Elsevier

Gene

Volume 312, 17 July 2003, Pages 297-303
Gene

Functional mapping of cannabinoid receptor homologs in mammals, other vertebrates, and invertebrates

https://doi.org/10.1016/S0378-1119(03)00638-3Get rights and content

Abstract

Over the past decade, several putative homologs of cannabinoid receptors (CBRs) have been identified by homology screening. Homology screening utilizes sequence alignment search engines to recognize homologs. We investigated these putative CBR homologs further by ‘functional mapping’ of their deduced amino acid sequences. The entire pharmacophore of a CBR has not yet been elucidated, but point-mutation studies have identified over 20 amino acid residues that impart CBR specificity for ligand recognition and/or signal transduction. Twenty point-mutation studies were used to construct a CBR functionality matrix. Sixteen putative CBR homologs were then mapped over the matrix. Several putative homologs did not hold up to this analysis: human GPR3, GPR6, GPR12, and Caenorhabditis elegans C02H7.2 expressed a series of crippling substitutions in the matrix, strongly suggesting they do not encode functional CBRs. Mapping the contested leech (Hirudo medicinalis) CBR sequence suggests that it encodes a functional CB1; it expresses fewer substitutions than the sea squirt (Ciona intestinalis) CB1 sequence. Mapping a putative CB2 ortholog in the puffer fish (Fugu rubripes T012234) suggests it may encode a CBR other than CB2. These findings are consistent with the lack of experimental data proving these putative CBRs have affinity for cannabinoid ligands. Matrix analysis also reveals that SR144528, a ‘CB2-specific’ synthetic antagonist, has affinity for non-mammalian CB1 receptors, and that L3.45 appears to be CB2-specific, its cognate in CB1 receptors is F3.45. In conclusion, functional mapping, utilizing point-mutation studies, may improve the specificity of homology screening performed by sequence alignment search engines.

Introduction

A homolog, in biological systematics, is defined as a similar structure, behavior, or other trait shared by different species. The concept of ‘homologous series’ was first described by Vavilov (1922). It was Vavilov's elucidation of homologous series that led to his clarification of Cannabis phylogenetics. In the field of molecular phylogenetics, homologs are divided into two groups. Orthologs are homologous genes found in a series of organisms, derived by descent from a common ancestor. Paralogs are a series of homologous genes found in a given organism, derived by gene duplication events.

Cannabinoid receptors (CBRs) bind tetrahydrocannabinol (THC) and other cannabimimetic compounds; the activation of CBRs by THC is responsible for most of the behavioral and physiological changes evoked by Cannabis (reviewed by Felder and Glass, 1998). The cDNA encoding a CBR was first cloned and sequenced from a rat cerebral cortex cDNA library; Gerard et al. (1991) subsequently identified the gene expressing CBRs in human brain tissue (now termed CB1). The human gene encoding CB1, CNR1, is a nucleotide sequence that translates (encodes) a protein consisting of 472 amino acids. The amino acid sequence winds into a series of seven transmembrane (TM) domains, consistent with the topography of a G-protein coupled receptor (GPCR).

A paralog of CNR1, called CNR2, was cloned and sequenced from a human leukemic cell HL60 cDNA library (Munro et al., 1993). CNR2 codes for a sequence 360 amino acids in length. When aligned with CNR1, the overall sequence of CNR2 shares 44% identity.

Orthologs of CNR1 have been identified in many animals, including 62 species of placental mammals, sampled across all extant orders within that clade (Murphy et al., 2001). CNR1 orthologs have been cloned and sequenced from earlier vertebrates, such as the zebra finch, Taeniopygia guttata, and the newt salamander, Taricha granulosa (Soderstrom et al., 2000). The puffer fish, Fugu rubripes, expresses a pair of CNR1 paralogous orthologs, FCB1A and FCB1B (Yamaguchi et al., 1996). Among the invertebrates, a putative CB1 ortholog was recently identified in the genome of the sea squirt (Ciona intestinalis) by homology screening (Elphick et al., 2003). A partial CB1 sequence (encoding 153 amino acids from the N-terminus through transmembrane helix 3) was cloned from the leech, Hirudo medicinalis (Stefano et al., 1997). However, according to Elphick (1998), the leech cDNA sequence may not be a bona fide CB receptor because it may be chimeric; the sequence contains a region that shares similarity with the bovine adrenocorticotropic hormone receptor. A putative CNR1 ortholog was identified in the nematode Caenorhabditis elegans genome by GENEFINDER (Wilson et al., 1994), which is an algorithm utilized by GenBank to annotate sequences that resemble known protein-coding genes.

Orthologs of CNR2 have not been as well characterized. They have only been cloned from the mouse, Mus musculus (Shire et al., 1996) and rat, Rattus norvegicus (Griffin et al., 2000, Brown et al., 2002). A putative puffer fish CB2 ortholog was identified by homology screening (Elphick, 2002), reportedly paralogous to FCB1A and FCB1B.

The relatively low sequence identity between CNR1 and CNR2 suggests the duplication event that created these paralogs occurred in the distant past. McPartland and Pruitt (2002) constructed a CBR gene tree, coupled it to paleontological evidence, and proposed the duplication event occurred 600 million years ago. The antiquity of the duplication event is supported by the wide separation of CNR1 and CNR2 in the human genome, on chromosomes 6q14–15 and 1p35–36, respectively.

CNR1 and CNR2 have diverged in their physiology: CB1 receptors are primarily expressed by cells in the central nervous system, whereas CB2 receptors are located in immune cells (e.g. B cells, monocytes, T cells) and immune tissues (e.g. tonsils, spleen). CB1 and CB2 also diverged in their affinity for ligands: The phytocannabinoid THC binds with 50% greater affinity for CB1, whereas cannabidiol binds with nearly 4-fold greater affinity for CB2 (reviewed by Felder and Glass, 1998). Among the synthetic cannabinoids, CP55,940 binds with 50% greater affinity for CB2, HU210 is 8-fold selective for CB1, and WIN55212-2 is 19-fold selective for CB2. The endocannabinoids 2-arachidonyl glycerol (2-AG) and N-arachidonyl ethanolamide (anandamide) bind with 3–4 times greater affinity to CB1 than to CB2 (Felder and Glass, 1998). Evidence suggests that anandamide may also serve as an endogenous ligand for vanilloid receptors (VRs) as well as CBRs (Zygmunt et al., 1999). If further experiments prove this to be true, anandamide may have first served as a VR ligand, because gene tree analysis indicated that VRs evolved before CBRs (McPartland and Pruitt, 2002).

Song et al. (1995) identified three human genes as possible CBR paralogs. The genes, GPR3, GPR6, and GPR12, were identified by their overall sequence identity and TM domain sequence identity. These ‘cannabinoid orphan receptors’ (Marchese et al., 1999) share 60–68% identity with each other, and share ∼35% identity with CBR genes in their TM domains (Lee et al., 2001, Pertwee and Ross, 2002). Homology is considered significant in the presence of ≧30% identity (McPartland and Pruitt, 2002).

Sequence analysis can now go beyond the measure of overall sequence identity and TM sequence identity. Computer modeling coupled with point-mutation analysis has identified specific amino acid residues that confer CBR specificity and functionality (reviewed by Reggio, 2002). Amino acid residue specificity has been assessed by measuring CBR affinity for cannabinoid ligands. For example, Song and Bonner (1996) demonstrated that CB1 binding of HU-210 and CP55,940 (but not WIN55212-2) depends on lysine 192. The functional role of specific amino acids has also been assessed by the ability of CBRs to activate G-proteins and activate or inhibit effector molecules. For example, McAllister et al. (2002) inhibited cAMP accumulation in HEK 293 cells whose tyrosine 275 in human CB1 was mutated to non-aromatic amino acids. Xenopus oocytes expressing similarly mutated CB1 lost functional coupling with G-protein-coupled inwardly rectifying channels (GIRK1 and GIRK4) (McAllister et al., 2002).

The purpose of this study is to take the analysis of putative CBR homologs beyond the current literature. The sequences of several putative CNR1 and CNR2 orthologs and paralogs will be mapped, utilizing the aforementioned functionality studies as a map matrix.

Section snippets

BLAST sequence alignment

The deduced amino acid sequences of curated human CNR1 and CNR2 were obtained from GenBank (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov), accession numbers g.i. 4502927 and g.i. 4502929, respectively. They were compared with the deduced amino acid sequences of orthologs and paralogs with the following accession (g.i.) numbers: rhesus monkey CB1, 9664881; rat CB1, 111475; mouse CB1, 733425; finch CB1, 8575561; newt CB1, 8575561; puffer fish CB1A, 2494952; puffer

Sequence alignment and functional mapping

CNR1 orthologs and paralogs included in this study are presented in Table 3, ranked by their percentage identity to the CNR1 sequence, as measured by BLAST 2.0. CNR2 orthologs included in this study consist of three sequences: rat CB2 gene (sharing 81% identity with CNR2), mouse CB2 gene (sharing 82% identity with CNR2), and puffer fish (sharing 50% identity with CNR2).

An example of functional mapping is provided in Fig. 1. It illustrates a segment of the CNR1 amino acid sequence corresponding

Analysis of putative CBR paralogs and orthologs

Our results demonstrate that the putative paralogs GPR3, GPR6, and GPR12 exhibited numerous substitutions at critical amino acid residues known to convey CBR specificity in ligand recognition and signal transduction. This suggests that GPR3, GPR6, and GPR12 are not CBR paralogs, although they continue to be characterized as such (Marchese et al., 1999, Lee et al., 2001, Pertwee and Ross, 2002).

Functional mapping does not support the characterization of the nematode sequence C. elegans CO2H7.2

Acknowledgements

This work was partially supported by an unrestricted grant from GW Pharmaceuticals, Salisbury, UK.

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