EP0542920A1 - Cannabinoid receptor - Google Patents

Cannabinoid receptor

Info

Publication number
EP0542920A1
EP0542920A1 EP91917229A EP91917229A EP0542920A1 EP 0542920 A1 EP0542920 A1 EP 0542920A1 EP 91917229 A EP91917229 A EP 91917229A EP 91917229 A EP91917229 A EP 91917229A EP 0542920 A1 EP0542920 A1 EP 0542920A1
Authority
EP
European Patent Office
Prior art keywords
receptor
cannabinoid receptor
cannabinoid
protein
recombinant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91917229A
Other languages
German (de)
French (fr)
Other versions
EP0542920A4 (en
Inventor
Lisa A. Matsuda
Michael J. Brownstein
Tom I. Bonner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
US Department of Commerce
Original Assignee
US Department of Health and Human Services
US Department of Commerce
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services, US Department of Commerce filed Critical US Department of Health and Human Services
Publication of EP0542920A1 publication Critical patent/EP0542920A1/en
Publication of EP0542920A4 publication Critical patent/EP0542920A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to a DNA segment encoding a mammalian cannabinoid receptor, the cannabinoid receptor encoded therein and recombinantly produced cannabinoid receptor protein.
  • Cannabinoids are the primary active constituents of marijuana, a preparation of the plant Cannabis Sativa , producing prominent effects on the central nervous system. They are responsible for effects such as changes in mood, perception, cognition, memory and psychomotor skills. Cannabinoids, well known for their psychoactivity, also have value as therapeutic agents. Cannabinoids have found use in the treatment of chemotherapy side effects, glaucoma, bronchial asthma and insomnia. They can also be used as analgesics, muscle relaxants or anticonvulsants.
  • cannabinoid action is that these drugs may be active at a specific neurotransmitter receptor site. This hypothesis is supported by the fact that the behavior of the drugs is dose dependent, pharma ⁇ cologically selective and stereospecific in man as well as a variety of animal idels. However, prior to the present invention, the structure of such a cannabinoid receptor was not known.
  • the present invention relates to a DNA segment encoding all, or a unique portion, of a mammalian cannabinoid receptor, or a DNA fragment comple ⁇ mentary to the DNA segment.
  • the present invention relates to a cannabinoid receptor protein substantially free of proteins with which it is normally associated. Further, the present invention relates to a recombinantly produced protein having all, or a unique portion of the amino acid sequence given in Figure 1 or Figure 5.
  • the present invention relates to a recombinant DNA molecule comprising the DNA segment encoding all, or a unique portion, of a mammalian cannabinoid receptor, or a DNA fragment complementary to the DNA segment and a vector.
  • the present invention also relates to a host cell stably transformed with the above- described recombinant DNA molecule in a manner allowing expression.
  • the present invention relates to a method of producing a recombinant cannabinoid receptor comprising culturing host cells of the present invention, in a manner allowing expression of the receptor and isolating the receptor.
  • the present invention relates to a method of screening compounds for their ability to bind to the recombinant cannabinoid receptor of the present invention comprising contacting the receptor with the compound under conditions such that binding can be effected and detecting the presence or absence of binding.
  • the present invention relates to a method of detecting cannabinoids or cannabinoid-like compounds in a biological sample compris ⁇ ing contacting the recombinant cannabinoid receptor of the present invention with the biological sample under condi ⁇ tions such that binding can be effected and detecting the presence or absence of binding.
  • the present invention relates to a method of identifying and isolating biomolecules which naturally interact with the cannabinoid receptor comprising contacting the recombinant receptor cannabinoid receptor of the present invention with a biological sample under conditions such that binding can be effected and detecting the presence or absence of binding.
  • the present invention relates to an assay for detection of cannabinoid receptor in tissue comprising contacting the tissue with the antibody of the present invention specific for the receptor and detecting the presence or absence of a complex formed between the antibodies and proteins present in the tissue.
  • Figure 1 shows the partial nucleotide sequence of SKR6 cDNA. Indicated above and below the sequence, respectively, are the predicted hydrophobic domains (I through VII) and the translated primary structure of the receptor. The initial stretch of guanine nucleotides represents the G tail produced during cDNA synthesis.
  • the substance K receptor derived probe sequence is indicated by dots (bases identical to SKR6) beginn i ng at base number 449; non-identical bases are provide, above the cDNA sequence and a single nucleotide gap (hyphen) has been introduced to align the probe with the cDNA sequence. Underlined amino acids are those which are highly con ⁇ served among other G protein-coupled receptors.
  • Figure 2 shows the results of a northern blot analysis of total RNAs from N18TG-2 (lane a) , NG108-15 (lane b) and C6BU-1 (lane c) cell lines.
  • N18TG-2 and C6BU-1 cells are the neuroblastoma (mouse) and glioma (rat) parents of the NG108-15 hybrid cell line, respec ⁇ tively.
  • the single hybridizing band present in lanes a and b is ca. 6.0 kb.
  • Figure 3 shows the localization of SKR6 mRNA in rat brain.
  • Cx cerebral cortex
  • DG dentate gyrus
  • Hi hippocampus
  • VMH ventromedial hypothalamic nucleus.
  • Figures 4a and 4b show cannabinoid-induced inhibition of forskolin-stimulated cAMP production in SKR6-trans- fected CHO-K1 cells.
  • Fig. 4a shows stereospecific inhibi ⁇ tion by d9THC.
  • Fig. 4b shows the dose-response curves of various cannabinoids and cannabinoid analogs. Data represent the average percent inhibition + Standard Error of the Mean (S.E.M.) of cAMP accumulation for 3-5 experi ⁇ ments each performed in triplicate. Curves were generated using Graph-Pad InPlot regression analysis program.
  • Figure 5 shows the partial nucleotide sequence of the human cannabinoid receptor gene. Indicated below is the deduced amino acid sequence of the receptor.
  • the present invention relates to a DNA sequence encoding all, or a unique portion, of a mammalian cannabinoid receptor and to the protein (or polypeptide) encoded therein, or allelic variations thereof.
  • a "unique portion” as used herein is defined as consisting of at least five (or six) amino acids or correspondingly, at least 15 (or 18) nucleotides.
  • the invention also relates to recombinant molecules containing such DNA segments and to cells transformed therewith.
  • the present invention relates to DNA segments that encode the entire amino acid sequence given in Figure 1 or Figure 5 (the specific DNA segments given in Figure 1 and Figure 5 being only two such exam- pie) i or any unique portion thereof.
  • DNA segments to which the invention relates also include those encoding substantially the same receptor as that of Figure 1 or Figure 5 which includes, for example, allelic forms of the Figure 1 or Figure 5 amino acid sequence.
  • the invention also relates to DNA fragments complementary to such sequences. A unique portion of the DNA segment or the complementary fragment thereof of the present invention can be used as probes for detecting the presence of its complementary strand in a DNA or RNA sample.
  • the present invention relates to a ribonucleotide segment (that is, a riboprobe) which encodes the entire amino acid sequence given in Figure 1 or Figure 5, or any unique portion thereof.
  • a ribonucleotide segment that is, a riboprobe
  • the riboprobes of the present invention can be used to detect the presence of its complementary strand of DNA or RNA in a sample.
  • the present invention relates to a cannabinoid receptor protein substantially free of proteins with which it is normally associated. (One skilled in the art can easily purify the cannabinoid receptor protein using standard methodologies for protein purification.)
  • the present invention also relates to peptide fragments unique to the cannabinoid receptor.
  • the protein and/or peptides of the present invention may be chemically synthesized using known methods.
  • the present invention relates to a recombinantly produced or chemically synthe- sized cannabinoid receptor with the amino acid sequence given in Figure 1, Figure 5 or an allelic variation thereof.
  • the receptor has several potential N-linked glycosylation sites. Accordingly, the recombinantly produced protein may be unglycosylated or glycosylated (the glycosylation pattern may differ from that of the naturally occurring receptor) .
  • the present invention further relates to proteins having amino acid sequences sufficiently similar to that given in Figure l or Figure 5 to afford the binding characteristics as the naturally occurring molecule.
  • the present invention relates to a recombinant DNA molecule and to a host cell transformed therewith.
  • a recombinant DNA molecule comprising a DNA segment encoding all or a unique portion of the mammalian cannabinoid receptor of the present invention and a vector can be constructed.
  • Possible vectors for use in the present invention include, but are not limited to, Okayama-Berg pCDl mammalian vector, ptk-muARS or pAcY l(baculovirus) and pGEX-3X(GST gene fusion system) .
  • the DNA segment can be present in the vector operably linked to regulatory elements, including, for example, a promoter.
  • Suitable host cells can be transformed with the recombinant molecule using standard methods well known in the art.
  • Possible host cells include eukaryotic cells such as CHO-K1 (ATCC accession member CCL 61) , mouse L cells or Sf9 (Spodoptera frugiperda) and yeast (schizo- saccharomyces pombe) and prokaryotic cells such as E. coli(JMlOl) , which are all publically available.
  • the present invention relates to antibodies specific for the cannabinoid recep ⁇ tor of the present invention.
  • the present invention relates to a method of screening compounds for their ability to bind to the recombinant cannabinoid receptor of the present invention.
  • Drugs which bind to the receptor may be useful in the treatment of cannabinoid-treatable conditions.
  • New drugs lacking the legal complications associated with marijuana, for treating, reversing or eliminating conditions such as chemotherapy side effects, glaucoma, bronchial asthma, insomnia, eating di ⁇ .>... ers and/or weight problems can be identified by contacting the receptor with a compound under conditions such that binding can be effected. The presence or absence of bound compound to receptor is then detected using methods well known in the art.
  • the present invention relates to a method of detecting cannabinoids or cannabinoid-like compounds (that is, exogeneous sub ⁇ stances) in a biological sample.
  • the presence or absence of the compound can be determined by contacting the recombinant cannabinoid receptor of the present invention with a biological sample such as for example, tissue extracts, cerebrospinal fluid, blood, urine or other body fluids, under conditions such that binding between a drug with the receptor can be effected and detecting the presence or absence of bound drug to the receptor using methods well known in the art.
  • This method can be incorporated into drug testing programs such as those used by companies and athletics clubs.
  • the present invention relates to a method of identifying and isolating natural biomolecules (such as, a neurotransmitter, hormone or peptide) which naturally interacts with the receptor in the body (that is, an endogeneous ligand) .
  • the receptor's natural ligand can be identified and isolated by contact- ing the recombinant cannabinoid receptor of the present invention with a biological sample under conditions such that binding can be effected.
  • the presence of a bound biomolecule can then be detected and identified using methods well known in the art.
  • the present invention relates to an assay for detection of the cannabinoid receptor in tissue. The presence or absence of the receptor can be determined by contacting the tissue with an antibody specific for the receptor and detecting formation of antibody-receptor complex.
  • EXAMPLE ISOLATION OF RAT RECEPTOR GENE SKR6 was isolated from a rat cerebral cortex cDNA library constructed in the mammalian expression vector pCDl as described by Okayama et al. [Mol. Cell. Biol. , 3: 280-289 (1983)]. Screening was performed as described previously for cloning muscarinic receptor subtype cDNAs [Bonner et al., Science, 237: 527-532 (1984)]. Sequencing was performed by dideoxynucleotide-chain termination of single-stranded DNA obtained from restriction fragments inserted into M13 mpl8 or 19 [Yanisch-Perron, Gene, 33: 103 (1985)]. The deduced amino acid sequence, shown in Figure
  • the probe sequence was derived from the sequence of the substance K receptor, however, less than 25% homology overall exists between the amino acid sequence of SKR6 and the substance K receptor. Although characteristic of the G protein-coupled class of membrane-located receptors, the amino acid sequence of SKR6 is not obviously similar to that of the substance K receptor nor any other receptor cloned to date.
  • LH-CG receptor (lacks the corresponding proline [McFarland et al., Science, 245: 494-499 (1989) and Loosfelt et al.,
  • SKR6 N-linked glycosylation sites are enclosed within boxes.
  • the entire SKR6 cDNA (5.7 kb) includes an additional circa 4100 bases 3' of the given sequence.
  • SKR14 a second clone was isolated whose coding region, although incomplete, was identical to SKR6.
  • the 3' untranslated sequence of SKR14 was circa 2900 bases shorter than that of SKR6. Comparison of the sequences of these clones indicates that SKR14 is the product of an alternatively poly-adenylated mRNA.
  • RNAs were isolated from cultured N18TG-2, NG108-15 and C6BU-1 cells using the guanidinium thiocyanate method as described by Chomczynski et al. [Anal. Biochem., 162: 156-159 (1987)], and loaded (lO ⁇ g/lane) into a 1% aga ⁇ rose/formaldehyde gel. Following electrophoresis and electrotransfer the filter was hybridized to a nick- translated fragment of the SKR6 cDNA, washed (0.1 x SSPE/0.1% SDS, 60°C) and exposed to x-ray sensitive film for 6 days (-80°C) (see Figure 2) .
  • SKR6 As a cannabinoid receptor, other ligands were tried such as neurotensin, d-ala-d-leu enkephalin, various adenosine analogs, somatostatin, bradykinin, secretion and PGE.
  • the recep ⁇ tors for these had all been described in either N18TG-2 or NG108-15 cells [Howlett et al., Mol. Pharmcol., 26: 532- 538 (1984) and Nirenburg et al. , Science, 222: 794-799 (1983)].
  • Transfection and selection of cells were performed as described [Chen et al. , Mol. Cell. Biol., 7: 2745-2752 (1987) ] .
  • a monoclonal line expressing the SKR6 cDNA was obtained by limiting-dilution cloning of cells expressing the corresponding mRNA as determined by Northern blot analysis. Functional expression studies were similar to experiments designed by Howlett et al. [Howlett et al., Mol. Pharmacol., 29: 307-313 (1986)].
  • Transfected cells were resuspended (1.25 x 10 6 cells/ml) in culture media containing fatty-acid free BSA (0.25%). Cells were incubated (37°C) for five min.
  • EC 50 values mean nM ⁇ Standard Error of the Mean (S.E.M.) for the inhibition of stimulated cAMP accumulation were 13.5 ⁇ 2.7 for (-)d9THC, 773 ⁇ 187 for (+) d9THC, 0.87 ⁇ 0.20 for CP 55940, 8.9 ⁇ 1.8 for 11-OH d9THC and 16.6 ⁇ 4.9 for nabilone.
  • CP 55940 and nabilone are synthesized by Pfizer and Lilly Research laboratories, respectively. other cannabinoics are compounds distributed by the National Institute of Drug Abuse.
  • CHO cells were transfected with an ⁇ - adrenergic receptor, a muscarinic receptor or SKR6.
  • ⁇ -adrenergic receptor a muscarinic receptor or SKR6.
  • Both the ⁇ -adrenergic nor muscarinic receptors responded to (-) ⁇ 9THC or CP 55940 (see Table I below) . Both these receptors, however, reduced cAMP production in response to their respective agonists.
  • both the muscarinic and adrenergic receptors are Gi-coupled, the cannabinoid-induced inhibition of adenylate cyclase activity observed in SKR6-transfected cells was not due to the interaction of cannabinoids with this class of recep ⁇ tors and was clearly specified to SKR6. TABLE I
  • Values represent the average accumulation of cAMP + S.E.M. as percent of forskolin-stimulated controls.
  • the effects of the various agonists were examined in 3-5 experiments (each performed in triplicate) .
  • Numbers in parentheses are the absolute values of cAMP as deter ⁇ mined by RIA (p oles cAMP/10 6 cells/5 min) .
  • Final concen- trations of forskolin were 500 nM for all cell lines except NG108-15; forskolin concentration for this cell line was 250 nM.
  • the muscarinic and adrenergic receptor- transfected cells were assayed under conditions identical to those routinely used to test the SKR6-transfected cells (see figure 3) .
  • Final concentrations of carbachol (ago ⁇ nist for muscarinic receptors) and clonidine (agonist for ⁇ -adrenergic receptors) were 100 ⁇ M and 10 ⁇ M, respec ⁇ tively.
  • carbachol ago ⁇ nist for muscarinic receptors
  • clonidine agonist for ⁇ -adrenergic receptors
  • a human cosmid library purchased from Stratagene [(catalog no. 951-202)], was screened with a probe specific for the rat cannabinoid receptor.
  • the probe con- stituted of EcoRV-Xbal SKR6 restriction fragment corre ⁇ sponding to bases 97 to 1370 of Figure 1.
  • a positive clone was sequenced as described for the rat cDNA clone.
  • the partial nucleotide sequence of the DNA and deduced amino acid sequence are shown in Figure 5. There is about 97% homology between the deduced amino acid sequence of the human receptor and the rat receptor.
  • the human cannabinoid receptor gene was inserted into a ptk-muARS vector [Hoist et al.. Cell, 52: 355-365 (1988) and Zastrow et al., Nucl. Acid Res., 17: 1867-1879 (1989)].
  • the recombinant molecule was then transfected into L-M(tk-) L cells (American Type Culture Collection Accession number ATCC CCL 1.3) by methods which are reproducible and known in the art. Expression of the receptor protein was detected using described methods [Devane et al., Mol. Pharmacol., 34: 605-613 (1988)]. This stably transformed cell line enables binding assays to be preformed on immortal tissue culture cells.
  • This cell line is unique in that the same cell line exists without the cannabinoid receptor (non- transfected L-cells) and thus can be used to demonstrate ligand-receptor interactions which are specific to the cannabinoid receptor.
  • Some cannabinoid receptor assays can be performed on tissue preparations, but without antagonist ligands (none currently available) , demonstra ⁇ tions of specificity are limited (particularly in assays which measure a functional response) .
  • This cell line expresses approximately 10 fold more cannabinoid receptor mRNA than the neuroblastoma cell line, N18TG-2.

Abstract

La présente invention concerne un segment d'ADN codant pour un récepteur de cannabinoïdes chez les mammifères et la protéine codée dans ce récepteur. Ladite invention concerne également une molécule d'ADN recombiné codant pour le récepteur dans des cellules hôtes transfectées avec ladite molécule et une protéine de récepteur produite par recombinaison.The present invention relates to a DNA segment encoding a cannabinoid receptor in mammals and the protein encoded therein. The invention also relates to a recombinant DNA molecule encoding the receptor in host cells transfected with said molecule and a recombinantly produced receptor protein.

Description

CANNABINOID RECEPTOR
BACKGROUND OF THE INVENTION Field of the Invention
The present invention relates to a DNA segment encoding a mammalian cannabinoid receptor, the cannabinoid receptor encoded therein and recombinantly produced cannabinoid receptor protein. Background Information
Cannabinoids are the primary active constituents of marijuana, a preparation of the plant Cannabis Sativa , producing prominent effects on the central nervous system. They are responsible for effects such as changes in mood, perception, cognition, memory and psychomotor skills. Cannabinoids, well known for their psychoactivity, also have value as therapeutic agents. Cannabinoids have found use in the treatment of chemotherapy side effects, glaucoma, bronchial asthma and insomnia. They can also be used as analgesics, muscle relaxants or anticonvulsants.
One important deficiency in the knowledge of cannabinoid pharmacology is that regarding their mechanism of action. Elucidation of the mechanisms underlying the actions of marijuana has been difficult due to the hydro- phobic nature of the cannabinoids, lack of specific antagonists and qualitative differences in the effects induced by individual compounds [Martin, Pharmacol. Rev., 38: 45-74 (1986)].
One hypothesis of cannabinoid action is that these drugs may be active at a specific neurotransmitter receptor site. This hypothesis is supported by the fact that the behavior of the drugs is dose dependent, pharma¬ cologically selective and stereospecific in man as well as a variety of animal idels. However, prior to the present invention, the structure of such a cannabinoid receptor was not known. SUMMARY OF THE INVENTION
It is one object of the present invention to provide a mammalian cannabinoid receptor.
It is another object of the present invention to provide a means of testing for cannabinoid agonists and antagonists.
It is a further object of the present invention to provide a means of screening for alternative drugs for use in the treatment of cannabinoid-treatable conditions.
Various other objects and advantages of the present invention will become obvious from the drawings and the following description of the invention.
In one embodiment, the present invention relates to a DNA segment encoding all, or a unique portion, of a mammalian cannabinoid receptor, or a DNA fragment comple¬ mentary to the DNA segment.
In another embodiment, the present invention relates to a cannabinoid receptor protein substantially free of proteins with which it is normally associated. Further, the present invention relates to a recombinantly produced protein having all, or a unique portion of the amino acid sequence given in Figure 1 or Figure 5.
In further embodiment, the present invention relates to a recombinant DNA molecule comprising the DNA segment encoding all, or a unique portion, of a mammalian cannabinoid receptor, or a DNA fragment complementary to the DNA segment and a vector. The present invention also relates to a host cell stably transformed with the above- described recombinant DNA molecule in a manner allowing expression.
In another embodiment, the present invention relates to a method of producing a recombinant cannabinoid receptor comprising culturing host cells of the present invention, in a manner allowing expression of the receptor and isolating the receptor.
In a further embodiment, the present invention relates to a method of screening compounds for their ability to bind to the recombinant cannabinoid receptor of the present invention comprising contacting the receptor with the compound under conditions such that binding can be effected and detecting the presence or absence of binding. In another embodiment, the present invention relates to a method of detecting cannabinoids or cannabinoid-like compounds in a biological sample compris¬ ing contacting the recombinant cannabinoid receptor of the present invention with the biological sample under condi¬ tions such that binding can be effected and detecting the presence or absence of binding.
In another embodiment, the present invention relates to a method of identifying and isolating biomolecules which naturally interact with the cannabinoid receptor comprising contacting the recombinant receptor cannabinoid receptor of the present invention with a biological sample under conditions such that binding can be effected and detecting the presence or absence of binding.
In yet a further embodiment, the present invention relates to an assay for detection of cannabinoid receptor in tissue comprising contacting the tissue with the antibody of the present invention specific for the receptor and detecting the presence or absence of a complex formed between the antibodies and proteins present in the tissue.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the partial nucleotide sequence of SKR6 cDNA. Indicated above and below the sequence, respectively, are the predicted hydrophobic domains (I through VII) and the translated primary structure of the receptor. The initial stretch of guanine nucleotides represents the G tail produced during cDNA synthesis. The substance K receptor derived probe sequence is indicated by dots (bases identical to SKR6) beginning at base number 449; non-identical bases are provide, above the cDNA sequence and a single nucleotide gap (hyphen) has been introduced to align the probe with the cDNA sequence. Underlined amino acids are those which are highly con¬ served among other G protein-coupled receptors.
Figure 2 shows the results of a northern blot analysis of total RNAs from N18TG-2 (lane a) , NG108-15 (lane b) and C6BU-1 (lane c) cell lines. N18TG-2 and C6BU-1 cells are the neuroblastoma (mouse) and glioma (rat) parents of the NG108-15 hybrid cell line, respec¬ tively. The single hybridizing band present in lanes a and b is ca. 6.0 kb.
Figure 3 shows the localization of SKR6 mRNA in rat brain. Cx, cerebral cortex; DG, dentate gyrus; Hi, hippocampus; VMH, ventromedial hypothalamic nucleus.
Figures 4a and 4b show cannabinoid-induced inhibition of forskolin-stimulated cAMP production in SKR6-trans- fected CHO-K1 cells. Fig. 4a shows stereospecific inhibi¬ tion by d9THC. Fig. 4b shows the dose-response curves of various cannabinoids and cannabinoid analogs. Data represent the average percent inhibition + Standard Error of the Mean (S.E.M.) of cAMP accumulation for 3-5 experi¬ ments each performed in triplicate. Curves were generated using Graph-Pad InPlot regression analysis program.
Figure 5 shows the partial nucleotide sequence of the human cannabinoid receptor gene. Indicated below is the deduced amino acid sequence of the receptor.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a DNA sequence encoding all, or a unique portion, of a mammalian cannabinoid receptor and to the protein (or polypeptide) encoded therein, or allelic variations thereof. A "unique portion" as used herein is defined as consisting of at least five (or six) amino acids or correspondingly, at least 15 (or 18) nucleotides. The invention also relates to recombinant molecules containing such DNA segments and to cells transformed therewith.
A complementary DNA segment (SKR6) from rat cerebral cortex and a DNA segment from a human cosmid library, each encoding a cannabinoid receptor, were cloned, sequenced and the amino acid sequences deduced therefrom. The deduced amino acid sequence of the rat DNA segment revealed the presence of seven hydrophobic domains. Furthermore, located throughout the translated sequence of SKR6, are a number of amino acids which are highly conserved among other G protein-coupled receptors [Bonner, Trends in Neurosci., 12: 148-151 (1989); Emorine et al.. Science, 245: 1118-1121 (1989); Zeng et al., Proc. Natl. Acad. Sci., 87: 3102-3106 (1990); Shigemoto et al., J. Biol Chem., 265: 623-628 (1990); Parmentier et al., Science, 246: 1620-1622 (1990)] as well as several poten¬ tial glycosylation sites in the amino-terminal portion of the protein (see Figure 1) . With only minimal similarity with other known receptors, the ligand for SKR6 could not be predicted from the sequence.
In one embodiment, the present invention relates to DNA segments that encode the entire amino acid sequence given in Figure 1 or Figure 5 (the specific DNA segments given in Figure 1 and Figure 5 being only two such exam- pie) i or any unique portion thereof. DNA segments to which the invention relates also include those encoding substantially the same receptor as that of Figure 1 or Figure 5 which includes, for example, allelic forms of the Figure 1 or Figure 5 amino acid sequence. The invention also relates to DNA fragments complementary to such sequences. A unique portion of the DNA segment or the complementary fragment thereof of the present invention can be used as probes for detecting the presence of its complementary strand in a DNA or RNA sample. In another embodiment, the present invention relates to a ribonucleotide segment (that is, a riboprobe) which encodes the entire amino acid sequence given in Figure 1 or Figure 5, or any unique portion thereof. The riboprobes of the present invention can be used to detect the presence of its complementary strand of DNA or RNA in a sample.
In another embodiment, the present invention relates to a cannabinoid receptor protein substantially free of proteins with which it is normally associated. (One skilled in the art can easily purify the cannabinoid receptor protein using standard methodologies for protein purification.) The present invention also relates to peptide fragments unique to the cannabinoid receptor. The protein and/or peptides of the present invention may be chemically synthesized using known methods.
In another embodiment, the present invention relates to a recombinantly produced or chemically synthe- sized cannabinoid receptor with the amino acid sequence given in Figure 1, Figure 5 or an allelic variation thereof. The receptor has several potential N-linked glycosylation sites. Accordingly, the recombinantly produced protein may be unglycosylated or glycosylated (the glycosylation pattern may differ from that of the naturally occurring receptor) . The present invention further relates to proteins having amino acid sequences sufficiently similar to that given in Figure l or Figure 5 to afford the binding characteristics as the naturally occurring molecule.
In another embodiment, the present invention relates to a recombinant DNA molecule and to a host cell transformed therewith. Using standard methodology well known in the art, a recombinant DNA molecule comprising a DNA segment encoding all or a unique portion of the mammalian cannabinoid receptor of the present invention and a vector can be constructed. Possible vectors for use in the present invention include, but are not limited to, Okayama-Berg pCDl mammalian vector, ptk-muARS or pAcY l(baculovirus) and pGEX-3X(GST gene fusion system) . The DNA segment can be present in the vector operably linked to regulatory elements, including, for example, a promoter. Suitable host cells can be transformed with the recombinant molecule using standard methods well known in the art. Possible host cells include eukaryotic cells such as CHO-K1 (ATCC accession member CCL 61) , mouse L cells or Sf9 (Spodoptera frugiperda) and yeast (schizo- saccharomyces pombe) and prokaryotic cells such as E. coli(JMlOl) , which are all publically available. In another embodiment, the present invention relates to antibodies specific for the cannabinoid recep¬ tor of the present invention. It is routine for one of ordinary skill in the art to generate antibodies against the canrabinoid receptor by, for example, using a peptide corresponding to all, or a unique portion, of the amino acid sequence defined in Figure 1 or Figure 5. Methods for this are well known in the art, such as those de- scribed in Antibodies: a laboratory manual by Ed Harlow and David Lane, Cold Spring Harbor (1988) . Both monoclonal and polyclonal antibodies can be raised against the cannabinoid receptor.
In another embodiment, the present invention relates to a method of screening compounds for their ability to bind to the recombinant cannabinoid receptor of the present invention. Drugs which bind to the receptor may be useful in the treatment of cannabinoid-treatable conditions. New drugs, lacking the legal complications associated with marijuana, for treating, reversing or eliminating conditions such as chemotherapy side effects, glaucoma, bronchial asthma, insomnia, eating di<.>... ers and/or weight problems can be identified by contacting the receptor with a compound under conditions such that binding can be effected. The presence or absence of bound compound to receptor is then detected using methods well known in the art.
In another embodiment, the present invention relates to a method of detecting cannabinoids or cannabinoid-like compounds (that is, exogeneous sub¬ stances) in a biological sample. The presence or absence of the compound can be determined by contacting the recombinant cannabinoid receptor of the present invention with a biological sample such as for example, tissue extracts, cerebrospinal fluid, blood, urine or other body fluids, under conditions such that binding between a drug with the receptor can be effected and detecting the presence or absence of bound drug to the receptor using methods well known in the art. This method can be incorporated into drug testing programs such as those used by companies and athletics clubs.
In another embodiment, the present invention relates to a method of identifying and isolating natural biomolecules (such as, a neurotransmitter, hormone or peptide) which naturally interacts with the receptor in the body (that is, an endogeneous ligand) . The receptor's natural ligand can be identified and isolated by contact- ing the recombinant cannabinoid receptor of the present invention with a biological sample under conditions such that binding can be effected. The presence of a bound biomolecule can then be detected and identified using methods well known in the art. In another embodiment, the present invention relates to an assay for detection of the cannabinoid receptor in tissue. The presence or absence of the receptor can be determined by contacting the tissue with an antibody specific for the receptor and detecting formation of antibody-receptor complex.
The following non-limiting Example describes the invention in greater detail.
EXAMPLE ISOLATION OF RAT RECEPTOR GENE SKR6 was isolated from a rat cerebral cortex cDNA library constructed in the mammalian expression vector pCDl as described by Okayama et al. [Mol. Cell. Biol. , 3: 280-289 (1983)]. Screening was performed as described previously for cloning muscarinic receptor subtype cDNAs [Bonner et al., Science, 237: 527-532 (1984)]. Sequencing was performed by dideoxynucleotide-chain termination of single-stranded DNA obtained from restriction fragments inserted into M13 mpl8 or 19 [Yanisch-Perron, Gene, 33: 103 (1985)]. The deduced amino acid sequence, shown in Figure
1, reveals the presence of seven hydrophobic domains. The probe sequence was derived from the sequence of the substance K receptor, however, less than 25% homology overall exists between the amino acid sequence of SKR6 and the substance K receptor. Although characteristic of the G protein-coupled class of membrane-located receptors, the amino acid sequence of SKR6 is not obviously similar to that of the substance K receptor nor any other receptor cloned to date.
The results are shown in Figure l. Notably absent from SKR6 is a proline residue within the fifth hydropho- bic domain and a cysteine just prior to hydrophobic domain III. In terms of structure, these substitutions may indicate interesting similarities between SKR6 and the
LH-CG receptor (lacks the corresponding proline [McFarland et al., Science, 245: 494-499 (1989) and Loosfelt et al.,
Science, 245: 525-528 (1989)]) or the mas oncogene or angiotensin receptor (lacks the same cysteine residue
[Young et al., Cell, 45: 711-719 (1986) and Jackson et al., Nature, 335: 437-440 (1988)]. Indeed, the homologous cysteine has been found to be essential in functional rhodopsin [Karnik et al., Proc. Natl. Acad. Sci., 85: 8459-8463 (1988)].
Potential N-linked glycosylation sites are enclosed within boxes. The entire SKR6 cDNA (5.7 kb) includes an additional circa 4100 bases 3' of the given sequence. In addition to SKR6, a second clone (SKR14) was isolated whose coding region, although incomplete, was identical to SKR6. The 3' untranslated sequence of SKR14, however, was circa 2900 bases shorter than that of SKR6. Comparison of the sequences of these clones indicates that SKR14 is the product of an alternatively poly-adenylated mRNA.
IDENTIFICATION OF SKR6
RNAs were isolated from cultured N18TG-2, NG108-15 and C6BU-1 cells using the guanidinium thiocyanate method as described by Chomczynski et al. [Anal. Biochem., 162: 156-159 (1987)], and loaded (lOμg/lane) into a 1% aga¬ rose/formaldehyde gel. Following electrophoresis and electrotransfer the filter was hybridized to a nick- translated fragment of the SKR6 cDNA, washed (0.1 x SSPE/0.1% SDS, 60°C) and exposed to x-ray sensitive film for 6 days (-80°C) (see Figure 2) .
Prior to identification of SKR6 as a cannabinoid receptor, other ligands were tried such as neurotensin, d-ala-d-leu enkephalin, various adenosine analogs, somatostatin, bradykinin, secretion and PGE. The recep¬ tors for these had all been described in either N18TG-2 or NG108-15 cells [Howlett et al., Mol. Pharmcol., 26: 532- 538 (1984) and Nirenburg et al. , Science, 222: 794-799 (1983)].
In addition to the expression of SKR6 mRNA in cell lines, hybridization patterns in rat brain were also considered in selecting candidate ligands for SKR6. Substance P, vasoactive intestinal peptide and cholecysto- kinin were tested due to the similar localizations of the receptors for these peptides (and/or the peptide them¬ selves) and the distribution of SKR6 mRNA as shown here in a representative coronal section (see Figure 3) .
Brain tissue from a male, Sprague-Dawley rat (200- 250 gm) was cut to 12-μm sections and thaw mounted to gelatin-coated slides and in situ hybridization histochem- istry performed as described [Young et al. Proc. Natl.
Acad. Sci., 83: 9827-0831 (1986) and Young et al.,
Neuroendocrinol; 46: 439-444 (1987)]. The section labelled with a 35S-tailed oligonucleotide probe (complementary to bases 349 to 396 of the cDNA sequence) was then exposed to x-ray sensitive film (25°C) for 16 days. Under similar hybridization conditions, this probe hybridized to a 6.2 kb band in preparations of rat cerebral cortex and hippocampal RNA.
Transfection and selection of cells were performed as described [Chen et al. , Mol. Cell. Biol., 7: 2745-2752 (1987) ] . A monoclonal line expressing the SKR6 cDNA was obtained by limiting-dilution cloning of cells expressing the corresponding mRNA as determined by Northern blot analysis. Functional expression studies were similar to experiments designed by Howlett et al. [Howlett et al., Mol. Pharmacol., 29: 307-313 (1986)]. Transfected cells were resuspended (1.25 x 106 cells/ml) in culture media containing fatty-acid free BSA (0.25%). Cells were incubated (37°C) for five min. with and without various concentrations of cannabinoids (final ethanol concentra¬ tions were less than or equal to 0.2%) and the reaction terminated with the addition of 0.1N HCI/0.1 mM CaCl2. Samples were frozen at -20°C and thawed just prior to determination of cAMP by RIA [Brooker et al., Adv. Cyclic Nucl. Res., 10: 1-33 (1979) and Borst et al.. Life Sci., 43: 1021-1030 (1988)].
Forskolin increased cAMP ca. 20 fold above basal concentrations; absolute values in forskolin-stimulated controls ranged from 9.5 to 17.7 pmole/106 cells/5 min. Cannabinoids did not significantly inhibit cAMP accumula- tion in non-transfected cells. EC50 values (mean nM ± Standard Error of the Mean (S.E.M.) for the inhibition of stimulated cAMP accumulation were 13.5 ± 2.7 for (-)d9THC, 773 ± 187 for (+) d9THC, 0.87 ± 0.20 for CP 55940, 8.9 ± 1.8 for 11-OH d9THC and 16.6 ± 4.9 for nabilone. CP 55940 and nabilone are synthesized by Pfizer and Lilly Research laboratories, respectively. other cannabinoics are compounds distributed by the National Institute of Drug Abuse.
Further, CHO cells were transfected with an α- adrenergic receptor, a muscarinic receptor or SKR6. Neither the α-adrenergic nor muscarinic receptors responded to (-) Δ9THC or CP 55940 (see Table I below) . Both these receptors, however, reduced cAMP production in response to their respective agonists. Since both the muscarinic and adrenergic receptors are Gi-coupled, the cannabinoid-induced inhibition of adenylate cyclase activity observed in SKR6-transfected cells was not due to the interaction of cannabinoids with this class of recep¬ tors and was clearly specified to SKR6. TABLE I
Effect of Δ9THC and CP55940 on f orskolin-sti ulated accumulation of cAMP in CHO-K1 cells transfected with SKR6 , muscarinic and α-adrenergic receptor cDNAs .
receptor/ ΔgTHC CP55940 carbachol/ cDNA forskolin < lOOnM) ( lOnM-) clonidine
SKR6 100 + 4 61 + 5
( 12. 6 + 0.5 )
SKR6 100 + 5 44 + 11
( 12.1 + 1.4) muscarinic 100 + 5 104 + 8 104 + 10 8 + 1 m2 ( 18.0 + 0.9 ) adrenergic 100 + 4 100 + 4 96 + 7 73 + 5 α2d ( 13.9 + 0.5 ) 100 + 10 61 + 8 16 + 2
( 44.4 + 4.3 ) 100 + 4 91 + 7 57 + 3
(320. 5 + 11.7 )
Values represent the average accumulation of cAMP + S.E.M. as percent of forskolin-stimulated controls. In each cell line, the effects of the various agonists were examined in 3-5 experiments (each performed in triplicate) . Numbers in parentheses are the absolute values of cAMP as deter¬ mined by RIA (p oles cAMP/106 cells/5 min) . Final concen- trations of forskolin were 500 nM for all cell lines except NG108-15; forskolin concentration for this cell line was 250 nM. The muscarinic and adrenergic receptor- transfected cells were assayed under conditions identical to those routinely used to test the SKR6-transfected cells (see figure 3) . Final concentrations of carbachol (ago¬ nist for muscarinic receptors) and clonidine (agonist for α-adrenergic receptors) were 100 μM and 10 μM, respec¬ tively. Clearly, the extent to which a receptor can inhibit cAMP accumulation varies considerably across different cell lines. The moderate effect of clonidine to inhibit cAMP accumulation reported here is lower than normally observed in this transfected cell line (inhibits cAMP accumulation to 50-25% of forskolin-stimulated control) . This difference, however, is due to the BSA which is included in our assay. ISOLATION OF HUMAN RECEPTOR GENE
A human cosmid library purchased from Stratagene [(catalog no. 951-202)], was screened with a probe specific for the rat cannabinoid receptor. The probe con- stituted of EcoRV-Xbal SKR6 restriction fragment corre¬ sponding to bases 97 to 1370 of Figure 1. A positive clone was sequenced as described for the rat cDNA clone.
The partial nucleotide sequence of the DNA and deduced amino acid sequence are shown in Figure 5. There is about 97% homology between the deduced amino acid sequence of the human receptor and the rat receptor.
The human cannabinoid receptor gene was inserted into a ptk-muARS vector [Hoist et al.. Cell, 52: 355-365 (1988) and Zastrow et al., Nucl. Acid Res., 17: 1867-1879 (1989)]. The recombinant molecule was then transfected into L-M(tk-) L cells (American Type Culture Collection Accession number ATCC CCL 1.3) by methods which are reproducible and known in the art. Expression of the receptor protein was detected using described methods [Devane et al., Mol. Pharmacol., 34: 605-613 (1988)]. This stably transformed cell line enables binding assays to be preformed on immortal tissue culture cells. This is the only cell line, known at present, which expresses enough receptor protein to allow the detection of radio- labelled ligand binding to the receptor in the membrane preparation. This cell line is unique in that the same cell line exists without the cannabinoid receptor (non- transfected L-cells) and thus can be used to demonstrate ligand-receptor interactions which are specific to the cannabinoid receptor. Some cannabinoid receptor assays can be performed on tissue preparations, but without antagonist ligands (none currently available) , demonstra¬ tions of specificity are limited (particularly in assays which measure a functional response) . This cell line expresses approximately 10 fold more cannabinoid receptor mRNA than the neuroblastoma cell line, N18TG-2. This was determined as follows. Cultured cells were adhered to a nitrocellulose filter under vacuum by aliquoting a suspension of cells, at known densities, into a multi-welled manifold. The filter was baked 90 min at 80°C, and hybridized under standard Northern blot conditions with 32p-labelled probes for the cannabinoid receptor mRNA (same as those used to produce the image shown in Figure 3) . The washed filter was then exposed to X-ray sensitive film and the intensity of the autoradiographic images produced by equal numbers of cells from different cell lines was used to estimate the amount of cannabinoid receptor mRNA present in each respective cell line. mRNA for the cannabinoid receptor has also been identified in rat lung tissue.
All publications mentioned hereinabove are hereby incorporated in their entirety by reference. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

Claims

WHAT IS CLAIMED IS:
I. A DNA segment encoding a mammalian cannabinoid receptor, or a DNA fragment complementary to said DNA segment.
2. The DNA segment according to claim 1 encoding a unique portion of the mammalian cannabinoid receptor or the complementary DNA fragment.
3. The DNA segment according to claim 1 wherein said mammal is a rat.
4. The DNA segment according to claim 1 wherein said mammal is a human.
5. The DNA segment according to claim 1 wherein said segment has a sequence as defined in Figure 1, Figure 5 or a unique portion thereof.
6. A ribonucleotide segment encoding all, or a unique portion, of the amino acid sequence as defined in Figure 1 or Figure 5.
7. A cannabinoid receptor protein substantially free of proteins with which it is normally associated.
8. The protein according to claim 7 having all, or a unique portion of the amino acid sequence given in Figure 1 or Figure 5.
9. A recombinantly produced or chemically synthesized protein having all, or a unique portion of the amino acid sequence given in Figure 1 or Figure 5.
10. The protein according to claim 9 wherein said protein is glycosylated.
II. A substantially pure cannabinoid receptor protein.
12. A recombinant DNA molecule comprising: i) said DNA segment according to claim 1; and ii) a vector.
13. The recombinant DNA molecule according to claim 12 wherein said vector is Okayama-Berg pCDl mammali¬ an expression vector.
14. A host cell stably transformed with said recombinant DNA molecule according to claim 12, in a manner allowing expression of said protein encoded in said recombinant DNA molecule.
15. The host cell according to claim 14 which does not naturally express a cannabinoid receptor.
16. The host cell according to claim 14 which is
CHO-K1 or mouse L cell.
17. A method of producing a recombinant cannabinoid receptor comprising culturing said host cells according to claim 14, in a manner allowing expression of said receptor and isolating said receptor.
18. A purified form of an antibody specific for a cannabinoid receptor.
19. The antibody according to claim 18 which is monoclonal.
20. The antibody according to claim 18 which is polyclonal.
21. A method of screening compounds for their ability to bind to said recombinant cannabinoid receptor according to claim 9 comprising contacting said receptor with said compound under conditions such that binding can be effected and detecting the presence or absence of said binding.
22. The method according to claim 21 wherein said receptor is expressed on a cell.
23. A method of detecting cannabinoids or cannabinoid-like compounds in a biological sample compris¬ ing contacting said recombinant cannabinoid receptor according to claim 9 with said biological sample under conditions such that binding can be effected and detecting the presence or absence of said binding.
24. The method according to claim 23 wherein said biological sample is tissue extract, cerebrospinal fluid, blood or urine.
25. A method of identifying and isolating biomolecules which naturally interact with cannabinoid receptor comprising contacting said recombinant cannabinoid receptor according to claim 9 with a biologi¬ cal sample under conditions such that binding can be effected and detecting the presence or absence of said binding.
26. An assay for detection of cannabinoid recep¬ tor in tissue comprising contacting said tissue with said antibody according to claim 18 and detecting the presence or absence of a complex formed between said antibodies and proteins present in said tissue.
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NUCLEIC ACIDS RESEARCH. vol. 18, no. 23, 11 December 1990, ARLINGTON, VIRGINIA US page 7142 Gerard C;Mollereau C;Vassart G;Parmentier M; 'Nucleotide sequence of a human cannabinoid receptor cDNA.' *
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