehensive studies are required to define mechanisms of LP signaling and explore the potential use as a therapeutic target.

There is increasing evidence that different phospholipids are involved in regulation of various cell processes and cell-cell interactions. Lysophospholipids (lysophosphatidic acid, lysophosphatidylcholine) and a number of lysosphingolipids play particular roles in these regulations. Their effects are mediated by specific G-protein-coupled receptors. G-Protein coupled signal transduction to the cell nucleus involving a chain of intracellular protein kinases induces the main effects in cells--growth, proliferation, survival, or apoptosis. This review summarizes recent data on various groups of lysophospholipid receptors and their cell signal transduction pathways.

Over the past decade the importance of lipids for cancer cell metabolism and cancer-related processes such as proliferation, metastasis and chemotherapy resistance has become more apparent. The mechanisms by which lipid signals are transduced are poorly understood, but frequently involve G-protein Coupled Receptors (GPCRs), which can be explored as druggable targets. Here, we discuss how GPCRs recognize four classes of cancer-relevant lipids (lysophospholipids, phospholipids, fatty acids and eicosanoids). We compare the ligand-binding properties of >50 lipid receptors, we examine how their dysregulation contributes to tumorigenesis and how they may be therapeutically exploited.

Nearly two decades have passed since it was first proposed that regulation of the interconvertible sphingolipid metabolites, ceramide and sphingosine-1-phosphate (S1P), and their opposing signaling pathways are major determinants of cell fate, a concept referred to as the “sphingolipid rheostat”. Since then, many reports have substantiated the role of the sphingolipid rheostat in cell fate determination and in the initiation, progression, and drug sensitivity of cancer. Thus, modulation of the rheostat has emerged as a focus for treatment strategies to battle cancer. S1P regulates numerous processes important for cancer including proliferation, transformation, angiogenesis, metastasis, survival, and drug resistance. Ceramide on the other hand has been linked to cell growth arrest and cell death. With the increased understanding of sphingolipid metabolism and signaling, as well as the present focus on therapies designed to modulate the levels of sphingolipids in cancer, it is an appropriate time to re-examine the sphingolipid rheostat concept and determine how it fits within the current knowledge of sphingolipid signaling in cancer.

Lysophosphatidyl-L-serine (lysoPS) is thought to be an immunological regulator because it dramatically augments the degranulation of rat peritoneal mast cells (RPMCs). This stimulatory effect may be mediated by a lysoPS receptor, but its molecule has not been identified yet. During a ligand fishing study for the orphan G-protein-coupled receptor 34 (GPR34), we found that lysoPS caused a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in human GPR34-expressing Chinese hamster ovary (CHO/hGPR34) cells. The CHO/hGPR34 cells were unresponsive to other structurally related phospholipids examined. Quantitative real-time-PCR demonstrated that mRNAs of GPR34 are particularly abundant in mast cells. The effective lysoPS concentration for RPMC degranulation was similar to that required for GPR34 activation, and the structural requirement of lysoPS for RPMC degranulation was in good agreement with that observed in CHO/hGPR34 cells. These results suggest that GPR34 is the functional mast cell lysoPS receptor.

Directed cloning approaches and large-scale sequencing of several vertebrate genomes unveiled many new members of the G-protein-coupled receptor (GPCR) superfamily, among them GPR34. Initial studies showed that GPR34 is an evolutionarily old GPCR structurally related to a group of ADP-like receptors. To gain insight into the genomic organization, regulation of expression, and supragenomic diversification of GPR34 several vertebrate species were analyzed. In contrast to the obviously intronless coding region GPR34 displays an evolutionary preserved 5′ noncoding intron–exon structure. Further, an alternatively used cryptic intron was identified within the coding region, which shortens the N terminus by 47 amino acids. Ubiquitous expression of GPR34 is driven by genomic sequences upstream of at least two transcriptional start regions in mouse and rat but only one region in human. In rodents, both promoters are active in all tissues investigated, but the level of activity is tissue-specific. At the translational level, several conserved in-frame AUGs within the first 150 bp of the coding region may serve as start points for translation in human and other mammals. Combinatory mutagenesis and expression of reporter constructs confirmed these multiple translational start points and revealed a preference for the second in-frame AUG in human GPR34. Our data show that multiple translation initiation starts and alternative splicing contribute to the supragenomic diversification of GPR34.

The phospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via transmembrane receptors S1P 1-5 and LPA 1-3, respectively. Both have been implicated in inflammatory responses. S1P and LPA receptor profiles on neutrophils of patients with pneumonia compared with healthy subjects were determined by PCR and Western blotting. Chemotaxis studies were performed to assess functional differences. S1P or LPA receptors were immunoprecipitated from neutrophils to assess receptor heterodimerization with CXCR1, an IL-8 receptor, by Western blotting. Receptors S1P 1, 4, and 5 and LPA 2 were expressed on neutrophils from both subject groups, but S1P 3 and LPA 1 receptor expression was mainly confined to neutrophils of patients with pneumonia. Chemotaxis of neutrophils from patients with pneumonia compared with control subjects was significantly increased in response to S1P and LPA. Pretreatment with S1P or LPA reduced IL-8-induced neutrophil chemotaxis and transcriptional expression of the CXCR1 receptor. Receptors S1P 3 and 4 and LPA 1 formed constitutive heterodimers with CXCR1. LPA treatment reduced the amount of LPA 1/CXCR1 heterodimer. Therefore, profiles of S1P and LPA receptors differ between neutrophils of patients with pneumonia and control subjects, with consequences for neutrophil function.

Lysophospholipid mediators, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), play important roles in diverse biological processes, including cell proliferation, survival, cytoskeleton changes, migration, wound healing, angiogenesis, tumor invasion, and embryonic development. The recent breakthrough in cloning and identification of several G-protein-coupled receptors for LPA and S1P has provided tools to dissect the complex mechanisms by which these lysophospholipids exert their (patho)physiological functions. Here, strategies and efforts in cloning and identifying the Edg family receptors for LPA and S1P are reviewed. Reporter gene assays used to take advantage of signaling properties of LPA and S1P, and to overcome intrinsic difficulties unique to this system, are described. From these experimental approaches, important lessons can be learned and applied to future studies of signal transduction of lysophospholipid receptors.

We investigated the molecular mechanism by which Sph-1-P affects the FN-dependent haptotactic motility of serum-starved mouse melanoma B16/F10 cells. We found that EDG-5-induced Rho activation followed by enhanced tyrosine phosphorylation of FAK and paxillin, and beta 1-integrin activation leading to overexpression of focal adhesion sites, as well as increment of stress fiber formation, must be the molecular basis of inhibition of haptotactic cell motility by Sph-1-P.

The bioactive lipid sphingosine-1-phosphate (SPP) is abundantly formed and released during the activation of platelets by thrombotic stimuli. Once exported, SPP interacts with the G-protein-coupled receptors (GPCR) of the EDG-1 family. SPP binds to EDG-1 with the dissociation constant of approximately 8 nM and induces signal transduction events such as mitogen-activated protein kinase (MAP kinase) activation, decrease of cAMP levels, remodeling of the actin cytoskeleton, among others. EDG-1 is a prototypical member of a large family of GPCRs that interact with glycero- and sphingolysolipid phosphates, namely, SPP and lysophosphatidic acid (LPA). Three other GPCRs, trivially termed EDG-3, EDG-5, and EDG-8, are also high-affinity receptors for SPP. The four SPP receptor subtypes regulate different intracellular signal transduction pathways. In vascular endothelial cells, cooperative signaling between EDG-1 and EDG-3 subtypes of SPP receptors results in adherens junction assembly, cell survival, morphogenesis into capillary-like networks, and angiogenesis. SPP acts distinctly, albeit cooperatively, with polypeptide angiogenic factors, resulting in the formation of mature neovessels. Thus SPP signaling as an extracellular mediator via the EDG-1 family of GPCRs may be a heretofore unrecognized mechanism for the regulation of angiogenesis and vascular endothelial cell function.

Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, cell growth, and survival. Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide, and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. This ceramide/SPP rheostat is an evolutionarily conserved stress regulatory mechanism influencing growth and survival of yeast. In addition, SPP also has been identified as the ligand for the G-protein-coupled receptors EDG-1, -3, -5, and -6. Binding of SPP to EDG-1 regulates chemotaxis and in vitro angiogenesis of endothelial cells, whereas EDG-5, and possibly EDG-3, are likely the cell surface receptors responsible for cell rounding and neurite retractions induced by SPP. Hence, the studies identify a family of highly specific SPP receptors that are capable of mediating different biological responses. Thus, it is suggested that SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers. Recently, sphingosine kinase was purified to homogeneity and the first mammalian sphingosine kinase, the enzyme responsible for the formation of SPP, was cloned. The studies should provide the necessary tools to develop insight into the biological roles of this important bioactive sphingolipid.

New therapies directed at ameliorating or altering autoimmune diseases represent an area of significant medical need. Included amongst autoimmune diseases are problems related to transplantation rejection, as well as a number of neurological diseases such as Multiple Sclerosis (MS). A new group of molecular targets that may lead to novel therapies are lysophospholipid (LP) receptors. A large range of biological activities has been attributed to the actions of these simple phospholipids that include well-studied members lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Documented cellular effects of these lipid molecules encompass growth-factor-like influences on cells, including but not limited to survival, migration, adhesion differentiation, as well as pathophysiological actions associated with cancer. In turn, these cellular effects have roles in developing and adult organ systems such as the nervous system, cardiovascular system, reproductive system and, of relevance here, the immune system. The mechanisms for these actions can be attributed to a growing family of cognate, 7-transmembrane G protein-coupled receptors (GPCRs), with documented validation through studies utilizing pharmacology, molecular genetics and an enlarging repertoire of chemical tools having agonist or antagonist properties. The growing literature on immunological effects of LP receptors, particularly those mediating the effects of S1P, has suggested possible therapeutic roles for this class of receptors. In particular, entry into humans of a non-selective S1P receptor agonist, FTY720, for kidney transplantation and possibly other indications (e.g., Multiple Sclerosis), has raised prospects for efficacious treatment of human diseases based on LP receptor targets. Here we provide a brief introduction to receptor-mediated lysophospholipid signaling and discuss its basic and potential therapeutic roles in autoimmune-related diseases.

Vascular stabilization, a process by which nascent vessels are invested with mural cells, is important in angiogenesis. Here we describe the molecular basis of vascular stabilization regulated by sphingosine 1-phosphate (S1P), a platelet-derived lipid mediator. S1P1 receptor-dependent cell-surface trafficking and activation of the cell-cell adhesion molecule N-cadherin is essential for interactions between endothelial and mural cells. Endothelial cell S1P1/Gi/Rac pathway induces microtubule polymerization, resulting in trafficking of N-cadherin to polarized plasma membrane domains. S1P treatment modulated the phosphorylation of N-cadherin as well as p120-catenin and induced the formation of cadherin/catenin/actin complexes containing novel regulatory and trafficking factors. The net result of endothelial cell S1P1 receptor activation is the proper trafficking and strengthening of N-cadherin-dependent cell-cell adhesion with mural cells. Perturbation of N-cadherin expression with small interfering RNA profoundly attenuated vascular stabilization in vitro and in vivo. S1P-induced trafficking and activation of N-cadherin provides a novel mechanism for the stabilization of nascent blood vessels by mural cells and may be exploited to control angiogenesis and vascular diseases.