Novel clusters of receptors for sphingosine-1-phosphate, sphingosylphosphorylcholine, and (lyso)-phosphatidic acid: new receptors for "old" ligands

The (lyso)phospholipid mediators sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC), and phosphatidic acid (PA) regulate diverse cellular responses such as proliferation, survival and death, cytoskeletal rearrangements, cell motility, and differentiation among many others. Signaling is complex and many signaling events are mediated through the activation of cell surface seven transmembrane (7TM) G protein coupled receptors. Five high affinity receptors for S1P have been identified so far and named S1P(1, 2,3,4,5) (formerly referred to as endothelial differentiation gene (edg)1, 5, 3, 6, 8). Recently, the orphan receptor GPR63 was identified a low affinity S1P receptor structurally distant from the S1P(1-5) family. The orphan GPR3, 6, 12 cluster, phylogenetically related to the edg and melanocortin receptors appears to be subject to modulation by S1P and SPC although all three receptors are strong constitutive stimulators of the Galphas-adenylyl cyclase (AC) pathway and would not require additional ligand stimulation but rather inverse agonism to control activity. Ovarian cancer G protein coupled receptor 1 (OGR1) and GPR4, two structurally closely related receptors were assigned in functional and binding studies as high affinity molecular targets for SPC. Very recently, however, both OGR1 and GPR4 were described as receptors endowed with the ability to signal cells in response to protons. LPA exerts its biological effects through the activation of G protein coupled LPA(1-3) receptors (formerly referred to as edg2, 4, 7). A fourth high affinity LPA receptor has been identified: P2Y9 (GPR23) structurally related to nucleotide receptors and phylogenetically quite distant from the high affinity LPA(1-3) cluster. This review attempts to give an overview about the existing families of lysophosholipid receptors and the spectrum of lipid agonists they use as high or low affinity ligands to relay extracellular signals into intracellular responses. Recently deorphaned lipid receptors, within and outside the known lipid receptor clusters will receive particular attention.     

Biological roles of lysophospholipid receptors revealed by genetic null mice: an update

Two lysophospholipids (LPs), lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), are known to affect various cellular events. Their actions are mediated by binding to at least ten bona fide high-affinity G protein-coupled receptors referred to as LPA(1-5) and S1P(1-5). These LPs are expressed throughout the body and are involved in a range of biological activities including normal development, as well as functioning in most organ systems. A growing number of biological functions have been uncovered in vivo using single- or multiple-null mice for each LP receptor. This review will focus on findings from in vivo as well as in vitro studies using genetic null mice for the LP receptors, LPA(1,2,3) and S1P(1,2,3,5), and for the LP producing enzymes, autotaxin and sphingosine kinase 1/2.     

Cell surface receptors in lysophospholipid signaling

The lysophospholipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), regulate various signaling pathways within cells by binding to multiple G protein-coupled receptors. Receptor-mediated LPA and S1P signaling induces diverse cellular responses including proliferation, adhesion, migration, morphogenesis, differentiation and survival. This review will focus on major components of lysophospholipid signaling: metabolism, identification and expression of LPA and S1P receptors, general signaling pathways and specific signaling mechanisms in mouse embryonic fibroblasts. Finally, in vivo effects of LP receptor gene deletion in mice will be discussed.     

A single amino acid determines lysophospholipid specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P(1)/EDG1 and LPA(1)/EDG2, respectively. We have developed a model of LPA(1) that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA(1)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P(1) study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P(1) and LPA(1) validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA(1), producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors.     

Two novel Xenopus homologs of mammalian LP(A1)/EDG-2 function as lysophosphatidic acid receptors in Xenopus oocytes and mammalian cells

Lysophosphatidic acid (LPA) induces diverse biological responses in many types of cells and tissues by activating its specific G protein-coupled receptors (GPCRs). Previously, three cognate LPA GPCRs (LP(A1)/VZG-1/EDG-2, LP(A2)/EDG-4, and LP(A3)/EDG-7) were identified in mammals. By contrast, an unrelated GPCR, PSP24, was reported to be a high affinity LPA receptor in Xenopus laevis oocytes, raising the possibility that Xenopus uses a very different form of LPA signaling. Toward addressing this issue, we report two novel Xenopus genes, xlp(A1)-1 and xlp(A1)-2, encoding LP(A1) homologs (approximately 90% amino acid sequence identity with mammalian LP(A1)). Both xlp(A1)-1 and xlp(A1)-2 are expressed in oocytes and the nervous system. Overexpression of either gene in oocytes potentiated LPA-induced oscillatory chloride ion currents through a pertussis toxin-insensitive pathway. Injection of antisense oligonucleotides designed to inhibit xlp(A1)-1 and xlp(A1)-2 expression in oocytes eliminated their endogenous response to LPA. Furthermore, retrovirus-mediated heterologous expression of xlp(A1)-1 or xlp(A1)-2 in B103 rat neuroblastoma cells that are unresponsive to LPA conferred LPA-induced cell rounding and adenylyl cyclase inhibition. These results indicate that XLP(A1)-1 and XLP(A1)-2 are functional Xenopus LPA receptors and demonstrate the evolutionary conservation of LPA signaling over a range of vertebrate phylogeny.     

The LPA receptors

Lysophosphatidic acid (LPA) is a growth factor-like lipid that produces many cellular responses. These responses, including actin cytoskeletal rearrangements, cell proliferation and inhibition of gap junction communication, have been documented in many cell types over the last 2 decades. Both non-receptor and receptor-mediated mechanisms had been implicated to explain these responses. A clear advance in this field was the cloning and functional identification of LPA receptors, and there are currently three high-affinity members, LP(A1), LP(A2) and LP(A3) (synonymous with orphan receptor names edg-2, edg-4 and edg-7, respectively). Here we review the gene structure, expression and functions of LPA receptors. We also discuss the in vivo roles mediated by a single LPA receptor type, based on studies of the nervous system, a major locus of LPA receptor expression.     

Lysophospholipid receptors: Signalling, pharmacology and regulation by lysophospholipid metabolism

The lysophospholipids, sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), activate diverse groups of G-protein-coupled receptors that are widely expressed and regulate decisive cellular functions. Receptors of the endothelial differentiation gene family are activated by S1P (S1P_1-5) or LPA (LPA_1-3); two more distantly related receptors are activated by LPA (LPA_4/5); the GPR_3/6/12 receptors have a high constitutive activity but are further activated by S1P and/or SPC; and receptors of the OGR1 cluster (OGR1, GPR4, G2A, TDAG8) appear to be activated by SPC, LPC, psychosine and/or protons. G-protein-coupled lysophospholipid receptors regulate cellular Ca²⁺ homoeostasis and the cytoskeleton, proliferation and survival, migration and adhesion. They have been implicated in development, regulation of the cardiovascular, immune and nervous systems, inflammation, arteriosclerosis and cancer. The availability of S1P and LPA at their G-protein-coupled receptors is regulated by enzymes that generate or metabolize these lysophospholipids, and localization plays an important role in this process. Besides FTY720, which is phosphorylated by sphingosine kinase-2 and then acts on four of the five S1P receptors of the endothelial differentiation gene family, other compounds have been identified that interact with more ore less selectivity with lysophospholipid receptors.     

EDG receptors and hepatic pathophysiology of LPA and S1P: EDG-ology of liver injury

The biological roles of phospholipid growth factors lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been broadly investigated. The cellular effects of LPA and S1P are mediated predominantly via endothelial differentiation gene (EDG) receptors. Yet, the biological significance of LPA, S1P and their EDG receptors in cells of the liver remains unclear. Recent data demonstrate the presence of EDG2 and EDG4 mRNA for LPA receptor in a murine hepatocyte cell line transformed with human TGF-alpha, and in primary mouse hepatocytes. EDG2 receptor protein is expressed in mouse liver, where it appears to be located in nonparenchymal cells. Moreover, we have obtained data suggesting that proliferation of small hepatocyte-progenitors and stem (oval) cells during liver injury is associated with the expression of EDG2 and EDG4 receptors. LPA, and possibly S1P, appear to be essential factors that control proliferation and motility of hepatic stellate cells (HSC) and hepatoma cells. It is proposed that LPA, S1P and their respective EDG receptors play important roles in pathophysiology of chronic liver injury and fibrogenesis. The underlying mechanisms recruited by LPA and S1P in pathogenesis of liver injury remain to be investigated.     

In vivo roles of lysophospholipid receptors revealed by gene targeting studies in mice

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are extracellular ligands for a family of G protein-coupled receptors (GPCRs), LPA1/2/3 and S1P1/2/3/4/5. Through coupling to multiple classes of G proteins and activating multiple signaling pathways, LPA/S1P receptors have been shown to be integral players for many essential cellular and physiological processes. Generation and analysis of mice deficient in each of LPA1, LPA2, S1P1, S1P2, and S1P3 have provided valuable information on the in vivo roles of these receptors. This review is focussed on expression patterns of each receptor gene in wild-type mice, targeted deletion approaches for generating mutant animals, main phenotypes of receptor-null mice, and alterations in signaling characteristics in receptor-deficient primary cells. Altogether, these data give insights to the importance of LPA/S1P receptors at the cellular and organismal level.     

Complete cDNA Sequence, Genomic Structure, and Chromosomal Localization of the LPA Receptor Gene,lpA1/vzg-1/Gpcr26

Thelp_A1/Gpcr26locus encodes the first cloned and identified G-protein-coupled receptor that specifically interacts with lysophosphatidic acid. A murine full-length cDNA of size consistent with that seen on Northern blots (3.7 kb) was determined using 3′ rapid amplification of cDNA ends. Analysis of genomic clones revealed that the gene is divided into five exons, with one intron inserted in the coding region for transmembrane domain VI and one exon encoding the divergent 5′ sequence in another published cDNA clone variant (orphan receptor mrec1.3). This structure differs from the intronless coding region for a homologous receptor,Edg1,but is identical to another more similar orphan receptor (lp_A2) that has been deposited with GenBank. Using backcross analysis, both exons 1 and 4 mapped to a proximal region of murine Chromosome 4 indistinguishable from the vacillans gene. Exon 4 also mapped to a second locus on proximal Chromosome 6 inMus spretus,and this partial duplication was confirmed by Southern blot. The genomic structure indicates a distinct, divergent evolutionary lineage for thevzg-1/lp_A1subfamily of receptors compared to those of homologous orphan receptor genes.     

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y₁₀ receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y₁₀ receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca²⁺ increases in the CHO cells stably expressing the P2Y₁₀ fused with a G_16α protein. These Ca²⁺ responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y₁₀ receptor into the P2Y₁₀-CHO cells effectively blocked both S1P- and LPA-induced Ca²⁺ increases. RT-PCR analysis showed that the mouse P2Y₁₀ was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y₁₀ receptor is the first receptor identified as a dual lysophospholipid receptor.     

Developmental changes in lysophospholipid receptor expression in rodent heart from near-term fetus to adult

Lysophospholipids (LPs) are small signaling lipids that regulate diverse physiological and pathological processes through G protein-coupled receptors. To investigate the function of LP signaling in heart organogenesis and maturation, we measured the expression of 10 confirmed LP receptors (Lpar1-5 and S1pr1-5) in rat heart from embryonic day 19.5 (E19.5d) to postnatal week 12 (P12w). The expression of Lpar3 mRNA peaked at 37-fold higher than adult expression at P1d, while the expression levels of Lpar1 and Lpar4 increased markedly after P1d and peaked at 19- and 48-folds of adult expression on P7d. The expression levels of all three receptor mRNAs were significantly reduced by P21d and remained low thereafter. Expression of the corresponding receptor proteins also peaked during the early postnatal period but the subsequent decline was less dramatic from P14d to P12w compared to mRNA expression. In contrast, S1pr1 and S1pr3 exhibited more gradual developmental changes. Although early expression was higher than mature expression (3- to 6-fold), these receptors were still strongly expressed at P12w. The other isotypes examined, Lpar2, Lpar5, S1pr4, and S1pr5, were very weakly expressed at all developmental stages. Analysis of receptor distribution within the developing heart (P1d) revealed that Lpar1, Lpar3, and Lpar4 were expressed in the myocardium of all four chambers but not in valves, while Lpar3 was also uniquely expressed in the aorta and coronary vessels. Western blots revealed that the developmental changes in Lpar1, Lpar3, and Lpar4 protein expression mirrored changes in β-actin and β-tubulin expression. The increase in Lpar1 and Lpar4 receptors from P1d to P7d corresponds to the period of rapid myocardial growth and functional maturation. Moreover, the relatively high expression of Lpar1, Lpar3, and Lpar4 in the late prenatal rat heart suggests that these LPA receptors may also contribute to organogenesis. The increase in Lpar3 and Lpar4 expression concomitant with rising expression of cytoskeleton proteins further suggests a possible role for LPA signaling in cytoskeletal remodeling during cardiac development.     

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

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

Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein-coupled receptors to mediate similar actions.     

Sharpening the edges of understanding the structure/function of the LPA1 receptor: expression in cancer and mechanisms of regulation

Since the molecular cloning of the vzg-1/Edg-2/LPA1 gene, studies have attempted to characterize LPA1 receptor functionality into a single categorical role, different from the other Edg-family LPA receptors. The desire to categorize LPA1 function has highlighted its complexity and demonstrated that the LPA1 receptor does not have one absolute function throughout every system. The central nervous system is highly enriched in the LPA1 receptor, suggesting an integral role in neuronal processes. Metastatic and invasive breast cancer also appears to have LPA-mediated LPA1 receptor functions that enhance phenotypes associated with tumorigenesis. LPA1 possesses a number of motifs conserved among G protein-coupled receptors (GPCRs): a DRY-like motif, a PDZ domain, Ser/Thr predicted sites of phosphorylation, a di-leucine motif, double cysteines in the tail and conserved residues that stabilize structure and determine ligand binding. The third intracellular loop of the LPA1 receptor may be the crux of receptor signaling and attenuation with phosphorylation of Thr-236 potentially a key determinant of basal LPA1 signaling. Mutagenesis data supports the notion that Thr-236 regulates this process since mutating Thr-236 to Ala-236 increased basal and LPA-mediated serum response factor (SRF) signaling activity and Lys-236 further increased this basal signaling. Here we describe progress on defining the major functions of the LPA1 receptor, discuss a context dependent dualistic role as both a negative regulator in cancer and a proto-oncogene, outline its structural components at the molecular amino acid level and present mutagenesis data on the third intracellular loop of the receptor.     

Targeted deletion of LPA5 identifies novel roles for lysophosphatidic acid signaling in development of neuropathic pain

Lysophosphatidic acid (LPA) is a bioactive lipid that serves as an extracellular signaling molecule acting through cognate G protein-coupled receptors designated LPA(1-6) that mediate a wide range of both normal and pathological effects. Previously, LPA(1), a G(αi)-coupled receptor (which also couples to other G(α) proteins) to reduce cAMP, was shown to be essential for the initiation of neuropathic pain in the partial sciatic nerve ligation (PSNL) mouse model. Subsequent gene expression studies identified LPA(5), a G(α12/13)- and G(q)-coupled receptor that increases cAMP, in a subset of dorsal root ganglion neurons and also within neurons of the spinal cord dorsal horn in a pattern complementing, yet distinct from LPA(1), suggesting its possible involvement in neuropathic pain. We therefore generated an Lpar5 null mutant by targeted deletion followed by PSNL challenge. Homozygous null mutants did not show obvious base-line phenotypic defects. However, following PSNL, LPA(5)-deficient mice were protected from developing neuropathic pain. They also showed reduced phosphorylated cAMP response element-binding protein expression within neurons of the dorsal horn despite continued up-regulation of the characteristic pain-related markers Caα(2)δ(1) and glial fibrillary acidic protein, results that were distinct from those previously observed for LPA(1) deletion. These data expand the influences of LPA signaling in neuropathic pain through a second LPA receptor subtype, LPA(5), involving a mechanistically distinct downstream signaling pathway compared with LPA(1).     

Biochemical regulation of breast cancer cell expression of S1P2 (Edg-5) and S1P3 (Edg-3) G protein-coupled receptors for sphingosine 1-phosphate

G protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) transduce signals to many functions of normal cells. Most human cancer cells upregulate S1P and LPA GPCRs, in patterns distinctive for each type of tumor. The findings that 1-alpha, 25-dihydroxy-vitamin D(3) (VD3) and all-trans retinoic acid (RA) differentially alter expression of the predominant S1P(3) (Edg-3) R and S1P(2) (Edg-5) R in human breast cancer cells (BCCs) permitted analyses of their individual activities, despite a lack of selective pharmacological probes. S1P-evoked increases in [Ca(2+)](i) in S1P(3) R-predominant BCCs were suppressed by concentrations of VD3 and RA which decreased expression of S1P(3) Rs, despite RA-induced increases in S1P(2) Rs. S1P-elicited chemokinetic migration of S1P(3) R-predominant BCCs across a type IV collagen-coated micropore filter also was inhibited by concentrations of VD3 and RA which decreased expression of S1P(3) Rs. The RA-induced increase in expression of S1P(2) Rs did not prevent suppression by RA of S1P-elicited chemokinesis, which appears to be mediated by S1P(3) Rs, but instead exposed S1P(2) R-mediated inhibition of epidermal growth factor-stimulated chemotaxis of BCCs. In contrast, expression of the predominant LPA(2) Rs, LPA-evoked increase in [Ca(2+)](i) and LPA-stimulated chemokinetic migration were suppressed concomitantly by RA but not VD3. Thus two structurally-homologous S1P Rs of BCCs differ in coupling to [Ca(2+)](i) signaling and have opposite effects on protein growth factor-stimulated chemotaxis.