Note: Descriptions are shown in the official language in which they were submitted.
W092/02~0 2 ~ ~ 7 3 '1 ~ PCT/USgl/056~6
CANNABINOID RECEPTOR
BACKG~OUND OF THE INVENTION
Field of the Invention
The present invention relates to a DNA segment
encoding a mammalian cannabinoid receptor, the cannabinoid
receptor encoued therein and recombinantly produced
cannabinoid receptor protein.
Backqround Information -~ -
Cannabinoids are the primary active constituents
of mari~uana, 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
-2S induced by individual compounds [Martin, Pharmacol. Rev.,
38: 45-74 (198~)].
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, ~harma-
cologically selective and stereospecific in man as well as
a variety of animal models. 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 ~
' ~
W092/02~0 2 0 8 7 ~ ~ ~ PCT/US91/0~656
provide a means of testing for cannabinoid agonists and
antagonists~
It is a further object of the present invention to
provide a means of scrsening for alternative drugs for use
in the treatment of cannabinoid-treat`able 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 allowingexpression.
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.
28~73~
~9~/02~0 PCT/~S91/OS6S6
-- 3
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 recombinank 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 l 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 provided 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 Nl8TG-2 (lane a), NGl08-15
.~' ''
W0 92/0264~ 2 0 ~ 7 ~
PCT/US91/05656
(lane b) and C6BU-1 (lane c) cell lines. Nl8TG-2 and
C6BU-l cells are the neuroblastoma (mouse) and glioma
(rat) parents of the NGl08-15 hybrid cell line, respec-
tively. Th~ 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; VMX, 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 bv d9THC. Fig. 4b shows the dose-response curves of
various cannabinoids and cannabinoid analogs. Data
represent the average percent inhibitlon + 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.
D~TAILED 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 aells 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
~v~2/02~0 2 0 ~ 7 ~ PCT/US91/05656
- 5 -
highly conserved among other G prote: ~oupled receptors
; [Bonner, Trends in Neurosci., 12: 1~8-151 (19~9); Emorine
et al., Science, 2~5: 1118-1121 (1989); Zeng et al., Proc.
Natl. ~cad. Sci., 87: 3102-3106 (1990); Shigemoto et al.,
~. Biol Chem., 265: 623-62~ (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 SXR6 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-
ple), 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 o~ 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 uni~ue to the cannabinoid receptor. The
:"' .' " '
r . .
WO92/Q2~0 2 ~ ~7 ~ PCT/US91/D5656
protein and/or peptides of the present invention may be
chemical~y synthesized using known methods.
In another embodiment, the present invention
relates to a recombinantly produced or chemically synthe-
si~ed 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 1 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 pCD1 mammalian vector, ptk-muARS or
pAcYml(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(JM101), 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
wn ~2/02~0 2 ~ ~ 7 8 4 ~ PCT/US~l/05S56
: - 7 -
- the cannabinoid receptor by, ~or example, using a pepti~e
corresponding to all, or a unique portion, of the amino
acid sequence defined in Figure l 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 usPful 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 disorders
andlor weight problems can be identified by contacting the
receptor with a compound under conditions such that
~ 20 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, tis~ue
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
W~92~02~0 2 ~ l L~ - 8 - PCT/US91/05656
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 li~and can be identified and isolated by contact-
ing the recombinant cannabinoid receptor of the presentinvention 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
pCD1 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 clas~s 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
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2~37~
W~2/02~0 PCT/VS91/05656
_ g _
cloned to date.
The results are shown in Figure 1. Notably absent
from SXR6 is a proline residue within the fifth hydropho-
bic domain and a cysteine just prior to hydrophobic domain
III. In t~rms 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 (SKRl4) was
isolated whose coding region, although incomplete, was
identical to SKR6. The 3' untranslated sequence of SKR14,
howevPr, 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-l cells using the guanidinium thiocyanate method
as described by Chomczynski et al. [Anal. Biochem., 162:
156-159 (1987)], and loaded (lO~q/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
SSPE/0.1% SDS, 60C) and exposed to x-ray sensitive film
for 6 days (-80C) (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,
.. i ,~
.. . ~ - - -
2~37~
W0~2/02~0 PCT/US91/056~6
- 1 0 -
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 (198~) and Nirenburg et al., Scienca, 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-~m 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 (25C) 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 (37C) 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
. ~ .
_ .. . . ... ... . , .. , , ...... . . . :
W~92/02~n ~ PCT/US91/05656
-- 11 -- . ,
terminated with the addition of 0.lN HC1/0.1 mM CaCl2.
Samples were frozen at -20~C and thawed just prior to
determination of cAMP by RIA EBrooker et al., Adv. Cyclic
Nucl. Res., 10: 1-33 (1979) and Borst et al., Life Sci.,
S ~3: 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. ECso 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 cannabinoids are
compounds distributed by the National Institute of Drug
Abuse.
Further, CHO cells were transfected with an ~-
adrenergic receptor, a muscarinic receptor or SKR6.Neither the ~-adrenergic nor muscarinic raceptors
responded to ~ 9THC 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 o recep-
tors and was clearly speciied to SKR6.
~: .
2~7~
W~92t~2~0 PCT/US91/05656
- 12 - ;
TABLE I
Effect o~ ~9THC and CP55940 on forskolin-stimulated
accumulation of cAMP in CHO-~l cells transfected with
SKR6, muscarinic and ~-adrenergic receptor cDNAs.
cell receptor/ ~qTHC CP55940 ca~bachol/ :
line cDNAforskolin ~lOOnM) (lOnM) clonidine - .~
CHO SKR6100 + 4 61 + 5 ----~- ------ : :
(12.6 + 0.5) .. -
CHO SKR6 100 + 5 ---------- ~4 + 11 ------ :
~12.1 + 1.4) :.
CHO muscarinic 100 + 5 104 + 8104 + 10 8 t 1 '
m2 (18.0 + 0.9) .: :
CHO adrenergic 100 + 4 100 + 4 96 + ~ 73 + 5 :
a2d (13.9 + 0.5) - - :
N18TG-2 ----------- 100 + 10 61 + 8 16 + 2 ------ .
(44.4 + 4.3) ::
NG108-15 ----------- 100 + 491 + 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~(pmoles cAMP/lO6 cells/5 min). Final concen-
trations of forskolin were 500 nM for all cell lines
except NGl08-15; 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 lO0 ~M and lO ~M, 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 lowar 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.
..
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~V~2/02~0 2 ~ $ 7 3 ~ ~ PCT/~S91/0~656
- 13 -
ISOLATION OF HUMAN RECF.PTOR_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 SXR6 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 [Holst 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 r ,~
- 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
; 20 [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 speci~ic 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 réceptor mRNA than the neuroblastoma cell
line, N18TG-2. This was determined as follows. Cultured
cells were adhered to a nitrocellulose filter under vacuum
~ .. " _, . .. . . .. ... . . . . .. . .. .. .. .. . .
.... . . . . . . . . .
,: , . :, , .. . . ~
W~92/02~o 2 ~ PCT/US9~/~56~6
by aliquoting a suspension of cells, at known densities,
into a multi-welled manifold. The filter was baked 90 min
at 80C, 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 clalms.
, ' ~
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