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Toward the three-dimensional structure and lysophosphatidic acid binding characteristics of the LPA(4)/p2y(9)/GPR23 receptor: a homology modeling study.

ABSTRACT: Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that initiates a broad array of biological processes, including those involved in cell proliferation, survival and migration via activation of specific G protein-coupled receptors located on the cell surface. To date, at least five receptor subtypes (LPA(1-5)) have been identified. The LPA(1-3) receptors are members of the endothelial cell differentiation gene (Edg) family. LPA(4), a member of the purinergic receptor family, and the recently identified LPA(5) are structurally distant from the canonical Edg LPA(1-3) receptors. LPA(4) and LPA(5) are linked to G(q), G(12/13) and G(s) but not G(i), while LPA(1-3) all couple to G(i) in addition to G(q) and G(12/13). There is also evidence that LPA(4) and LPA(5) are functionally different from the Edg LPA receptors. Computational modeling has provided useful information on the structure-activity relationship (SAR) of the Edg LPA receptors. In this work, we focus on the initial analysis of the structural and ligand-binding properties of LPA(4), a prototype non-Edg LPA receptor. Three homology models of the LPA(4) receptor were developed based on the X-ray crystal structures of the ground state and photoactivated bovine rhodopsin and the recently determined human beta(2)-adrenergic receptor. Docking studies of LPA in the homology models were then conducted, and plausible LPA binding loci were explored. Based on these analyses, LPA is predicted to bind to LPA(4) in an orientation similar to that reported for LPA(1-3), but through a different network of hydrogen bonds. In LPA(1-3), the ligand polar head group is reported to interact with residues at positions 3.28, 3.29 and 7.36, whereas three non-conserved amino acid residues, S114(3.28), T187(EL2) and Y265(6.51), are predicted to interact with the polar head group in the LPA(4) receptor models.

SUBMITTER: Li G

PROVIDER: S-EPMC4373609 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Toward the three-dimensional structure and lysophosphatidic acid binding characteristics of the LPA(4)/p2y(9)/GPR23 receptor: a homology modeling study.

Li Guo G Mosier Philip D PD Fang Xianjun X Zhang Yan Y

Journal of molecular graphics & modelling 20090419 1

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that initiates a broad array of biological processes, including those involved in cell proliferation, survival and migration via activation of specific G protein-coupled receptors located on the cell surface. To date, at least five receptor subtypes (LPA(1-5)) have been identified. The LPA(1-3) receptors are members of the endothelial cell differentiation gene (Edg) family. LPA(4), a member of the purinergic receptor family, and t ...[more]

PMID: 19423373

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Project description:Human P2Y receptors encompass at least eight subtypes of Class A G protein-coupled receptors (GPCRs), responding to adenine and/or uracil nucleotides. Using a BLAST search against the Homo sapiens subset of the SWISS-PROT and TrEMBL databases, we identified 68 proteins showing high similarity to P2Y receptors. To address the problem of low sequence identity between rhodopsin and the P2Y receptors, we performed a multiple-sequence alignment of the retrieved proteins and the template bovine rhodopsin, combining manual identification of the transmembrane domains (TMs) with automatic techniques. The resulting phylogenetic tree delineated two distinct subgroups of P2Y receptors: Gq-coupled subtypes (e.g., P2Y1) and those coupled to Gi (e.g., P2Y12). On the basis of sequence comparison we mutated three Tyr residues of the putative P2Y1 binding pocket to Ala and Phe and characterized pharmacologically the mutant receptors expressed in COS-7 cells. The mutation of Y306 (7.35, site of a cationic residue in P2Y12) or Y203 in the second extracellular loop selectively decreased the affinity of the agonist 2-MeSADP, and the Y306F mutation also reduced antagonist (MRS2179) affinity by 5-fold. The Y273A (6.48) mutation precluded the receptor activation without a major effect on the ligand-binding affinities, but the Y273F mutant receptor still activated G proteins with full agonist affinity. Thus, we have identified new recognition elements to further define the P2Y1 binding site and related these to other P2Y receptor subtypes. Following sequence-based secondary-structure prediction, we constructed complete models of all the human P2Y receptors by homology to rhodopsin. Ligand docking on P2Y1 and P2Y12 receptor models was guided by mutagenesis results, to identify the residues implicated in the binding process. Different sets of cationic residues in the two subgroups appeared to coordinate phosphate-bearing ligands. Within the P2Y1 subgroup these residues are R3.29, K/R6.55, and R7.39. Within the P2Y12 subgroup, the only residue in common with P2Y1 is R6.55, and the role of R3.29 in TM3 seems to be fulfilled by a Lys residue in EL2, whereas the R7.39 in TM7 seems to be substituted by K7.35. Thus, we have identified common and distinguishing features of P2Y receptor structure and have proposed modes of ligand binding for the two representative subtypes that already have well-developed ligands.

Project description:BACKGROUND:In the extracellular environment, lysophosphatidic acid (LPA) species are generated via autotaxin (ATX)-mediated hydrolysis of lysophospholipid precursors. Members of the LPA family are potent lipid mediators transmitting signals via six different G protein-coupled LPA receptors (LPAR1-6). The LPA signaling axis is indispensable for brain development and function of the nervous system; however, during damage of the central nervous system, LPA levels can increase and aberrant signaling events counteract brain function. Here, we investigated regulation of the ATX/LPA/LPAR axis in response to lipopolysaccharide-induced systemic inflammation in mice and potential neurotoxic polarization programs in LPA-activated primary murine microglia. METHODS:In vivo, LPAR1-6 expression was established by qPCR in whole murine brain homogenates and in FACS-sorted microglia. ELISAs were used to quantitate LPA concentrations in the brain and cyto-/chemokine secretion from primary microglia in vitro. Transcription factor phosphorylation was analyzed by immunoblotting, and plasma membrane markers were analyzed by flow cytometry. We used MAPK inhibitors to study signal integration by the JNK, p38, and ERK1/2 branches in response to LPA-mediated activation of primary microglia. RESULTS:Under acute and chronic inflammatory conditions, we observed a significant increase in LPA concentrations and differential regulation of LPAR, ATX (encoded by ENPP2), and cytosolic phospholipase A2 (encoded by PLA2G4A) gene expression in the brain and FACS-sorted microglia. During pathway analyses in vitro, the use of specific MAPK antagonists (SP600125, SB203580, and PD98059) revealed that JNK and p38 inhibition most efficiently attenuated LPA-induced phosphorylation of proinflammatory transcription factors (STAT1 and -3, p65, and c-Jun) and secretion of IL-6 and TNF?. All three inhibitors decreased LPA-mediated secretion of IL-1?, CXCL10, CXCL2, and CCL5. The plasma membrane marker CD40 was solely inhibited by SP600125 while all three inhibitors affected expression of CD86 and CD206. All MAPK antagonists reduced intracellular COX-2 and Arg1 as well as ROS and NO formation, and neurotoxicity of microglia-conditioned media. CONCLUSION:In the present study, we show that systemic inflammation induces aberrant ATX/LPA/LPAR homeostasis in the murine brain. LPA-mediated polarization of primary microglia via MAPK-dependent pathways induces features reminiscent of a neurotoxic phenotype.

Dataset Information

Toward the three-dimensional structure and lysophosphatidic acid binding characteristics of the LPA(4)/p2y(9)/GPR23 receptor: a homology modeling study.

Publications

Toward the three-dimensional structure and lysophosphatidic acid binding characteristics of the LPA(4)/p2y(9)/GPR23 receptor: a homology modeling study.

Similar Datasets

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