©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Amino-terminal Variant of the Central Cannabinoid Receptor Resulting from Alternative Splicing (*)

(Received for publication, September 8, 1994; and in revised form, December 2, 1994)

David Shire (§) Christine Carillon Mourad Kaghad Bernard Calandra Murielle Rinaldi-Carmona (1) Gérard Le Fur Daniel Caput Pascual Ferrara

From the From Sanofi Recherche, Labège-Innopole Voie 1, BP 137, 31676 Labège Cedex 04 and Sanofi Recherche, 371 rue du Professeur Blayac, 34184 Montpellier Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The cDNA sequences encoding the central cannabinoid receptor, CB1, are known for two species, rat and human. However, little information concerning the flanking, noncoding regions is presently available. We have isolated two overlapping clones from a human lung cDNA library with CB1 cDNA inserts. One of these, cann7, contains a short stretch of the CB1 coding region and 4 kilobase pairs (kb) of the 3`-untranslated region (UTR), including two polyadenylation signals. The other, cann6, is identical to cann7 upstream from the first polyadenylation signal, and in addition, it contains the whole coding region and extends for 1.8 kb into the 5`-UTR. Comparison of cann6 with the published sequence (Gérard, C. M., Mollereau, C., Vassart, G., and Parmentier, M.(1991) Biochem. J. 279, 129-134) shows the coding regions to be identical, but reveals important differences in the flanking regions. Notably, the cann6 sequence appears to be that of an immature transcript, containing 1.8 kb of an intronic sequence in the 5`-UTR.

In addition, polymerase chain reaction amplification of the CB1 coding region in the IM-9 cell line cDNA resulted in two fragments, one containing the whole CB1 coding region and the second lacking a 167-base pair intron within the sequence encoding the amino-terminal tail of the receptor. This alternatively spliced form would translate to an NH(2)-terminal modified isoform (CB1A) of the receptor, shorter than CB1 by 61 amino acids. In addition, the first 28 amino acids of the putative truncated receptor are completely different from those of CB1, containing more hydrophobic residues. Rat CB1 mRNA is similarly alternatively spliced. A study of the distribution of the human CB1 and CB1A mRNAs by reverse transcription-polymerase chain reaction analysis showed the presence of both CB1 and CB1A throughout the brain and in all the peripheral tissues examined, with CB1A being present in amounts of up to 20% of CB1.


INTRODUCTION

The pharmacological activities of cannabis, its most active principle, Delta^9-tetrahydrocannabinol, an endogenous agonist, anandamide(1) , and numerous synthetic agonists are mediated by the cannabinoid receptor. Partial cDNA sequences encoding the receptor have been isolated from rat (2) and human (3) brain tissues. The deduced protein sequence of the human receptor shows that it comprises 472 amino acids, with the seven highly hydrophobic regions typical of the G protein-coupled receptor superfamily. The rat receptor shares 97.3% identity with the human receptor. This central form of the cannabinoid receptor, CB1, mediates both the inhibition of adenylyl cyclase via a pertussis toxin-sensitive GTP-binding regulatory protein (4) and the inhibition of N-type calcium channels(5) . Although CB1 and its mRNA are predominantly found in the brain(2, 6, 7, 8, 9) , the mRNA has also been detected in peripheral tissues (3, 10) by Northern analysis and by PCR(^1)(11) . PCR fragments within the coding region of CB1 cDNA from the human testis (3) and from the myeloid cell line U937 (11) were identical in sequence to the brain-derived CB1 cDNA, suggesting that the peripheral and brain sequences were identical. However, the cannabinoid receptor story became more complicated with the subsequent cloning from the human promyelocytic leukemic cell line HL-60 and from macrophages in the marginal zone of human spleen of a distinct, exclusively peripheral receptor, designated CB2(12) . CB2, although exhibiting only 51% identity to CB1 in the transmembrane regions, strongly resembles the latter in its interactions with various ligands and also mediates the inhibition of adenylyl cyclase(13) . Many questions arise from this apparent redundancy, among them the exact cellular localization and regulation of the two receptors and of their transcripts. To start answering these questions, we have undertaken the cloning of the full-length human CB1 cDNA. In the course of this investigation, we discovered the presence of two introns in the CB1 gene, one in the 5`-UTR and the second in the coding region of the receptor. Here we show that translation of the fully matured coding region would lead to a truncated and amino-terminal modified form of CB1, designated CB1A. Interestingly, during expression studies of rat CB1 cDNA in a baculovirus expression system, Pettit et al.(14) recently detected short variants of CB1, which they attributed to protein degradation, but which were more probably translation products of the CB1A mRNA. Finally, using PCR we have established the relative distribution of the CB1 and CB1A mRNAs in central and peripheral tissues and show that both mRNAs are to be found in the majority of these tissues.


EXPERIMENTAL PROCEDURES

cDNA Libraries and RNA Preparations

Human brain cDNA libraries in ZAP vectors were from Stratagene (Ozyme, France). A human lung cDNA library was made as described(15) . Cell lines from the American Type Culture Collection, the IM-9 human lymphoblastoid cell line (CCL 159), the U937 promonocyte cell line (CRL 1593), and the U373 astrocytoma cell line (HTB 17), together with peripheral blood mononuclear cells, were cultured as described previously(16) . Total RNAs from human heart, liver, kidney, lung, pancreas, placenta, and stomach (all adult) were from CLONTECH (Ozyme, France). Other RNAs were isolated from tissues (human infant inferior hemisphere, brain stem, temporal lobe, and cortex + cerebellum) and cellular sources using the guanidinium isothiocyanate procedure(17) . RNAs from rat hippocampus and astrocytes were supplied by T. Gautier (Sanofi Recherche, Toulouse, France). All RNA preparations were passed through Bio-Gel P-10 (Bio-Rad, Vitry-Sur-Seine, France) before being treated with DNase I (Boehringer Mannheim, Meylan, France). cDNA was obtained from RNA using Superscript II reverse transcriptase (Life Technologies, Inc., Eragny, France) according to the manufacturer's instructions.

PCR, Cloning, and Sequence Analysis of CB1

PCR was carried out using oligonucleotide primers (100 ng of each) flanking the coding region of CB1 (5`-CUACUACUACUAATGAAGTCGATCCTAGATGGC (NH(2)-terminal sense primer in Fig. 2b) and 5`-CAUCAUCAUCAUTCACAGAGCCTCGGCAG) in 50 µl containing 75 ng of IM-9 cDNA, 7 mM Tris-HCl, 20 mM ammonium sulfate, 0.02% (w/v) gelatin, 0.015% (v/v) Triton X-100, 2 mM MgCl(2), 0.2 mM each deoxyribonucleoside triphosphate, and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer, Roissy CDG France) for 35 cycles at 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2.5 min. The mixture was electrophoretically separated on 2% agarose gel, and the two slowest migrating bands were coexcised, electroeluted, purified by DE52 chromatography, precipitated, and cloned in pAmp1 using the CloneAmp system (Life Technologies, Inc.). Positive transformants in Escherichia coli DH5alphaF`IQ (Life Technologies, Inc.) were sequenced using the dideoxynucleotide method. A lung cDNA library of 4 times 10^5 colonies distributed on 10 duplicated Biodyne B membranes (Pall Industrie, St. Germain-en-Láye, France) was screened with the 5`-peroxidase-labeled probe TGATCAACACCACCAGGATC (50 ng/ml) with hybridization for 2 h at 42 °C, followed by chemiluminescent detection as described previously(18) . About 50 positive colonies were detected. Colonies from one membrane were recovered, grown up, redistributed, and rescreened with the probe. Several colonies were selected, and plasmids were recovered using the Magic Prep system (Promega, Coger, France) and digested with HindIII/BamHI. Two classes of inserts were identified, both of 4 kb, one of which had an internal HindIII site giving a second fragment of 1200 base pairs. One example of each class, cann6 and cann7, was sequenced by the dideoxynucleotide method. Sequences were analyzed using the University of Wisconsin Genetics Computer Group programs(19) .


Figure 2: The structure of the 5`-extremity of the coding region of the human cannabinoid receptor CB1 cDNA. a, schematic representation of the 5`-extremity of the coding region of CB1. CB1 is initiated at ATG1 from an mRNA that is unspliced in the coding region. CB1A results from a 167-nucleotide excision between donor (D) and acceptor (A), using ATG2 as the initiation codon. TM1 codes for the first transmembrane region. The stripedbox represents sequences common to CB1 and CB1A, between acceptor (A) and the end of the coding region (represented by the asterisk). b, open reading frames of the two human cannabinoid receptor isoform cDNAs and their deduced amino-terminal sequences. The nucleotide sequences in boldface indicate the consensus donor and acceptor sequences, with the splice junctions indicated by arrows. The singlyunderlined sequences are the amino-terminal sense and antisense PCR primer regions, and the doublyunderlined sequence is that of the NH(2)-terminal probe. The asterisks indicate in-phase stop codons, with the sequence in parentheses being that of cann6/BS08. The amino acids common to the two sequences are in boldface. TMI is the first transmembrane region. The glycosable asparagines are indicated by .



Tissue Distribution

PCR was carried out on tissue- and cell culture-derived cDNAs (500 ng) or cDNA libraries (200 ng) using the NH(2)-terminal sense (see above) and NH(2)-terminal antisense (see Fig. 2b) (5`-CAUCAUCAUCAUGTTCTCCCCACACTGGATG) primers (100 ng of each), common to both CB1 and CB1A, in the above PCR buffer with AmpliTaq (2.5 units) for 35 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 0.7 min. Southern blotting was carried out using the 5`-coupled horseradish peroxidase oligonucleotide 5`-TACAGGAGGTCAGTGGTGAT (NH(2)-terminal probe in Fig. 2b) as described previously(18) . Chemiluminescence signals were quantitated using the National Institutes of Health Image program, version 1.47 (Wayne Rasband, Research Service Branch, NIMH). In the case of RNA, control PCR experiments were carried out in the absence of reverse transcriptase. Amplicon cloning from certain tissues (see ``Results'') was carried out using the CloneAmp system as described above.


RESULTS

Human Lung CB1 cDNA

Using an oligonucleotide probe corresponding to codons in the sixth transmembrane region of CB1, we identified two clones (cann6 and cann7, both containing inserts of 4 kb) in a human lung cDNA library. Partial sequence analysis (^2)of cann7 showed that it contained 0.34 kb of the end of the published CB1 coding region (3) and 4.0 kb of the 3`-UTR, including two polyadenylation signals 0.6 and 4.0 kb downstream from the stop codon (Fig. 1a).


Figure 1: The human central cannabinoid receptor CB1 cDNA. a, schematic structure of clones cann6, cann7, and BS08(3) . The shaded boxes represent the coding region; the arrows represent the polyadenylation sites; and the bentlines in BS08 are divergent sequences, discussed in the text. b, partial sequence of clone cann6. The CB1 coding region (shadedarea) is identical for cann6 and BS08(3) . Nucleotides common to both cann6 and BS08 are in upper-case letters; divergences are in lower-case letters for cann6 and in italic letters for BS08. Only the last 28 nucleotides of the 5`-intron in cann6 are given to show the splice acceptor sequence (doubly underlined). The totality of the 3`-UTR of cann6 is shown, with the canonical polyadenylation signal boxed. A possible splice donor sequence, which may account for the difference with BS08 downstream from nucleotide 1546, is singly underlined.



The 3`-extremity of cann6 overlapped with the 5`-extremity of cann7 over 0.95 kb, the sequences being identical in this region. In contrast to cann7, cann6 contained only a 0.6-kb 3`-UTR, ending shortly after a polyadenylation signal (Fig. 1b), the first to be found in cann7, but contained the entire coding region of CB1 plus an upstream region of 1.82 kb.^2 The coding region of cann6 was identical to that of the original BS08 clone from a human brain stem library(3) . Taken together, the 1.5 kb of the BS08 clone plus the 4-kb 3`-UTR identified in cann7 corresponds well with the 6-kb mRNA identified for CB1 by Northern analysis of RNA from dog, rat, and guinea pig tissues(2, 3) . Matsuda et al.(2) reported the existence of two alternatively polyadenylated rat CB1 mRNAs with 3`-UTRs 4.1 and 1.2 kb in length; the sequences have not been published, but the general structures of the rat and human sequences are clearly similar.

Two significant differences were seen between the extremities of BS08 and the cDNA sequences described here (Fig. 1). The cann6 and cann7 sequences diverge from the BS08 sequence 123 nucleotides after the stop codon, just after what appears to be a splice donor consensus sequence (singlyunderlined in Fig. 1b). Since the 64 nucleotides remaining in BS08 have not yet been found farther downstream in the cann7 sequence,^2 these nucleotides may be at the start of a nonmatured intron. More interestingly, cann6 diverges from BS08 63 nucleotides upstream from the initiation codon. Examination of the cann6 sequence at this point reveals the presence of a splice acceptor site (doublyunderlined in Fig. 1b). The 1.76 kb at the 5`-extremity of the cann6 sequence would appear to be part of a nonexcised intron, only the 3`-extremity of which is shown in lower-case letters in Fig. 1b. Therefore, the cann6 clone most probably results from an immature transcript. The sequence of the BS08 clone upstream from the splice site (in italic letters in Fig. 1b) is not to be found anywhere in the cann6 sequence.^2 The 3`-extremity of this part of the BS08 clone, AAGG, could be part of a splice donor site. The full length of the 5`-intron in the human DNA is not known, but is currently being investigated in our laboratory. Interestingly, the rat CB1 sequence (2) is very similar to the human sequence starting 20 nucleotides downstream from the putative splice acceptor site, but diverges markedly upstream from this point (not shown), perhaps indicating that the rat gene also contains an intron in its 5`-flanking region.

Identification of Alternatively Spliced Central Cannabinoid Receptor mRNAs

Previous work had shown the presence of the cannabinoid receptor mRNA in B cells(11) . PCR amplification of cDNA from the human lymphoblastoid cell line IM-9 using primers flanking the coding regions of CB1 (3) and subsequent purification on agarose gel resulted in the expected amplicon of 1400 base pairs, with a fainter band migrating slightly faster; both bands were positive on Southern analysis (data not shown). Sequence analysis of several of the cloned bands revealed the presence of two cDNA species, one having a coding region identical to that of the published cannabinoid receptor (3) and the other containing a 167-base pair deletion near the 5`-extremity of the coding region.

Examination of this region of the CB1 sequence revealed the presence of consensus splice donor and acceptor sequences in the precursor mRNA (D and A in Fig. 2, a and b). The shorter, deleted sequence we found corresponded to a spliced form of the receptor mRNA. In this spliced form, the translational start codon, AUG1, the first to be encountered after an upstream stop codon in the cannabinoid receptor gene (CB1 in Fig. 2b), is no longer in frame with the rest of the coding region, and its use would result in a peptide of only 35 amino acids. A putative initiation codon, AUG2, for the foreshortened form of the receptor is a little further downstream, immediately preceded by a stop codon. Initiation from AUG2 could occur only if AUG1 were a ``leaky'' codon according to the Kozak scanning model of transcription initiation (20) , thereby allowing the transcription complex to initiate from the next downstream AUG triplet. In fact, AUG1, although preceded by G at position -3, is followed by A at position +3, giving a leaky initiation site, for which there are numerous precedents(21, 22) . AUG2 has G at position +4, but T at position -3, again constituting only a moderately favorable context for translation(23) . If AUG2 initiates translation of this shorter receptor, it would involve a single frameshift from the reading frame of CB1. The deduced form of the receptor would be shorter than CB1 by 61 amino acids, and in addition, the 28 amino acids at its NH(2)-terminal extremity would be quite different from those of the longer form, being, in particular, hydrophobic and relatively rich in Pro.

As a result of the excision, the putative isoform, designated CB1A, lacks two of the three potential Asn-linked glycosylation sites present in CB1. After the splice junction, the reading frame of CB1 is restored, and the remaining amino acids of CB1 and CB1A are identical, including the remaining 27 amino acids of the NH(2)-terminal region (Fig. 2b and Fig. 6). Experimental evidence that CB1A protein indeed exists has very recently come from the expression of the rat CB1 cDNA from a baculovirus expression vector in Sf9 cells(14) . Western immunoblotting and S metabolic labeling revealed the presence of a predominant species with a relative molecular mass of 55 kDa, corresponding to CB1 (calculated molecular mass of 53 kDa), together with cannabinoid-specific bands at 45 and 50 kDa, which could well correspond to non-glycosylated CB1A (calculated molecular mass of 46 kDa) and to a glycosylated form, respectively.


Figure 6: The deduced NH(2)-terminal extremities of CB1 and CB1A of the human and rat cannabinoid receptors. The human sequences are inside the circles; the differences found in the rat receptor are shown at the relevant positions outside the circles. The Gly insert in the rat sequence is shown by the arrow. The amino acids common to the two forms are lightlyshaded, and the glycosable asparagines are darklyshaded. TM1 is the first transmembrane region beneath the thickline representing the membrane surface.



Distribution of Human CB1 and CB1A

To discover whether the splice variant was expressed in other tissues, PCR primers corresponding to regions on either side of the excised sequence in the CB1 mRNA, amino-terminal sense and antisense primers (underlined in Fig. 2b), were designed. The CB1 and CB1A amplicons were of 327 and 160 base pairs, respectively. Since only one primer pair was used for the two variant mRNAs simultaneously in the same tube and since it has been shown that priming efficiency is the predominating factor in PCR amplification(18, 24) , the relative quantities of the two amplicons are directly related to the initial relative quantities of the two splice versions. We carried out PCR analysis of cDNA libraries and genomic DNA and reverse transcription-PCR of total RNA from a wide range of tissues and cell cultures. Simultaneous Southern analysis of the amplicons made use of a peroxidase-linked probe (NH(2)-terminal probe) complementary to a sequence shared by both amplicons (Fig. 2b). As can be seen from Fig. 3a, CB1 was found to be the predominant form in all the peripheral organs tested, the relative quantities of the two forms being fairly uniform (Table 1), although the truncated mRNA was barely detectable in kidney. Notably, both mRNAs were present in spleen, where CB2 has also been detected(12) . In accordance with a post-transcriptional splicing event, only CB1 was detected in genomic DNA (Fig. 3b, lane10). The amplicons obtained from stomach tissue RNA were sequenced and were identical to the CB1 and CB1A cDNAs in the B cell line IM-9. CB1A was present in low quantities in astrocytoma U373 cells, but was found in larger amounts in cells of the immune system (Table 1), in peripheral blood lymphocytes, in U937 cells, and in IM-9 cells (Fig. 4b). Both forms were also present throughout the central nervous system (Fig. 3b and Fig. 4a). The quantity of the shorter form varied considerably, being hardly detectable in the brain stem and temporal lobes of a newborn and rising to 20% in the hippocampus.


Figure 3: Tissue distribution of CB1 and CB1A transcripts determined by PCR. The upperpanels are ethidium bromide-stained 2% agarose gels; the lowerpanels are the respective Southern blots with the bands revealed using the NH(2)-terminal probe, followed by chemiluminescence. a: lane 1, size markers; lane2, heart; lane3, colon; lane4, stomach; lane5, liver; lane6, pancreas; lane7, placenta; lane8, lung; lane9, kidney. b: lane1, brain stem; lane2, cortex + cerebellum; lane3, inferior hemisphere; lane4, temporal lobe; lane5, size markers; lane6, IM-9; lane7, U937; lane8, U373; lane9, peripheral blood leukocytes; lane10, genomic DNA.






Figure 4: Distribution of CB1 and CB1A transcripts in human and rat brain determined by PCR. a and b (upperpanel), ethidium bromide-stained 2% agarose gel of amplicons resulting from PCR with NH(2)-terminal sense and antisense primers of human cDNA libraries. a: lane1, size markers; lane2, brain stem; lane3, frontal cortex; lane4, cerebellum; lane5, hippocampus; lane6, occipital cortex; lane7, striatum; lane8, substantia nigra; lane9, temporal cortex. b: lane1, rat astrocytes; lane2, rat hippocampus; lane3, size markers. The lowerpanel in b is a Southern blot of the upperpanel revealed using the NH(2)-terminal probe detected by chemiluminescence.



Presence of Isoforms in the Rat

Since the primers used for the human cannabinoid receptor cDNA (Fig. 2) were fully complementary to the rat cDNA sequence, they were used to determine whether the equivalent of CB1A existed in rat mRNA. As for the human RNA, reverse transcription-PCR of mRNA from rat astrocytes and hippocampus gave rise to two amplicons (Fig. 4b), which were cloned and sequenced. The rat CB1 sequence between the two primers (Fig. 5) was identical to the published rat sequence(2) . Sequence analysis of the shorter rat amplicon revealed that splicing had occurred (Fig. 2a), showing the existence of rat CB1A cDNA (Fig. 5). Between the amino-terminal sequences of the full-length human CB1 and rat CB1, there are seven amino acid differences out of the total 116 amino acids composing this region, i.e. six amino acid differences plus an insertion of a Gly in the rat sequence (Fig. 6). In the putative CB1A receptor, however, the human and rat amino termini are identical in length (55 amino acids) since the insertion has disappeared, but there are now nine amino acid differences (Fig. 6). As in the human sequence, the two potential asparagine-linked glycosylation sites in the rat isoform have disappeared from this region of the receptor, leaving just one a little farther downstream.


Figure 5: The structure of the 5`-extremity of the coding region of the rat cannabinoid receptor CB1 cDNA. See legend to Fig. 2b for details. The 5`-UTR sequence in parentheses is identical in both rat and human.




DISCUSSION

A distinguishing feature of the central cannabinoid receptor is its long extracellular extremity. The discovery that CB1 mRNA exists in two forms differing in the amino-terminal coding region not only poses the question as to the existence and function of two CB1 isoforms, but also opens up an opportunity to assess the importance of this region of the receptor for its interaction with its ligands. In addition to the small coding region intron, a large intron of at least 1.8 kb in length is to be found just upstream of the initiation codon in the human gene (Fig. 1) and, possibly, in the genes of other species. This presents the possibility of further alternative splicing. In view of the potential of cannabinomimetics for the treatment of a variety of ailments, to ensure drug specificity, it is clearly important to ascertain whether other subtypes or isoforms of the receptor exist.

Most of the receptors for small nonpeptidic molecules have small amino-terminal regions and ligand-binding sites constituted by pockets formed by the heptahelical transmembrane regions(25, 26) . The metabotropic glutamate receptor is unique in that its unusually large extracellular extremity has been shown to be directly implicated in small molecule selectivity(27) . All the known cannabinoids, including an endogenous ligand, anandamide(1) , are small, nonpeptidic molecules. We have evidence that the truncated and modified isoform CB1A exhibits somewhat different ligand binding properties than CB1 when expressed in Chinese hamster ovary cells. (^3)At the present time, we do not know if the amino-terminal region plays any structural or functional role. As a result of the splicing, CB1A lacks two of the three potential glycosylation sites present in CB1. We cannot assess the importance of this observation at present. However, Howlett et al.(28) reported that treatment of N18TG2 neuroblastoma cells with tunicamycin failed to alter either agonist-induced inhibition of cAMP accumulation or desensitization processes in the presence of cannabinoids, suggesting that glycosylation is unimportant for activity, but the authors cautioned that the rate of receptor synthesis and degradation had not been taken into consideration. Unfortunately, no agonist binding data to correlate activation with signal transduction were presented. This point remains to be clarified, as it has been shown that glycosylation can be important either for signal transduction, as shown in rhodopsin(29) , or for directing subcellular distribution, as observed for the hamster beta(2)-adrenergic receptor(30) .

We recently reported the presence of CB1 mRNA in leukocytes using reverse transcription-PCR(11) . Here, using the same technique, we show the ubiquitous distribution of both CB1 and CB1A mRNAs in relative amounts covering a wide range of values (Table 1). CB1A mRNA exists as a minor transcript since at its highest level it represents only 20% of the total central cannabinoid receptor mRNA content. In most of the peripheral tissues, CB1A mRNA was consistently found at 10% of CB1 levels, falling to <1% in the kidney. The relative levels in the brain were more variable. CB1A mRNA was almost totally absent in the brain stem and temporal lobe of the human infant (Table 1), but was found at a higher level in a cortex-cerebellum fraction prepared from the same donor. In contrast, in a human brain stem cDNA library derived from a 2-year-old, CB1A mRNA was present at 12% of CB1 mRNA levels. The overall age-related levels of the central cannabinoid receptor and its mRNA have been studied in rat brain(31, 32, 33) . In at least some brain regions, CB1 levels rise just after birth to attain adult levels, thereafter decreasing, the decrease perhaps being attributable to a lower rate of CB1 transcription and/or to messenger instability(33) ; within these global fluctuations, we do not know if the CB1/CB1A ratio also varies. It is perhaps relevant that CB1 mRNA exists in at least two 3`-UTR variants, of 0.6 and 4 kb, which may contain regulatory signals^2 important for mRNA stability and for the control of the translation process (see (34) for a recent review). It follows that a detailed knowledge of the age-related brain distribution and control of these 3`-UTR variants and of the amino-terminal isoforms would help toward our understanding of this receptor.

The existence of two subtypes of the cannabinoid receptor, CB1 and CB2, the first of which exists as two isoforms, will facilitate the discovery and development of cannabinoids having high selectivity and possibly therapeutic potential. One such molecule is the recently described antagonist SR 141716A(13) , which has high affinity and specificity for the central forms of the receptor. In return, these specific ligands will be invaluable tools for facilitating a detailed investigation of the structural features of the various cannabinoid receptors and their physiological significance.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X81120[GenBank], X81121[GenBank], and X81122[GenBank].

§
To whom correspondence should be addressed. Tel.: 33-61-39-96-00; Fax: 33-61-39-86-37.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; UTR, untranslated region; kb, kilobase pair(s).

(^2)
The complete cann6 and cann7 sequences will be published elsewhere (D. Shire, C. Carillon, M. Kaghad, B. Calandra, M. Rinaldi-Carmona, G. Le Fur, D. Caput, and P. Ferrara, manuscript in preparation).

(^3)
D. Shire, C. Carillon, M. Kaghad, B. Calandra, M. Rinaldi-Carmona, G. Le Fur, D. Caput, and P. Ferrara, manuscript in preparation.


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