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Evolution of cannabinoid receptors in vertebrates: identification of a [CB.sub.2] gene in the puffer fish Fugu rubripes.

Following the discovery of cannabinoid receptors in mammals (1, 2, 3), endogenous ligands for these receptors ("endocannabinoids") have also been identified; these ligands include arachidonylethanolamide ("anandamide") and 2-arachidonylglycerol (2-AG) (4, 5, 6). Endocannabinoids mediate retrograde signaling at synapses in the brain by diffusing from postsynaptic sites of synthesis to act on presynaptic [CB.sub.1] receptors, which results in an inhibition of "classical" neurotransmitter release (7, 8, 9, 10, 11, 12). The physiological roles of [CB.sub.2] are less well understood than those of [CB.sub.1], although analysis of [CB.sub.2]-knockout mice indicates that the [CB.sub.2] receptor mediates immunomodulatory actions of cannabinoids in mammals (13). The emergence of a cannabinoid signaling system has aroused interest in the physiological roles of endocannabinoids and in the potential of cannabinoids to be therapeutic agents in humans (14).

With the discovery of endocannabinoid signaling mechanisms in mammals, the evolution of cannabinoid receptors has become of interest because non-mammalian species provide important models for analysis of gene function. Orthologs of mammalian [CB.sub.1] and [CB.sub.2] genes do not, however, appear to be present in protostomian invertebrate species, such as Drosophila melanogaster and Caenorhabditis elegans, for which complete genome sequences are known (see ref. 7). Thus these cannabinoid receptor genes may have evolved in the deuterostomian, chordate, or vertebrate clades of the animal kingdom. To date, orthologs of mammalian [CB.sub.1] or [CB.sub.2] receptors have also not been identified in any invertebrate deuterostomes (e.g., cephalochordates, urochordates, hemichordates, and echinoderms); but complete genome sequences have not yet been obtained for species representative of these phyla. [CB.sub.1]-type genes have, however, been identified in several vertebrate species, including a bird (15), an amphibian (16), and the puffer fish Fugu rubripes, which has two [CB.sub.1]-like genes ([FCB.sub.1A], [FCB.sub.1B]) (17). These discoveries indicate that the [CB.sub.1]-type cannabinoid receptor can be traced back at least as far as the common ancestor of teleosts (bony fish) and tetrapod vertebrates (amphibians, reptiles, birds, and mammals). In contrast, [CB.sub.2]-type receptor genes have so far been identified only in mammalian species. Yamaguchi et al. (17) attempted to clone a [CB.sub.2]-type gene in Fugu rubripes, using a probe for human [CB.sub.2] (HCB2), but reported that "hybridization with HCR2 failed to identify a Fugu homologue." With the recent announcement that 99% of the genome of Fugu rubripes has been sequenced (Nature, 414, 1 November 2001, page 8; see also ref. 18), it has become possible to search again for a [CB.sub.2]-type gene in this species using techniques for genome sequence analysis.

To search the Fugu genome for a [CB.sub.2] gene, the Basic Local Alignment Search Tool (BLAST; 19) was employed, using the Fugu BLAST server at http://fugu.hgmp.mrc.ac.uk/blastl. With the human [CB.sub.2] receptor protein sequence as the search query, three clones were identified (T012234, T017853, and T002576) that shared 49%, 45%, and 43% amino-acid identity with human [CB.sub.2], respectively, with corresponding BLAST E-values of 4e-78, 6e-78, and 1e-67. Further analysis of T017853 and T002576 revealed that these contained the DNA sequences of the previously discovered Fugu [CB.sub.1A] and [CB.sub.1B] genes, respectively (17). Having eliminated these [CB.sub.1]-type genes, the sequence of T012234 was examined in more detail. BLAST analysis of the putative cannabinoid receptor-encoding region of T012234 using the BLAST server at http://www.ncbi.nlm.nih.gov/BLAST/ (nr database) revealed that this sequence displayed more similarity with the human [CB.sub.2] receptor (50% amino-acid identity) than with the hum an [CB.sub.1] receptor (47% amino-acid identity). This suggested that clone T012234 may contain the sequence of a Fugu [CB.sub.2] gene. To determine the complete sequence of the protein-encoding region of the putative [CB.sub.2] gene, the DNA sequence of T012234 was translated in all three possible frames for both the forward (+) and reverse (-) strands using the transeq program at http://www.ebi.ac.uk/emboss/transeq/. This revealed that the putative Fugu [CB.sub.2] gene was in the -2 frame of clone T012234 and enabled identification of the probable positions of the 5' start codon and the 3' stop codon. Based on this analysis, the putative Fugu [CB.sub.2]-like receptor protein has a predicted length of 379 amino-acid residues, which is within the range for [CB.sub.2] receptors in mammals (e.g., mouse, 347; human, 360; rat, 410). To establish whether the Fugu [CB.sub.2]-like protein is an ortholog of mammalian [CB.sub.2] receptors, it was aligned with human [CB.sub.2], mouse [CB.sub.2], and [CB.sub.1] receptor sequences from several vertebrate species including Fugu ([CB.sub.1A] and [CB.sub.1B]), the amphibian Taricha granulosa, the zebra finch Taeniopygia guttata, human, rat, mouse, and cat (Fig. 1) using the ClustalX multiple alignment program (20). Phylogenetic trees of the aligned sequences were then constructed using the neighbor-joining method (21); human lysophospholipid receptors were used as an outgroup because these receptors are more closely related to cannabinoid receptors than other G-protein-coupled receptors in mammals (see ref. 7). Trees were constructed using the unedited aligned sequences (Fig. 2) or using truncated sequences lacking the N-terminal and C-terminal regions, where there is sequence divergence (not shown). The branching structure of the two trees was identical, with bootstrap values of 980-1000 for all of the major branches. Analysis of the tree shown in Figure 2 reveals that cannabinoid receptors fall into two distinct clades, a [CB.sub.1] clade and a [CB.sub.2] clade; and, importan tly, the Fugu [CB.sub.2]-like sequence is positioned in the [CB.sub.2] clade, indicating orthology to mammalian [CB.sub.2] receptors.

Based on the data available (99% of the Fugu genome sequence), only one [CB.sub.2]-type gene occurs in the Fugu genome. This is of interest because in Fugu and in other teleosts there often are two orthologs for each single-copy gene in mammals (22). For example, Fugu contains two [CB.sub.1] genes ([CB.sub.1A] and [CB.sub.1B]) whereas mammalian genomes contain only one. The existence of two genes in teleosts for each single-copy gene in mammals is thought to reflect a genome duplication that occurred in the ancestor of teleost fish 300 to 450 million years ago (22). Presumably, however, the duplicates of some genes have subsequently been lost, and this probably explains the existence of only one [CB.sub.2] gene in Fugu. Whether other teleosts such as the zebrafish Danjo rerio have the same number and distribution of [CB.sub.1] and [CB.sub.2] genes remains to be determined, but to date neither [CB.sub.1] nor [CB.sub.2] genes have been sequenced in Danio. With the ongoing sequencing of the Danio genome, a compr ehensive search will probably be practical soon.

The [CB.sub.2] gene in Fugu is the first [CB.sub.2] gene to be identified in a non-mammalian species. The discovery of this gene indicates that the gene duplication event that gave rise to [CB.sub.1] and [CB.sub.2] receptors occurred before teleosts and tetrapods diverged from a common ancestor. Thus, [CB.sub.2]-type cannabinoid receptors, like [CB.sub.1] receptors, are likely to be also present in non-mammalian tetrapod vertebrates (amphibians, reptiles, birds). Non-mammalian vertebrate species may provide useful model systems with which to explore the physiological roles of the [CB.sub.2] receptor. In particular, because the [CB.sub.2] receptor appears to be principally involved in immunoregulation in mammals, it may have a related role in the more "primitive" immune systems of fish and other non-mammalian vertebrates.

Acknowledgments

[FIGURE 2 OMITTED]

I am grateful to Greg Elgar (Fugu Genomics Project, MRC Human Genome Mapping Project Resource Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK) for permission to publish this analysis of Fugu genomic sequence data obtained by the International Fugu Genome Consortium. I am also grateful to Michaela Egertova (Queen Mary, University of London) and three anonymous referees for constructive criticism of the manuscript.

Received 21 December 2001; accepted 28 February 2002.

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To whom correspondence should be addressed: E-mail: M.R.Elphick@qmul.ac.uk
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Author:Elphick, Maurice R.
Publication:The Biological Bulletin
Geographic Code:4EUUK
Date:Apr 1, 2002
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