Elsevier

Gene

Volume 302, Issues 1–2, 2 January 2003, Pages 95-101
Gene

The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis

https://doi.org/10.1016/S0378-1119(02)01094-6Get rights and content

Abstract

The G-protein coupled cannabinoid receptors CB1 and CB2 are activated by Δ9-tetrahydrocannabinol, the psychoactive ingredient of cannabis, and mediate physiological effects of endogenous cannabinoids (‘endocannabinoids’). CB1 genes have been identified in mammals, birds, amphibians and fish, whilst CB2 genes have been identified in mammals and in the puffer fish Fugu rubripes. Therefore, both CB1 and CB2 receptors probably occur throughout the vertebrates. However, cannabinoid receptor genes have yet to be identified in any invertebrate species and the evolutionary origin of cannabinoid receptors is unknown. Here we report the identification of CiCBR, a G-protein coupled receptor in a deuterostomian invertebrate – the urochordate Ciona intestinalis – that is orthologous to vertebrate cannabinoid receptors. The CiCBR cDNA encodes a protein with a predicted length (423 amino-acids) that is the intermediate of human CB1 (472 amino-acids) and human CB2 (360-amino-acid) receptors. Interestingly, the protein-coding region of the CiCBR gene is interrupted by seven introns, unlike in vertebrate cannabinoid receptor genes where the protein-coding region is typically intronless. Phylogenetic analysis revealed that CiCBR forms a clade with vertebrate cannabinoid receptors but is positioned outside the CB1 and CB2 clades of a phylogenetic tree, indicating that the common ancestor of CiCBR and vertebrate cannabinoid receptors predates a gene (genome) duplication event that gave rise to CB1- and CB2-type receptors in vertebrates. Importantly, the discovery of CiCBR and the absence of orthologues of CiCBR in protostomian invertebrates such as Drosophila melanogaster and Caenorhabditis elegans indicate that the ancestor of vertebrate CB1 and CB2 cannabinoid receptors originated in a deuterostomian invertebrate.

Introduction

Cannabinoid receptors mediate the effects of the main psychoactive ingredient of cannabis, Δ9-tetrahydrocannabinol (Howlett et al., 2002). To date, two related G-protein coupled cannabinoid receptors have been identified in humans and other mammals, known as CB1 and CB2 (Matsuda et al., 1990, Munro et al., 1993). CB1 is predominantly expressed in the central nervous system, whilst CB2 is associated with cells of the immune system (Lutz, 2002). Endogenous ligands for these receptors (‘endocannabinoids’) have also been identified, including arachidonoylethanolamide (‘anandamide’), 2-arachidonoylglycerol (2-AG) and 2-arachidonyl glyceryl ether (‘noladin ether’) (Devane et al., 1992, Mechoulam et al., 1995, Sugiura et al., 1995, Hanus et al., 2001). At present, little is known about the physiological roles and relevance of CB2-receptors in the immune system. However, there is growing evidence that endocannabinoids function as intercellular signalling molecules by activating cannabinoid receptors on target cells (Elphick and Egertová, 2001). Importantly, it has recently been demonstrated that endocannabinoids act as retrograde signalling molecules at synapses in the brain (Elphick and Egertová, 2001, Maejima et al., 2001, Wilson and Nicoll, 2002, Kreitzer and Regehr, 2002).

The discovery of endocannabinoid signalling mechanisms in mammals has prompted investigation of the occurrence of cannabinoid receptors in non-mammalian vertebrates and in invertebrates (Elphick and Egertová, 2001). CB1-type receptors have been identified in several vertebrate species including the zebra finch Taeniopygia guttata, the amphibian Taricha granulosa and the puffer fish Fugu rubripes (Soderstrom and Johnson, 2001, Soderstrom et al., 2000, Yamaguchi et al., 1996). Recently, a gene encoding the first non-mammalian CB2-type receptor to be identified was also discovered in Fugu (Elphick, 2002). Thus, it appears that both CB1-type and CB2-type cannabinoid receptors may occur throughout the vertebrates.

A number of studies have investigated the occurrence of endocannabinoids and cannabinoid receptors in a variety of invertebrate species. Anandamide and 2-AG are present in primitive animals such as the cnidarian Hydra vulgaris (De Petrocellis et al., 1999), indicating that the ability of cells to produce these substances may be an evolutionarily ancient phenomenon. The occurrence of specific receptors for cannabinoids in invertebrates has been investigated using radiolabelled cannabinoids but these studies have yielded both positive and negative data. For example, the presence of specific binding sites for cannabinoids has been reported in sperm from the sea urchin Strongylocentrotus purpuratus (Chang et al., 1993), in the central nervous system of the leech Hirudo medicinalis (Stefano et al., 1997) and in whole-animal homogenates of Hydra (De Petrocellis et al., 1999). In contrast, absence of cannabinoid binding sites has been reported in the brain of the locust Schistocerca gregaria (Elphick and Egertová, 2001) and in other insects (McPartland et al., 2001). Where cannabinoid binding sites have been detected in invertebrate species, further molecular characterization of the putative cannabinoid receptors is now required. So far there has been just one report where molecular characterization of a putative invertebrate cannabinoid receptor was attempted. Stefano et al. (1997) isolated a cDNA fragment from the leech Hirudo, which shares sequence similarity with mammalian CB1-type cannabinoid receptors. However, subsequent analysis of this partial cDNA sequence revealed that it may be an artefact because it is chimeric, with one region that shares 98% amino-acid identity with the bovine adrenocorticotropic hormone receptor and two regions that share 68% and 65% amino-acid identity with mammalian CB1 receptors (Elphick, 1998). It is unlikely, therefore, that this sequence represents part of a bona fide leech cannabinoid receptor.

Although attempts to clone and sequence cannabinoid receptor genes from invertebrates have so far been unsuccessful, with the complete sequencing of a several invertebrate genomes it has become possible to perform genome-wide searches for orthologues of vertebrate CB1 and CB2 receptors. Using this approach, no orthologues of vertebrate cannabinoid receptors were identified in the genomes of the insect Drosophila melanogaster or the nematode Caenorhabditis elegans (Elphick and Egertová, 2001, McPartland et al., 2001). Moreover, Drosophila and C. elegans also lack orthologues of other vertebrate G-protein coupled receptors that are closely related to cannabinoid receptors (e.g. lysophospholipid receptors and melanocortin receptors) (Elphick and Egertová, 2001). These findings have led to the hypothesis that CB1- and CB2-related cannabinoid receptors may be absent from all protostomian invertebrates, which include arthropods, nematodes, molluscs, annelids, platyhelminthes and several other phyla (Elphick and Egertová, 2001).

It remains to be established at what stage in the evolution of the animal kingdom G-protein coupled cannabinoid receptors related to vertebrate CB1 and CB2 receptors first appeared. If, as suggested above, cannabinoid receptors of the CB1/CB2-type are absent in protostomian invertebrates, then receptors of this type may have evolved in deuterostomian invertebrates, which share a more recent common ancestry with vertebrates than protostomian invertebrates do (Adoutte et al., 2000). The deuterostomian invertebrates include echinoderms, hemichordates, urochordates and cephalochordates but, to the best of our knowledge, the occurrence of cannabinoid receptors has only been investigated in echinoderms: Chang et al. (1993) reported the presence of cannabinoid binding sites in sea urchin sperm. However, the molecular nature of the protein(s) that confer cannabinoid binding sites on sea urchin sperm is as yet unknown. Therefore, it remains to be established whether orthologues of the vertebrate cannabinoid receptors CB1 and CB2 occur in deuterostomian invertebrates. To address this issue, here we have investigated the occurrence of a cannabinoid receptor gene in a urochordate species, the sea-squirt Ciona intestinalis.

Section snippets

BLAST analysis of Ciona ESTs

A database containing both 5′ and 3′ sequences (expressed sequence tags, ESTs) of cDNAs from several C. intestinalis cDNA libraries (Satou et al., 2001, Kusakabe et al., 2002, Ogasawara et al., 2002) was analysed using the Basic Local Alignment Search Tool (BLAST; Altschul et al., 1990), available at http://ghost.zool.kyoto-u.ac.jp/indexr1.html. Using the Fugu CB1A receptor protein sequence (Yamaguchi et al., 1996) as the search query, a 723 base 5′ EST (ciad70l24) encoding a partial protein

Analysis of the sequence of ciad70l24

Sequencing of cDNA ciad70l24 revealed that it is a 1551 bp sequence with a probable start codon (ATG) at bases 75–77 and a stop codon (TGA) at bases 1344–1346 (Fig. 1; DDBJ/EMBL/GenBank accession no. AB087259). The protein encoded by ciad70l24 therefore has a predicted length of 423 amino-acids (Fig. 1). Alignment of this 423 amino-acid sequence with vertebrate cannabinoid receptor sequences, showed that for 53 of the 423 residues in the Ciona protein there is amino-acid identity across all of

Discussion

Here we report the discovery of CiCBR, the first putative cannabinoid receptor gene to be identified in an invertebrate species – the urochordate C. intestinalis. CiCBR encodes a protein with a predicted length of 423 amino-acids that shares 28% sequence identity with the human CB1 receptor and 24% sequence identity with the human CB2 receptor. Despite these relatively low levels of sequence identity, phylogenetic analysis demonstrated that CiCBR is an orthologue of vertebrate cannabinoid

Acknowledgements

This research was supported by a Grant-in-Aid for Priority Area (No. 12202001) from the MEXT, Japan to N.S. and by a Wellcome Trust (UK) grant (057058) to M.R.E.

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