Molecular characteristics of cannabinoid receptors Cnrs

Marijuana, Cnr gene had been elusive to clone but evidence for the existence of the receptor had been demonstrated since the 1980s (Howlett et al., 1988; Devane et al., 1988). It has now been shown and recognized that cannabinoids have specific receptors with endogenous ligands and inhibit adenylate cyclase. The CB1 receptors also modulate the activities of calcium and potassium channels. Although a number of approaches are now available for the cloning of genes encoding different receptors, the most common methods previously available, which involved the purification to homogeneity of the gene protein product, did not work for the cannabinoid receptors.

Despite the wealth of information and major advances that have transformed cannabinoid research into mainstream science, little information is available at the molecular level about Cnr gene structure, regulation and polymorphisms. Therefore, much research remains to be conducted at the molecular level about the 5' untranslated regions, particularly cannabinoid promoter structure and regulation and the 3' untranslated regions, which apart from containing several polyadenyla-tion signals may also play important regulatory roles (Shire et al., 1999). In order to start to characterize the genomic structure of the Cnrs, we have cloned, sequenced, (Chakrabarti et al., 1995) constructed the 3D model (Onaivi et al., 1998) and localized the mouse CB1 Cnr gene to chromosome 4 (Stubbs et al., 1996). In addition, a EMBL3SP6/T7 library of C57BL/6N genomic DNA was screened by a full length (2.2kb) CB i cDNA and obtained some clones. From these clones AAC21 was used and restriction enzyme digestion produced three bands from the CB1 genomic insert. The bands were 9kb, 6.5kb and 1.5kb in size. Southern hybridization of these bands with CB1 cDNA lights up the 9 kb and 1.5 kb bands. Thus, the 9 kb and 1.5kb bands are linked and host the cDNA. The 6.5kb band is flanking either the C-terminal or in the ^-terminal. The 9kb band showed stronger hybridization than the 1.5kb band. In the 2.2kb band the long sfi I site is 0.8kb apart from the ^-terminal fragment of the cDNA. Compilation of these data result in the following two possibilities for the structure of the CB1 genomic DNA insert (Figure 1.1). To characterize the insert in further detail long PCR (XLPCR) amplification was performed with ^-terminal GSP and universal primers which amplified a band of about 7 kb. This indicates that the 6.5 kb band flanks the structural part of the gene at its ^-terminal. Hence the map of the genomic DNA insert in AAC21 is as shown in (Figure 1.1). The currently available information on the genomic structure of CB1 and CB2 is sketchy and the regulation of these genes is poorly

Figure I.I Initial characterization of the murine CB| genomic DNA using CB| cDNA. It was determined that the 6.5 kb band flanks the structural part of the gene at its N-terminal.

understood, the emerging putative structure for the CB1 gene is depicted in Figure 1.2. As discussed below, the CB1 Cnr gene structure is polymorphic with implication, not only for substance abuse but also in other neuropsychiatric disorders.

In order to determine whether there are introns in the coding sequences of human, rat and mouse CB1 and CB2 Cnr genes, primer pairs spanning the cDNA sequences of these genes were generated to test whether the DNA fragments amplified by these primer pairs were identical with both genomic DNA and cDNA templates. The hypothesis we tested was that if the DNA fragment sizes were identical with both templates, then the gene was intronless, if not, the intron location, size and structure can be determined. To determine whether the structure of the testis CB1 receptor gene or its transcripts is different from those in the brain, we isolated DNA and RNA from rat testes and brain and used them as templates for PCR with primer pairs. There was no difference in sizes of the PCR amplified DNA bands between brain DNA and RNA, between testes DNA and RNA or between the brain and testis DNA or RNA. Specified amplified DNA bands were identified by Southern hybridization with human CB1 cDNA as probe, and data conform with those expected. Cannabinoids are known to have effects on male fertility by their ability to lower testosterone levels in testis (Iversen, 1993), CB1 gene was also found to be expressed in testis (Gerard et al., 1991). Northern analysis of CB1 mRNA levels in rat brain and testis expresses this gene about 20-25 fold

Cannabinoid Receptor Diagram
Figure 1.2 Structure of CB| Cnr gene. The emerging structure of the CB| Cnr gene, containing the single coding exon and 112 kb published sequence from the 3' region on chromosome 6ql4-ql5. A number of polymorphisms indicated have been identified in the CB| Cnr gene.

© 2002 Taylor & Francis less than the brain tissue (Gerard et al., 1991). The data obtained indicated that there was no apparent CB1 rearrangement in testis and there are no size variants of this gene in rat brain and testis. Such variations are known for dopamine D2 receptor transcripts, which arise from alternate splicing (O'Dowd, 1993). If there is any testis-specific subtype of the Cnr, it is likely to be coded by a largely dissimilar gene. CB2 and an unknown Cnr subtype may be a good candidate for a Cnr subtype in the testis. In our study, primer pairs specific for human CB2 cDNA failed to amplify specific DNA molecules from rat testis DNA or RNA templates under conditions when human DNA or RNA yielded positive results. Similar experiments were performed with DNA and RNA isolated from brains of three mouse strains, the C57BL/6, DBA/2 and ICR. These mouse strains show similarities and marked differences in cannabinoid-induced neurobehavioral patterns (Onaivi et al., 1995). We have investigated, whether the mouse CBX Cnr gene is also intronless, whether the mouse strains differ from each other in the Cnr gene (CBX) structure or whether there are any size variants in the CBX transcripts in their brains. The DNA PCR data shows that the CBX Cnr gene in the three strains appears to be identical and intronless. We also tested the CBX and CB2 Cnr gene structures in human blood cells. The data indicated that the human CBX Cnr gene might also be intronless, at least in its coding region. Similar observations indicated that the CB2 Cnr gene in human cells might also be intronless at least in the coding region. Furthermore, the rat and human CB1 cDNA sequences are very similar (Matsuda et al., 1990; Gerard et al., 1991). Unlike the CBX Cnr, which is highly conserved across the human, rat and mouse species, the CB2 Cnr is much more divergent (Griffin et al., 1999). This divergence in mouse, rat and human CB2 Cnr leading to differences in functional assays may be related to species specificity.

Although many of the GPCRs are found to be intronless (O'Dowd, 1993), there are exceptions, such as some dopamine receptors. In common with many of the genes encoding members of the GPCRs, the genes encoding the dopamine, D1 and D5 lack introns in their coding regions (O'Dowd, 1993). But the dopamine D1 receptor gene is reported to have an intron at the 5' non-coding region. However, the dopamine, D2, D3 and D4 receptor genes which have large introns, are distinguishable from many members of the GPCR family, where the entire protein is encoded by just one exon. It is interesting to find that both of the subtypes of the Cnrs may be coded by single-exon genes. There is of course the possibility of these genes having intron(s) at the upstream or downstream non-coding regions. We have not tested the possibility, but Shire et al. (1995) have found the presence of two introns in the CBX gene, one in the 5' UTR and the second in the coding region of the receptor. The advantages or disadvantages of being intronless are subject to speculation (Lambowitz and Belfort, 1993). One obvious advantage is that the expression of these genes has a major RNA processing event to skip, thus making the conditions of their expression relatively quick and simple. This advantage may have implications related to the biological functions of these Cnr proteins. This issue may seem to be complex at the moment, because the structural features of the Cnr genes are currently incompletely understood.

The existence of a subtype of CBX Cnr gene, originally designated as CBXA (now designated CBXB and described by Shire et al. (1995)), has not been detected in any species in vivo. Therefore, while it is unlikely and doubtful that CBjB exists in the form described by Shire et al. (1995), this does not mean that other Cnr subtypes may not exist. For example, the molecular cloning of two cannabinoid type 1-like receptor genes from the puffer fish has been characterized by Yamaguchi et al. (1996). They characterized two putative G-protein coupled receptor encoding genes, FCBXA and FCBXB, obtained by degenerate PCR and low-stringency hybridization of a Fugu genomic library. It was found that these two genes showed high homology to the human CBX, but very low homology to the CB2 Cnr gene. The amino acid sequences of the FCBXA and FCBXB genes are 66.2% identical, and the homology of each gene to human CBX is 72.2% and 59%, respectively. The transcripts of both the FCBXA and FCBXB receptors are abundant in the brain. No CB2 Cnr gene could be cloned from the puffer fish. Therefore, the puffer fish has two subtypes of CBX Cnrs, which is distinct from the CB2 subtype. The primary structure of the CBX and CB2 Cnrs are similar to those of other G-protein coupled receptors with the characteristic features of a typical seven hydrophobic domains with some highly conserved amino acid residues. A detailed comparison of the molecular properties of the human, rat and mouse CB ! and where applicable, CB2 Cnrs had been previously reviewed (Onaivi et al., 1996; Matsuda, 1997). These receptors mediate their intracellular actions by a pathway that involves activation of one or more guanine nucleotide-binding regulatory proteins, which responds to cannabinoids including the endocanna-binoids. The conservatism of the CBX Cnr sequence contrasts with the variability seen with the CB2 Cnr as discussed by Shire in this volume. The composition and amino acid sequence alignments of CBX and CB2 Cnrs show considerable structural homology and distribution in the CNS between species with substantial amino acid conservation but with significant differences with the CB2 Cnr whose presence in the CNS is controversial. Like other GPCRs, the primary structures of the Cnr are characterized by the seven hydrophobic stretches of 20-25 amino acids predicted to form transmembrane a helices, connected by alternating extracellular and intracellular loops. In comparing the composition of the ^-terminal 28 amino acids between human CBX and CB2 Cnrs and also between human, rat and mouse, it has been reported by Onaivi et al. (1996), that: (1) the human and rat ^-terminal 28 amino acids in the CBX Cnrs were similar in the total number of non-polar, polar, acidic and basic amino acids; (2) the mouse ^-terminal 28 amino acids differed from the rat and human CBX Cnrs in number and composition of the total non-polar and polar amino acids; (3) there are significant differences in the total non-polar, polar, acidic and basic amino acid composition of the ^-terminal 28 amino acids between human CBX and CB2 Cnrs; and (4) the molecular weights of human, rat and mouse CB1 Cnrs are similar. Therefore, the amino acid composition of the mammalian CBX Cnrs shows strong conservatism in contrast to molecular weights and amino acid composition of CB2 Cnrs.

The three dimensional (3D) model, helix bundle arrangement of human, rat and mouse CBX and CB2 receptors have been constructed and compared (Bramblett et al., 1995; Onaivi et al., 1996) and extensively reviewed in this book by Reggio. The transmembrane helix bundle arrangement obtained for the CBX Cnrs is consistent with that obtained for other GPCRs. Potential sites for N-glycosylation, and the action of protein kinase C, cAMP-dependent protein kinase and Ca-calmodulin-dependent protein kinase II in the derived amino acid sequence of the Cnr proteins have been identified (see Figure 1.3) (Onaivi et al., 1996). Most but not all GPCRs are glycoproteins and consensus sites for W-glycosylation are mainly concentrated at the W-terminus of the protein. There are three potential W-glycosylation sites highly conserved in human, rat and mouse. The rodent CB1 Cnr protein has an additional potential W-glycosylation site at the C-terminal segment that is absent in the human CBX Cnr protein. One potential W-glycosylation site is present in human and rat CB1 Cnr protein but that site is missing in mouse CB1 Cnr. Whether all of these potential W-glycosylation sites are naturally glycosylated in CB 1 Cnr proteins or whether these W-glycosylation are essential for Cnr function and whether additional W-glycosylation in the CBX Cnr of different mammalian species imparts differential activity of this protein are yet to be determined. However, mutation of W-glycosylation sites in similar GPCRs, e.g. ^-adrenergic receptors and muscarinic receptors, abolishes glycosylation, but has essentially no effect on receptor expression and function (Dohlman et al., 1991). The human Cnr subtypes CBj, and CB2 with some similarities and differences in their receptor function, appear to differ in the number and distribution of their potential W-glycosylation sites. Due to the modification of the W-terminal region, CB2 has only one potential W-glycosylation site whereas CB1 Cnr has five. There is no potential W-glycosylation site at the C-terminal segment of CB2 Cnr. The biological significance (if any) of these differences is yet to be determined.

The C-terminal regions and the third intracellular loop of GPCRs are known to be rich in serine and threonine residues. In the case of rhodopsin, ^-adrenergic and some muscarinic receptors, some of these residues are targets of cAMP-dependent protein kinase and other protein kinases (Strada et al., 1994). These phosphorylations are often agonist dependent and result in desensitization and coupling of the receptor from the G-protein. There are four clusters of potential cAMP-dependent protein kinase and Ca-calmodulin-dependent-protein kinase sites in CB x Cnr that are conserved in human, rat and mouse proteins. There is a single potential protein kinase C site that is also conserved in all these CB1 Cnrs whereas CB2 has no such site. The W-terminal potential cAMP clusters present in CB 1 appears to be conserved and the CB2 Cnr has two such potential sites. None of the Cnrs have any potential protein kinase site at the C-terminal regions. The biological significance of these potential protein phosphorylation sites in these receptor molecules is yet to be determined. In addition, many members of GPCRs are known to contain conserved cysteine residues that appear to stabilize the tertiary structure of the receptor because of their involvement in an intra-molecular disul-fide bridge. In most receptors, these cysteines occur in the extracellular domains that lie between hydrophobic domains two and three, and hydrophobic domains four and five (second and third extra-cellular domains, on the assumption that W-terminal domain is also extra-cellular). In CBj and CB2 Cnrs, no cysteines are found within the second extra-cellular domain, but the third extra-cellular domain contains two or more cysteines. One other deviation from most other GPCRs is that CBX and CB2 Cnrs lack a highly conserved proline residue in the fifth hydro-phobic domain (Matsuda, 1997). The structural features of these proteins that is critical for ligand binding and functional properties have been evaluated in in vivo and in vitro models (Akinshola et al., 1999). Heterologously, Cnrs bind tritriated synthetic ligands like WIN 55,212-2 in a saturable and competitive manner. The

Figure 1.3 Potential modification sites of the CB| Cnr protein. Comparison of potential N-glyco-sylation (N-glycos) and protein kinase (PKC: Protein kinase C; camp-Kin: camp-dependent protein kinase; Ca-KinII: Ca-dependent protein kinase II) sites in human (A), rat (B) and mouse CB1 Cnr proteins obtained using the MacVector sequence analysis software.

Figure 1.3 Potential modification sites of the CB| Cnr protein. Comparison of potential N-glyco-sylation (N-glycos) and protein kinase (PKC: Protein kinase C; camp-Kin: camp-dependent protein kinase; Ca-KinII: Ca-dependent protein kinase II) sites in human (A), rat (B) and mouse CB1 Cnr proteins obtained using the MacVector sequence analysis software.

binding activities of CBX and CB2 Cnrs have been determined after transfection into CHO cells, COS-7 cells, and mouse AtT20 cells (Felder et al., 1995; Slipetz etal., 1995). In other expression systems, the CB1 Cnrs have been examined in insect Sfi cells, (Pettit et al., 1994), Xenopus oocytes (Henry and Chavkin, 1995), mouse L cells, human embryonic kidney 293 cells, (Song and Bonner, 1996) and dissociated rat superior cervical ganglion neurons (Pan et al., 1996). Furthermore, the performance of mutated Cnrs with that of wild-type receptors have been compared in a number of functional assays and reviewed by Matsuda, 1997. In those studies, mutant Cnrs containing point mutation (a single amino acid substitution) or ones that have been modified by replacement of a series of amino acids from one receptor with that of another (chimeric receptor) have been expressed and studied (Matsuda, 1997).

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