farnesyl-pyrophosphate and lysophosphatidic-acid

Lysophosphatidic acid (LPA) is the umbrella term for lipid signaling molecules that share structural homology and activate the family of LPA receptors. Farnesyl Pyrophosphate (FPP) is commonly known as an intermediate in the synthesis of steroid hormones; however, its function as a signaling lipid is beginning to be explored. FPP was recently shown to an activator of the G-protein coupled receptor 92 (also known as LPA5) of the calcium channel TRPV(3). The LPA receptors (including GPR92) are associated with the signal transduction of noxious stimuli, however, very little is known about the distribution of their signaling ligands (LPAs and FPP) in the brain. Here, using HPLC/MS/MS, we developed extraction and analytical methods for measuring levels of FPP and 4 species of LPA (palmitoyl, stearoyl, oleoyl and arachidonoyl-sn-glycerol-3 phosphate) in rodent brain. Relative distributions of each of the five compounds was significantly different across the brain suggesting divergent functionality for each as signaling molecules based on where and how much of each is being produced. Brainstem, midbrain, and thalamus contained the highest levels measured for each compound, though none in the same ratios while relatively small amounts were produced in cortex and cerebellum. These data provide a framework for investigations into functional relationships of these lipid ligands in specific brain areas, many of which are associated with the perception of pain.

Topics: Animals; Brain; Female; Lysophospholipids; Organ Specificity; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Sesquiterpenes

P2Y5 is a G protein-coupled receptor that binds and is activated by lysophosphatidic acid (LPA). We determined that P2Y5 transcript is expressed along the intestinal mucosa and investigated the intracellular pathways induced by P2Y5 activation, which could contribute to LPA effects on intestinal cell adhesion. P2Y5 heterologously expressed in CHO and small intestinal hBRIE 380i cells was activated by LPA resulting in an increase in intracellular calcium ([Ca(2+)](i)) when the cells concurrently expressed G(alpha)(Delta6qi5myr). P2Y5 activation also increased the phosphorylation of ERK1/2 that was sensitive to pertussis toxin. Together these indicate that P2Y5 activation by LPA induces an increase in [Ca(2+)](i) and ERK1/2 phosphorylation through G(alpha)(i). We discovered that P2Y5 was activated by farnesyl pyrophosphate (FPP) without a detectable change in [Ca(2+)](i). The activation of P2Y5 by LPA or FPP induced the activity of a serum response element (SRE)-linked luciferase reporter that was inhibited by the RGS domain of p115RhoGEF, C3 exotoxin, and Y-27632, suggesting the involvement of G(alpha)(12/13), Rho GTPase, and ROCK, respectively. However, only LPA-mediated induction of SRE reporter activity was sensitive to inhibitors targeting p38 MAPK, PI3K, PLC, and PKC. In addition, only LPA transactivated the epidermal growth factor receptor, leading to an induction of ERK1/2 phosphorylation. These observations correlate with our subsequent finding that P2Y5 activation by LPA, and not FPP, reduced intestinal cell adhesion. This study elucidates a mechanism whereby LPA can act as a luminal and/or serosal cue to alter mucosal integrity.

Topics: Animals; Calcium Signaling; Cell Adhesion; CHO Cells; Cricetinae; Cricetulus; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; ErbB Receptors; G-Protein-Coupled Receptor Kinase 2; GTP-Binding Protein alpha Subunits, G12-G13; GTP-Binding Protein alpha Subunits, Gi-Go; Guanine Nucleotide Exchange Factors; HeLa Cells; Humans; Intestinal Mucosa; Lysophospholipids; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Receptor Cross-Talk; Receptors, Purinergic P2; Recombinant Fusion Proteins; rho GTP-Binding Proteins; Rho Guanine Nucleotide Exchange Factors; rho-Associated Kinases; RNA, Messenger; Serum Response Element; Sesquiterpenes; Transfection

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G(q/11)- and G(s)-mediated signaling pathways, whereas NAG activated only the G(q/11)-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr(97), Gly(98), Phe(101), and Arg(267) of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca(2+) levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Topics: Arachidonic Acids; Calcium; Extracellular Signal-Regulated MAP Kinases; Glycine; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Inositol Phosphates; Ligands; Lysophospholipids; Models, Biological; Neurons; Polyisoprenyl Phosphates; Receptors, Lysophosphatidic Acid; Sesquiterpenes; Signal Transduction; Tissue Distribution

Oligoprenyl phosphates are key metabolic intermediates for the biosynthesis of steroids, the side chain of ubiquinones, and dolichols and the posttranslational isoprenylation of proteins. Farnesyl phosphates are isoprenoid phosphates that resemble polyunsaturated fatty alcohol phosphates, which we have recently shown to be the minimal pharmacophores of lysophosphatidic acid (LPA) receptors. Here we examine whether farnesyl phosphates can interact with the cell surface and nuclear receptors for LPA. Both farnesyl phosphate and farnesyl diphosphate potently and specifically antagonized LPA-elicited intracellular Ca(2+)-mobilization mediated through the LPA(3) receptor, while causing only modest inhibition at the LPA(2) receptor and no measurable effect at the LPA(1) receptor. Farnesol also inhibited LPA(3) but was much less effective. The estimated dissociation constant of LPA(3) for farnesyl phosphate is 48+/-12 nM and 155+/-30 nM for farnesyl diphosphate. The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) binds to and is activated by LPA and its analogs including fatty alcohol phosphates. We found that both farnesyl phosphate and diphosphate, but not farnesol, compete with the binding of the synthetic PPARgamma agonist [(3)H]rosiglitazone and activate the PPARgamma-mediated gene transcription. Farnesyl monophosphate at 1 microM, but not diphosphate, activated PPARalpha and PPARbeta/delta reporter gene expression. These results indicate new potential roles for the oligoprenyl phosphates as potential endogenous modulators of LPA targets and show that the polyisoprenoid chain is recognized by some LPA receptors.

Topics: Calcium Signaling; Cell Line; Humans; In Vitro Techniques; Kinetics; Ligands; Lysophospholipids; Peroxisome Proliferator-Activated Receptors; Polyisoprenyl Phosphates; Receptors, Lysophosphatidic Acid; Sesquiterpenes; Transfection