ABSTRACT
GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.
Subject(s)
Fatty Acids , Lysophospholipids , Humans , Cryoelectron Microscopy , Fatty Acids/metabolism , Ligands , Lysophospholipids/metabolism , Receptors, Lysophospholipid/agonists , Receptors, Lysophospholipid/metabolismABSTRACT
Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.
Subject(s)
Lysophospholipids , Osteogenesis , Receptors, Lysophospholipid , Tooth Loss , Animals , Mice , Cell Differentiation/physiology , Lysophospholipids/metabolism , Osteoblasts/metabolism , Periodontal Ligament/metabolism , Transforming Growth Factor beta1/metabolism , Receptors, Lysophospholipid/metabolismABSTRACT
GPR34 is a rhodopsin-like class G protein-coupled receptor (GPCR) that is involved in the development and progression of several diseases. Despite its importance, effective targeting strategies are lacking. We herein report a series of (S)-3-(4-(benzyloxy)phenyl)-2-(2-phenoxyacetamido)propanoic acid derivatives as a new class of GPR34 antagonists. Structure-activity relationship (SAR) studies led to the identification of the most potent compound, 5e, which displayed an IC50 value of 0.680 µM in the GloSensor cAMP assay and 0.059 µM in the Tango assay. 5e demonstrated low cytotoxicity and high selectivity in vitro, and it was able to dose-dependently inhibit Lysophosphatidylserine-induced ERK1/2 phosphorylation in CHO cells expressing GPR34. Furthermore, 5e displayed excellent efficacy in a mouse model of neuropathic pain without any apparent signs of toxicity. Collectively, this study has identified a promising compound, which shows great potential in the development of potent antagonists with a new chemical scaffold targeting GPR34.
Subject(s)
Propionates , Receptors, Lysophospholipid , Animals , Cricetinae , Mice , CHO Cells , Cricetulus , Receptors, Lysophospholipid/antagonists & inhibitors , Receptors, Lysophospholipid/chemistry , Structure-Activity RelationshipABSTRACT
Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator involved in various physiological and pathological processes especially those related to the immune system. GPR34, GPR174, and P2Y10 have been identified as the receptors for LysoPS, and its analogues have been developed as agonists or antagonists for these receptors. However, the lack of structural information hinders the drug development with novel characteristics, such as nonlipid ligands and allosteric modulators. Here, we determined the structures of human GPR34 and GPR174 in complex with LysoPS and G protein by cryo-EM. Combined with structural analysis and functional studies, we elucidated the lipid-binding modes of these receptors. By structural comparison, we identified the structural features of GPR34 and GPR174 in active state. Taken together, our findings provide insights into ligand recognition and signaling of LysoPS receptors and will facilitate the development of novel therapeutics for related inflammatory diseases and autoimmune diseases.
Subject(s)
Lysophospholipids , Receptors, G-Protein-Coupled , Humans , Ligands , Lysophospholipids/pharmacology , Lysophospholipids/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Lysophospholipid/agonists , Receptors, Lysophospholipid/metabolismABSTRACT
Our group has reported various derivatives of lysophosphatidylserine (LysoPS) as potent and subtype-selective agonists for G-protein-coupled receptors (GPCRs). However, the ester linkage between the glycerol moiety and fatty acid or fatty acid surrogate is present in all of them. In order to develop these LysoPS analogs as drug candidates, appropriate pharmacokinetic consideration is essential. Here, we found that the ester bond of LysoPS is highly susceptible to metabolic degradation in mouse blood. Accordingly, we examined isosteric replacement of the ester linkage with heteroaromatic rings. The resulting compounds showed excellent retention of potency and receptor subtype selectivity, as well as increased metabolic stability in vitro.
Subject(s)
Lysophospholipids , Receptors, G-Protein-Coupled , Mice , Animals , Receptors, Lysophospholipid/agonists , Receptors, Lysophospholipid/metabolism , Lysophospholipids/chemistry , Lysophospholipids/metabolism , Receptors, G-Protein-Coupled/agonists , Fatty Acids/metabolism , Glycerol/chemistryABSTRACT
Lysophosphatidylserine (LysoPS) is an endogenous pan-agonist of three G-protein coupled receptors (GPCRs): LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, and we previously reported a series of LysoPS-based agonists of these receptors. Interestingly, we found that LPS1 agonist activity was very sensitive to structural change at the hydrophobic fatty acid moiety, whereas LPS2 agonist activity was not. Here, to probe the molecular basis of LPS2 agonist binding, we developed a new class of hydrophobic fatty acid surrogates having a biphenyl-ether scaffold. The LPS2 agonist activity of these compounds proved sensitive to molecular modification of the hydrophobic skeleton. Thus, we next constructed an LPS2 model by homology modeling and docking/molecular dynamics (MD) simulation, and validated it by means of SAR studies together with point mutations of selected receptor amino-acid residues. The putative ligand-binding site of LPS2 is Γ-shaped, with a hydrophilic site horizontally embedded in the receptor transmembrane helix bundles and a perpendicular hydrophobic groove adjoining transmembrane domains 4 and 5 that is open to the membrane bilayer. The binding poses of LPS2 agonists to this site are consistent with easy incorporation of various kinds of fatty acid surrogates. Structural development based on this model afforded a series of potent and selective LPS2 full agonists, which showed enhanced in vitro actin stress fiber formation effect.
Subject(s)
Lipopolysaccharides , Molecular Dynamics Simulation , Receptors, Lysophospholipid/agonists , Receptors, Lysophospholipid/genetics , Receptors, Lysophospholipid/metabolism , Lipopolysaccharides/pharmacology , Receptors, G-Protein-Coupled/agonists , Binding Sites , Fatty Acids , LigandsABSTRACT
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.
Subject(s)
Colorectal Neoplasms , Phosphatidylinositol 3-Kinases , Receptors, Lysophospholipid , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Colorectal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic/genetics , Humans , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Lysophospholipid/genetics , Receptors, Lysophospholipid/metabolism , Signal Transduction/geneticsABSTRACT
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.
Subject(s)
Lymphoma, B-Cell, Marginal Zone , Receptors, Lysophospholipid , Humans , Ligands , Lymphoma, B-Cell, Marginal Zone/pathology , Phosphatidylserines , Phospholipases , Receptors, Lysophospholipid/metabolism , Salivary Glands/metabolism , Salivary Glands/pathologyABSTRACT
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.
Subject(s)
Apoptosis/immunology , Immunity, Innate/immunology , Lymphocytes/immunology , Lysophospholipids/immunology , Neutrophils/immunology , Receptors, Lysophospholipid/immunology , Animals , Cells, Cultured , Colitis/immunology , Colon/immunology , Homeostasis/immunology , Humans , Interleukins/immunology , Intestinal Mucosa/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphatidylinositol 3-Kinases/immunology , Interleukin-22ABSTRACT
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.
Subject(s)
MicroRNAs , Receptors, Lysophospholipid/genetics , Uterine Cervical Neoplasms/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cervix Uteri/metabolism , Female , Humans , Middle Aged , Receptors, Lysophospholipid/metabolism , Uterine Cervical Neoplasms/pathologyABSTRACT
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.
Subject(s)
Cardiovascular Diseases/metabolism , Cardiovascular System/metabolism , Lysophospholipids/metabolism , Receptors, Lysophospholipid/metabolism , Animals , Cardiovascular Diseases/pathology , Cardiovascular Diseases/physiopathology , Cardiovascular System/pathology , Cardiovascular System/physiopathology , Endothelial Cells/metabolism , Humans , Ligands , Morphogenesis , Receptors, Lysophosphatidic Acid/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine-1-Phosphate Receptors/metabolism , Wnt Signaling PathwayABSTRACT
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.
Subject(s)
Lysophospholipids/pharmacology , Purinergic P2 Receptor Agonists/pharmacology , Receptors, Lysophospholipid/agonists , Receptors, Purinergic P2/metabolism , HEK293 Cells , Humans , Isomerism , Lysophospholipids/chemical synthesis , Molecular Conformation , Molecular Docking Simulation , Purinergic P2 Receptor Agonists/chemical synthesis , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Cell Membrane/metabolism , Lysophospholipids/metabolism , Receptors, Lysophospholipid/metabolism , Amino Acid Motifs , Cell Membrane/chemistry , Cell Membrane/genetics , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Lysophospholipids/chemistry , Protein Binding , Protein Domains , Receptors, Lysophospholipid/chemistry , Receptors, Lysophospholipid/geneticsABSTRACT
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.
Subject(s)
Microglia/metabolism , Neuralgia/metabolism , Neuralgia/pathology , Receptors, Lysophospholipid/metabolism , Spinal Cord/pathology , Analysis of Variance , Animals , Calcium-Binding Proteins/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation/genetics , Interferon Regulatory Factors/metabolism , Lysophospholipids/therapeutic use , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/metabolism , Neuralgia/drug therapy , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Pain Measurement , Pain Threshold/physiology , Protein Kinase C/metabolism , RNA, Messenger/metabolism , Receptors, Lysophospholipid/antagonists & inhibitors , Receptors, Lysophospholipid/genetics , Time FactorsABSTRACT
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), S1P1-5 and LPA1-6, have expanded their known involvement in all mammalian physiological systems. Resolution of the S1P1, LPA1, and LPA6 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 toward understanding actual biological interactions and phenotypes.
Subject(s)
Receptors, Lysophospholipid/chemistry , Animals , Humans , Ligands , Lysophospholipids/metabolism , Protein Conformation , Receptors, Lysophospholipid/metabolismABSTRACT
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 (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 ß-arrestin-mediated receptor desensitization and internalization. Screening of these newly identified mutations, together with previously defined genetic changes, revealed distinct mutation profiles in MALT lymphoma of various sites, with those of salivary gland characterized 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 co-operation between receptor signaling and genetic changes.
Subject(s)
Lymphoma, B-Cell, Marginal Zone/genetics , Mutation , Receptors, CCR6/genetics , Receptors, Lysophospholipid/genetics , Genetic Profile , Humans , Lymphoma, B-Cell, Marginal Zone/pathology , Salivary Gland Neoplasms/genetics , Thyroid Neoplasms/genetics , Exome SequencingABSTRACT
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 20â¯years.
Subject(s)
Receptors, Lysophospholipid/physiology , Animals , Evolution, Molecular , Genomics , History, 20th Century , History, 21st Century , Humans , Phenotype , Receptors, Lysophospholipid/chemistry , Receptors, Lysophospholipid/historyABSTRACT
Haemostasis is a defence mechanism that has evolved to protect organisms from losing their circulating fluid. We have previously introduced zebrafish as a model to study the genetics of haemostasis to identify novel genes that play a role in haemostasis. Here, we identify a zebrafish mutant that showed prolonged time to occlusion (TTO) in the laser injury venous thrombosis assay. By linkage analysis and fine mapping, we found a mutation in the orphan G protein-coupled receptor 34 like gene (gpr34l) causing a change of Val to Glu in the third external loop of Gpr34l. We have shown that injection of zebrafish gpr34l RNA rescues the prolonged TTO defect. The thrombocytes from the mutant showed elevated levels of cAMP that supports the defective thrombocyte function. We also have demonstrated that knockdown of this gene by intravenous Vivo-Morpholino injections yielded a phenotype similar to the gpr34l mutation. These results suggest that the lack of functional Gpr34l leads to increased cAMP levels that result in defective thrombocyte aggregation.
Subject(s)
Blood Platelets/metabolism , Mutation , Receptors, Lysophospholipid/genetics , Animals , Breeding , DNA Mutational Analysis , Gene Expression , Phenotype , ZebrafishABSTRACT
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), LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, were found to react specifically with LysoPS, raising the possibility that LysoPS exerts its roles through these receptors. In this study, we show that LPS3 is expressed in various T cell subtypes and is involved in suppression of Interleukin-2 (IL-2) production in CD4 T cells. We found that LysoPS suppressed the IL-2 production from activated T cells at the mRNA and protein levels. In addition, LysoPS did not have such an effect on the splenocytes and CD4 T cells isolated from LPS3-deficient mice. In LPS3-deficient splenocytes and CD4 T cells, anti-CD3/anti-CD28-triggered IL-2 production is somewhat increased. Interestingly, LysoPS with various fatty acids was up-regulated upon T cell activation. The present study raised the possibility that LysoPS exerts its immunosuppressive roles by down-regulating IL-2 production through a LysoPS-LPS3 axis in T cells.