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Genomic insights into mediator lipidomics
Prostaglandins & other Lipid Mediators 77 (2005) 197–209
Genomic insights into mediator lipidomics Timothy Hla ∗ Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA Received 1 June 2005; received in revised form 2 June 2005; accepted 7 June 2005
Abstract G protein-coupled receptors (GPCR) are used ubiquitously and widely for signal transduction across the plasma membrane. The ligands for GPCRs are structurally diverse and include peptides, odorants, photon, ions and lipids. It is thought that GPCRs evolved by gene duplication and mutational events that diversified the ligand binding and signaling properties, thereby resulting in paralogues in various organisms. Genomic sequencing efforts of various organisms indicate that GPCRs evolved very early in evolution; for example, unicellular eukaryotes use GPCRs for mating, differentiation and sporulation responses and prokarotes utilize these receptors for phototransduction, as exemplified by the bacteriorhodopsin, a photon sensor. Many GPCRs fall into subfamilies, usually determined by structural similarity to their ligands. Bioactive lipids such as lysophospholipids, eicosanoids, ether lipids and endocannabinoids, which are produced widely in evolution, also signal through GPCRs. Thus, distinct subfamilies of bioactive lipid GPCRs, such as prostanoid receptors, lysophosphatidic, sphingosine 1-phosphate, leukotrienes, hydroxy fatty acids, endocannabinoids and ether lipids exist in the mammalian genome. With the increasing availability of genomic information throughout the phylogenetic tree, orthologues of bioactive lipid receptors are found in the genomes of vertebrates and chordates but not in worms, flies or other lower organisms. This is in contrast to GPCRs for biogenic amines and polypeptide growth factors, which are conserved in invertebrates as well. Thus, it appears that with the evolution of chordates, lipids may have acquired novel roles in cellcell communication events via GPCRs. This hypothesis will be discussed using the prostanoid and lysophospholipid signaling systems. Since such bioactive lipids play critical roles in immune, vascular and nervous systems, this suggests that lipid metabolite signaling via the GPCRs co-evolved
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1098-8823/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2005.06.008
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with the development of sophisticated vascular, immune and nervous systems in chordates and vertebrates. © 2005 Elsevier Inc. All rights reserved. Keywords: Prostaglandins; Cyclooxygenase; COX; Sphingosine; Sphingolipid mediators; Receptors; G proteincoupled receptors; Mammals; Vertebrates; Invertebrates; Chordates; Ciona intestinalis; Evolution
Contents 1. 2. 3. 4.
Bioactive lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptors for bioactive lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prostanoid signaling system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lysosphingolipid signaling system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Bioactive lipids Lipids are essential molecules for the structure of cells and organelles. Cellular membranes (plasma- and organelle-membranes) require specific structural motifs for optimum functionality under physiological conditions and to withstand cellular stresses. Thus, complex metabolic pathways are needed to properly synthesize and degrade membranes, depending on the needs of the cell. However, in multicellular organisms, a large fraction of the organism’s effort must be spent on cell–cell communication, so that different cells and tissues function in synchrony. Membrane lipids are used in higher organisms in cell–cell communication events. Examples include eicosanoids, lysophospholipids, ether lipids and endocannabinoids. These molecules are collectively referred to as lipid mediators or bioactive lipids. In many circumstances, they are used in autocrine and/or paracrine signaling paradigms, perhaps due to their limited stability and physicochemical properties. Most of the receptors on cells that respond to lipid mediators belong to the G protein-coupled receptor (GPCR) superfamily of plasma membrane receptors [1]. GPCRs are a large family of plasma membrane receptors that are activated by a wide variety of ligands, including, photon, H+ ion, small molecules, odorants, peptides, proteins and lipids [2]. These receptors are encoded by seven membrane spanning helix containing proteins. The basic structural motif of these receptors form an extracellular and/or transmembrane ligand binding pocket and an intracellular domain that interact with signal transducer molecules, most prominent family being that of heterotrimeric G proteins. Ligand binding results in conformational changes of the receptor that allows for productive interaction with the G proteins and other effector molecules. It is thought the basic structural motif of seven transmembrane helices is well suited in cell–cell communication events that utilize diffusible mediators. Most lipid receptors belong to the Rhodopsin superfamily of GPCRs [1]. Historically, lipid mediators were discovered by investigators utilizing specific biological assays, such as contraction of smooth muscle preparation, platelet aggregation, neutrophil
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migration, tissue inflammation, among others. Examples include prostaglandins, platelet activating factor, leukotrienes (slow reacting substance of anaphylaxis) and thromboxane A2 (rabbit aorta contracting substance) [3]. More recently, tissue culture systems of mammalian fibroblasts have led to the discovery of another family of lipid mediators, that of lysophospholipids [4]. It was significantly later that the molecular basis of lipid mediator action was revealed, when molecular pharmacologists cloned and defined the receptors for bioactive lipid mediators [1]. It is now well-established as a general principle that most bioactive lipid mediators act on GPCRs. Intriguingly, other widely utilized membranebound structural motifs, such as receptor tyrosine kinases, receptor serine kinases have not been identified as responders to bioactive lipids. However, it is worth noting that several lipid activated transcription factors respond to intracellular lipids, for example fatty acids, hydroxy fatty acids and prostanoids [5]. This review will not consider these family of molecules, which has received significant attention recently. Instead, I will focus on the GPCR family of bioactive lipid mediators.
2. Receptors for bioactive lipids The first GPCRs to be molecularly characterized were rhodopsin, -adrenergic, serotonin and neuropeptide receptors [6]. It soon became apparent that primary sequences of such receptors were related, suggesting that such receptors may have evolved by gene duplication events. The genomic structures of GPCRs also supported this notion, as many of these receptors are encoded by compact genes with few exons [7]. In many cases, a single exon encodes the entire open reading frame of the GPCR. It also became clear that receptors that bind to the same ligand, i.e., receptor subtypes, are closer in sequence similarity than those that bind to unrelated ligands. Therefore, ␣- and -adrenergic receptors are more closely related to each other in primary sequence than the sequence similarity between ␣-adrenergic and bombesin receptors. Thus, sequence alignment of GPCR sequences and representation of the data with an evolutionary tree diagram (dendrogram) shows that receptor subtypes cluster into subfamilies of sequence relatedness. With the advent of the polymerase chain reaction (PCR) technology, it became apparent that a large number of GPCR sequences exist. Through degenerate PCR approaches, numerous receptor sequences were isolated [8]. In some cases, high level of sequence relatedness provided an essential clue to the ligand identification of such receptors. However, in some cases, expression patterns of the receptor transcript led to the correct identification of the ligand. Such was the case in the identification of the CNR1 (CB1) cannabinoid receptor by Matsuda et al. [9]. Early efforts to characterize the bioactive lipid GPCRs followed classical approaches of protein purification, followed by protein microsequencing and cDNA library-based cloning efforts. Such was the example of the cloning of the thromboxane A2 receptor, a prototypical member of the prostanoid receptor subfamily [10]. This was rapidly followed by the cloning of other prostanoid receptors, for example for PGE2 , PGF2␣ , PGD2 and PGI2 , since these receptors are highly related in primary sequence [11]. Currently, we know of the following receptor subfamilies; prostanoid, hydroxy eicosatetraenoate, lipoxin, leukotriene B4 , cystenieyl leukotrienes, lysophosphatidic acid, sphingosine 1-phosphate, endocannabinoid, platelet activating factor and free fatty acids [1]. There are also some
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receptor sequences which have been classified as “orphan receptors”, as the ligands for such receptors remain to be characterized. Collectively, these receptor sequences indicate the diversity of the extracellular lipidome, because all the ligands for these receptors are present in the extracellular environment and are capable of intercellular communication events. In some rare cases, immunoreactive receptor epitopes have been detected in the nucleus [12]. The significance and generality of such data are not clear. However, it is clear that the vast majority of GPCRs for bioactive lipids function at the plasma membrane. It is worth considering whether bioactive lipids are freely secreted into the extracellular milieu and function as freely diffusible mediators. Clearly, structural diversity exists among bioactive lipids and more hydrophilic mediators, such as cysteinyl leukotrienes may be freely diffusible. Many lipid mediators are found complexed to carrier proteins, such as serum albumin and serum lipoproteins, as has been shown for sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) [13]. The mode of action of receptor activation is also worthy of consideration. It is not known if bioactive lipids interact with the plasma membrane, followed by lateral binding to the GPCR. Alternatively, carrier protein/bioactive lipid complex could interact with the receptor to exchange the ligand. It is noteworthy that the Kd of ligand binding to GPCRs is in the nanomolar (10−9 M) range whereas that of binding to albumin is in the 10−7 to 10−6 M range [14,15]. Presumably, the association of bioactive lipids with plasma membranes will be lower affinity and higher capacity. However, due to energetic constraints, the bioactive lipid (such as LPA or S1P) may not be able to efficiently flip between the bilayers. Specific transporters and/or carriers for bioactive lipids are needed to efficiently uptake bioactive lipids from one cellular compartment to the other. For example, the prostanoid transporter functions to uptake classical prostaglandins from the extracellular environment into the cytosol [16]. Although the diversity of ligands necessitates the heterogeneity in structure of ligand binding pockets of lipid GPCRs, the signaling properties of such receptors follow the classical receptor/G protein signaling paradigms. Thus, most, if not all of the bioactive lipid GPCRs interact with one or more of classical heterotrimeric G proteins (Gs , Gi/o , Gq/11 , G12/13 and G16 ) [6]. Such interactions lead to down-stream signaling events, such as small GTPase activation, cytosolic and nuclear phosphorylation events (MAP kinases), and eventually changes in the activity of transcription factors. In many cases, non-transcriptional changes in cell behavior are also seen, for example, those that change the activity of cell–cell and cell–matrix adhesion molecules [17]. Thus, the bioactive lipidome modulates cell proliferation, migration, differentiation, death and fate via the GPCR repertoire of the organism.
3. Prostanoid signaling system Prostanoids are ubiquitous mediators synthesized by the cyclooxygenase (COX) pathway which oxidizes polyunsaturated fatty acids such as arachidonic acid (20:4), linoleic acid (18:2), eicosapentaenoid acid (20:5), docosahexaenoic acid (22:6) as well as derivatives such as 2-arachidonyl glycerol (2-AG) [18]. They have the prostanoic ring structure and diversity in double bond placement, hydroxyl and ketone moieties, which determine the biologically distinct prostanoids, such as prostaglandin E2 (PGE2 ), thromboxane A2 (TXA2 ), etc. Prostanoids are highly unstable and act as local hormones or autocoids.
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The major, rate-limiting enzyme in the biosynthesis of prostanoids is cyclooxygenase, which is also known as PG endoperoxide synthase or PG H synthase [18]. Two genes, commonly referred to as COX-1 and COX-2 encode the COX enzyme. The enzymes are localized in the luminal compartment of the endoplasmic reticulum. This enzyme catalyzes the formation of PGH2 , which is subsequently converted into bioactive prostanoids by the action of specific synthases, for example, thromboxane synthase, prostacyclin synthase, PGE synthase, etc. The subcellular location of these synthases are not well understood. However, once formed, prostanoids are rapidly secreted by cells. Although prostanoid transporters exist, their function appears to be involved in the uptake of prostanoids by cells [16]. Once secreted, prostanoids are thought to act on target cells by binding to specific cell surface receptors. Many prostanoid receptors are molecularly characterized. Each major prostanoid, for example, PGE2 , PGF2␣ , TXA2 , PGI2 and PGD2 act on structurally related GPCR isoforms [11]. For example, PGE2 acts on four related GPCRs termed as EP1-4 , PGF2␣ , PGI2 and TXA2 acts on single FP, IP and TP receptors, whereas PGD2 acts on DP and CRTH2 family of structurally unrelated receptors. Prostanoid receptors are expressed in different cell types and exhibit differential signaling properties. For example, the TP receptor is expressed in platelets and is coupled to the Gq , G12/13 and Gi family of G proteins whereas the IP receptor on the platelets is coupled mainly to the Gs family of heterotrimeric G proteins. By acting on prostanoid receptors, this family of bioactive lipid mediators modulates the activity of most organ systems in higher vertebrates. Well-studied effects of prostanoids include vascular smooth muscle contraction, platelet aggregation, pain, inflammation, febrile response, uterine contraction and immune modulation [11]. The COX enzymes were initially cloned from mammalian sources [18]. Structurally these enzymes show distant similarity to animal and plant peroxidases. The COX enzyme catalyzes the formation of PGG2 by bis-oxygenation of arachidonic acid and then reduction to PGH2 . Although the COX-1 isoenzyme prefers arachidonic acid, the COX-2 isoform can utilize a broader array of substrates, including 2-arachidonyl glycerol and anadamide. The COX isoenzymes are the targets of non-steroidal anti-inflammatory drugs and are widely studied for their physiological and pathological roles. Recent genomic sequencing efforts have indicated that COX-1 and COX-2 genes are conserved in the vertebrate genome, as homologous sequences have been isolated from mammalian, avian, reptilian, amphibian sources. Interestingly, in the bony fishes, zebrafish (Danio rario) and pufferfish (Tetraodon nigroviridis) both COX isoenzyme orthologues were found. In the cartiligenous fish (dogfish shark: Squalus acanthias) only one COX sequence was characterized, which may be due to the fact that only limited information of the genome of this organism is available at the present time [19]. However, in the chordate Ciona intestinalis (commonly referred to as the seasquirt), two genes that are highly related to the COX isoenzymes were observed (Figs. 1 and 2). In contrast, the COX enzyme homologues were not found widely in invertebrates, for example in the completely characterized genomes of Drosophila melanogaster, C. elegans, S. cerevesiae and D. discoidium. In some specialized cases, for example in some sea corals, in which prostanoids are used as defense molecules, the COX enzymes were expressed; however, in this case, a single COX enzyme was necessary. This supports the notion that the general use of prostanoids in cell–cell communication events in multiple organ systems evolved with the development of the chordates.
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Fig. 1. Alignment of COX-1, COX-2 and COX-like sequences from various vertebrates, the chordate Ciona intestinalis and the coral Gersemia fruticosa. The primary amino acid sequences from various organisms were obtained from the Genbank and ENSEBL databases (http://www.ensembl.org/) and aligned with the program MultAlin program (http://prodes.toulouse.inra.fr/multalin/multalin.html). Conserved residues are indicated in red and a consensus sequence is indicated in the last line. Note that vertebrate COX-1 and -2 sequences clustered into groups whereas chordate and coral COX sequences showed considerable divergence, even though essential catalytic residues are conserved.
On the other hand, the PG receptor sequences are only present in vertebrates and chordates. As shown in Fig. 3, clustal analysis and Dendrogram representation of PG receptor sequences show that receptor orthologues cluster in closely related tree branches. Therefore, mouse and human orthologues of EP, FP, TP, IP and DP receptor sequences are closest to each other, as indicated by the distal branch points of the tree. Several PG receptorrelated sequences from the pufferfish Tetraodon appear to be orthologues of mammalian PG receptors. For example, TetPRL2, TetPRL3 and TetPRL4 appear to be orthologues of mammalian TP, EP3 and EP4 receptors, respectively. Some zebrafish prostanoid receptors may be orthologues, as they are closely related to Tetraodon receptors. An example of this is that zPRL3 maybe a zebrafish orthologue of the mammalian EP4 receptor. The genome of the cartiligenous fish are not yet complete and PG receptor sequences are not available from this organism at this time. Interestingly, one PG receptor-related sequence was found in the Ciona intestinalis genome. For example, the Ciona EP4-like sequence is highly related to three Tetraodon PRL sequences, zPRL1 and somewhat distantly to mammalian EP4 receptors. Whether such sequences are indeed bona fide PGE receptor sequences must be determined experimentally. However, it is known that Ciona synthesize prostanoids and different prostanoids are known to exert biological effects in this organism [20,21]. The genomic data provides additional support for the presence of the classical prostanoid signaling system in this organism. In contrast, extensive searches in the genomes of Drosophila, C. elegans, S. cerevesiae and D. discoidium did not yield homologous sequences, suggesting
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Fig. 2. Dendrogram of COX polypeptide sequences. The COX-1, -2 and COX-like sequences from Fig. 1 (above) were analyzed by the CLUSTAL program (http://clustalw.genome.jp/) for relatedness [34]. An unrooted dendrogram is shown. h: human; m: murine; c: chicken; z: zebrafish; x: Xenopus; df: dogfish shark. Note that the cloned dfCOX is closer to the COX-1 group. The coral and Ciona COX-like sequences cluster into its own group, as they are distant in primary sequence to vertebrate COX enzymes.
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Fig. 2. (Continued )
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Fig. 2. (Continued ).
Fig. 3. Dendrogram of PG receptor sequences. Amino acid sequences of prostaglandin E2 (EP1-4), I2 (IP), D2 (DP and CRTH2), F2␣ (FP) and thromboxane A2 (TP) from human, mouse, Tetraodon, zebrafish as well as a related Ciona sequence were analyzed by the CLUSTAL program and represented as an unrooted tree. H: human; m: murine; Tet: Tetraodon, z: zebrafish; PRL: prostanoid receptor-like.
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that prostanoid receptors evolved with the occurrence of the chordates in the animal kingdom. This is also supported by the presence of COX genes in the chordates and vertebrates as discussed above. As chordates and vertebrates have sophisticated nervous, circulatory and immune systems, it is possible that lipid mediator signaling systems are needed to fine tune these complicated organ systems. In contrast, polypeptide signaling systems are also present in lower metazoans such as C. elegans and Drosophila. Thus, the full complement of lipid-based and peptide-based signaling pathways in higher animals may be needed. Although the prostanoids are involved in the regulatory processes in most, if not all organ systems in mammals, the evolutionary analysis of prostanoid synthase enzymes and receptors suggest that in the early stages of its evolution, prostanoids may have been utilized primarily in neuronal, immune and vascular systems. This is supported by the fact that even in mammals, prostanoids are major regulators of neuronal (e.g. pain), vascular (e.g. thrombosis, tone control, permeability) and immune (e.g. inflammation) systems.
4. Lysosphingolipid signaling system Sphingomyelin (SM) is an abundant membrane phospholipid that is critical for the integrity of membrane function in eukaryotes. It is metabolized by the sphingomyelinase (SMase) pathway to generate sphingolipid mediators such as sphingosine, ceramide and sphingosine 1-phosphate (S1P) [22]. Key enzymes in this pathway include sphingomyelinases, ceramidases, sphingosine kinases, S1P phosphatases and S1P lyase. Sphingolipid mediators are thought to regulate various intracellular processes in both unicellular eukaryotes and metazoans, even though the manner in which they mediate such effects are poorly understood. For example, in the yeast S. cerevesiae, loss of function of sphingolipid metabolic enzymes, sphingosine kinase, S1P lyase or phosphatase, results in alterations in cellular growth, stress response, protein synthesis and endocytosis responses [23]. Similar findings were also found in invertebrates such as C. elegans and Drosophila, suggesting the widespread conservation of sphingolipid function in evolution [24]. However, recent studies in mammalian cells have shown that S1P binds to the GPCRs of the EDG-1 family with high affinity and transduces signals into cells [25,26]. Currently, it is established that five GPCRs (S1P1 to S1P5 ) are S1P receptors that mediate various biological functions of S1P across cells. Interestingly, tissue expression of these receptors are divergent and many tissues express one or more of S1P receptors. The function of S1P receptors probably explain the multiple biological effects of S1P, which include heart function, vascular tone, vascular permeability, angiogenesis, vascular maturation, immune cell trafficking, mast cell activation, neuronal cell survival, oocyte survival and sperm viability [13]. These receptors are coupled to intracellular signal transduction pathways via the heterotrimeric G proteins. This aspect of S1P receptors will not be reviewed here; the reader is referred to a number of recent reviews published on this subject [17,27,28]. Evolutionary conservation of S1P metabolic enzymes and receptors may provide some insights into the workings of this lipid mediator system. All the sphingolipid metabolic enzymes are conserved throughout evolution from unicellular eukaryotes to mammals. For example, orthologues of sphingosine kinase-1 and -2 have been found from S. cerevesiae to humans [26]. Functional studies have documented that these enzymes carry out similar
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Fig. 4. Dendrogram of S1P, LPA and cannabinoid (CNR) receptor sequences. Amino acid sequences of human, Tetraodon, zebrafish, Fugu, and Ciona sequences were analyzed by the CLUSTAL program and an unrooted dendrogram is shown. T: Tetraodon; h: human; z: zebrafish; c: Ciona.
enzymatic reactions. In addition, genetic studies have shown that sphingosine kinases play important roles in unicellular eukaryotic and metazoan invertebrate biology. Distinct phenotypes were shown in organisms with sphingosine kinase genes deleted. For example, in Drosophila, deletion of the sphingosine kinase gene leads to egg laying and muscle defects [24]. In sharp contrast, S1P receptor genes were found only in vertebrates and perhaps in chordates but lacking in invertebrates. This is illustrated in Fig. 4. The five S1PRs cluster into a distinct group, which is closely related to the lysophosphatidic acid (LPA) and cannabinoid (CNR) receptor subfamilies. All mammalian genomes examined possess S1P receptor orthologues. The five paralogues are also represented in mammalian genome, suggesting that gene duplication events of S1PRs occurred earlier in evolution. Indeed, all five receptor orthologues were found in the genome data banks of bony fishes, namely, Takafugu rubipres, D. rario and T. nigroviridis, as shown in Fig. 3. Although not shown in the figure, other vertebrate species, namely Xenopus, also contain S1P receptor orthologues. However, related orthologous sequences were not found in invertebrate genome databases. Similarly, LPA receptor sequences were seen in the vertebrate but not invertebrate genomes. These data support the notion that lysophospolipid receptors evolved as the vertebrate species developed. Interestingly, the release of the complete genome sequence of the chordate, Ciona intestinalis (seasquirt) [29], suggested that lysophospholipid receptors may have occurred in the phylum chordata as well. A single open reading frame was seen in the Ciona genome, and homology analysis suggests that this GPCR-like sequence is distantly related to the cannabinoid receptors of the seasquirt and the lysophospholipid receptors from vertebrates
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(Fig. 4). Although experimental evidence is lacking, this finding supports the notion that lysophospholipid signaling may have begun with the development of chordates in evolution. Studies in vertebrates have shown that S1P is present abundantly in the blood and plasma. In humans, 100–400 nM of S1P is present in normal plasma, which is much higher than the binding constant for the ligand for its receptors (Kd ∼ 5–50 nM) [30]. Indeed, S1P receptor dynamics are critical for its effects on angiogenesis and immune cell trafficking. Induction and internalization/recycling of receptors for S1P are essential for these biological effects [31–33]. Since S1P is a major regulator of neuronal, vascular and immune systems, it is possible that the development of complexity in those systems that occurred in the chordates and vertebrates necessitated the S1P signaling via its plasma membrane receptors. Why this simple lipid mediator was selected in the evolutionary process is an interesting question for further consideration and analysis. Genome informatics of the extracellular lipid mediators has provided interesting insights into the function of such systems in the mammals. As exemplified in this review, prostanoid synthases and receptors seem to have evolved in the chordates. In contrast, the sphingolipid mediators were present throughout the eukaryotic kingdom as their metabolic enzymes are widely expressed. However, the receptors for S1P appear to be vertebrate and perhaps chordate-specific. Through the analysis of genomic information, unique insights on the extracellular lipidome may be forthcoming. Furthermore, structure function studies on receptors and metabolic enzymes of lipid mediators can be facilitated, by examining conserved as well as divergent residues. Ultimately, genome informatics may provide a unique tool in the understanding of the extracellular lipidome.
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Genomic insights into mediator lipidomics Timothy Hla ∗ Center for Vascular Biology, Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030, USA Received 1 June 2005; received in revised form 2 June 2005; accepted 7 June 2005
Abstract G protein-coupled receptors (GPCR) are used ubiquitously and widely for signal transduction across the plasma membrane. The ligands for GPCRs are structurally diverse and include peptides, odorants, photon, ions and lipids. It is thought that GPCRs evolved by gene duplication and mutational events that diversified the ligand binding and signaling properties, thereby resulting in paralogues in various organisms. Genomic sequencing efforts of various organisms indicate that GPCRs evolved very early in evolution; for example, unicellular eukaryotes use GPCRs for mating, differentiation and sporulation responses and prokarotes utilize these receptors for phototransduction, as exemplified by the bacteriorhodopsin, a photon sensor. Many GPCRs fall into subfamilies, usually determined by structural similarity to their ligands. Bioactive lipids such as lysophospholipids, eicosanoids, ether lipids and endocannabinoids, which are produced widely in evolution, also signal through GPCRs. Thus, distinct subfamilies of bioactive lipid GPCRs, such as prostanoid receptors, lysophosphatidic, sphingosine 1-phosphate, leukotrienes, hydroxy fatty acids, endocannabinoids and ether lipids exist in the mammalian genome. With the increasing availability of genomic information throughout the phylogenetic tree, orthologues of bioactive lipid receptors are found in the genomes of vertebrates and chordates but not in worms, flies or other lower organisms. This is in contrast to GPCRs for biogenic amines and polypeptide growth factors, which are conserved in invertebrates as well. Thus, it appears that with the evolution of chordates, lipids may have acquired novel roles in cellcell communication events via GPCRs. This hypothesis will be discussed using the prostanoid and lysophospholipid signaling systems. Since such bioactive lipids play critical roles in immune, vascular and nervous systems, this suggests that lipid metabolite signaling via the GPCRs co-evolved
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1098-8823/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2005.06.008
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with the development of sophisticated vascular, immune and nervous systems in chordates and vertebrates. © 2005 Elsevier Inc. All rights reserved. Keywords: Prostaglandins; Cyclooxygenase; COX; Sphingosine; Sphingolipid mediators; Receptors; G proteincoupled receptors; Mammals; Vertebrates; Invertebrates; Chordates; Ciona intestinalis; Evolution
Contents 1. 2. 3. 4.
Bioactive lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Receptors for bioactive lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prostanoid signaling system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lysosphingolipid signaling system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Bioactive lipids Lipids are essential molecules for the structure of cells and organelles. Cellular membranes (plasma- and organelle-membranes) require specific structural motifs for optimum functionality under physiological conditions and to withstand cellular stresses. Thus, complex metabolic pathways are needed to properly synthesize and degrade membranes, depending on the needs of the cell. However, in multicellular organisms, a large fraction of the organism’s effort must be spent on cell–cell communication, so that different cells and tissues function in synchrony. Membrane lipids are used in higher organisms in cell–cell communication events. Examples include eicosanoids, lysophospholipids, ether lipids and endocannabinoids. These molecules are collectively referred to as lipid mediators or bioactive lipids. In many circumstances, they are used in autocrine and/or paracrine signaling paradigms, perhaps due to their limited stability and physicochemical properties. Most of the receptors on cells that respond to lipid mediators belong to the G protein-coupled receptor (GPCR) superfamily of plasma membrane receptors [1]. GPCRs are a large family of plasma membrane receptors that are activated by a wide variety of ligands, including, photon, H+ ion, small molecules, odorants, peptides, proteins and lipids [2]. These receptors are encoded by seven membrane spanning helix containing proteins. The basic structural motif of these receptors form an extracellular and/or transmembrane ligand binding pocket and an intracellular domain that interact with signal transducer molecules, most prominent family being that of heterotrimeric G proteins. Ligand binding results in conformational changes of the receptor that allows for productive interaction with the G proteins and other effector molecules. It is thought the basic structural motif of seven transmembrane helices is well suited in cell–cell communication events that utilize diffusible mediators. Most lipid receptors belong to the Rhodopsin superfamily of GPCRs [1]. Historically, lipid mediators were discovered by investigators utilizing specific biological assays, such as contraction of smooth muscle preparation, platelet aggregation, neutrophil
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migration, tissue inflammation, among others. Examples include prostaglandins, platelet activating factor, leukotrienes (slow reacting substance of anaphylaxis) and thromboxane A2 (rabbit aorta contracting substance) [3]. More recently, tissue culture systems of mammalian fibroblasts have led to the discovery of another family of lipid mediators, that of lysophospholipids [4]. It was significantly later that the molecular basis of lipid mediator action was revealed, when molecular pharmacologists cloned and defined the receptors for bioactive lipid mediators [1]. It is now well-established as a general principle that most bioactive lipid mediators act on GPCRs. Intriguingly, other widely utilized membranebound structural motifs, such as receptor tyrosine kinases, receptor serine kinases have not been identified as responders to bioactive lipids. However, it is worth noting that several lipid activated transcription factors respond to intracellular lipids, for example fatty acids, hydroxy fatty acids and prostanoids [5]. This review will not consider these family of molecules, which has received significant attention recently. Instead, I will focus on the GPCR family of bioactive lipid mediators.
2. Receptors for bioactive lipids The first GPCRs to be molecularly characterized were rhodopsin, -adrenergic, serotonin and neuropeptide receptors [6]. It soon became apparent that primary sequences of such receptors were related, suggesting that such receptors may have evolved by gene duplication events. The genomic structures of GPCRs also supported this notion, as many of these receptors are encoded by compact genes with few exons [7]. In many cases, a single exon encodes the entire open reading frame of the GPCR. It also became clear that receptors that bind to the same ligand, i.e., receptor subtypes, are closer in sequence similarity than those that bind to unrelated ligands. Therefore, ␣- and -adrenergic receptors are more closely related to each other in primary sequence than the sequence similarity between ␣-adrenergic and bombesin receptors. Thus, sequence alignment of GPCR sequences and representation of the data with an evolutionary tree diagram (dendrogram) shows that receptor subtypes cluster into subfamilies of sequence relatedness. With the advent of the polymerase chain reaction (PCR) technology, it became apparent that a large number of GPCR sequences exist. Through degenerate PCR approaches, numerous receptor sequences were isolated [8]. In some cases, high level of sequence relatedness provided an essential clue to the ligand identification of such receptors. However, in some cases, expression patterns of the receptor transcript led to the correct identification of the ligand. Such was the case in the identification of the CNR1 (CB1) cannabinoid receptor by Matsuda et al. [9]. Early efforts to characterize the bioactive lipid GPCRs followed classical approaches of protein purification, followed by protein microsequencing and cDNA library-based cloning efforts. Such was the example of the cloning of the thromboxane A2 receptor, a prototypical member of the prostanoid receptor subfamily [10]. This was rapidly followed by the cloning of other prostanoid receptors, for example for PGE2 , PGF2␣ , PGD2 and PGI2 , since these receptors are highly related in primary sequence [11]. Currently, we know of the following receptor subfamilies; prostanoid, hydroxy eicosatetraenoate, lipoxin, leukotriene B4 , cystenieyl leukotrienes, lysophosphatidic acid, sphingosine 1-phosphate, endocannabinoid, platelet activating factor and free fatty acids [1]. There are also some
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receptor sequences which have been classified as “orphan receptors”, as the ligands for such receptors remain to be characterized. Collectively, these receptor sequences indicate the diversity of the extracellular lipidome, because all the ligands for these receptors are present in the extracellular environment and are capable of intercellular communication events. In some rare cases, immunoreactive receptor epitopes have been detected in the nucleus [12]. The significance and generality of such data are not clear. However, it is clear that the vast majority of GPCRs for bioactive lipids function at the plasma membrane. It is worth considering whether bioactive lipids are freely secreted into the extracellular milieu and function as freely diffusible mediators. Clearly, structural diversity exists among bioactive lipids and more hydrophilic mediators, such as cysteinyl leukotrienes may be freely diffusible. Many lipid mediators are found complexed to carrier proteins, such as serum albumin and serum lipoproteins, as has been shown for sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) [13]. The mode of action of receptor activation is also worthy of consideration. It is not known if bioactive lipids interact with the plasma membrane, followed by lateral binding to the GPCR. Alternatively, carrier protein/bioactive lipid complex could interact with the receptor to exchange the ligand. It is noteworthy that the Kd of ligand binding to GPCRs is in the nanomolar (10−9 M) range whereas that of binding to albumin is in the 10−7 to 10−6 M range [14,15]. Presumably, the association of bioactive lipids with plasma membranes will be lower affinity and higher capacity. However, due to energetic constraints, the bioactive lipid (such as LPA or S1P) may not be able to efficiently flip between the bilayers. Specific transporters and/or carriers for bioactive lipids are needed to efficiently uptake bioactive lipids from one cellular compartment to the other. For example, the prostanoid transporter functions to uptake classical prostaglandins from the extracellular environment into the cytosol [16]. Although the diversity of ligands necessitates the heterogeneity in structure of ligand binding pockets of lipid GPCRs, the signaling properties of such receptors follow the classical receptor/G protein signaling paradigms. Thus, most, if not all of the bioactive lipid GPCRs interact with one or more of classical heterotrimeric G proteins (Gs , Gi/o , Gq/11 , G12/13 and G16 ) [6]. Such interactions lead to down-stream signaling events, such as small GTPase activation, cytosolic and nuclear phosphorylation events (MAP kinases), and eventually changes in the activity of transcription factors. In many cases, non-transcriptional changes in cell behavior are also seen, for example, those that change the activity of cell–cell and cell–matrix adhesion molecules [17]. Thus, the bioactive lipidome modulates cell proliferation, migration, differentiation, death and fate via the GPCR repertoire of the organism.
3. Prostanoid signaling system Prostanoids are ubiquitous mediators synthesized by the cyclooxygenase (COX) pathway which oxidizes polyunsaturated fatty acids such as arachidonic acid (20:4), linoleic acid (18:2), eicosapentaenoid acid (20:5), docosahexaenoic acid (22:6) as well as derivatives such as 2-arachidonyl glycerol (2-AG) [18]. They have the prostanoic ring structure and diversity in double bond placement, hydroxyl and ketone moieties, which determine the biologically distinct prostanoids, such as prostaglandin E2 (PGE2 ), thromboxane A2 (TXA2 ), etc. Prostanoids are highly unstable and act as local hormones or autocoids.
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The major, rate-limiting enzyme in the biosynthesis of prostanoids is cyclooxygenase, which is also known as PG endoperoxide synthase or PG H synthase [18]. Two genes, commonly referred to as COX-1 and COX-2 encode the COX enzyme. The enzymes are localized in the luminal compartment of the endoplasmic reticulum. This enzyme catalyzes the formation of PGH2 , which is subsequently converted into bioactive prostanoids by the action of specific synthases, for example, thromboxane synthase, prostacyclin synthase, PGE synthase, etc. The subcellular location of these synthases are not well understood. However, once formed, prostanoids are rapidly secreted by cells. Although prostanoid transporters exist, their function appears to be involved in the uptake of prostanoids by cells [16]. Once secreted, prostanoids are thought to act on target cells by binding to specific cell surface receptors. Many prostanoid receptors are molecularly characterized. Each major prostanoid, for example, PGE2 , PGF2␣ , TXA2 , PGI2 and PGD2 act on structurally related GPCR isoforms [11]. For example, PGE2 acts on four related GPCRs termed as EP1-4 , PGF2␣ , PGI2 and TXA2 acts on single FP, IP and TP receptors, whereas PGD2 acts on DP and CRTH2 family of structurally unrelated receptors. Prostanoid receptors are expressed in different cell types and exhibit differential signaling properties. For example, the TP receptor is expressed in platelets and is coupled to the Gq , G12/13 and Gi family of G proteins whereas the IP receptor on the platelets is coupled mainly to the Gs family of heterotrimeric G proteins. By acting on prostanoid receptors, this family of bioactive lipid mediators modulates the activity of most organ systems in higher vertebrates. Well-studied effects of prostanoids include vascular smooth muscle contraction, platelet aggregation, pain, inflammation, febrile response, uterine contraction and immune modulation [11]. The COX enzymes were initially cloned from mammalian sources [18]. Structurally these enzymes show distant similarity to animal and plant peroxidases. The COX enzyme catalyzes the formation of PGG2 by bis-oxygenation of arachidonic acid and then reduction to PGH2 . Although the COX-1 isoenzyme prefers arachidonic acid, the COX-2 isoform can utilize a broader array of substrates, including 2-arachidonyl glycerol and anadamide. The COX isoenzymes are the targets of non-steroidal anti-inflammatory drugs and are widely studied for their physiological and pathological roles. Recent genomic sequencing efforts have indicated that COX-1 and COX-2 genes are conserved in the vertebrate genome, as homologous sequences have been isolated from mammalian, avian, reptilian, amphibian sources. Interestingly, in the bony fishes, zebrafish (Danio rario) and pufferfish (Tetraodon nigroviridis) both COX isoenzyme orthologues were found. In the cartiligenous fish (dogfish shark: Squalus acanthias) only one COX sequence was characterized, which may be due to the fact that only limited information of the genome of this organism is available at the present time [19]. However, in the chordate Ciona intestinalis (commonly referred to as the seasquirt), two genes that are highly related to the COX isoenzymes were observed (Figs. 1 and 2). In contrast, the COX enzyme homologues were not found widely in invertebrates, for example in the completely characterized genomes of Drosophila melanogaster, C. elegans, S. cerevesiae and D. discoidium. In some specialized cases, for example in some sea corals, in which prostanoids are used as defense molecules, the COX enzymes were expressed; however, in this case, a single COX enzyme was necessary. This supports the notion that the general use of prostanoids in cell–cell communication events in multiple organ systems evolved with the development of the chordates.
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Fig. 1. Alignment of COX-1, COX-2 and COX-like sequences from various vertebrates, the chordate Ciona intestinalis and the coral Gersemia fruticosa. The primary amino acid sequences from various organisms were obtained from the Genbank and ENSEBL databases (http://www.ensembl.org/) and aligned with the program MultAlin program (http://prodes.toulouse.inra.fr/multalin/multalin.html). Conserved residues are indicated in red and a consensus sequence is indicated in the last line. Note that vertebrate COX-1 and -2 sequences clustered into groups whereas chordate and coral COX sequences showed considerable divergence, even though essential catalytic residues are conserved.
On the other hand, the PG receptor sequences are only present in vertebrates and chordates. As shown in Fig. 3, clustal analysis and Dendrogram representation of PG receptor sequences show that receptor orthologues cluster in closely related tree branches. Therefore, mouse and human orthologues of EP, FP, TP, IP and DP receptor sequences are closest to each other, as indicated by the distal branch points of the tree. Several PG receptorrelated sequences from the pufferfish Tetraodon appear to be orthologues of mammalian PG receptors. For example, TetPRL2, TetPRL3 and TetPRL4 appear to be orthologues of mammalian TP, EP3 and EP4 receptors, respectively. Some zebrafish prostanoid receptors may be orthologues, as they are closely related to Tetraodon receptors. An example of this is that zPRL3 maybe a zebrafish orthologue of the mammalian EP4 receptor. The genome of the cartiligenous fish are not yet complete and PG receptor sequences are not available from this organism at this time. Interestingly, one PG receptor-related sequence was found in the Ciona intestinalis genome. For example, the Ciona EP4-like sequence is highly related to three Tetraodon PRL sequences, zPRL1 and somewhat distantly to mammalian EP4 receptors. Whether such sequences are indeed bona fide PGE receptor sequences must be determined experimentally. However, it is known that Ciona synthesize prostanoids and different prostanoids are known to exert biological effects in this organism [20,21]. The genomic data provides additional support for the presence of the classical prostanoid signaling system in this organism. In contrast, extensive searches in the genomes of Drosophila, C. elegans, S. cerevesiae and D. discoidium did not yield homologous sequences, suggesting
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Fig. 2. Dendrogram of COX polypeptide sequences. The COX-1, -2 and COX-like sequences from Fig. 1 (above) were analyzed by the CLUSTAL program (http://clustalw.genome.jp/) for relatedness [34]. An unrooted dendrogram is shown. h: human; m: murine; c: chicken; z: zebrafish; x: Xenopus; df: dogfish shark. Note that the cloned dfCOX is closer to the COX-1 group. The coral and Ciona COX-like sequences cluster into its own group, as they are distant in primary sequence to vertebrate COX enzymes.
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Fig. 2. (Continued )
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Fig. 2. (Continued ).
Fig. 3. Dendrogram of PG receptor sequences. Amino acid sequences of prostaglandin E2 (EP1-4), I2 (IP), D2 (DP and CRTH2), F2␣ (FP) and thromboxane A2 (TP) from human, mouse, Tetraodon, zebrafish as well as a related Ciona sequence were analyzed by the CLUSTAL program and represented as an unrooted tree. H: human; m: murine; Tet: Tetraodon, z: zebrafish; PRL: prostanoid receptor-like.
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that prostanoid receptors evolved with the occurrence of the chordates in the animal kingdom. This is also supported by the presence of COX genes in the chordates and vertebrates as discussed above. As chordates and vertebrates have sophisticated nervous, circulatory and immune systems, it is possible that lipid mediator signaling systems are needed to fine tune these complicated organ systems. In contrast, polypeptide signaling systems are also present in lower metazoans such as C. elegans and Drosophila. Thus, the full complement of lipid-based and peptide-based signaling pathways in higher animals may be needed. Although the prostanoids are involved in the regulatory processes in most, if not all organ systems in mammals, the evolutionary analysis of prostanoid synthase enzymes and receptors suggest that in the early stages of its evolution, prostanoids may have been utilized primarily in neuronal, immune and vascular systems. This is supported by the fact that even in mammals, prostanoids are major regulators of neuronal (e.g. pain), vascular (e.g. thrombosis, tone control, permeability) and immune (e.g. inflammation) systems.
4. Lysosphingolipid signaling system Sphingomyelin (SM) is an abundant membrane phospholipid that is critical for the integrity of membrane function in eukaryotes. It is metabolized by the sphingomyelinase (SMase) pathway to generate sphingolipid mediators such as sphingosine, ceramide and sphingosine 1-phosphate (S1P) [22]. Key enzymes in this pathway include sphingomyelinases, ceramidases, sphingosine kinases, S1P phosphatases and S1P lyase. Sphingolipid mediators are thought to regulate various intracellular processes in both unicellular eukaryotes and metazoans, even though the manner in which they mediate such effects are poorly understood. For example, in the yeast S. cerevesiae, loss of function of sphingolipid metabolic enzymes, sphingosine kinase, S1P lyase or phosphatase, results in alterations in cellular growth, stress response, protein synthesis and endocytosis responses [23]. Similar findings were also found in invertebrates such as C. elegans and Drosophila, suggesting the widespread conservation of sphingolipid function in evolution [24]. However, recent studies in mammalian cells have shown that S1P binds to the GPCRs of the EDG-1 family with high affinity and transduces signals into cells [25,26]. Currently, it is established that five GPCRs (S1P1 to S1P5 ) are S1P receptors that mediate various biological functions of S1P across cells. Interestingly, tissue expression of these receptors are divergent and many tissues express one or more of S1P receptors. The function of S1P receptors probably explain the multiple biological effects of S1P, which include heart function, vascular tone, vascular permeability, angiogenesis, vascular maturation, immune cell trafficking, mast cell activation, neuronal cell survival, oocyte survival and sperm viability [13]. These receptors are coupled to intracellular signal transduction pathways via the heterotrimeric G proteins. This aspect of S1P receptors will not be reviewed here; the reader is referred to a number of recent reviews published on this subject [17,27,28]. Evolutionary conservation of S1P metabolic enzymes and receptors may provide some insights into the workings of this lipid mediator system. All the sphingolipid metabolic enzymes are conserved throughout evolution from unicellular eukaryotes to mammals. For example, orthologues of sphingosine kinase-1 and -2 have been found from S. cerevesiae to humans [26]. Functional studies have documented that these enzymes carry out similar
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Fig. 4. Dendrogram of S1P, LPA and cannabinoid (CNR) receptor sequences. Amino acid sequences of human, Tetraodon, zebrafish, Fugu, and Ciona sequences were analyzed by the CLUSTAL program and an unrooted dendrogram is shown. T: Tetraodon; h: human; z: zebrafish; c: Ciona.
enzymatic reactions. In addition, genetic studies have shown that sphingosine kinases play important roles in unicellular eukaryotic and metazoan invertebrate biology. Distinct phenotypes were shown in organisms with sphingosine kinase genes deleted. For example, in Drosophila, deletion of the sphingosine kinase gene leads to egg laying and muscle defects [24]. In sharp contrast, S1P receptor genes were found only in vertebrates and perhaps in chordates but lacking in invertebrates. This is illustrated in Fig. 4. The five S1PRs cluster into a distinct group, which is closely related to the lysophosphatidic acid (LPA) and cannabinoid (CNR) receptor subfamilies. All mammalian genomes examined possess S1P receptor orthologues. The five paralogues are also represented in mammalian genome, suggesting that gene duplication events of S1PRs occurred earlier in evolution. Indeed, all five receptor orthologues were found in the genome data banks of bony fishes, namely, Takafugu rubipres, D. rario and T. nigroviridis, as shown in Fig. 3. Although not shown in the figure, other vertebrate species, namely Xenopus, also contain S1P receptor orthologues. However, related orthologous sequences were not found in invertebrate genome databases. Similarly, LPA receptor sequences were seen in the vertebrate but not invertebrate genomes. These data support the notion that lysophospolipid receptors evolved as the vertebrate species developed. Interestingly, the release of the complete genome sequence of the chordate, Ciona intestinalis (seasquirt) [29], suggested that lysophospholipid receptors may have occurred in the phylum chordata as well. A single open reading frame was seen in the Ciona genome, and homology analysis suggests that this GPCR-like sequence is distantly related to the cannabinoid receptors of the seasquirt and the lysophospholipid receptors from vertebrates
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(Fig. 4). Although experimental evidence is lacking, this finding supports the notion that lysophospholipid signaling may have begun with the development of chordates in evolution. Studies in vertebrates have shown that S1P is present abundantly in the blood and plasma. In humans, 100–400 nM of S1P is present in normal plasma, which is much higher than the binding constant for the ligand for its receptors (Kd ∼ 5–50 nM) [30]. Indeed, S1P receptor dynamics are critical for its effects on angiogenesis and immune cell trafficking. Induction and internalization/recycling of receptors for S1P are essential for these biological effects [31–33]. Since S1P is a major regulator of neuronal, vascular and immune systems, it is possible that the development of complexity in those systems that occurred in the chordates and vertebrates necessitated the S1P signaling via its plasma membrane receptors. Why this simple lipid mediator was selected in the evolutionary process is an interesting question for further consideration and analysis. Genome informatics of the extracellular lipid mediators has provided interesting insights into the function of such systems in the mammals. As exemplified in this review, prostanoid synthases and receptors seem to have evolved in the chordates. In contrast, the sphingolipid mediators were present throughout the eukaryotic kingdom as their metabolic enzymes are widely expressed. However, the receptors for S1P appear to be vertebrate and perhaps chordate-specific. Through the analysis of genomic information, unique insights on the extracellular lipidome may be forthcoming. Furthermore, structure function studies on receptors and metabolic enzymes of lipid mediators can be facilitated, by examining conserved as well as divergent residues. Ultimately, genome informatics may provide a unique tool in the understanding of the extracellular lipidome.
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