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Biol. Bull. 202: 104-107. (April 2002)
© 2002 Marine Biological Laboratory

Evolution of Cannabinoid Receptors in Vertebrates: Identification of a CB2 Gene in the Puffer Fish Fugu rubripes

Maurice R. Elphick

School of Biological Sciences, Queen Mary, University of London, London E1 4NS, UK

To whom correspondence should be addressed: E-mail: M.R.Elphick{at}qmul.ac.uk

{Delta}9-tetrahydrocannabinol, the psychoactive ingredient of cannabis, exerts effects in humans by binding to the G-protein-coupled cannabinoid receptors CB1 and CB2, which are expressed in the nervous and immune systems, respectively. Genes encoding CB1 receptors have also been discovered in non-mammalian vertebrates, including the puffer fish Fugu rubripes. Here the identification of a Fugu gene that encodes an ortholog of mammalian CB2 receptors is reported. This is the first CB2 gene to be identified in a non-mammalian vertebrate, and it indicates that the gene duplication event that gave rise to CB1 and CB2 receptors occurred before the divergence of teleosts and tetrapods. Moreover, non-mammalian vertebrate species can now be considered as potential model systems in which the physiological roles of the CB2 receptor can be investigated.

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 CB1 receptors, which results in an inhibition of "classical" neurotransmitter release (7,8,9,10,11,12). The physiological roles of CB2 are less well understood than those of CB1, although analysis of CB2-knockout mice indicates that the CB2 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 CB1 and CB2 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 CB1 or CB2 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. CB1-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 CB1-like genes (FCB1A, FCB1B) (17). These discoveries indicate that the CB1-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, CB2-type receptor genes have so far been identified only in mammalian species. Yamaguchi et al. (17) attempted to clone a CB2-type gene in Fugu rubripes, using a probe for human CB2 (HCB2), but reported that "hybridization with HCB2 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 CB2-type gene in this species using techniques for genome sequence analysis.

To search the Fugu genome for a CB2 gene, the Basic Local Alignment Search Tool (BLAST; 19) was employed, using the Fugu BLAST server at http://fugu.hgmp.mrc.ac.uk/blast/. With the human CB2 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 CB2, 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 CB1A and CB1B genes, respectively (17). Having eliminated these CB1-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 CB2 receptor (50% amino-acid identity) than with the human CB1 receptor (47% amino-acid identity). This suggested that clone T012234 may contain the sequence of a Fugu CB2 gene. To determine the complete sequence of the protein-encoding region of the putative CB2 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 CB2 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 CB2-like receptor protein has a predicted length of 379 amino-acid residues, which is within the range for CB2 receptors in mammals (e.g., mouse, 347; human, 360; rat, 410). To establish whether the Fugu CB2-like protein is an ortholog of mammalian CB2 receptors, it was aligned with human CB2, mouse CB2, and CB1 receptor sequences from several vertebrate species including Fugu (CB1A and CB1B), 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 CB1 clade and a CB2 clade; and, importantly, the Fugu CB2-like sequence is positioned in the CB2 clade, indicating orthology to mammalian CB2 receptors.



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  		Figure 1. Alignment of the predicted amino-acid sequence of the Fugu CB2 receptor with other cannabinoid receptors including human CB2 (3), mouse CB2 (23), Fugu CB1B (17), Fugu CB1A (17), newt Taricha granulosa CB1 (16), zebra finch (z-finch) Taeniopygia guttata CB1 (15), human CB1 (24), rat CB1 (2), mouse CB1 (25), and cat CB1 (D. Gebremedhin et al., unpubl.; accession number AAB53440). The sequences were aligned using ClustalX (1.8) multiple alignment program (20). The symbol * indicates the positions of residues that are identical in all of the sequences; the symbols: and . indicate the positions of strongly and weakly conserved residues, respectively.

 


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  		Figure 2. Phylogenetic tree of CB1-type and CB2-type cannabinoid receptors with human lysophospholipid receptors as an outgroup (LPA1, LPA2, LPB1, LPB3). The tree was constructed using the neighbor-joining method (21) with bootstrapping (1000 bootstrap trials) and then viewed in NJ Plot. The tree shows that the Fugu CB2-like receptor forms a clade with human CB2 and mouse CB2 and is therefore an ortholog of mammalian CB2 receptors.

 
Based on the data available (99% of the Fugu genome sequence), only one CB2-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 CB1 genes (CB1A and CB1B) 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 CB2 gene in Fugu. Whether other teleosts such as the zebrafish Danio rerio have the same number and distribution of CB1 and CB2 genes remains to be determined, but to date neither CB1 nor CB2 genes have been sequenced in Danio. With the ongoing sequencing of the Danio genome, a comprehensive search will probably be practical soon.

The CB2 gene in Fugu is the first CB2 gene to be identified in a non-mammalian species. The discovery of this gene indicates that the gene duplication event that gave rise to CB1 and CB2 receptors occurred before teleosts and tetrapods diverged from a common ancestor. Thus, CB2-type cannabinoid receptors, like CB1 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 CB2 receptor. In particular, because the CB2 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
 
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 Egertová (Queen Mary, University of London) and three anonymous referees for constructive criticism of the manuscript.


   Footnotes
 
Received 21 December 2001; accepted 28 February 2002.


   Literature Cited
TOP
 Literature Cited
 

  1. Devane, W. A., F. A. I. Dysarz, M. R. Johnson, L. S. Melvin, and A. C. Howlett. Determination and characterization of a cannabinoid receptor in rat brain. Mol. Pharmacol. 1998; 34: 605–613.[Abstract]
  2. Matsuda, L. A., S. J. Lolait, M. J. Brownstein, A. C. Young, and T. I. Bonner. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. 1990. Nature 346: 561–564.[Medline]
  3. Munro, S., K. L. Thomas and M. Abu-Shaar. Molecular characterization of a peripheral receptor for cannabinoids. Nature 365: 61–65.
  4. Devane, W. A., L. Hanus, A. Breuer, R. G. Pertwee, L. A. Stevenson, G. Griffin, D. Gibson, A. Mandelbaum, A. Etinger, and R. Mechoulam. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992. 258: 1946–1948.[Abstract/Free Full Text]
  5. Mechoulam, R., S. Ben-Shabat, L. Hanus, M. Ligumsky, N. E. Kaminski, A. R. Schatz, A. Gopher, S. Almog, B. R. Martin, D. R. Compton, R. G. Pertwee, G. Griffin, M. Bayewitch, J. Barg, and Z. V. I. Vogel. Identification of an endogenous 2-monoglyceride, present in canine gut that binds to cannabinoid receptors. 1995. Biochem. Pharmacol. 50: 83–90.[ISI][Medline]
  6. Sugiura, T., S. Kondo, A. Sukagawa, S. Nakane, A. Shinoda, K. Itoh, A. Yamashita, and K. Waku. 2-arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. 1995. Biochem. Biophys. Res. Commun. 215: 89–97.[ISI][Medline]
  7. Elphick, M. R., and M. Egertová. The neurobiology and evolution of cannabinoid signalling. 2001. Philos. Trans. R. Soc. Lond. B 356: 381–408.[ISI][Medline]
  8. Egertová, M., D. K. Giang, B. F. Cravatt, and M. R. Elphick. A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain. 1998. Proc. R. Soc. Lond. B 265: 2081–2085.[Medline]
  9. Kreitzer, A. C., and W. G. Regehr. Retrograde inhibition of presynaptic calcium influx by endogenous cannabinoids at excitatory synapses onto Purkinje cells. 2001. Neuron 29: 717–727.[ISI][Medline]
  10. Ohno-Shosaku, T., T. Maejima, and M. Kano. Endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals. 2001. Neuron 29: 729–738.[ISI][Medline]
  11. Wilson, R. I., and R. A. Nicoll. Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses. 2001. Nature 410: 588–592.[Medline]
  12. Wilson, R. I., G. Kunos, and R. A. Nicoll. Presynaptic specificity of endocannabinoid signaling in the hippocampus. 2001. Neuron 31: 453–462.[ISI][Medline]
  13. Buckley, N. E., K. L. McCoy, E. Mezey, T. Bonner, A. Zimmer, C. C. Felder, M. Glass, and A. Zimmer. Immunomodulation by cannabinoids is absent in mice deficient for the cannabinoid CB2 receptor. 2000. Eur. J. Pharmacol. 396: 2–3.
  14. British Medical Association. Therapeutic Uses of Cannabis. Harwood Academic Publishers, Amsterdam. 1997.
  15. Soderstrom, K., and F. Johnson. Zebra finch CB1 cannabinoid receptor: pharmacology and in vivo and in vitro effects of activation. 2001. J. Pharmacol. Exp. Ther. 297: 189–197.[Abstract/Free Full Text]
  16. Soderstrom, K., M. Leid, F. L. Moore, and T. F. Murray. Behavioural, pharmacological and molecular characterization of an amphibian cannabinoid receptor. 2000. J. Neurochem. 75: 413–423.[ISI][Medline]
  17. Yamaguchi, F., A. D. Macrae, and S. Brenner. Molecular cloning of two cannabinoid type 1-like receptor genes from the puffer fish Fugu rubripes. 1996. Genomics 35: 603–605.[ISI][Medline]
  18. Elgar, G., M. S. Clark, S. Meek, S. Smith, S. Warner, Y. J. Edwards, N. Bouchireb, A. Cottage, G. S. Yeo, Y. Umrania, G. Williams, and S. Brenner. Generation and analysis of 25 Mb of genomic DNA from the pufferfish Fugu rubripes by sequence scanning. 1999. Genome Res. 10: 960–971.
  19. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. Basic local alignment search tool. 1990. J. Mol. Biol. 215: 403–410.[ISI][Medline]
  20. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. 1997. Nucleic Acids Res. 24: 4876–4882.
  21. Saitou, N., and M. Nei. The neighbor-joining method: a method for reconstructing phylogenetic trees. 1987. Mol. Biol. Evol. 4: 406–425.[Abstract]
  22. Taylor, J. S., Y. Van de Peer, I. Braasch, and A. Meyer. Comparative genomics provides evidence for an ancient genome duplication event in fish. 2001. Philos. Trans. R. Soc. Lond. B 356: 1661–1679.[ISI][Medline]
  23. Shire, D., B. Calandra, M. Rinaldi-Carmona, D. Oustric, B. Pessegue, O. Bonnin-Cabanne, G. Le Fur, D. Caput, and P. Ferrara. Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor. 1996. Biochim. Biophys. Acta 1307: 132–136.[Medline]
  24. Gérard, C. M., C. Mollereau, G. Vassart, and M. Parmentier. Molecular cloning of a human cannabinoid receptor which is also expressed in testis. 1991. J. Biochem. 279: 129–134.
  25. Chakrabarti, A., E. S. Onaivi, and G. Chaudhuri. Cloning and sequencing of a cDNA encoding the mouse brain-type cannabinoid receptor protein. 1995. DNA Seq. 5: 385–388.[ISI][Medline]




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