U.S. patent application number 10/282958 was filed with the patent office on 2003-06-12 for transgenic gpcr expressing animals.
This patent application is currently assigned to MILLENNIUM PHARMACEUTICALS, INC.. Invention is credited to Glucksmann, M. Alexandra, Goodearl, Andrew D.J..
Application Number | 20030110519 10/282958 |
Document ID | / |
Family ID | 26719616 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030110519 |
Kind Code |
A1 |
Goodearl, Andrew D.J. ; et
al. |
June 12, 2003 |
Transgenic GPCR expressing animals
Abstract
The invention provides isolated nucleic acids molecules,
designated muscarinic acetylcholine receptor 6 ("mACHR-6") nucleic
acid molecules, which encode polypeptides involved in the
modulation of acetylcholine responses in acetylcholine responsive
cells. The invention also provides antisense nucleic acid
molecules, expression vectors containing mACHR-6 nucleic acid
molecules, host cells into which the expression vectors have been
introduced, and non-human transgenic animals in which an mACHR-6
gene has been introduced or disrupted. The invention still further
provides isolated mACHR-6 polypeptides, fusion polypeptides,
antigenic peptides, and anti-mACHR-6 antibodies. Diagnostic,
screening, and therapeutic methods utilizing compositions of the
invention are also provided.
Inventors: |
Goodearl, Andrew D.J.;
(Natick, MA) ; Glucksmann, M. Alexandra;
(Lexington, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
MILLENNIUM PHARMACEUTICALS,
INC.
75 Sidney Street
Cambridge
MA
02139
|
Family ID: |
26719616 |
Appl. No.: |
10/282958 |
Filed: |
October 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10282958 |
Oct 28, 2002 |
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09349755 |
Jul 8, 1999 |
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09349755 |
Jul 8, 1999 |
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09042780 |
Mar 17, 1998 |
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09042780 |
Mar 17, 1998 |
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08985090 |
Dec 4, 1997 |
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5882893 |
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Current U.S.
Class: |
800/8 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/70571 20130101; A61K 38/00 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
800/8 ; 435/69.1;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
A01K 067/00; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/705 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule which encodes a
polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) a
nucleic acid molecule which encodes a polypeptide comprising the
amino acid sequence of SEQ ID NO:5; c) a nucleic acid molecule
which encodes a polypeptide comprising the amino acid sequence of
SEQ ID NO:32; d) a nucleic acid molecule which encodes a fragment
of a polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:2; e) a nucleic acid molecule which encodes a fragment
of a polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:5; f) a nucleic acid molecule which encodes a fragment
of a polypeptide comprising the amino acid sequence of SEQ ID
NO:32, wherein the fragment comprises at least 15 contiguous amino
acids of SEQ ID NO:32; g) a nucleic acid molecule which encodes a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:2, wherein the nucleic acid
molecule hybridizes to a nucleic acid molecule comprising SEQ ID
NO:1 under stringent conditions; and h) a nucleic acid molecule
which encodes a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the nucleic acid molecule hybridizes to a nucleic
acid-molecule comprising SEQ ID NO:4 under stringent conditions;
and i) a nucleic acid molecule which encodes a naturally occurring
allelic variant of a polypeptide comprising the amino acid sequence
of SEQ ID NO:32, wherein the nucleic acid molecule hybridizes to a
nucleic acid molecule comprising SEQ ID NO:31 under stringent
conditions.
2. The nucleic acid molecule of claim 1 further comprising vector
nucleic acid sequences.
3. The nucleic acid molecule of claim 1 further comprising nucleic
acid sequences encoding a heterologous polypeptide.
4. A host cell which contains the nucleic acid molecule of claim
1.
5. The host cell of claim 4 which is a mammalian host cell.
6. A non-human mammalian host cell containing the nucleic acid
molecule of claim 1.
7. The isolated nucleic acid molecule of claim 1, which is selected
from the group consisting of: a) the coding region of SEQ ID NO:1;
b) the coding region of SEQ ID NO:4; c) the coding region of SEQ ID
NO:31; d) a nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:7; e) a nucleic
acid molecule which encodes a polypeptide comprising the amino acid
sequence of SEQ ID NO:8; f) a nucleic acid molecule which encodes a
polypeptide comprising the amino acid sequence of SEQ ID NO:9; g) a
nucleic acid molecule which encodes a polypeptide comprising the
amino acid sequence of SEQ ID NO:10; h) a nucleic acid molecule
which encodes a polypeptide comprising the amino acid sequence of
SEQ ID NO:11; i) a nucleic acid molecule which encodes a
polypeptide comprising the amino acid sequence of SEQ ID NO:12; and
j) a nucleic acid molecule which encodes a polypeptide comprising
the amino acid sequence of SEQ ID NO:13.
8. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2;
b) a polypeptide comprising the amino acid sequence of SEQ ID NO:5;
c) a polypeptide comprising the amino acid sequence of SEQ ID
NO:32; d) a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, wherein the fragment comprises at least 15
contiguous amino acids of SEQ ID NO:2; e) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:5; f) a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO:32, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO:32; g) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by
a nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:1 under stringent conditions; h) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:5, wherein the polypeptide is encoded by
a nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:4 under stringent conditions; and i) a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:32, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a nucleic
acid molecule comprising SEQ ID NO:31 under stringent
conditions.
9. The polypeptide of claim 8, further comprising heterologous
amino acid sequences.
10. An antibody which selectively binds to a polypeptide of claim
8.
11. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2; b) a polypeptide comprising the amino acid sequence
of SEQ ID NO:5; c) a polypeptide comprising the amino acid sequence
of SEQ ID NO:32; d) a fragment of a polypeptide comprising the
amino acid sequence of SEQ ID NO:2, wherein the fragment comprises
at least 15 contiguous amino acids of SEQ ID NO:2; e) a fragment of
a polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:5; f) a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO:32, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO:32; g) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by
a nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:1 under stringent conditions; h) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:5, wherein the polypeptide is encoded by
a nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:4 under stringent conditions; and i) a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:32, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a nucleic
acid molecule comprising SEQ ID NO:31 under stringent conditions;
the method comprising the step of culturing the host cell of claim
4 under conditions in which the nucleic acid molecule is
expressed.
12. A method for detecting the presence of a polypeptide selected
from the group consisting of: a) a polypeptide comprising the amino
acid sequence of SEQ ID NO:2; b) a polypeptide comprising the amino
acid sequence of SEQ ID NO:5; c) a polypeptide comprising the amino
acid sequence of SEQ ID NO:32; d) a fragment of a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, wherein the
fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2; e) a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:5, wherein the fragment comprises at least 15
contiguous amino acids of SEQ ID NO:5; f) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:32,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:32; g) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 under
stringent conditions; h) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 under
stringent conditions; and i) a naturally occurring allelic variant
of a polypeptide comprising the amino acid sequence of SEQ ID
NO:32, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO:31 under stringent conditions; in a sample, the method
comprising the steps of: i) contacting the sample with a compound
which selectively binds to the polypeptide; and ii) determining
whether the compound binds to the polypeptide in the sample.
13. The method of claim 12, wherein the compound which binds to the
polypeptide is an antibody.
14. A kit comprising reagents used for the method of claim 12,
wherein the reagents comprise a compound which selectively binds to
a polypeptide selected from the group consisting of: a) a
polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) a
polypeptide comprising the amino acid sequence of SEQ ID NO:5; c) a
polypeptide comprising the amino acid sequence of SEQ ID NO:32; d)
a fragment of a polypeptide comprising the amino acid sequence of
SEQ ID NO:2, wherein the fragment comprises at least 15 contiguous
amino acids of SEQ ID NO:2; e) a fragment of a polypeptide
comprising the amino acid sequence of SEQ ID NO:5, wherein the
fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:5; f) a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:32, wherein the fragment comprises at least
15 contiguous amino acids of SEQ ID NO:32; g) a naturally occurring
allelic variant of a polypeptide comprising the amino acid sequence
of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic
acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:1 under stringent conditions; h) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:5, wherein the polypeptide is encoded by
a nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:4 under stringent conditions; and i) a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:32, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a nucleic
acid molecule comprising SEQ ID NO:31 under stringent
conditions.
15. A method for detecting the presence of a nucleic acid molecule
selected from the group consisting of: a) a polypeptide comprising
the amino acid sequence of SEQ ID NO:2; b) a polypeptide comprising
the amino acid sequence of SEQ ID NO:5; c) a polypeptide comprising
the amino acid sequence of SEQ ID NO:32; d) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:2; e) a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO:5, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO:5; f) a fragment of a
polypeptide comprising the amino-acid sequence of SEQ ID NO:32,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:32; g) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 under
stringent conditions; h) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 under
stringent conditions; and i) a naturally occurring allelic variant
of a polypeptide comprising the amino acid sequence of SEQ ID
NO:32, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO:31 under stringent conditions; in a sample, the method
comprising the steps of: i) contacting the sample with a nucleic
acid probe or primer which selectively hybridizes to the nucleic
acid molecule; and ii) determining whether the nucleic acid probe
or primer binds to a nucleic acid molecule in the sample.
16. The method of claim 15, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
17. A kit comprising reagents used for the method of claim 15,
wherein the reagents comprise a compound which selectively
hybridizes to a nucleic acid molecule selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2; b) a polypeptide comprising the amino acid sequence
of SEQ ID NO:5; c) a polypeptide comprising the amino acid sequence
of SEQ ID NO:32; d) a fragment of a polypeptide comprising the
amino acid sequence of SEQ ID NO:2, wherein the fragment comprises
at least 15 contiguous amino acids of SEQ ID NO:2; e) a fragment of
a polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:5; f) a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO:32, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO:32; g) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by
a nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:1 under stringent conditions; h) a naturally
occurring allelic variant of a polypeptide comprising the amino
acid sequence of SEQ ID NO:5, wherein the polypeptide is encoded by
a nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising SEQ ID NO:4 under stringent conditions; and i) a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:32, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a nucleic
acid molecule comprising SEQ ID NO:31 under stringent
conditions.
18. A method for identifying a compound which binds to a
polypeptide selected from the group consisting of: a) a polypeptide
comprising the amino acid sequence of SEQ ID NO:2; b) a polypeptide
comprising the amino acid sequence of SEQ ID NO:5; c) a polypeptide
comprising the amino acid sequence of SEQ ID NO:32; d) a fragment
of a polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:2; e) a fragment of a polypeptide comprising the amino
acid sequence of SEQ ID NO:5, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO:5; f) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:32,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:32; g) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 under
stringent conditions; h) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 under
stringent conditions; and i) a naturally occurring allelic variant
of a polypeptide comprising the amino acid sequence of SEQ ID
NO:32, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO:31 under stringent conditions, the method comprising the
steps of: i) contacting the polypeptide, or a cell expressing the
polypeptide with a test compound; and ii) determining whether the
polypeptide binds to the test compound.
19. The method of claim 18, wherein the binding of the test
compound to the polypeptide is detected by a method selected from
the group consisting of: a) detection of binding by direct
detecting of test compound/polypeptide binding; b) detection of
binding using a competition binding assay; c) detection of binding
using an assay for mACHR-6 activity.
20. A method for modulating the activity of a polypeptide selected
from the group consisting of: a) a polypeptide comprising the amino
acid sequence of SEQ ID NO:2; b) a polypeptide comprising the amino
acid sequence of SEQ ID NO:5; c) a polypeptide comprising the amino
acid sequence of SEQ ID NO:32; d) a fragment of a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, wherein the
fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2; e) a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:5, wherein the fragment comprises at least 15
contiguous amino acids of SEQ ID NO:5; f) a fragment of a
polypeptide comprising the amino acid sequence of SEQ ID NO:32,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:32; g) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 under
stringent conditions; h) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:5,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 under
stringent conditions; and i) a naturally occurring allelic variant
of a polypeptide comprising the amino acid sequence of SEQ ID
NO:32, wherein the polypeptide is encoded by a nucleic acid
molecule which hybridizes to a nucleic acid molecule comprising SEQ
ID NO:31 under stringent conditions, the method comprising the
steps of: i) contacting a cell expressing the polypeptide with a
compound which binds to the polypeptide in a sufficient
concentration to modulate the activity of the polypeptide.
21. The method of claim 20, wherein the activity is a
phosphatidylinositol activity.
22. The method of claim 20, wherein the method results in an
increase in phosphatidylinositol metabolism.
23. The method of claim 20, wherein the method results in a
decrease in phosphatidylinositol metabolism.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. Ser. No. 08/985,090, filed Dec. 4, 1997, now pending. The
contents of this co-pending patent application are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Muscarinic receptors, so named because the actions of
acetylcholine on such receptors are similar to those produced by
the mushroom alkaloid muscarine, mediate most of the inhibitory and
excitatory effects of the neurotransmitter acetylcholine in the
heart, smooth muscle, glands and in neurons (both presynaptic and
postsynaptic) in the autonomic and the central nervous system
(Eglen, R. and Watson, N. (1996) Pharmacology & Toxicology
78:59-68). The muscarinic receptors belong to the G protein-coupled
receptor superfamily (Wess, J. et al. (1990) Comprehensive
Medicinal Chemistry 3:423-491). Like all other G protein-coupled
receptors, the muscarinic receptors are predicted to conform to a
generic protein fold consisting of seven hydrophobic transmembrane
helices joined by alternative intracellular and extracellular
loops, an extracellular amino-terminal domain, and a cytoplasmic
carboxyl-terminal domain. The mammalian muscarinic receptors
display a high degree of sequence identity, particularly in the
transmembrane domains, sharing approximately 145 invariant amino
acids (Wess, J. (1993) TIPS 14:308-313). Moreover, all of the
mammalian muscarinic receptors contain a very large third
cytoplasmic loop which, except for the membrane-proximal portions,
displays virtually no sequence identity among the different family
members (Bonner, T. I. (1989) Trends Neurosci. 12:148-151). Ligand
binding to the receptor is believed to trigger conformational
changes within the helical bundle, which are then transmitted to
the cytoplasmic domain, where the interaction with specific G
proteins occurs.
[0003] Molecular cloning studies have revealed the existence of
five molecularly distinct mammalian muscarinic receptor proteins,
termed the M.sub.1-M.sub.5 receptors (Bonner, T. I. (1989) Trends
Neurosci. 12:148-151; and Hulme, E. C. et al. (1990) Annu. Rev.
Pharmacol. Toxicol. 30:633-673). The M.sub.1 receptor is expressed
primarily in the brain (cerebral cortex, olfactory bulb, olfactory
tubercle, basal forebrain/septum, amygdala, and hippocampus) and in
exocrine glands (Buckley, N. J. et al. (1988) J. Neurosci.
8:4646-4652). The M.sub.2 receptor is expressed in the brain
(olfactory bulb, basal forebrain/septum, thalamus and amygdala),
and in the ileum and the heart. The M.sub.3 receptor is expressed
in the brain (cerebral cortex, olfactory tubercle, thalamus and
hippocampus) the lung, the ileum, and in exocrine glands. The
M.sub.4 receptor is expressed in the brain (olfactory bulb,
olfactory tubercle, hippocampus and striatum) and in the lung.
Finally, the M.sub.5 receptor is expressed primarily in the brain
(substantia nigra) (Hulme, E. C. et al. (1990) A. Rev. Pharmac.
Toxic. 30:633-673).
[0004] The two enzymes with which muscarinic receptors interact
most directly are adenylate cyclase and phospholipase C. Studies
with cloned receptors have shown that the M.sub.1, M.sub.3, and
M.sub.5 muscarinic receptors are coupled to the types of G proteins
known as Go (a stimulatory protein linked to phospholipase C) or Gq
and that their activation results in the activation of
phospholipase C. The M.sub.2 and M.sub.4 muscarinic receptors are
coupled to a Gi protein (an inhibitory protein linked to adenylate
cyclase), and their activation results in the inhibition of
adenylate cyclase. Through these signal transduction pathways, the
muscarinic receptors are responsible for a variety of physiological
functions including the regulation of neurotransmitter release
(including acetylcholine release) from the brain, the regulation of
digestive enzyme and insulin secretion in the pancreas, the
regulation of amylase secretion by the parotid gland, and the
regulation of contraction in cardiac and smooth muscle (Caulfield,
M. P. (1993) Pharmac. Ther. 58:319-379).
SUMMARY OF THE INVENTION
[0005] This invention provides a novel nucleic acid molecule which
encodes a polypeptide, referred to herein as muscarinic
acetylcholine receptor 6 ("mACHR-6") polypeptide or protein, which
is capable of, for example, modulating the effects of acetylcholine
on acetylcholine responsive cells e.g., by modulating phospholipase
C signaling/activity. Nucleic acid molecules encoding an mACHR-6
polypeptide are referred to herein as mACHR-6 nucleic acid
molecules. In a preferred embodiment, the mACHR-6 polypeptide
interacts with (e.g., binds to) a protein which is a member of the
G family of proteins. Examples of such proteins include Go, Gi, Gs,
Gq and Gt. These proteins are described in Lodish H. et al.
Molecular Cell Biology, (Scientific American Books Inc., New York,
N.Y., 1995); Dolphin A. C. et al. (1987) Trends Neurosci. 10:53;
and Birnbaumer L. et al. (1992) Cell 71:1069, the contents of which
are expressly incorporated herein by reference.
[0006] In a preferred embodiment, the mACHR-6 polypeptide interacts
with (e.g., binds to) acetylcholine. Acetylcholine is the
predominant neurotransmitter in the sympathetic and parasympathetic
preganglionic synapses, as well as in the parasympathetic
postganglionic synapses and in some sympathetic postganglionic
synapses. Synapses in which acetylcholine is the neurotransmitter
are called cholinergic synapses. Acetylcholine acts to regulate
smooth muscle contraction, heart rate, glandular function such as
gastric acid secretion, and neural function such as release of
neurotransmitters from the brain. The mACHR-6 polypeptide of the
present invention binds to acetylcholine and serves to mediate the
acetylcholine induced signal to the cell. Thus, mACHR-6 molecules
can be used as targets to modulate acetylcholine induced functions
and thus to treat disorders associated with, for example, abnormal
acetylcholine levels, or abnormal or aberrant mACHR-6 polypeptide
activity or nucleic acid expression.
[0007] Accordingly, one aspect of the invention pertains to
isolated nucleic acid molecules (e.g., cDNAs) comprising a
nucleotide sequence encoding an mACHR-6 polypeptide or biologically
active portions thereof, as well as nucleic acid fragments suitable
as primers or hybridization probes for the detection of
mACHR-6-encoding nucleic acid (e.g., mRNA). In particularly
preferred embodiments, the isolated nucleic acid molecule comprises
the nucleotide sequence of SEQ ID NO:1, 4, or 31, the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC.RTM.
as Accession Number ______, or the coding region or a complement of
either of these nucleotide sequences. In other particularly
preferred embodiments, the isolated nucleic acid molecule of the
invention comprises a nucleotide sequence which encodes naturally
occurring allelic variants, genetically altered variants and
non-human and non-rat homologues of the mACHR-6 polypeptides
described herein. Such nucleic acid molecules are identifiable as
being able to hybridize to or which are at least about 30-35%,
preferably at least about 40-45%, more preferably at least about
50-55%, even more preferably at least about 60-65%, yet more
preferably at least about 70-75%, still more preferably at least
about 80-85%, and most preferably at least about 90-95% or more
homologous to the nucleotide sequence shown in SEQ ID NO:1, 4, or
31, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC.RTM. as Accession Number ______, or a portion
of either of these nucleotide sequences. In other preferred
embodiments, the isolated nucleic acid molecule encodes the amino
acid sequence of SEQ ID NO:2, 5, or 32 or an amino acid sequence
encoded by the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC.RTM. as Accession Number ______. The preferred
mACHR-6 polypeptides of the present invention also preferably
possess at least one of the mACHR-6 activities described
herein.
[0008] In another embodiment, the isolated nucleic acid molecule
encodes a polypeptide or portion thereof wherein the polypeptide or
portion thereof includes an amino acid sequence which is
sufficiently homologous to an amino acid sequence of SEQ ID NO:2,
5, or 32, e.g., sufficiently homologous to an amino acid sequence
of SEQ ID NO:2, 5, or 32 such that the polypeptide or portion
thereof maintains an mACHR-6 activity. Preferably, the polypeptide
or portion thereof encoded by the nucleic acid molecule maintains
the ability to modulate an acetylcholine response in an
acetylcholine responsive cell. In one embodiment, the polypeptide
encoded by the nucleic acid molecule is at least about 30-35%,
preferably at least about 40-45%, more preferably at least about
50-55%, even more preferably at least about 60-65%, yet more
preferably at least about 70-75%, still more preferably at least
about 80-85%, and most preferably at least about 90-95% or more
homologous to the amino acid sequence of SEQ ID NO:2, 5, or 32
(e.g., the entire amino acid sequence of SEQ ID NO:2, 5, or 32) or
the amino acid sequence encoded by the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC.RTM. as Accession
Number ______. In another preferred embodiment the nucleic acid
molecule encodes a polypeptide fragment comprising at least 15
contiguous amino acids of SEQ ID NO:2, 5, or 32. In yet another
preferred embodiment, the polypeptide is a full length
human-polypeptide which is substantially homologous to the entire
amino acid sequence of SEQ ID NO:2, 5, or 32 (encoded by the open
reading frame shown in SEQ ID NO:3, 6, or 33, respectively). In
still another preferred embodiment, the nucleic acid molecule
encodes a naturally occurring allelic variant of the polypeptide of
SEQ ID NO:2, 5, or 32 and hybridizes under stringent conditions to
a nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO:1, 4, or 31, respectively.
[0009] In yet another embodiment, the isolated nucleic acid
molecule is derived from a human and encodes a portion of a
polypeptide which includes a transmembrane domain. Preferably, the
transmembrane domain encoded by the human nucleic acid molecule is
at least about 50-55%, preferably at least about 60-65%, more
preferably at least about 70-75%, even more preferably at least
about 80-85%, and most preferably at least about 90-95% or more
homologous to any of the human transmembrane domains (i.e., amino
acid residues 34-59, 109-130, 152-174, 197-219, or 396416) of SEQ
ID NO:2 which are shown as separate sequences designated SEQ ID
NOs:7, 9, 10, 11, and 13, respectively, or to any of the rat
transmembrane domains (i.e., amino acid residues 34-59, 73-91,
109-130, 152-174, 197-219, 360-380, or 396-416 of SEQ ID NO:5 which
are shown as separate sequences designated SEQ ID NOs:14, 15, 16,
17, 18, 19, and 20, respectively or amino acid residues 1-8, 26-47,
69-91, 114-136, 277-297, or 313-333 of SEQ ID NO:32 which are shown
as separate sequences designated SEQ ID NOs:34, 35, 36, 37, 38, or
39, respectively). More preferably, the transmembrane domain
encoded by the human nucleic acid molecule is at least about
75-80%, preferably at least about 80-85%, more preferably at least
about 85-90%, and most preferably at least about 90-95% or more
homologous to the transmembrane domain (i.e., amino acid residues
360-380) of SEQ ID NO:2 which is shown as a separate sequence
designated SEQ ID NO:12, or at least about 80-85%, more preferably
at least about 85-90%, and most preferably at least about 90-95% or
more homologous to the transmembrane domain (i.e., amino acid
residues 73-91) of SEQ ID NO:2 which is shown as a separate
sequence designated SEQ ID NO:8.
[0010] In another preferred embodiment, the isolated nucleic acid
molecule is derived from a human and encodes a polypeptide (e.g.,
an mACHR-6 fusion polypeptide such as an mACHR-6 polypeptide fused
with a heterologous polypeptide) which includes a transmembrane
domain which is at least about 75% or more homologous to SEQ ID
NO:7-13, or to the corresponding rat sequences shown as SEQ ID
NOs:14-20 and has one or more of the following mACHR-6 activities:
1) it can interact with (e.g., bind to) acetylcholine; 2) it can
interact with (e.g., bind to) a G protein or another protein which
naturally binds to mACHR-6; 3) it can modulate the activity of an
ion channel (e.g., a potassium channel or a calcium channel); 4) it
can modulate cytosolic ion, e.g., calcium, concentration; 5) it can
modulate the release of a neurotransmitter, e.g., acetylcholine,
from a neuron, e.g., a presynaptic neuron; 6) it can modulate an
acetylcholine response in an acetylcholine responsive cell (e.g., a
smooth muscle cell or a gland cell) to, for example, beneficially
affect the acetylcholine responsive cell, e.g., a neuron; 7) it can
signal ligand binding via phosphatidylinositol turnover; and 8) it
can modulate, e.g., activate or inhibit, phospholipase C
activity.
[0011] In another embodiment, the isolated nucleic acid molecule is
at least 15 nucleotides, e.g., at least 15 contiguous nucleotides,
in length and hybridizes under stringent conditions to a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 4,
or 31 or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC.RTM. as Accession Number ______. Preferably,
the isolated nucleic acid molecule corresponds to a
naturally-occurring nucleic acid molecule. More preferably, the
isolated nucleic acid encodes naturally-occurring human mACHR-6 or
a biologically active portion thereof. Moreover, given the
disclosure herein of an mACHR-6-encoding cDNA sequence (e.g., SEQ
ID NO:1, 4, or 31), antisense nucleic acid molecules (e.g.,
molecules which are complementary to the coding strand of the
mACHR-6 cDNA sequence) are also provided by the invention.
[0012] Another aspect of the invention pertains to vectors, e.g.,
recombinant expression vectors, containing the nucleic acid
molecules of the invention and host cells into which such vectors
have been introduced. In one embodiment, such a host cell is used
to produce an mACHR-6 polypeptide by culturing the host cell in a
suitable medium. If desired, the mACHR-6 polypeptide can then be
isolated from the medium or the host cell.
[0013] Yet another aspect of the invention pertains to transgenic
non-human animals in which an mACHR-6 gene has been introduced or
altered. In one embodiment, the genome of the non-human animal has
been altered by introduction of a nucleic acid molecule of the
invention encoding mACHR-6 as a transgene. In another embodiment,
an endogenous mACHR-6 gene within the genome of the non-human
animal has been altered, e.g., functionally disrupted, by
homologous recombination.
[0014] Still another aspect of the invention pertains to an
isolated mACHR-6 polypeptide or a portion, e.g., a biologically
active portion, thereof. In a preferred embodiment, the isolated
mACHR-6 polypeptide or portion thereof can modulate an
acetylcholine response in an acetylcholine responsive cell. In
another preferred embodiment, the isolated mACHR-6 polypeptide or
portion thereof is sufficiently homologous to an amino acid
sequence of SEQ ID NO:2, 5, or 32 such that the polypeptide or
portion thereof maintains the ability to modulate an acetylcholine
response in an acetylcholine responsive cell.
[0015] In one embodiment, the biologically active portion of the
mACHR-6 polypeptide includes a domain or motif, preferably a domain
or motif which has an mACHR-6 activity. The domain can be
transmembrane domain. If the active portion of the polypeptide
which comprises the transmembrane domain is isolated or derived
from a human, it is preferred that the transmembrane domain be at
least about 75-80%, preferably at least about 80-85%, more
preferably at least about 85-90%, and most preferably at least
about 90-95% or more homologous to SEQ ID NO:7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 34, 35, 36, 37, 38, or 39.
Preferably, the biologically active portion of the mACHR-6
polypeptide which includes a transmembrane domain also has one of
the following mACHR-6 activities: 1) it can interact with (e.g.,
bind to) acetylcholine; 2) it can interact with (e.g., bind to) a G
protein or another protein which naturally binds to mACHR-6; 3) it
can modulate the activity of an ion channel (e.g., a potassium
channel or a calcium channel); 4) it can modulate cytosolic ion,
e.g., calcium, concentration; 5) it can modulate the release of a
neurotransmitter, e.g., acetylcholine, from a neuron, e.g., a
presynaptic neuron; 6) it can modulate an acetylcholine response in
an acetylcholine responsive cell (e.g., a smooth muscle cell or a
gland cell) to, for example, beneficially affect the acetylcholine
responsive cell, e.g., a neuron; 7) it can signal ligand binding
via phosphatidylinositol turnover; and 8) it can modulate, e.g.,
activate or inhibit, phospholipase C activity.
[0016] The invention also provides an isolated preparation of an
mACHR-6 polypeptide. In preferred embodiments, the mACHR-6
polypeptide comprises the amino acid sequence of SEQ ID NO:2, 5, or
32 or an amino acid sequence encoded by the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC.RTM. as Accession
Number ______. In another preferred embodiment, the invention
pertains to an isolated full length polypeptide which is
substantially homologous to the entire amino acid sequence of SEQ
ID NO:2, 5, or 32 (encoded by the open reading frame shown in SEQ
ID NO:3, 6, or 33, respectively) such as a naturally occurring
allelic variant of the mACHR-6 polypeptides described herein. In
yet another embodiment, the polypeptide is at least about 30-35%,
preferably at least about 40-45%, more preferably at least about
50-55%, even more preferably at least about 60-65%, yet more
preferably at least about 70-75%, still more preferably at least
about 80-85%, and most preferably at least about 90-95% or more
homologous to the entire amino acid sequence of SEQ ID NO:2, 5, or
32 such as a non-human or non-rat homologue of the mACHR-6
polypeptides described herein. In other embodiments, the isolated
mACHR-6 polypeptide comprises an amino acid sequence which is at
least about 30-40% or more homologous to the amino acid sequence of
SEQ ID NO:2, 5, or 32 and has an one or more of the following
mACHR-6 activities: 1) it can interact with (e.g., bind to)
acetylcholine; 2) it can interact with (e.g., bind to) a G protein
or another protein which naturally binds to mACHR-6; 3) it can
modulate the activity of an ion channel (e.g., a potassium channel
or a calcium channel); 4) it can modulate cytosolic ion, e.g.,
calcium, concentration; 5) it can modulate the release of a
neurotransmitter, e.g., acetylcholine, from a neuron, e.g., a
presynaptic neuron; 6) it can modulate an acetylcholine response in
an acetylcholine responsive cell (e.g., a smooth muscle cell or a
gland cell) to, for example, beneficially affect the acetylcholine
responsive cell, e.g., a neuron; 7) it can signal ligand binding
via phosphatidylinositol turnover; and 8) it can modulate, e.g.,
activate or inhibit, phospholipase C activity.
[0017] Alternatively, the isolated mACHR-6 polypeptide can comprise
an amino acid sequence which is encoded by a nucleotide sequence
which hybridizes, e.g., hybridizes under stringent conditions, or
is at least about 30-35%, preferably at least about 40-45%, more
preferably at least about 50-55%, even more preferably at least
about 60-65%, yet more preferably at least about 70-75%, still more
preferably at least about 80-85%, and most preferably at least
about 90-95% or more homologous to the nucleotide sequence of SEQ
ID NO:1, 4, or 31 or the nucleotide sequence of the DNA insert of
the plasmid deposited with ATCC.RTM. as Accession Number ______,
such as the allelic variants and non-human and non-rat homologues
of the mACHR-6 polypeptides described herein as well as genetically
altered variants generated by recombinant DNA methodologies. It is
also preferred that the preferred forms of mACHR-6 also have one or
more of the mACHR-6 activities described herein.
[0018] The mACHR-6 polypeptide (or protein) or a biologically
active portion thereof can be operatively linked to a non-mACHR-6
polypeptide (e.g., a polypeptide comprising heterologous amino acid
sequences) to form a fusion polypeptide. In addition, the mACHR-6
polypeptide or a biologically active portion thereof can be
incorporated into a pharmaceutical composition comprising the
polypeptide and a pharmaceutically acceptable carrier.
[0019] The mACHR-6 polypeptide of the invention, or portions or
fragments thereof, can be used to prepare anti-mACHR-6 antibodies.
Accordingly, the invention also provides an antigenic peptide of
mACHR-6 which comprises at least 8 amino acid residues of the amino
acid sequence shown in SEQ ID NO:2, 5, or 32 and encompasses an
epitope of mACHR-6 such that an antibody raised against the peptide
forms a specific immune complex with mACHR-6. Preferably, the
antigenic peptide comprises at least 10 amino acid residues, more
preferably at least 15 amino acid residues, even more preferably at
least 20 amino acid residues, and most preferably at least 30 amino
acid residues. The invention further provides an antibody that
specifically binds mACHR-6. In one embodiment, the antibody is
monoclonal. In another embodiment, the antibody is coupled to a
detectable substance. In yet another embodiment, the antibody is
incorporated into a pharmaceutical composition comprising the
antibody and a pharmaceutically acceptable carrier.
[0020] Another aspect of the invention pertains to methods for
modulating a cell activity mediated by mACHR-6, e.g., biological
processes mediated by phosphatidylinositol turnover and signaling;
secretion of a molecule, e.g., a neurotransmitter from a brain
cell, or an enzyme from a gland cell; or contraction of a smooth
muscle cell, e.g., an ileum smooth muscle cell or a cardiac cell,
e.g., a cardiomyocyte. Such methods include contacting the cell
with an agent which modulates mACHR-6 polypeptide activity or
mACHR-6 nucleic acid expression such that an mACHR-6-mediated cell
activity is altered relative to the same cellular activity which
occurs in the absence of the agent. In a preferred embodiment, the
cell (e.g., a smooth muscle cell or a neural cell) is capable of
responding to acetylcholine through a signaling pathway involving
an mACHR-6 polypeptide. The agent which modulates mACHR-6 activity
can be an agent which stimulates mACHR-6 polypeptide activity or
mACHR-6 nucleic acid expression. Examples of agents which stimulate
mACHR-6 polypeptide activity or mACHR-6 nucleic acid expression
include small molecules, active mACHR-6 polypeptides, and nucleic
acids encoding mACHR-6 that have been introduced into the cell.
Examples of agents which inhibit mACHR-6 activity or expression
include small molecules, antisense mACHR-6 nucleic acid molecules,
and antibodies that specifically bind to mACHR-6. In a preferred
embodiment, the cell is present within a subject and the agent is
administered to the subject.
[0021] The present invention also pertains to methods for treating
subjects having various disorders, e.g., disorders mediated by
abnormal mACHR-6 polypeptide activity, such as conditions caused by
over, under, or inappropriate expression of mACHR-6. For example,
the invention pertains to methods for treating a subject having a
disorder characterized by aberrant mACHR-6 polypeptide activity or
nucleic acid expression such as a nervous system disorder, e.g., a
cognitive disorder, a sleep disorder, a movement disorder, a
schizo-effective disorder, a disorder affecting pain generation
mechanisms, a drinking disorder, or an eating disorder; a smooth
muscle related disorder, e.g., irritable bowel syndrome, a cardiac
muscle related disorder, e.g., bradycardia, or a gland related
disorder, e.g., xerostomia. These methods include administering to
the subject an mACHR-6 modulator (e.g., a small molecule) such that
treatment of the subject occurs.
[0022] In other embodiments, the invention pertains to methods for
treating a subject having a disorder mediated by abnormal mACHR-6
polypeptide activity, such as conditions caused by over, under, or
inappropriate expression of mACHR-6, e.g., a nervous system
disorder, e.g., a cognitive disorder, a sleep disorder, a movement
disorder, a schizo-effective disorder, a disorder affecting pain
generation mechanisms, a drinking disorder, or an eating disorder;
a smooth muscle related disorder, e.g., irritable bowel syndrome; a
cardiac muscle related disorder, e.g., bradycardia; or a gland
related disorder, e.g., xerostomia. The method includes
administering to the subject an mACHR-6 polypeptide or portion
thereof such that treatment occurs. A nervous system disorder,
smooth muscle related disorder, cardiac muscle related disorder or
a gland related disorder can also be treated according to the
invention by administering to the subject having the disorder a
nucleic acid encoding an mACHR-6 polypeptide or portion thereof
such that treatment occurs.
[0023] The invention also pertains to methods for detecting
naturally occurring and recombinantly created genetic mutations in
an mACHR-6 gene, thereby determining if a subject with the mutated
gene is at risk for (or is predisposed to have) a disorder
characterized by aberrant or abnormal mACHR-6 nucleic acid
expression or mACHR-6 polypeptide activity, e.g., a nervous system
disorder, a smooth muscle related disorder, a cardiac muscle
related disorder or a gland related disorder. In preferred
embodiments, the methods include detecting, in a sample of cells
from the subject, the presence or absence of a genetic mutation
characterized by an alteration affecting the integrity of a gene
encoding an mACHR-6 polypeptide, or the misexpression of the
mACHR-6 gene, such as that caused by a nucleic acid base
substitution, deletion or addition, or gross sequence changes
caused by a genetic translation, inversion or insertion.
[0024] Another aspect of the invention pertains to methods for
detecting the presence of mACHR-6, or allelic variants thereof, in
a biological sample. In a preferred embodiment, the methods involve
contacting a biological sample (e.g., a brain or smooth muscle cell
sample) with a compound or an agent capable of detecting mACHR-6
polypeptide or mACHR-6 mRNA such that the presence of mACHR-6 is
detected in the biological sample. The compound or agent can be,
for example, a labeled or labelable nucleic acid probe capable of
hybridizing to mACHR-6 mRNA or a labeled or labelable antibody
capable of binding to mACHR-6 polypeptide. The invention further
provides methods for diagnosis of a subject with, for example, a
nervous system disorder, a smooth muscle related disorder, a
cardiac muscle related disorder or a gland related disorder, based
on detection of mACHR-6 polypeptide or mRNA. In one embodiment, the
method involves contacting a cell or tissue sample (e.g., a brain
or smooth muscle cell sample) from the subject with an agent
capable of detecting mACHR-6 polypeptide or mRNA, determining the
amount of mACHR-6 polypeptide or mRNA expressed in the cell or
tissue sample, comparing the amount of mACHR-6 polypeptide or mRNA
expressed in the cell or tissue sample to a control sample and
forming a diagnosis based on the amount of mACHR-6 polypeptide or
mRNA expressed in the cell or tissue sample as compared to the
control sample. Preferably, the cell sample is a brain cell sample.
Kits for detecting mACHR-6 in a biological sample which include
agents capable of detecting mACHR-6 polypeptide or mRNA are also
within the scope of the invention.
[0025] Still another aspect of the invention pertains to methods,
e.g., screening assays, for identifying a compound, e.g., a test
compound, for treating a disorder characterized by aberrant mACHR-6
nucleic acid expression or polypeptide activity, e.g., a nervous
system disorder, a smooth muscle related disorder, a cardiac muscle
related disorder or a gland related disorder. These methods
typically include assaying the ability of the compound or agent to
modulate the expression of the mACHR-6 gene or the activity of the
mACHR-6 polypeptide thereby identifying a compound for treating a
disorder characterized by aberrant mACHR-6 nucleic acid expression
or polypeptide activity. In a preferred embodiment, the method
involves contacting a biological sample, e.g., a cell or tissue
sample, e.g., a brain or smooth muscle cell sample, obtained from a
subject having the disorder with the compound or agent, determining
the amount of mACHR-6 polypeptide expressed and/or measuring the
activity of the mACHR-6 polypeptide in the biological sample,
comparing the amount of mACHR-6 polypeptide expressed in the
biological sample and/or the measurable mACHR-6 biological activity
in the cell to that of a control sample. An alteration in the
amount of mACHR-6 polypeptide expression or mACHR-6 activity in the
cell exposed to the compound or agent in comparison to the control
is indicative of a modulation of mACHR-6 expression and/or mACHR-6
activity.
[0026] The invention also pertains to methods for identifying a
compound or agent, e.g., a test compound or agent, which interacts
with (e.g., binds to) an mACHR-6 polypeptide. These methods can
include the steps of contacting the mACHR-6 polypeptide with the
compound or agent under conditions which allow binding of the
compound to the mACHR-6 polypeptide to form a complex and detecting
the formation of a complex of the mACHR-6 polypeptide and the
compound in which the ability of the compound to bind to the
mACHR-6 polypeptide is indicated by the presence of the compound in
the complex.
[0027] The invention further pertains to methods for identifying a
compound or agent, e.g., a test compound or agent, which modulates,
e.g., stimulates or inhibits, the interaction of the mACHR-6
polypeptide with a target molecule, e.g., acetylcholine, or a
cellular protein involved in phosphatidylinositol turnover and
signaling. In these methods, the mACHR-6 polypeptide is contacted,
in the presence of the compound or agent, with the target molecule
under conditions which allow binding of the target molecule to the
mACHR-6 polypeptide to form a complex. An alteration, e.g., an
increase or decrease, in complex formation between the mACHR-6
polypeptide and the target molecule as compared to the amount of
complex formed in the absence of the compound or agent is
indicative of the ability of the compound or agent to modulate the
interaction of the mACHR-6 polypeptide with a target molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 depicts the human mACHR-6 nucleotide (SEQ ID NO:1)
and amino acid (SEQ ID NO:2) sequences. The coding region without
the 5' and 3' untranslated region of the human mACHR-6 gene is
shown in SEQ ID NO:3.
[0029] FIG. 2 depicts the rat mACHR-6 nucleotide (SEQ ID NO:4) and
amino acid (SEQ ID NO:5) sequences. The coding region without the
5'0 and 3' untranslated region of the rat mACHR-6 gene is shown in
SEQ ID NO:6.
[0030] FIG. 3 depicts the partial rat mACHR-6 nucleotide (SEQ ID
NO:31.) and amino acid (SEQ ID NO:32) sequences. The partial coding
region without the 3' untranslated region of the rat mACHR-6 gene
is shown in SEQ ID NO:33.
DETAILED DESCRIPTION OF THE INVENTION
[0031] The present invention is based on the discovery of novel
molecules, referred to herein as mACHR-6 nucleic acid and
polypeptide molecules, which play a role in or function in
acetylcholine signaling pathways. In one embodiment, the mACHR-6
molecules modulate the activity of one or more proteins involved in
a neurotransmitter signaling pathway, e.g., an acetylcholine
signaling pathway. In a preferred embodiment, the mACHR-6 molecules
of the present invention are capable of modulating the activity of
proteins involved in the acetylcholine signaling pathway to thereby
modulate the effects of acetylcholine on acetylcholine responsive
cells.
[0032] As used herein, the phrase "acetylcholine responsive cells"
refers to cells which have a function which can be modulated (e.g.,
stimulated or inhibited) by the neurotransmitter acetylcholine.
Examples of such functions include mobilization of intracellular
molecules which participate in a signal transduction pathway, e.g.,
phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) or inositol
1,4,5-triphosphate (IP.sub.3), polarization of the plasma membrane,
production or secretion of molecules, alteration-in the structure
of a cellular component, cell proliferation, cell migration, cell
differentiation, and cell survival. Acetylcholine responsive cells
preferably express an acetylcholine receptor, e.g., a muscarinic
receptor. Examples of acetylcholine responsive cells include neural
cells, e.g., central nervous system and peripheral nervous system
cells (such as sympathetic and parasympathetic neurons); smooth
muscle cells, e.g., smooth muscle cells in the digestive tract, the
urinary tract, the blood vessels, the airways and the lungs, or the
uterus; cardiac muscle cells, e.g., cardiomyocytes; and gland cells
such as exocrine gland cells, e.g., pancreatic gland cells, e.g.,
pancreatic beta cells, tear gland cells, sweat gland cells, or
parotid gland cells.
[0033] Depending on the type of cell, the response elicited by
acetylcholine is different. For example, in neural cells,
acetylcholine regulates ion channels, and neural signal to noise
ratio. Inhibition or over stimulation of the activity of proteins
involved in the acetylcholine signaling pathway or misexpression of
acetylcholine can lead to hypo- or hyperpolarization of the neural
plasma membrane and to perturbed neural signal to noise ratio,
which can in turn lead to nervous system related disorders.
Examples of nervous system related disorders include cognitive
disorders, e.g., memory and learning disorders, such as amnesia,
apraxia, agnosia, amnestic dysnomia, amnestic spatial
disorientation, Kluver-Bucy syndrome, Alzheimer's related memory
loss (Eglen R. M. (1996) Pharmacol. and Toxicol. 78(2):59-68; Perry
E. K. (1995) Brain and Cognition 28(3):240-58) and learning
disability; disorders affecting consciousness, e.g., visual
hallucinations, perceptual disturbances, or delerium associated
with Lewy body dementia; schitzo-effective disorders (Dean B.
(1996) Mol. Psychiatry 1(1):54-8), schizophrenia with mood swings
(Bymaster F. P. (1997) J. Clin. Psychiatry 58 (suppl. 10):28-36;
Yeomans J. S. (1995) Neuropharmacol. 12(1):3-16; Reimann D. (1994)
J. Psychiatric Res. 28(3):195-210), depressive illness (primary or
secondary); affective disorders (Janowsky D. S. (1994) Am. J. Med.
Genetics 54(4):335-44); sleep disorders (Kimura F. (1997) J.
Neurophysiol. 77(2):709-16), e.g., REM sleep abnormalities in
patients suffering from, for example, depression (Riemann D. (1994)
J. Psychosomatic Res. 38 Suppl. 1:15-25; Bourgin P. (1995)
Neuroreport 6(3): 532-6), paradoxical sleep abnormalities (Sakai K.
(1997) Eur. J. Neuroscience 9(3):415-23), sleep-wakefulness, and
body temperature or respiratory depression abnormalities during
sleep (Shuman S. L. (1995) Am. J. Physiol. 269(2 Pt 2):R308-17;
Mallick B. N. (1997) Brain Res. 750(1-2):311-7). Other examples of
nervous system related disorders include disorders affecting pain
generation mechanisms, e.g., pain related to irritable bowel
syndrome (Mitch C. H. (1997) J. Med. Chem. 40(4):538-46; Shannon H.
E. (1997) J. Pharmac. and Exp. Therapeutics 281(2):884-94; Bouaziz
H. (1995) Anesthesia and Analgesia 80(6):1140-4; or Guimaraes A. P.
(1994) Brain Res. 647(2):220-30) or chest pain; movement disorders
(Monassi C. R. (1997) Physiol. and Behav. 62(1):53-9), e.g.,
Parkinson's disease related movement disorders (Finn M. (1997)
Pharmacol. Biochem. & Behavior 57(1-2):243-9; Mayorga A. J.
(1997) Pharmacol. Biochem. & Behavior 56(2):273-9); eating
disorders, e.g., insulin hypersecretion related obesity (Maccario
M. (1997) J. Endocrinol. Invest. 20(1):8-12; Premawardhana L. D.
(1994) Clin. Endocrinol. 40(5): 617-21); or drinking disorders,
e.g., diabetic polydipsia (Murzi E. (1997) Brain Res.
752(1-2):184-8; Yang X. (1994) Pharmacol. Biochem. & Behavior
49(1):1-6).
[0034] In smooth muscle, acetylcholine regulates (e.g., stimulates
or inhibits) contraction. Inhibition or overstimulation of the
activity of proteins involved in the acetylcholine signaling
pathway or misexpression of acetylcholine can lead to smooth muscle
related disorders such as irritable bowel syndrome, diverticular
disease, urinary incontinence, oesophageal achalasia, or chronic
obstructive airways disease.
[0035] In cardiac muscle, acetylcholine induces a reduction in the
heart rate and in cardiac contractility. Inhibition or
overstimulation of the activity of proteins involved in the
acetylcholine signaling pathway or misexpression of acetylcholine
can lead to heart muscle related disorders such as pathologic
bradycardia or tachycardia, arrhythmia, flutter or
fibrillation.
[0036] In glands such as exocrine glands, acetylcholine regulates
the secretion of enzymes or hormones, e.g., in the parotid gland
acetylcholine induces the release of amylase, and in the pancreas
acetylcholine induces the release of digestive enzymes and insulin.
Inhibition or over stimulation of the activity of proteins involved
in the acetylcholine signaling pathway or misexpression of
acetylcholine can lead to gland related disorders such as
xerostomia, or diabetes mellitus.
[0037] In a particularly preferred embodiment, the mACHR-6
molecules are capable of modulating the activity of G proteins, as
well as phosphatidylinositol metabolism and turnover in
acetylcholine responsive cells. As used herein, a "G protein" is a
protein which participates, as a secondary signal, in a variety of
intracellular signal transduction pathways, e.g., in the
acetylcholine signaling pathway primarily through
phosphatidylinositol metabolism and turnover. G proteins represent
a family of heterotrimeric proteins composed of .alpha., .beta. and
.gamma. subunits, which bind guanine nucleotides. These proteins
are usually linked to cell surface receptors, e.g., receptors
containing seven transmembrane domains, such as the muscarinic
receptors. Following ligand binding to the receptor, a
conformational change is transmitted to the G protein, which causes
the .alpha.-subunit to exchange a bound GDP molecule for a GTP
molecule and to dissociate from the .beta..gamma.-subunits. The
GTP-bound form of the .alpha.-subunit typically functions as an
effector-modulating moiety, leading to the production of second
messengers, such as cyclic AMP (e.g., by activation of adenylate
cyclase), diacylglycerol or inositol phosphates. Greater than 20
different types of .alpha.-subunits are known in man, which
associate with a smaller pool of .beta. and .gamma. subunits.
Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G
proteins are described extensively in Lodish H. et al. Molecular
Cell Biology, (Scientific American Books Inc., New York, N.Y.,
1995).
[0038] As used herein, "phosphatidylinositol turnover and
metabolism" refers to the molecules involved in the turnover and
metabolism of phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) as
well as to the activities of these molecules. PIP.sub.2 is a
phospholipid found in the cytosolic leaflet of the plasma membrane.
Binding of acetylcholine to a muscarinic receptor activates the
plasma-membrane enzyme phospholipase C which in turn can hydrolyze
PIP.sub.2 to produce 1,2-diacylglycerol (DAG) and inositol
1,4,5-triphosphate (IP.sub.3). Once formed IP.sub.3 can diffuse to
the endoplasmic reticulum surface where it can bind an IP.sub.3
receptor, e.g., a calcium channel protein containing an IP.sub.3
binding site. IP.sub.3 binding can induce opening of the channel,
allowing calcium ions to be released into the cytoplasm. 1P.sub.3
can also be phosphorylated by a specific kinase to form inositol
1,3,4,5-tetraphosphate (IP.sub.4), a molecule which can cause
calcium entry into the cytoplasm from the extracellular medium.
IP.sub.3 and IP.sub.4 can subsequently be hydrolyzed very rapidly
to the inactive products inositol 1,4-biphosphate (IP.sub.2) and
inositol 1,3,4-triphosphate, respectively. These inactive products
can be recycled by the cell to synthesize PIP.sub.2. The other
second messenger produced by the hydrolysis of PIP.sub.2, namely
1,2-diacylglycerol (DAG), remains in the cell membrane where it can
serve to activate the enzyme protein kinase C. Protein kinase C is
usually found soluble in the cytoplasm of the cell, but upon an
increase in the intracellular calcium concentration, this enzyme
can move to the plasma membrane where it can be activated by DAG.
The activation of protein kinase C in different cells results in
various cellular responses such as the phosphorylation of glycogen
synthase, or the phosphorylation of various transcription factors,
e.g., NF-.kappa.B. The language "phosphatidylinositol activity", as
used herein, refers to an activity of PIP.sub.2 or one of its
metabolites.
[0039] mACHR-6 nucleic acid molecules were identified by screening
appropriate cDNA libraries (described in detail in Example 1). The
rat mACHR-6 nucleic acid molecule was identified by screening a rat
brain cDNA library. Positive clones were sequenced and the partial
sequences were analyzed by comparison with sequences in a nucleic
acid sequence data base. This analysis indicated that the sequences
were homologous to the muscarinic family of receptors. A longer rat
clone was then isolated and sequenced. The human mACHR-6 nucleic
acid molecule was identified by screening a human cerebellum cDNA
library using probes designed based on the rat sequence.
[0040] Because of its ability to interact with (e.g., bind to)
acetylcholine, G proteins and other proteins involved in the
acetylcholine signaling pathway, the mACHR-6 polypeptide is also a
polypeptide which functions in the acetylcholine signaling
pathway.
[0041] The nucleotide sequence of the isolated human mACHR-6 cDNA
and the predicted amino acid sequence of the human mACHR-6
polypeptide are shown in FIG. 1 and in SEQ ID NOs:1 and 2,
respectively. A plasmid containing the full length nucleotide
sequence encoding human mACHR-6 was deposited with ATCC.RTM. on
______ and assigned Accession Number ______. This deposit will be
maintained under the terms of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure. This deposit was made merely as a
convenience for those of skill in the art and is not an admission
that a deposit is required under 35 U.S.C. .sctn.112.
[0042] The nucleotide sequence of the isolated rat mACHR-6 cDNA and
the predicted amino acid sequence of the rat mACHR-6 polypeptide
are shown in FIG. 2 and in SEQ ID NOs:4 and 5, respectively. A
plasmid containing the full length nucleotide sequence encoding rat
mACHR-6 was deposited with ATCC.RTM. on and assigned Accession
Number ______. This deposit will be maintained under the terms of
the Budapest Treaty on the International Recognition of the Deposit
of Microorganisms for the Purposes of Patent Procedure. This
deposit was made merely as a convenience for those of skill in the
art and is not an admission that a deposit is required under 35
U.S.C. .sctn.112.
[0043] The nucleotide sequence of the isolated partial rat mACHR-6
cDNA and the predicted amino acid sequence of the partial rat
mACHR-6 polypeptide are shown in FIG. 3 and in SEQ ID NOs:31 and
32, respectively.
[0044] The human mACHR-6 gene, which is approximately 2689
nucleotides in length, encodes a full length polypeptide having a
molecular weight of approximately 51.2 KDa and which is
approximately 445 amino acid residues in length. The human mACHR-6
polypeptide is expressed at least in the brain, in particular,
regions of the brain such as the cerebellum, the cerebral cortex,
the medulla, the occipital pole, the frontal lobe, the temporal
lobe, the putamen, the corpus callosum the amygdala, the caudate
nucleus, the hippocampus, the substantia nigra, the subthalamic
nucleus and the thalamus; spinal cord, placenta, lungs, spleen,
liver, skeletal muscle, kidney, and testis. Based on structural
analysis, amino acid residues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID
NO:8), 109-130 (SEQ ID NO:9), 152-174 (SEQ ID NO:10), 197-219 (SEQ
ID NO:11), 360-380 (SEQ ID NO:12), and 396416 (SEQ ID NO:13)
comprise transmembrane domains. As used herein, the term
"transmembrane domain" refers to a structural amino acid motif
which includes a hydrophobic helix that spans the plasma membrane.
A transmembrane domain also preferably includes a series of
conserved serine, threonine, and tyrosine residues. For example,
the transmembrane domains between residues 109-130 (SEQ ID NO:9),
197-219 (SEQ ID NO:11), 360-380 (SEQ ID NO:12), and 396416 (SEQ ID
NO:13), contain threonine and tyrosine residues (located about 1-2
helical turns away from the membrane surface), which are important
for ligand, e.g., acetylcholine, binding. Other important residues
in the transmembrane domains include the conserved aspartate
residue in the transmembrane domain between residues 109-130 (SEQ
ID NO:9) and the conserved proline residue in the transmembrane
domain between residues 152-174 (SEQ ID NO:10), which are also
important for ligand, e.g., acetylcholine, binding. A skilled
artisan will readily appreciate that the beginning and ending amino
acid residue recited for various domains/fragments of mACHR-6 are
based on structural analysis and that the actual beginning/ending
amino acid for each may vary by a few amino acids from that
identified herein.
[0045] The rat mACHR-6 gene, which is approximately 3244
nucleotides in length, encodes a full length polypeptide having a
molecular weight of approximately 51.2 kDa and which is at least
about 445 amino acid residues in length. The rat mACHR-6
polypeptide is expressed in the brain. Amino acid residues 34-59
(SEQ ID NO:14), 73-91 (SEQ ID NO:15), 109-130 (SEQ ID NO:16),
152-174 (SEQ ID NO:17), 197-219 (SEQ ID NO:18), 360-380 (SEQ ID
NO:19) and 396-416 (SEQ ID NO:20) comprise transmembrane
domains.
[0046] The rat mACHR-6 gene, which is at least about 2218
nucleotides in length, encodes a full length polypeptide having a
molecular weight of at least about 41.6 kDa and which is at least
about 362 amino acid residues in length. The rat mACHR-6
polypeptide is expressed in the brain. Amino acid residues 1-8 (SEQ
ID NO:14), 26-47 (SEQ ID NO:15), 69-91 (SEQ ID NO:16), 114-136 (SEQ
ID NO:17), 277-297 (SEQ ID NO:18), and 313-333 (SEQ ID NO:19)
comprise transmembrane domains.
[0047] The partial rat mACHR-6 gene, which is at least about 2218
nucleotides in length, encodes a polypeptide having a molecular
weight of at least about 41.6 kDa and which is at least about 362
amino acid residues in length. The rat mACHR-6 polypeptide is
expressed in the brain. Amino acid residues 1-8 (SEQ ID NO:34),
26-47 (SEQ ID NO:35), 69-91 (SEQ ID NO:36), 114-136 (SEQ ID NO:37),
277-297 (SEQ ID NO:38), and 313-333 (SEQ ID NO:39) comprise
transmembrane domains.
[0048] The mACHR-6 polypeptide, a biologically active portion or
fragment of the polypeptide, or an allelic variant thereof can have
one or more of the following mACHR-6 activities: 1) it can interact
with (e.g., bind to) acetylcholine; 2) it can interact with (e.g.,
bind to) a G protein or another protein which naturally binds to
mACHR-6; 3) it can modulate the activity of an ion channel (e.g., a
potassium channel or a calcium channel); 4) it can modulate
cytosolic ion, e.g., calcium, concentration; 5) it can modulate the
release of a neurotransmitter, e.g., acetylcholine, from a neuron,
e.g., a presynaptic neuron; 6) it can modulate an acetylcholine
response in an acetylcholine responsive cell (e.g., a smooth muscle
cell or a gland cell) to, for example, beneficially affect the
acetylcholine responsive cell, e.g., a neuron; 7) it can signal
ligand binding via phosphatidylinositol turnover; and 8) it can
modulate, e.g., activate or inhibit, phospholipase C activity.
[0049] Various aspects of the invention are described in further
detail in the following subsections:
[0050] I. Isolated Nucleic Acid Molecules
[0051] One aspect of the invention pertains to isolated nucleic
acid molecules that encode mACHR-6 or biologically active portions
thereof, as well as nucleic acid fragments sufficient for use as
hybridization probes to identify mACHR-6-encoding nucleic acid
(e.g., mACHR-6 mRNA). As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or
genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA
or RNA generated using nucleotide analogs. The nucleic acid
molecule can be single-stranded or double-stranded, but preferably
is double-stranded DNA. An "isolated" nucleic acid molecule is one
which is separated from other nucleic acid molecules which are
present in the natural source of the nucleic acid. Preferably, an
"isolated" nucleic acid is free of sequences which naturally flank
the nucleic acid (i.e., sequences located at the 5' and 3' ends of
the nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For example, in various embodiments, the
isolated mACHR-6 nucleic acid molecule can contain less than about
5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide
sequences which naturally flank the nucleic acid molecule in
genomic DNA of the cell from which the nucleic acid is derived
(e.g., a hippocampal cell). Moreover, an "isolated" nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by
recombinant techniques, or chemical precursors or other chemicals
when chemically synthesized.
[0052] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of SEQ ID
NO:1, 4, or 31, or a portion thereof, can be isolated using
standard molecular biology techniques and the sequence information
provided herein. For example, a human mACHR-6 cDNA can be isolated
from a human hippocampus library using all or portion of SEQ ID
NO:1, 4, or 31 as a hybridization probe and standard hybridization
techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and
Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989). Moreover, a nucleic acid molecule
encompassing all or a portion of SEQ ID NO:1, 4, or 31 can be
isolated by the polymerase chain reaction using oligonucleotide
primers designed based upon the sequence of SEQ ID NO:1, 4, or 31.
For example, mRNA can be isolated from normal brain cells (e.g., by
the guanidinium-thiocyanate extraction procedure of Chirgwin et al.
(1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using
reverse transcriptase (e.g., Moloney MLV reverse transcriptase,
available from Gibco/BRL, Bethesda, Md.; or AMV reverse
transcriptase, available from Seikagaku America, Inc., St.
Petersburg, Fla.). Synthetic oligonucleotide primers for PCR
amplification can be designed based upon the nucleotide sequence
shown in SEQ ID NO:1, 4, or 31. A nucleic acid of the invention can
be amplified using cDNA or, alternatively, genomic DNA, as a
template and appropriate oligonucleotide primers according to
standard PCR amplification techniques. The nucleic acid so
amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to an mACHR-6 nucleotide sequence
can be prepared by standard synthetic techniques, e.g., using an
automated DNA synthesizer.
[0053] In a preferred embodiment, an isolated nucleic acid molecule
of the invention comprises the nucleotide sequence shown in SEQ ID
NO:1, 4, or 31 or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC.RTM. as Accession Number ______. The
sequence of SEQ ID NO:1 corresponds to the human mACHR-6 cDNA. This
cDNA comprises sequences encoding the human mACHR-6 polypeptide
(i.e., "the coding region", from nucleotides 291 to 1628 of SEQ ID
NO:1), as well as 5' untranslated sequences (nucleotides 1 to 290
of SEQ ID NO:1) and 3' untranslated sequences (nucleotides 1629 to
2689 of SEQ ID NO:1). Alternatively, the nucleic acid molecule can
comprise only the coding region of SEQ ID NO:1 (e.g., nucleotides
291 to 1628 of SEQ ID NO:1, shown separately as SEQ ID NO:3). The
sequence of SEQ ID NO:4 corresponds to the rat mACHR-6 cDNA. This
cDNA comprises sequences encoding the rat mACHR-6 polypeptide
(i.e., "the coding region", from nucleotides 778 to 2112 of SEQ ID
NO:4), as well as 5' untranslated sequences (nucleotides 1 to 777
of SEQ ID NO:4), and 3' untranslated sequences (nucleotides 2113 to
3244 of SEQ ID NO:4). Alternatively, the nucleic acid molecule can
comprise only the coding region of SEQ ID NO:4 (e.g., nucleotides
778 to 2112 of SEQ ID NO:4, shown separately as SEQ ID NO:6). The
sequence of SEQ ID NO:31 corresponds to the partial rat mACHR-6
cDNA. This cDNA comprises sequences encoding part of the rat
mACHR-6 polypeptide (i.e., part of "the coding region", from
nucleotides 1 to 1089 of SEQ ID NO:31), and 3' untranslated
sequences (nucleotides 1090 to 2218 of SEQ ID NO:31).
Alternatively, the nucleic acid molecule can comprise only the
partial coding region of SEQ ID NO:31 (e.g., nucleotides 1 to 1089,
shown separately as SEQ ID NO:33).
[0054] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
is a complement of the nucleotide sequence shown in SEQ ID NO:1, 4,
or 31, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC.RTM. as Accession Number ______, or a portion
of either of these nucleotide sequences. A nucleic acid molecule
which is complementary to the nucleotide sequence shown in SEQ ID
NO:1, 4, or 31 is one which is sufficiently complementary to the
nucleotide sequence shown in SEQ ID NO:1, 4, or 31 such that it can
hybridize to the nucleotide sequence shown in SEQ ID NO:1, 4, or
31, respectively, thereby forming a stable duplex.
[0055] In still another preferred embodiment, an isolated nucleic
acid molecule of the invention comprises a nucleotide sequence
which is at least about 30-35%, preferably at least about 40-45%,
more preferably at least about 50-55%, even more preferably at
least about 60-65%, yet more preferably at least about 70-75%,
still more preferably at least about 80-85%, and most preferably at
least about 90-95% or more homologous to the nucleotide sequence
shown in SEQ ID NO:1, 4, or 31, or to the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC.RTM. as Accession
Number ______, or a portion of these nucleotide sequences.
Preferably, such nucleic acid molecules encode functionally active
or inactive allelic variants of mACHR-6. In an additional preferred
embodiment, an isolated nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, e.g., hybridizes
under stringent conditions, to the nucleotide sequence shown in SEQ
ID NO:1, 4, or 31, or the nucleotide sequence of the DNA insert of
the plasmid deposited with ATCC.RTM. as Accession Number ______, or
a portion of either of these nucleotide sequences.
[0056] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the coding region of SEQ ID NO:1, 4, or
31, for example a fragment which can be used as a probe or primer
or a fragment encoding a biologically active portion of mACHR-6.
The nucleotide sequence determined from the cloning of the mACHR-6
gene from a mammal allows for the generation of probes and primers
designed for use in identifying and/or cloning mACHR-6 homologues
in other cell types, e.g., from other tissues, as well as mACHR-6
homologues from other mammals. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, preferably about
25, more preferably about 40, 50 or 75 consecutive nucleotides of
SEQ ID NO:1, 4, or 31 sense, an anti-sense sequence of SEQ ID NO:1,
4, or 31, or naturally occurring mutants thereof. Primers based on
the nucleotide sequence in SEQ ID NO:1, 4, or 31 can be used in PCR
reactions to clone mACHR-6 homologues. Probes based on the mACHR-6
nucleotide sequences can be used to detect transcripts or genomic
sequences encoding the same or homologous polypeptides. In
preferred embodiments, the probe further comprises a label group
attached thereto, e.g., the label group can be a radioisotope, a
fluorescent compound, an enzyme, or an enzyme co-factor. Such
probes can be used as a part of a diagnostic test kit for
identifying cells or tissue which misexpress an mACHR-6
polypeptide, such as by measuring a level of an mACHR-6-encoding
nucleic acid in a sample of cells from a subject e.g., detecting
mACHR-6 mRNA levels or determining whether a genomic mACHR-6 gene
has been mutated or deleted.
[0057] In one embodiment, the nucleic acid molecule of the
invention encodes a polypeptide or portion thereof which includes
an amino acid sequence which is sufficiently homologous to an amino
acid sequence of SEQ ID NO:2, 5, or 32 or an amino acid sequence
encoded by the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC.RTM. as Accession Number ______ such that the
polypeptide or portion thereof maintains the ability to modulate an
acetylcholine response in an acetylcholine responsive cell (e.g.,
naturally occurring allelic variants of the rat and human mACHR-6
polypeptides described herein). As used herein, the language
"sufficiently homologous" refers to polypeptides or portions
thereof which have amino acid sequences which include a minimum
number of identical or equivalent (e.g., an amino acid residue
which has a similar side chain as an amino acid residue in SEQ ID
NO:2, 5, or 32) amino acid residues to an amino acid sequence of
SEQ ID NO:2, 5, or 32 or an amino acid sequence encoded by the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC.RTM. as Accession Number ______ such that the polypeptide or
portion thereof is able to modulate an acetylcholine response in an
acetylcholine responsive cell or a skilled artisan would clearly
recognize it as a non-functional allelic variant of the rat and
human mACHR-6 polypeptides described herein. Acetylcholine, as
described herein, initiates a variety of responses in many
different cell types. Examples of such responses are also described
herein. In another embodiment, the polypeptide is at least about
30-35%, preferably at least about 4045%, more preferably at least
about 50-55%, even more preferably at least about 60-65%, yet more
preferably at least about 70-75%, still more preferably at least
about 80-85%, and most preferably at least about 90-95% or more
homologous to the amino acid sequence of SEQ ID NO:2, 5, or 32.
[0058] Portions of polypeptides encoded by the mACHR-6 nucleic acid
molecule of the invention are preferably biologically active
portions of the mACHR-6 polypeptide. As used herein, the term
"biologically active portion of mACHR-6" is intended to include a
portion, e.g., a domain/motif, of mACHR-6 that has one or more of
the following mACHR-6 activities: 1) it can interact with (e.g.,
bind to) acetylcholine; 2) it can interact with (e.g., bind to) a G
protein or another protein which naturally binds to mACHR-6; 3) it
can modulate the activity of an ion channel (e.g., a potassium
channel or a calcium channel); 4) it can modulate cytosolic ion,
e.g., calcium, concentration; 5) it can modulate the release of a
neurotransmitter, e.g., acetylcholine, from a neuron, e.g., a
presynaptic neuron; 6) it can modulate an acetylcholine response in
an acetylcholine responsive cell (e.g., a smooth muscle cell or a
gland cell) to, for example, beneficially affect the acetylcholine
responsive cell, e.g., a neuron; 7) it can signal ligand binding
via phosphatidylinositol turnover; and 8) it can modulate, e.g.,
activate or inhibit, phospholipase C activity.
[0059] Standard binding assays, e.g., immunoprecipitations and
yeast two-hybrid assays as described herein, can be performed to
determine the ability of an mACHR-6 polypeptide or a biologically
active portion thereof to interact with (e.g., bind to) a binding
partner such as a G protein. To determine whether an mACHR-6
polypeptide or a biologically active portion thereof can modulate
an acetylcholine response in an acetylcholine responsive cell, such
cells can be transfected with a construct driving the
overexpression of an mACHR-6 polypeptide or a biologically active
portion thereof. Methods for the preparation of acetylcholine
responsive cells, e.g., intact smooth muscle cells or extracts from
such cells are known in the art and described in Glukhova et al.
(1987) Tissue Cell 19 (5):657-63, Childs et al. (1992) J. Biol.
Chem. 267 (32):22853-9, and White et al. (1996) J. Biol. Chem. 271
(25):15008-17. The cells can be subsequently treated with
acetylcholine, and a biological effect of acetylcholine on the
cells, such as phosphatidylinositol turnover or cytosolic calcium
concentration can be measured using methods known in the art (see
Hartzell H. C. et al. (1988) Prog. Biophys. Mol. Biol. 52:165-247).
Alternatively, transgenic animals, e.g., mice overexpressing an
mACHR-6 polypeptide or a biologically active portion thereof, can
be used. Tissues from such animals can be obtained and treated with
acetylcholine. For example, methods for preparing detergent-skinned
muscle fiber bundles are known in the art (Strauss et al. (1992)
Am. J. Physiol. 262:1437-45). The contractility of these tissues in
response to acetylcholine can be determined using, for example,
isometric force measurements as described in Strauss et al., supra.
Similarly, to determine whether an mACHR-6 polypeptide or a
biologically active portion thereof can modulate an acetylcholine
response in an acetylcholine responsive cell such as a gland cell,
gland cells, e.g., parotid gland cells grown in tissue culture, can
be transfected with a construct driving the overexpression of an
mACHR-6 polypeptide or a biologically active portion thereof. The
cells can be subsequently treated with acetylcholine, and the
effect of the acetylcholine on amylase secretion from such cells
can be determined using, for example an enzymatic assay with a
labeled substrate. The preferred assays used for mACHR-6 activity
will be based on phosphatidylinositol turnover such as those
developed for the M1, M3 and M5 classes of receptors (see E. Watson
et al. The G Protein Linked Receptor: FactsBook (Academic Press,
Boston, Mass., 1994), the contents of which are incorporated herein
by reference).
[0060] In one embodiment, the biologically active portion of
mACHR-6 comprises a transmembrane domain. Preferably, the
transmembrane domain is encoded by a nucleic acid molecule derived
from a human and is at least about 50-55%, preferably at least
about 60-65%, more preferably at least about 70-75%, even more
preferably at least about 80-85%, and most preferably at least
about 90-95% or more homologous to any of the transmembrane domains
(i.e., amino acid residues 34-59, 109-130, 152-174, 197-219, or
396-416) of SEQ ID NO:2 which are shown as separate sequences
designated SEQ ID NOs:7, 9, 10, 11, and 13, respectively, or to the
rat transmembrane domains (i.e., amino acid residues 34-59, 73-91,
109-130, 152-174, 197-219, 360-380, or 396416 of SEQ ID NO:5 which
are shown as separate sequences designated SEQ ID NOs:14, 15, 16,
17, 18, 19, and 20, respectively or amino acid residues 1-8, 2647,
69-91, 114-136, 277-297, or 313-333 of SEQ ID NO:32 which are shown
as separate sequences designated SEQ ID NOs:34, 35, 36, 37, 38, or
39, respectively). More preferably, the transmembrane domain
encoded by the human nucleic acid molecule is at least about
75-80%, preferably at least about 80-85%, more preferably at least
about 85-90%, and most preferably at least about 90-95% or more
homologous to the transmembrane domain (i.e., amino acid residues
360-380) of SEQ ID NO:2 which is shown as a separate sequence
designated SEQ ID NO:12, or at least about 80-85%, more preferably
at least about 85-90%, and most preferably at least about 90-95% or
more homologous to the transmembrane domain (i.e., amino acid
residues 73-91) of SEQ ID NO:2 which is shown as a separate
sequence designated SEQ ID NO:8. In a preferred embodiment, the
biologically active portion of the polypeptide which includes the
transmembrane domain can modulate the activity of a G protein or
other binding partner in a cell and/or modulate an acetylcholine
response in an acetylcholine responsive cell, e.g., a brain cell,
to thereby beneficially affect the cell. In a preferred embodiment,
the biologically active portion comprises a transmembrane domain of
the human mACHR-6 as represented by amino acid residues 34-59 (SEQ
ID NO:7), 73-91 (SEQ ID NO:8), 109-130 (SEQ ID NO:9),152-174 (SEQ
ID NO:10), 197-219 (SEQ ID NO:11), 360-380 (SEQ ID NO:12), and
396416 (SEQ ID NO:13), a transmembrane domain of the full length
rat mACHR-6 as represented by amino acid residues 34-59 (SEQ ID
NO:14), 73-91 (SEQ ID NO:15), 109-130 (SEQ ID NO:16), 152-174 (SEQ
ID NO:17), 197-219 (SEQ ID NO:18), 360-380 (SEQ ID NO:19), and
396-416 (SEQ ID NO:20), or a transmembrane domain of the partial
rat mACHR-6 as represented by amino residues 1-8 (SEQ ID
NO:34),2647 (SEQ ID NO:35),69-91 (SEQ ID NO:36),114-136 (SEQ ID
NO:37),277-297 (SEQ ID NO:38), and 313-333 (SEQ ID NO:39).
Additional nucleic acid fragments encoding biologically active
portions of mACHR-6 can be prepared by isolating a portion of SEQ
ID NO:1, 4, or 31, expressing the encoded portion of mACHR-6
polypeptide or peptide (e.g., by recombinant expression in vitro)
and assessing the activity of the encoded portion of mACHR-6
polypeptide or peptide.
[0061] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1, 4,
or 31 (and portions thereof) due to degeneracy of the genetic code
and thus encode the same mACHR-6 polypeptide as that encoded by the
nucleotide sequence shown in SEQ ID NO:1, 4, or 31. In another
embodiment, an isolated nucleic acid molecule of the invention has
a nucleotide sequence encoding a polypeptide having an amino acid
sequence shown in SEQ ID NO:2, 5, or 32 or a polypeptide having an
amino acid sequence encoded by the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC.RTM. as Accession Number
______. In a still further embodiment, the nucleic acid molecule of
the invention encodes a full length human polypeptide which is
substantially homologous to the amino acid sequence of SEQ ID NO:2
or 4 (encoded by the open reading frame shown in SEQ ID NO:3, 6, or
33, respectively) or an amino acid sequence encoded by the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC.RTM. as Accession Number ______.
[0062] In addition to the mACHR-6 nucleotide sequence shown in SEQ
ID NO:1, 4, or 31, it will be appreciated by those skilled in the
art that DNA sequence polymorphisms that lead to changes in the
amino acid sequences of mACHR-6 may exist within a population
(e.g., the human population). Such genetic polymorphism in the
mACHR-6 gene may exist among individuals within a population due to
natural allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame encoding an mACHR-6 polypeptide, preferably a
mammalian mACHR-6 polypeptide. Such natural allelic variations can
typically result in 1-5% variance in the nucleotide sequence of the
mACHR-6 gene. Any and all such nucleotide variations and resulting
amino acid polymorphisms in mACHR-6 that are the result of natural
allelic variation are intended to be within the scope of the
invention. Such allelic variation includes both active allelic
variants as well as non-active or reduced activity allelic
variants, the later two types typically giving rise to a
pathological disorder. Moreover, nucleic acid molecules encoding
mACHR-6 polypeptides from other species, and thus which have a
nucleotide sequence which differs from the human sequence of SEQ ID
NO:1, are intended to be within the scope of the invention. Nucleic
acid molecules corresponding to natural allelic variants and
non-human homologues of the human mACHR-6 cDNA of the invention can
be isolated based on their homology to the human mACHR-6 nucleic
acid disclosed herein using the human cDNA, or a portion thereof,
as a hybridization probe according to standard hybridization
techniques under stringent hybridization conditions. Accordingly,
in another embodiment, an isolated nucleic acid molecule of the
invention is at least 15 nucleotides in length and hybridizes under
stringent conditions to the nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:1 or the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC.RTM. as Accession
Number ______. In other embodiments, the nucleic acid is at least
30, 50, 100, 250, 300, 400, 500, 600, 700, 800, 900, or 1000
nucleotides in length. As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
60% homologous to each other typically remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 65%, more preferably at least about 70%, and even more
preferably at least about 75% or more homologous to each other
typically remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found
in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of
stringent hybridization conditions are hybridization in
6.times.sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. Preferably, an isolated nucleic acid molecule of
the invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:1 corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
polypeptide). In one embodiment, the nucleic acid encodes a natural
human mACHR-6.
[0063] In addition to naturally-occurring allelic variants of the
mACHR-6 sequence that may exist in the population, the skilled
artisan will further appreciate that changes can be introduced by
mutation into the nucleotide sequence of SEQ ID NO:1, 4, or 31,
thereby leading to changes in the amino acid sequence of the
encoded mACHR-6 polypeptide, without altering the functional
ability of the mACHR-6 polypeptide. For example, nucleotide
substitutions leading to amino acid substitutions at
"non-essential" amino acid residues can be made in the sequence of
SEQ ID NO:1, 4, or 31. A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence of mACHR-6
(e.g., the sequence of SEQ ID NO:2, 5, or 32) without altering the
activity of mACHR-6, whereas an "essential" amino acid residue is
required for mACHR-6 activity. For example, conserved amino acid
residues, e.g., aspartates, prolines threonines and tyrosines, in
the transmembrane domains of mACHR-6 are most likely important for
binding to acetylcholine and are thus essential residues of
mACHR-6. Other amino acid residues, however, (e.g., those that are
not conserved or only semi-conserved in the transmembrane domain)
may not be essential for activity and thus are likely to be
amenable to alteration without altering mACHR-6 activity.
[0064] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding mACHR-6 polypeptides that contain
changes in amino acid residues that are not essential for mACHR-6
activity. Such mACHR-6 polypeptides differ in amino acid sequence
from SEQ ID NO:2, 5, or 32 yet retain at least one of the mACHR-6
activities described herein. In one embodiment, the isolated
nucleic acid molecule comprises a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 30-35%, preferably at least about 40-45%,
more preferably at least about 50-55%, even more preferably at
least about 60-65%, yet more preferably at least about 70-75%,
still more preferably at least about 80-85%, and most preferably at
least about 90-95% or more homologous to the amino acid sequence of
SEQ ID NO:2, 5, or 32.
[0065] To determine the percent homology of two amino acid
sequences (e.g., SEQ ID NO:2, 5, or 32 and a mutant form thereof)
or of two nucleic acids, the sequences are aligned for optimal
comparison purposes (e.g., gaps can be introduced in the sequence
of one polypeptide or nucleic acid for optimal alignment with the
other polypeptide or nucleic acid). The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in one sequence (e.g.,
SEQ ID NO:2, 5, or 32) is occupied by the same amino acid residue
or nucleotide as the corresponding position in the other sequence
(e.g., a mutant form of mACHR-6), then the molecules are homologous
at that position (i.e., as used herein amino acid or nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The percent homology between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of
positions.times.100).
[0066] The determination of percent homology between two sequences
can be accomplished using a mathematical algorithim. A preferred,
non-limiting example of a mathematical algorithim utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin
and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an
algorithm is incorporated into the NBLAST and XBLAST programs of
Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide
searches can be performed performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to mACHR-6 nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to mACHR-6
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402.
When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. See http://www.ncbi.nlm.nih.gov. Another preferred,
non-limiting example of a mathematical algorithim utilized for the
comparison of sequences is the algorithm of Myers and Miller,
CABIOS (1989). Such an algorithm is incorporated into the ALIGN
program (version 2.0) which is part of the GCG sequence alignment
software package. When utilizing the ALIGN program for comparing
amino acid sequences, a PAM120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 can be used.
[0067] An isolated nucleic acid molecule encoding an mACHR-6
polypeptide homologous to the polypeptide of SEQ ID NO:2, 5, or 32
can be created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of SEQ ID NO:1,
4, or 31, respectively, such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded polypeptide. Mutations can be introduced into SEQ ID NO:1,
4, or 31 by standard techniques, such as site-directed mutagenesis
and PCR-mediated mutagenesis. Preferably, conservative amino acid
substitutions are made at one or more predicted non-essential amino
acid residues., A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), non-polar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in mACHR-6 is preferably
replaced with another amino acid residue from the same side chain
family. Alternatively, in another embodiment, mutations can be
introduced randomly along all or part of an mACHR-6 coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be screened for an mACHR-6 activity described herein to
identify mutants that retain mACHR-6 activity. Following
mutagenesis of SEQ ID NO:1, 4, or 31, the encoded polypeptide can
be expressed recombinantly (e.g., as described in Examples 3 and 4)
and the activity of the polypeptide can be determined using, for
example, assays described herein.
[0068] In addition to the nucleic acid molecules encoding mACHR-6
polypeptides described above, another aspect of the invention
pertains to isolated nucleic acid molecules which are antisense
thereto. An "antisense" nucleic acid comprises a nucleotide
sequence which is complementary to a "sense" nucleic acid encoding
a polypeptide, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA sequence.
Accordingly, an antisense nucleic acid can hydrogen bond to a sense
nucleic acid. The antisense nucleic acid can be complementary to an
entire mACHR-6 coding strand, or to only a portion thereof. In one
embodiment, an antisense nucleic acid molecule is antisense to a
"coding region" of the coding strand of a nucleotide sequence
encoding mACHR-6.
[0069] The term "coding region" refers to the region of the
nucleotide sequence comprising codons which are translated into
amino acid residues, e.g., the entire coding region of SEQ ID NO:1
comprises nucleotides 291 to 1628 (shown separately as SEQ ID NO:3)
and the coding region of SEQ ID NO:4 comprises nucleotides 778 to
2112 (shown separately as SEQ ID NO:6). In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding
mACHR-6. The term "noncoding region" refers to 5' and 3' sequences
which flank the coding region that are not translated into amino
acids (i.e., also referred to as 5' and 3' untranslated
regions).
[0070] Given the coding strand sequences encoding mACHR-6 disclosed
herein (e.g., SEQ ID NOs:1, 4, and 31), antisense nucleic acids of
the invention can be designed according to the rules of Watson and
Crick base pairing. The antisense nucleic acid molecule can be
complementary to the entire coding region of mACHR-6 mRNA, but more
preferably is an oligonucleotide which is antisense to only a
portion of the coding or noncoding region of mACHR-6 mRNA. For
example, the antisense oligonucleotide can be complementary to the
region surrounding the translation start site of mACHR-6 mRNA. An
antisense oligonucleotide can be, for example, about 5, 10, 15, 20,
25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known
in the art. For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids, e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides which can be used to generate the
antisense nucleic acid include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil-2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0071] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding an mACHR-6 polypeptide to thereby inhibit expression of
the polypeptide, e.g., by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the
case of an antisense nucleic acid molecule which binds to DNA
duplexes, through specific interactions in the major groove of the
double helix. An example of a route of administration of an
antisense nucleic acid molecule of the invention includes direct
injection at a tissue site. Alternatively, an antisense nucleic
acid molecule can be modified to target selected cells and then
administered systemically. For example, for systemic
administration, an antisense molecule can be modified such that it
specifically binds to a receptor or an antigen expressed on a
selected cell surface, e.g., by linking the antisense nucleic acid
molecule to a peptide or an antibody which binds to a cell surface
receptor or antigen. The antisense nucleic acid molecule can also
be delivered to cells using the vectors described herein. To
achieve sufficient intracellular concentrations of the antisense
molecules, vector constructs in which the antisense nucleic acid
molecule is placed under the control of a strong pol II or pol III
promoter are preferred.
[0072] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0073] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave mACHR-6 mRNA transcripts to thereby
inhibit translation of mACHR-6 mRNA. A ribozyme having specificity
for an mACHR-6-encoding nucleic acid can be designed based upon the
nucleotide sequence of an mACHR-6 cDNA disclosed herein (i.e., SEQ
ID NO:1, 4, or 31). For example, a derivative of a Tetrahymena L-19
IVS RNA can be constructed in which the nucleotide sequence of the
active site is complementary to the nucleotide sequence to be
cleaved in an mACHR-6-encoding mRNA. See, e.g., Cech et al. U.S.
Pat. No. 4,987,071 and Cech et al. U.S. Pat. No. 5,116,742.
Alternatively, mACHR-6 mRNA can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science
261:1411-1418.
[0074] Alternatively, mACHR-6 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the mACHR-6 (e.g., the mACHR-6 promoter and/or enhancers)
to form triple helical structures that prevent transcription of the
mACHR-6 gene in target cells. See generally, Helene, C. (1991)
Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann.
N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays
14(12):807-15.
[0075] II. Recombinant Expression Vectors and Host Cells
[0076] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
mACHR-6 (or a portion thereof). As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked. One type of vector is a
"plasmid", which refers to a circular double stranded DNA loop into
which additional DNA segments can be ligated. Another type of
vector is a viral vector, wherein additional DNA segments can be
ligated into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively linked. Such vectors are referred to herein as
"expression vectors". In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. In
the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of
vector. However, the invention is intended to include such other
forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0077] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of polypeptide desired, etc.
The expression vectors of the invention can be introduced into host
cells to thereby produce polypeptides or peptides, including fusion
polypeptides or peptides, encoded by nucleic acids as described
herein (e.g., mACHR-6 polypeptides, mutant forms of mACHR-6, fusion
polypeptides, and the like).
[0078] The recombinant expression vectors of the invention can be
designed for expression of mACHR-6 in prokaryotic or eukaryotic
cells. For example, mACHR-6 can be expressed in bacterial cells
such as E. coli, insect cells (e.g., using baculovirus expression
vectors) yeast cells or mammalian cells. Suitable host cells are
discussed further in Goeddel, Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. (1990).
Alternatively, the recombinant expression vector can be transcribed
and translated in vitro, for example using T7 promoter regulatory
sequences and T7 polymerase.
[0079] Expression of polypeptides in prokaryotes is most often
carried out in E. coli with vectors containing constitutive or
inducible promoters directing the expression of either fusion or
non-fusion polypeptides. Fusion vectors add a number of amino acids
to a polypeptide encoded therein, usually to the amino terminus of
the recombinant polypeptide. Such fusion vectors typically serve
three purposes: 1) to increase expression of recombinant
polypeptide; 2) to increase the solubility of the recombinant
polypeptide; and 3) to aid in the purification of the recombinant
polypeptide by acting as a ligand in affinity purification. Often,
in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
polypeptide to enable separation of the recombinant polypeptide
from the fusion moiety subsequent to purification of the fusion
polypeptide. Such enzymes, and their cognate recognition sequences,
include Factor Xa, thrombin and enterokinase. Typical fusion
expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.
B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England
Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)
which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, to the target recombinant
polypeptide. In one embodiment, the coding sequence of the mACHR-6
is cloned into a pGEX expression vector to create a vector encoding
a fusion polypeptide comprising, from the N-terminus to the
C-terminus, GST-thrombin cleavage site-mACHR-6. The fusion
polypeptide can be purified by affinity chromatography using
glutathione-agarose resin. Recombinant mACHR-6 unfused to GST can
be recovered by cleavage of the fusion polypeptide with
thrombin.
[0080] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
.lambda. prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0081] One strategy to maximize recombinant polypeptide expression
in E. coli is to express the polypeptide in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
polypeptide (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be carried out by standard DNA synthesis
techniques.
[0082] In another embodiment, the mACHR-6 expression vector is a
yeast expression vector. Examples of vectors for expression in
yeast S. cerivisae include pYepSec1 (Baldari, et al., (1987) Embo
J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and
pYES2 (Invitrogen Corporation, San Diego, Calif.).
[0083] Alternatively, mACHR-6 can be expressed in insect cells
using, for example, baculovirus expression vectors. Baculovirus
vectors available for expression of polypeptides in cultured insect
cells (e.g., Sf 9 cells) include the pAc series (Smith et al.
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and
Summers (1989) Virology 170:31-39).
[0084] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0085] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) PNAS
86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-regulated
promoters are also encompassed, for example the murine hox
promoters (Kessel and Gruss (1990) Science 249:374-379) and the
.alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.
3:537-546).
[0086] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
which allows for expression (by transcription of the DNA molecule)
of an RNA molecule which is antisense to mACHR-6 mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0087] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0088] A host cell can be any prokaryotic or eukaryotic cell. For
example, mACHR-6 polypeptide can be expressed in bacterial cells
such as E. coli, insect cells, yeast or mammalian cells (such as
Chinese hamster ovary cells (CHO) or COS cells). Other suitable
host cells are known to those skilled in the art.
[0089] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calciumchloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0090] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding mACHR-6 or can be introduced on a separate vector.
Cells stably transfected with the introduced nucleic acid can be
identified by drug selection (e.g., cells that have incorporated
the selectable marker gene will survive, while the other cells
die).
[0091] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) mACHR-6 polypeptide. Accordingly, the invention further
provides methods for producing mACHR-6 polypeptide using the host
cells of the invention. In one embodiment, the method comprises
culturing the host cell of invention (into which a recombinant
expression vector encoding mACHR-6 has been introduced) in a
suitable medium until mACHR-6 is produced. In another embodiment,
the method further comprises isolating mACHR-6 from the medium or
the host cell.
[0092] The host cells of the invention can also be used to produce
non-human transgenic animals. The non-human transgenic animals can
be used in screening assays designed to identify agents or
compounds, e.g., drugs, pharmaceuticals, etc., which are capable of
ameliorating detrimental symptoms of selected disorders such as
nervous system disorders, smooth muscle related disorders, cardiac
muscle related disorders and gland related disorders. For example,
in one embodiment, a host cell of the invention is a fertilized
oocyte or an embryonic stem cell into which mACHR-6-coding
sequences have been introduced. Such host cells can then be used to
create non-human transgenic animals in which exogenous mACHR-6
sequences have been introduced into their genome or homologous
recombinant animals in which endogenous mACHR-6 sequences have been
altered. Such animals are useful for studying the function and/or
activity of mACHR-6 and for identifying and/or evaluating
modulators of mACHR-6 activity. As used herein, a "transgenic
animal" is a non-human animal, preferably a mammal, more preferably
a rodent such as a rat or mouse, in which one or more of the cells
of the animal include a transgene. Other examples of transgenic
animals include non-human primates, sheep, dogs, cows, goats,
chickens, amphibians, and the like. A transgene is exogenous DNA
which is integrated into the genome of a cell from which a
transgenic animal develops and which remains in the genome of the
mature animal, thereby directing the expression of an encoded gene
product in one or more cell types or tissues of the transgenic
animal. As used herein, a "homologous recombinant animal" is a
non-human animal, preferably a mammal, more preferably a mouse, in
which an endogenous mACHR-6 gene has been altered by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell of the animal, e.g., an embryonic
cell of the animal, prior to development of the animal.
[0093] A transgenic animal of the invention can be created by
introducing mACHR-6-encoding nucleic acid into the male pronuclei
of a fertilized oocyte, e.g., by microinjection, retroviral
infection, and allowing the oocyte to develop in a pseudopregnant
female foster animal. The human mACHR-6 cDNA sequence of SEQ ID
NO:1 can be introduced as a transgene into the genome of a
non-human animal. Furthermore, the rat mACHR-6 cDNA sequence of SEQ
ID NO:4 can be introduced as a transgene into the genome of a
non-rat animal. Moreover, a non-human homologue of the human
mACHR-6 gene, such as a mouse mACHR-6 gene, can be isolated based
on hybridization to the human or rat mACHR-6 cDNA (described
further in subsection I above) and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the
transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to the mACHR-6 transgene to direct expression of mACHR-6
polypeptide to particular cells. Methods for generating transgenic
animals via embryo manipulation and microinjection, particularly
animals such as mice, have become conventional in the art and are
described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009,
both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and
in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods
are used for production of other transgenic animals. A transgenic
founder animal can be identified based upon the presence of the
mACHR-6 transgene in its genome and/or expression of mACHR-6 mRNA
in tissues or cells of the animals. A transgenic founder animal can
then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene encoding mACHR-6
can further be bred to other transgenic animals carrying other
transgenes.
[0094] To create a homologous recombinant animal, a vector is
prepared which contains at least a portion of an mACHR-6 gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the mACHR-6 gene. The
mACHR-6 gene can be a human gene (e.g., from a human genomic clone
isolated from a human genomic library screened with the cDNA of SEQ
ID NO:1), but more preferably, is a rat mACHR-6 gene of SEQ ID NO:4
or 31, or another non-human homologue of a human mACHR-6 gene. For
example, a mouse mACHR-6 gene can be isolated from a mouse genomic
DNA library using the mACHR-6 cDNA of SEQ ID NO:1, 4, or 31 as a
probe. The mouse mACHR-6 gene then can be used to construct a
homologous recombination vector suitable for altering an endogenous
mACHR-6 gene in the mouse genome. In a preferred embodiment, the
vector is designed such that, upon homologous recombination, the
endogenous mACHR-6 gene is functionally disrupted (i.e., no longer
encodes a functional polypeptide; also referred to as a "knock out"
vector). Alternatively, the vector can be designed such that, upon
homologous recombination, the endogenous mACHR-6 gene is mutated or
otherwise altered but still encodes functional polypeptide (e.g.,
the upstream regulatory region can be altered to thereby alter the
expression of the endogenous mACHR-6 polypeptide). In the
homologous recombination vector, the altered portion of the mACHR-6
gene is flanked at its 5' and 3' ends by additional nucleic acid of
the mACHR-6 gene to allow for homologous recombination to occur
between the exogenous mACHR-6 gene carried by the vector and an
endogenous mACHR-6 gene in an embryonic stem cell. The additional
flanking mACHR-6 nucleic acid is of sufficient length for
successful homologous recombination with the endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3'
ends) are included in the vector (see for example, Thomas, K. R.
and Capecchi, M. R. (1987) Cell 51:503 for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation) and cells in
which the introduced mACHR-6 gene has homologously recombined with
the endogenous mACHR-6 gene are selected (see e.g., Li, E. et al.
(1992) Cell 69:915). The selected cells are then injected into a
blastocyst of an animal (e.g., a mouse) to form aggregation
chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,
Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted
into a suitable pseudopregnant female foster animal and the embryo
brought to term. Progeny harboring the homologously recombined DNA
in their germ cells can be used to breed animals in which all cells
of the animal contain the homologously recombined DNA by germline
transmission of the transgene. Methods for constructing homologous
recombination vectors and homologous recombinant animals are
described further in Bradley, A. (1991) Current Opinion in
Biotechnology 2:823-829 and in PCT International Publication Nos.
WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
[0095] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355. If a cre/loxP recombinase system is
used to regulate expression of the transgene, animals containing
transgenes encoding both the Cre recombinase and a selected
polypeptide are required. Such animals can be provided through the
construction of "double" transgenic animals, e.g., by mating two
transgenic animals, one containing a transgene encoding a selected
polypeptide and the other containing a transgene encoding a
recombinase.
[0096] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[0097] III. Isolated mACHR-6 polypeptides and Anti-mACHR-6
Antibodies
[0098] Another aspect of the invention pertains to isolated mACHR-6
polypeptides, and biologically active portions thereof, as well as
peptide fragments suitable for use as immunogens to raise
anti-mACHR-6 antibodies. An "isolated" or "purified" polypeptide or
biologically active portion thereof is substantially free of
cellular material when produced by recombinant DNA techniques, or
chemical precursors or other chemicals when chemically synthesized.
The language "substantially free of cellular material" includes
preparations of mACHR-6 polypeptide in which the polypeptide is
separated from cellular components of the cells in which it is
naturally or recombinantly produced. In one embodiment, the
language "substantially free of cellular material" includes
preparations of mACHR-6 polypeptide having less than about 30% (by
dry weight) of non-mACHR-6 polypeptide (also referred to herein as
a "contaminating polypeptide"), more preferably less than about 20%
of non-mACHR-6 polypeptide, still more preferably less than about
10% of non-mACHR-6 polypeptide, and most preferably less than about
5% non-mACHR-6 polypeptide. When the mACHR-6 polypeptide or
biologically active portion thereof is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the polypeptide preparation. The language "substantially
free of chemical precursors or other chemicals" includes
preparations of mACHR-6 polypeptide in which the polypeptide is
separated from chemical precursors or other chemicals which are
involved in the synthesis of the polypeptide. In one embodiment,
the language "substantially free of chemical precursors or other
chemicals" includes preparations of mACHR-6 polypeptide having less
than about 30% (by dry weight) of chemical precursors or
non-mACHR-6 chemicals, more preferably less than about 20% chemical
precursors or non-mACHR-6 chemicals, still more preferably less
than about 10% chemical precursors or non-mACHR-6 chemicals, and
most preferably less than about 5% chemical precursors or
non-mACHR-6 chemicals. In preferred embodiments, isolated
polypeptides or biologically active portions thereof lack
contaminating polypeptides from the same animal from which the
mACHR-6 polypeptide is derived. Typically, such polypeptides are
produced by recombinant expression of, for example, a human mACHR-6
polypeptide in a non-human cell.
[0099] An isolated mACHR-6 polypeptide or a portion thereof of the
invention can modulate an acetylcholine response in an
acetylcholine responsive cell or be a naturally occurring,
non-functional allelic variant of an mACHR-6 polypeptide. In
preferred embodiments, the polypeptide or portion thereof comprises
an amino acid sequence which is sufficiently homologous to an amino
acid sequence of SEQ ID NO:2, 5, or 32 such that the polypeptide or
portion thereof maintains the ability to modulate an acetylcholine
response in an acetylcholine responsive cell. The portion of the
polypeptide is preferably a biologically active portion as
described herein. In another preferred embodiment, the human
mACHR-6 polypeptide (i.e., amino acid residues 1-398 of SEQ ID
NO:2) or the rat mACHR-6 polypeptide (i.e., amino acid residues
1-445 of SEQ ID NO:5 or amino acid residues 1-401 of SEQ ID NO:32)
has an amino acid sequence shown in SEQ ID NO:2, 5, or 32,
respectively, or an amino acid sequence which is encoded by the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC.RTM. as Accession Number ______. In yet another preferred
embodiment, the mACHR-6 polypeptide has an amino acid sequence
which is encoded by a nucleotide sequence which hybridizes, e.g.,
hybridizes under stringent conditions, to the nucleotide sequence
of the DNA insert of the plasmid deposited with ATCC.RTM. as
Accession Number ______. In still another preferred embodiment, the
mACHR-6 polypeptide has an amino acid sequence which is encoded by
a nucleotide sequence that is at least about 30-35%, preferably at
least about 4045%, more preferably at least about 50-55%, even more
preferably at least about 60-65%, yet more preferably at least
about 70-75%, still more preferably at least about 80-85%, and most
preferably at least about 90-95% or more homologous to the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC.RTM. as Accession Number ______. The preferred mACHR-6
polypeptides of the present invention also preferably possess at
least one of the mACHR-6 activities described herein. For example,
a preferred mACHR-6 polypeptide of the present invention includes
an amino acid sequence encoded by a nucleotide sequence which
hybridizes, e.g., hybridizes under stringent conditions, to the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC.RTM. as Accession Number ______ and which can modulate an
acetylcholine response in an acetylcholine responsive cell.
[0100] In other embodiments, the mACHR-6 polypeptide is
substantially homologous to the amino acid sequence of SEQ ID NO:2,
5, or 32 and retains the functional activity of the polypeptide of
SEQ ID NO:2, 5, or 32 yet differs in amino acid sequence due to
natural allelic variation or mutagenesis, as described in detail in
subsection I above. Accordingly, in another embodiment, the mACHR-6
polypeptide is a polypeptide which comprises an amino acid sequence
which is at least about 30-35%, preferably at least about 40-45%,
more preferably at least about 50-55%, even more preferably at
least about 60-65%, yet more preferably at least about 70-75%,
still more preferably at least about 80-85%, and most preferably at
least about 90-95% or more homologous to the amino acid sequence of
SEQ ID NO:2, 5, or 32 and which has at least one of the mACHR-6
activities described herein. In still other embodiments, the
invention pertains to a full length human polypeptide which is
substantially homologous to the entire amino acid sequence of SEQ
ID NO:2, 5, or 32. In still another embodiment, the invention
pertains to nonfunctional, naturally occurring allelic variants of
the mACHR-6 polypeptides described herein. Such allelic variants
will typically contain a non-conservative substitution, a deletion,
or insertion or premature truncation of the amino acid sequence of
SEQ ID NO:2, 5, or 32.
[0101] Biologically active portions of the mACHR-6 polypeptide
include peptides comprising amino acid sequences derived from the
amino acid sequence of the mACHR-6 polypeptide, e.g., the amino
acid sequence shown in SEQ ID NO:2, 5, or 32 or the amino acid
sequence of a polypeptide homologous to the mACHR-6 polypeptide,
which include less amino acids than the full length mACHR-6
polypeptide or the full length polypeptide which is homologous to
the mACHR-6 polypeptide, and exhibit at least one activity of the
mACHR-6 polypeptide. Typically, biologically active portions
(peptides, e.g., peptides which are, for example, 5, 10, 15, 20,
30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length)
comprise a domain or motif, e.g., a transmembrane domain, with at
least one activity of the mACHR-6 polypeptide. Preferably, the
domain is a transmembrane domain derived from a human and is at
least about 75-80%, preferably at least about 80-85%, more
preferably at least about 85-90%, and most preferably at least
about 90-95% or more homologous to SEQ ID NO:7, 8, 9, 10, 11, 12,
or 13 or to the corresponding rat sequences. In a preferred
embodiment, the biologically active portion of the polypeptide
which includes the transmembrane domain can modulate the activity
of a G protein in a cell and/or modulate an acetylcholine response
in a cell, e.g., an acetylcholine responsive cell, e.g., a brain
cell, to thereby beneficially affect the acetylcholine responsive
cell. In a preferred embodiment, the biologically active portion
comprises a transmembrane domain of mACHR-6 as represented by amino
acid residues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID NO:8), 109-130
(SEQ ID NO:9), 152-174 (SEQ ID NO:10), 197-219 (SEQ ID NO:11),
360-380 (SEQ ID NO:12), and 396416 (SEQ ID NO:13), or the
corresponding rat sequences shown in SEQ ID NOs:14-20 amd 34-39.
Moreover, other biologically active portions, in which other
regions of the polypeptide are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
activities described herein. Preferably, the biologically active
portions of the mACHR-6 polypeptide include one or more selected
domains/motifs or portions thereof having biological activity.
[0102] mACHR-6 polypeptides are preferably produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
polypeptide is cloned into an expression vector (as described
above), the expression vector is introduced into a host cell (as
described above) and the mACHR-6 polypeptide is expressed in the
host cell. The mACHR-6 polypeptide can then be isolated from the
cells by an appropriate purification scheme using standard
polypeptide purification techniques. Alternative to recombinant
expression, an mACHR-6 polypeptide, protein, or peptide can be
synthesized chemically using standard peptide synthesis techniques.
Moreover, native mACHR-6 polypeptide can be isolated from cells
(e.g., hippocampal cells, substantia nigra cells, or parotid gland
cells), for example using an anti-mACHR-6 antibody (described
further below).
[0103] The invention also provides mACHR-6 chimeric or fusion
polypeptides. As used herein, an mACHR-6 "chimeric polypeptide" or
"fusion polypeptide" comprises an mACHR-6 polypeptide operatively
linked to a non-mACHR-6 polypeptide. An "mACHR-6 polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to mACHR-6, whereas a "non-mACHR-6 polypeptide" refers to a
heterologous polypeptide having an amino acid sequence
corresponding to a polypeptide which is not substantially
homologous to the mACHR-6 polypeptide, e.g., a polypeptide which is
different from the mACHR-6 polypeptide and which is derived from
the same or a different organism. Within the fusion polypeptide,
the term "operatively linked" is intended to indicate that the
mACHR-6 polypeptide and the non-mACHR-6 polypeptide are fused
in-frame to each other. The non-mACHR-6 polypeptide can be fused to
the N-terminus or C-terminus of the mACHR-6 polypeptide. For
example, in one embodiment the fusion polypeptide is a GST-mACHR-6
fusion polypeptide in which the mACHR-6 sequences are fused to the
C-terminus of the GST sequences. Other types of fusion polypeptides
include, but are not limited to, enzymatic fusion polypeptides, for
example beta-galactosidase fusions, yeast two-hybrid GAL fusions,
poly His fusions and Ig fusions. Such fusion polypeptides,
particularly poly His fusions, can facilitate the purification of
recombinant mACHR-6. In another embodiment, the fusion polypeptide
is an mACHR-6 polypeptide containing a heterologous signal sequence
at its N-terminus. In certain host cells (e.g., mammalian host
cells), expression and/or secretion of mACHR-6 can be increased
through use of a heterologous signal sequence.
[0104] Preferably, an mACHR-6 chimeric or fusion polypeptide of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. In another embodiment, the fusion
gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see, for example, Current
Protocols in Molecular Biology, eds. Ausubel et al. John Wiley
& Sons: 1992). Moreover, many expression vectors are
commercially available that already encode a fusion moiety (e.g., a
GST polypeptide). An mACHR-6-encoding nucleic acid can be cloned
into such an expression vector such that the fusion moiety is
linked in-frame to the mACHR-6 polypeptide.
[0105] The present invention also pertains to homologues of the
mACHR-6 polypeptides which function as either an mACHR-6 agonist
(mimetic) or an mACHR-6 antagonist. In a preferred embodiment, the
mACHR-6 agonists and antagonists stimulate or inhibit,
respectively, a subset of the biological activities of the
naturally occurring form of the mACHR-6 polypeptide. Thus, specific
biological effects can be elicited by treatment with a homologue of
limited function. In one embodiment, treatment of a subject with a
homologue having a subset of the biological activities of the
naturally occurring form of the polypeptide has fewer side effects
in a subject relative to treatment with the naturally occurring
form of the mACHR-6 polypeptide.
[0106] Homologues of the mACHR-6 polypeptide can be generated
by-mutagenesis, e.g., discrete point mutation or truncation of the
mACHR-6 polypeptide. As used herein, the term "homologue" refers to
a variant form of the mACHR-6 polypeptide which acts as an agonist
or antagonist of the activity of the mACHR-6 polypeptide. An
agonist of the mACHR-6 polypeptide can retain substantially the
same, or a subset, of the biological activities of the mACHR-6
polypeptide. An antagonist of the mACHR-6 polypeptide can inhibit
one or more of the activities of the naturally occurring form of
the mACHR-6 polypeptide, by, for example, competitively binding to
a downstream or upstream member of the mACHR-6 cascade which
includes the mACHR-6 polypeptide. Thus, the mammalian mACHR-6
polypeptide and homologues thereof of the present invention can be
either positive or negative regulators of acetylcholine responses
in acetylcholine responsive cells.
[0107] In an alternative embodiment, homologues of the mACHR-6
polypeptide can be identified by screening combinatorial libraries
of mutants, e.g., truncation mutants, of the mACHR-6 polypeptide
for mACHR-6 polypeptide agonist or antagonist activity. In one
embodiment, a variegated library of mACHR-6 variants is generated
by combinatorial mutagenesis at the nucleic acid level and is
encoded by a variegated gene library. A variegated library of
mACHR-6 variants can be produced by, for example, enzymatically
ligating a mixture of synthetic oligonucleotides into gene
sequences such that a degenerate set of potential mACHR-6 sequences
is expressible as individual polypeptides, or alternatively, as a
set of larger fusion polypeptides (e.g., for phage display)
containing the set of mACHR-6 sequences therein. There are a
variety of methods which can be used to produce libraries of
potential mACHR-6 homologues from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
performed in an automatic DNA synthesizer, and the synthetic gene
then ligated into an appropriate expression vector. Use of a
degenerate set of genes allows for the provision, in one mixture,
of all of the sequences encoding the desired set of potential
mACHR-6 sequences. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang, S. A.
(1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.
53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)
Nucleic Acid Res. 11:477).
[0108] In addition, libraries of fragments of the mACHR-6
polypeptide coding can be used to generate a variegated population
of mACHR-6 fragments for screening and subsequent selection of
homologues of an mACHR-6 polypeptide. In one embodiment, a library
of coding sequence fragments can be generated by treating a double
stranded PCR fragment of an mACHR-6 coding sequence with a nuclease
under conditions wherein nicking occurs only about once per
molecule, denaturing the double stranded DNA, renaturing the DNA to
form double stranded DNA which can include sense/antisense pairs
from different nicked products, removing single stranded portions
from reformed duplexes by treatment with S1 nuclease, and ligating
the resulting fragment library into an expression vector. By this
method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the mACHR-6 polypeptide.
[0109] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of mACHR-6 homologues. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recrusive ensemble mutagenesis
(REM), a new technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify mACHR-6 homologues (Arkin and Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
[0110] In one embodiment, cell based assays can be exploited to
analyze a variegated mACHR-6 library. For example, a library of
expression vectors can be transfected into a cell line ordinarily
responsive to acetylcholine. The transfected cells are then
contacted with acetylcholine and the effect of the mACHR-6 mutant
on signaling by acetylcholine can be detected, e.g., by measuring
intracellular calcium concentration. Plasmid DNA can then be
recovered from the cells which score for inhibition, or
alternatively, potentiation of acetylcholine induction, and the
individual clones further characterized.
[0111] An isolated mACHR-6 polypeptide, or a portion or fragment
thereof, can be used as an immunogen to generate antibodies that
bind mACHR-6 using standard techniques for polyclonal and
monoclonal antibody preparation. The full-length mACHR-6
polypeptide can be used or, alternatively, the invention provides
antigenic peptide fragments of mACHR-6 for use as immunogens. The
antigenic peptide of mACHR-6 comprises at least 8 amino acid
residues of the amino acid sequence shown in SEQ ID NO:2, 5, or 32
and encompasses an epitope of mACHR-6 such that an antibody raised
against the peptide forms a specific immune complex with mACHR-6.
Preferably, the antigenic peptide comprises at least 10 amino acid
residues, more preferably at least 15 amino acid residues, even
more preferably at least 20 amino acid residues, and most
preferably at least 30 amino acid residues. Preferred epitopes
encompassed by the antigenic peptide are regions of mACHR-6 that
are located on the surface of the polypeptide, e.g., hydrophilic
regions.
[0112] An mACHR-6 immunogen typically is used to prepare antibodies
by immunizing a suitable subject, (e.g., rabbit, goat, mouse or
other mammal) with the immunogen. An appropriate immunogenic
preparation can contain, for example, recombinantly expressed
mACHR-6 polypeptide or a chemically synthesized mACHR-6 peptide.
The preparation can further include an adjuvant, such as Freund's
complete or incomplete adjuvant, or similar immunostimulatory
agent. Immunization of a suitable subject with an immunogenic
mACHR-6 preparation induces a polyclonal anti-mACHR-6 antibody
response.
[0113] Accordingly, another aspect of the invention pertains to
anti-mACHR-6 antibodies. The term "antibody" as used herein refers
to immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site which specifically binds (immunoreacts with) an
antigen, such as mACHR-6. Examples of immunologically active
portions of immunoglobulin molecules include F(ab) and F(ab').sub.2
fragments which can be generated by treating the antibody with an
enzyme such as pepsin. The invention provides polyclonal and
monoclonal antibodies that bind mACHR-6. The term "monoclonal
antibody" or "monoclonal antibody composition", as used herein,
refers to a population of antibody molecules that contain only one
species of an antigen binding site capable of immunoreacting with a
particular epitope of mACHR-6. A monoclonal antibody composition
thus typically displays a single binding affinity for a particular
mACHR-6 polypeptide with which it immunoreacts.
[0114] Polyclonal anti-mACHR-6 antibodies can be prepared as
described above by immunizing a suitable subject with an mACHR-6
immunogen. The anti-mACHR-6 antibody titer in the immunized subject
can be monitored over time by standard techniques, such as with an
enzyme linked immunosorbent assay (ELISA) using immobilized
mACHR-6. If desired, the antibody molecules directed against
mACHR-6 can be isolated from the mammal (e.g., from the blood) and
further purified by well known techniques, such as protein A
chromatography to obtain the IgG fraction. At an appropriate time
after immunization, e.g., when the anti-mACHR-6 antibody titers are
highest, antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981)
J. Immunol. 127:53946; Brown et al. (1980) J. Biol. Chem.
255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al.
(1982) Int. J. Cancer 29:269-75), the more recent human B cell
hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the
EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma
techniques. The technology for producing monoclonal antibody
hybridomas is well known (see generally R. H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981)
Yale J. Biol. Med., 54:387402; M. L. Gefter et al. (1977) Somatic
Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a
myeloma) is fused to lymphocytes (typically splenocytes) from a
mammal immunized with an mACHR-6 immunogen as described above, and
the culture supernatants of the resulting hybridoma cells are
screened to identify a hybridoma producing a monoclonal antibody
that binds mACHR-6.
[0115] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-mACHR-6 monoclonal antibody (see,
e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited
supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC.RTM.. Typically, HAT-sensitive mouse myeloma
cells are fused to mouse splenocytes using polyethylene glycol
("PEG"). Hybridoma cells resulting from the fusion are then
selected using HAT medium, which kills unfused and unproductively
fused myeloma cells (unfused splenocytes die after several days
because they are not transformed). Hybridoma cells producing a
monoclonal antibody of the invention are detected by screening the
hybridoma culture supernatants for antibodies that bind mACHR-6,
e.g., using a standard ELISA assay.
[0116] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-mACHR-6 antibody can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with mACHR-6 to thereby isolate immunoglobulin library members that
bind mACHR-6. Kits for generating and screening phage display
libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT International Publication No. WO
92/18619; Dower et al. PCT International Publication No. WO
91/17271; Winter et al. PCT International Publication WO 92/20791;
Markland et al. PCT International Publication No. WO 92/15679;
Breitling et al. PCT International Publication WO 93/01288;
McCafferty et al. PCT International Publication No. WO 92/01047;
Garrard et al. PCT International Publication No. WO 92/09690;
Ladner et al. PCT International Publication No. WO 90/02809; Fuchs
et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins
et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991)
Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et
al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991)
Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) PNAS
88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
[0117] Additionally, recombinant anti-mACHR-6 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in Robinson et al. PCT International Application
No. PCT/US86/02269; Akira, et al. European Patent Application
184,187; Taniguchi, M., European Patent Application 171,496;
Morrison et al. European Patent Application 173,494; Neuberger et
al. PCT International Publication No. WO 86/01533; Cabilly et al.
U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol.
139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et
al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0118] An anti-mACHR-6 antibody (e.g., monoclonal antibody) can be
used to isolate mACHR-6 by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-mACHR-6 antibody can
facilitate the purification of natural mACHR-6 from cells and of
recombinantly produced mACHR-6 expressed in host cells. Moreover,
an anti-mACHR-6 antibody can be used to detect mACHR-6 polypeptide
(e.g., in a cellular lysate or cell supernatant) in order to
evaluate the abundance and pattern of expression of the mACHR-6
polypeptide or a fragment of an mACHR-6 polypeptide. The detection
of circulating fragments of an mACHR-6 polypeptide can be used to
identify mACHR-6 turnover in a subject. Anti-mACHR-6 antibodies can
be used diagnostically to monitor polypeptide levels in tissue as
part of a clinical testing procedure, e.g., to, for example,
determine the efficacy of a given treatment regimen. Detection can
be facilitated by coupling (i.e., physically linking) the antibody
to a detectable substance. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0119] IV. Pharmaceutical Compositions
[0120] The mACHR-6 nucleic acid molecules, mACHR-6 polypeptides
(particularly fragments of mACHR-6), mACHR-6 modulators, and
anti-mACHR-6 antibodies (also referred to herein as "active
compounds") of the invention can be incorporated into
pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically comprise the
nucleic acid molecule, polypeptide, modulator, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, such media can be
used in the compositions of the invention. Supplementary active
compounds can also be incorporated into the compositions.
[0121] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0122] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0123] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an mACHR-6 polypeptide or
anti-mACHR-6 antibody) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0124] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0125] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0126] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0127] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0128] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0129] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0130] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g. retroviral vectors, the pharmaceutical preparation can include
one or more cells which produce the gene delivery system.
[0131] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0132] V. Uses and Methods of the Invention
[0133] The nucleic acid molecules, polypeptides, polypeptide
homologues, modulators, and antibodies described herein can be used
in one or more of the following methods: a) drug screening assays;
b) diagnostic assays particularly in disease identification,
allelic screening and pharmocogenetic testing; c) methods of
treatment; d) pharmacogenomics; and e) monitoring of effects during
clinical trials. An mACHR-6 polypeptide of the invention has one or
more of the activities described herein and can thus be used to,
for example, modulate an acetylcholine response in an acetylcholine
responsive cell, for example by binding to acetylcholine or an
mACHR-6 binding partner making it unavailable for binding to the
naturally present mACHR-6 polypeptide. The isolated nucleic acid
molecules of the invention can be used to express mACHR-6
polypeptide (e.g., via a recombinant expression vector in a host
cell or in gene therapy applications), to detect mACHR-6 mRNA
(e.g., in a biological sample) or a naturally occurring or
recombinantly generated genetic mutation in an mACHR-6 gene, and to
modulate mACHR-6 activity, as described further below. In addition,
the mACHR-6 polypeptides can be used to screen drugs or compounds
which modulate mACHR-6 polypeptide activity as well as to treat
disorders characterized by insufficient production of mACHR-6
polypeptide or production of mACHR-6 polypeptide forms which have
decreased activity compared to wild type mACHR-6. Moreover, the
anti-mACHR-6 antibodies of the invention can be used to detect and
isolate an mACHR-6 polypeptide, particularly fragments of mACHR-6
present in a biological sample, and to modulate mACHR-6 polypeptide
activity.
[0134] a. Drug Screening Assays:
[0135] The invention provides methods for identifying compounds or
agents which can be used to treat disorders characterized by (or
associated with) aberrant or abnormal mACHR-6 nucleic acid
expression and/or mACHR-6 polypeptide activity. These methods are
also referred to herein as drug screening assays and typically
include the step of screening a candidate/test compound or agent to
be an agonist or antagonist of mACHR-6, and specifically for the
ability to interact with (e.g., bind to) an mACHR-6 polypeptide, to
modulate the interaction of an mACHR-6 polypeptide and a target
molecule, and/or to modulate mACHR-6 nucleic acid expression and/or
mACHR-6 polypeptide activity. Candidate/test compounds or agents
which have one or more of these abilities can be used as drugs to
treat disorders characterized by aberrant or abnormal mACHR-6
nucleic acid expression and/or mACHR-6 polypeptide activity.
Candidate/test compounds include, for example, 1) peptides such as
soluble peptides, including Ig-tailed fusion peptides and members
of random peptide libraries (see, e.g., Lam, K. S. et al. (1991)
Nature 354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L-configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang, Z. et al. (1993) Cell 72:767-778);
3) antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0136] In one embodiment, the invention provides assays for
screening candidate/test compounds which interact with (e.g., bind
to) mACHR-6 polypeptide. Typically, the assays are recombinant cell
based or cell-free assays which include the steps of combining an
mACHR-6 polypeptide or a bioactive fragment thereof, and a
candidate/test compound, e.g., under conditions which allow for
interaction of (e.g., binding of) the candidate/test compound to
the mACHR-6 polypeptide or fragment thereof to form a complex, and
detecting the formation of a complex, in which the ability of the
candidate compound to interact with (e.g., bind to) the mACHR-6
polypeptide or fragment thereof is indicated by the presence of the
candidate compound in the complex. Formation of complexes between
the mACHR-6 polypeptide and the candidate compound can be
quantitated, for example, using standard immunoassays.
[0137] In another embodiment, the invention provides screening
assays to identify candidate/test compounds which modulate (e.g.,
stimulate or inhibit) the interaction (and most likely mACHR-6
activity as well) between an mACHR-6 polypeptide and a molecule
(target molecule) with which the mACHR-6 polypeptide normally
interacts. Examples of such target molecules include polypeptides
in the same signaling path as the mACHR-6 polypeptide, e.g.,
polypeptides which may function upstream (including both
stimulators and inhibitors of activity) or downstream of the
mACHR-6 polypeptide in, for example, a cognitive function signaling
pathway or in a pathway involving mACHR-6 activity, e.g., a G
protein or other interactor involved in phosphatidylinositol
turnover and/or phospholipase C activation. Typically, the assays
are recombinant cell based or cell-free assays which include the
steps of combining a cell expressing an mACHR-6 polypeptide, or a
bioactive fragment thereof, an mACHR-6 target molecule (e.g., an
mACHR-6 ligand) and a candidate/test compound, e.g., under
conditions wherein but for the presence of the candidate compound,
the mACHR-6 polypeptide or biologically active portion thereof
interacts with (e.g., binds to) the target molecule, and detecting
the formation of a complex which includes the mACHR-6 polypeptide
and the target molecule or detecting the interaction/reaction of
the mACHR-6 polypeptide and the target molecule. Detection of
complex formation can include direct quantitation of the complex
by, for example, measuring inductive effects of the mACHR-6
polypeptide. A statistically significant change, such as a
decrease, in the interaction of the mACHR-6 and target molecule
(e.g., in the formation of a complex between the mACHR-6 and the
target molecule) in the presence of a candidate compound (relative
to what is detected in the absence of the candidate compound) is
indicative of a modulation (e.g., stimulation or inhibition) of the
interaction between the mACHR-6 polypeptide and the target
molecule. Modulation of the formation of complexes between the
mACHR-6 polypeptide and the target molecule can be quantitated
using, for example, an immunoassay.
[0138] To perform cell free drug screening assays, it is desirable
to immobilize either mACHR-6 or its target molecule to facilitate
separation of complexes from uncomplexed forms of one or both of
the polypeptides, as well as to accommodate automation of the
assay. Interaction (e.g., binding of) of mACHR-6 to a target
molecule, in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
polypeptide can be provided which adds a domain that allows the
polypeptide to be bound to a matrix. For example,
glutathione-S-transfera- se/mACHR-6 fusion polypeptides can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtitre plates, which are
then combined with the cell lysates (e.g., .sup.35S-labeled) and
the candidate compound, and the mixture incubated under conditions
conducive to complex formation (e.g., at physiological conditions
for salt and pH). Following incubation, the beads are washed to
remove any unbound label, and the matrix immobilized and radiolabel
determined directly, or in the supernatant after the complexes are
dissociated. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of mACHR-6-binding
polypeptide found in the bead fraction quantitated from the gel
using standard electrophoretic techniques.
[0139] Other techniques for immobilizing polypeptides on matrices
can also be used in the drug screening assays of the invention. For
example, either mACHR-6 or its target molecule can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated
mACHR-6 molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates
(Pierce Chemical). Alternatively, antibodies reactive with mACHR-6
but which do not interfere with binding of the polypeptide to its
target molecule can be derivatized to the wells of the plate, and
mACHR-6 trapped in the wells by antibody conjugation. As described
above, preparations of an mACHR-6-binding polypeptide and a
candidate compound are incubated in the mACHR-6-presenting wells of
the plate, and the amount of complex trapped in the well can be
quantitated. Methods for detecting such complexes, in addition to
those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
mACHR-6 target molecule, or which are reactive with mACHR-6
polypeptide and compete with the target molecule; as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the target molecule.
[0140] In yet another embodiment, the invention provides a method
for identifying a compound (e.g., a screening assay) capable of use
in the treatment of a disorder characterized by (or associated
with) aberrant or abnormal mACHR-6 nucleic acid expression or
mACHR-6 polypeptide activity. This method typically includes the
step of assaying the ability of the compound or agent to modulate
the expression of the mACHR-6 nucleic acid or the activity of the
mACHR-6 polypeptide thereby identifying a compound for treating a
disorder characterized by aberrant or abnormal mACHR-6 nucleic acid
expression or mACHR-6 polypeptide activity. Disorders characterized
by aberrant or abnormal mACHR-6 nucleic acid expression or mACHR-6
polypeptide activity are described herein. Methods for assaying the
ability of the compound or agent to modulate the expression of the
mACHR-6 nucleic acid or activity of the mACHR-6 polypeptide are
typically cell-based assays. For example, cells which are sensitive
to ligands which transduce signals via a pathway involving mACHR-6
can be induced to overexpress an mACHR-6 polypeptide in the
presence and absence of a candidate compound. Candidate compounds
which produce a statistically significant change in
mACHR-6-dependent responses (either stimulation or inhibition) can
be identified. In one embodiment, expression of the mACHR-6 nucleic
acid or activity of an mACHR-6 polypeptide is modulated in cells
and the effects of candidate compounds on the readout of interest
(such as phosphatidylinositol turnover) are measured. For example,
the expression of genes which are up- or down-regulated in response
to an mACHR-6-dependent signal cascade can be assayed. In preferred
embodiments, the regulatory regions of such genes, e.g., the 5'
flanking promoter and enhancer regions, are operably linked to a
detectable marker (such as luciferase) which encodes a gene product
that can be readily detected. Phosphorylation of mACHR-6 or mACHR-6
target molecules can also be measured, for example, by
immunoblotting.
[0141] Alternatively, modulators of mACHR-6 expression (e.g.,
compounds which can be used to treat a disorder characterized by
aberrant or abnormal mACHR-6 nucleic acid expression or mACHR-6
polypeptide activity) can be identified in a method wherein a cell
is contacted with a candidate compound and the expression of
mACHR-6 mRNA or polypeptide in the cell is determined. The level of
expression of mACHR-6 mRNA or polypeptide in the presence of the
candidate compound is compared to the level of expression of
mACHR-6 mRNA or polypeptide in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of mACHR-6 nucleic acid expression based on this
comparison and be used to treat a disorder characterized by
aberrant mACHR-6 nucleic acid expression. For example, when
expression of mACHR-6 mRNA or polypeptide is greater (statistically
significantly greater) in the presence of the candidate compound
than in its absence, the candidate compound is identified as a
stimulator of mACHR-6 nucleic acid expression. Alternatively, when
mACHR-6 nucleic acid expression is less (statistically
significantly less) in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of mACHR-6 nucleic acid expression. The level of mACHR-6
nucleic acid expression in the cells can be determined by methods
described herein for detecting mACHR-6 mRNA or polypeptide.
[0142] In yet another aspect of the invention, the mACHR-6
polypeptides, or fragments thereof, can be used as "bait proteins"
in a two-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos
et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO
94/10300), to identify other proteins, which bind to or interact
with mACHR-6 ("mACHR-6-binding proteins" or "mACHR-6-bp") and
modulate mACHR-6 polypeptide activity. Such mACHR-6-binding
proteins are also likely to be involved in the propagation of
signals by the mACHR-6 polypeptides as, for example, upstream or
downstream elements of the mACHR-6 pathway.
[0143] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Bartel, P. et al. "Using the Two-Hybrid System
to Detect Protein-Protein Interactions" in Cellular Interactions in
Development: A Practical Approach, Hartley, D. A. ed. (Oxford
University Press, Oxford, 1993) pp. 153-179. Briefly, the assay
utilizes two different DNA constructs. In one construct, the gene
that codes for mACHR-6 is fused to a gene encoding the DNA binding
domain of a known transcription factor (e.g., GAL-4). In the other
construct, a DNA sequence, from a library of DNA sequences, that
encodes an unidentified protein ("prey" or "sample") is fused to a
gene that codes for the activation domain of the known
transcription factor. If the "bait" and the "prey" proteins are
able to interact, in vivo, forming an mACHR-6-dependent complex,
the DNA-binding and activation domains of the transcription factor
are brought into close proximity. This proximity allows
transcription of a reporter gene (e.g., LacZ) which is operably
linked to a transcriptional regulatory site responsive to the
transcription factor. Expression of the reporter gene can be
detected and cell colonies containing the functional transcription
factor can be isolated and used to obtain the cloned gene which
encodes the protein which interacts with mACHR-6.
[0144] Modulators of mACHR-6 polypeptide activity and/or mACHR-6
nucleic acid expression identified according to these drug
screening assays can be used to treat, for example, nervous system
disorders, smooth muscle related disorders, cardiac muscle related
disorders, and gland related disorders. These methods of treatment
include the steps of administering the modulators of mACHR-6
polypeptide activity and/or nucleic acid expression, e.g., in a
pharmaceutical composition as described in subsection IV above, to
a subject in need of such treatment, e.g., a subject with a
disorder described herein.
[0145] b. Diagnostic Assays:
[0146] The invention further provides a method for detecting the
presence of mACHR-6, or fragment thereof, in a biological sample.
The method involves contacting the biological sample with a
compound or an agent capable of detecting mACHR-6 polypeptide or
mRNA such that the presence of mACHR-6 is detected in the
biological sample. A preferred agent for detecting mACHR-6 mRNA is
a labeled or labelable nucleic acid probe capable of hybridizing to
mACHR-6 mRNA. The nucleic acid probe can be, for example, the
full-length mACHR-6 cDNA of SEQ ID NO:1, 4, or 31, or a portion
thereof, such as an oligonucleotide of at least 15, 30, 50, 100,
250 or 500 nucleotides in length and sufficient to specifically
hybridize under stringent conditions to mACHR-6 mRNA. A preferred
agent for detecting mACHR-6 polypeptide is a labeled or labelable
antibody capable of binding to mACHR-6 polypeptide. Antibodies can
be polyclonal, or more preferably, monoclonal. An intact antibody,
or a fragment thereof (e.g., Fab or F(ab').sub.2) can be used. The
term "labeled or labelable", with regard to the probe or antibody,
is intended to encompass direct labeling of the probe or antibody
by coupling (i.e., physically linking) a detectable substance to
the probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect mACHR-6 mRNA or
polypeptide in a biological sample in vitro as well as in vivo. For
example, in vitro techniques for detection of mACHR-6 mRNA include
Northern hybridizations and in situ hybridizations. In vitro
techniques for detection of mACHR-6 polypeptide include enzyme
linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, mACHR-6
polypeptide can be detected in vivo in a subject by introducing
into the subject a labeled anti-mACHR-6 antibody. For example, the
antibody can be labeled with a radioactive marker whose presence
and location in a subject can be detected by standard imaging
techniques. Particularly useful are methods which detect the
allelic variant of mACHR-6 expressed in a subject and methods which
detect fragments of an mACHR-6 polypeptide in a sample.
[0147] The invention also encompasses kits for detecting the
presence of mACHR-6 in a biological sample. For example, the kit
can comprise a labeled or labelable compound or agent capable of
detecting mACHR-6 polypeptide or mRNA in a biological sample; means
for determining the amount of mACHR-6 in the sample; and means for
comparing the amount of mACHR-6 in the sample with a standard. The
compound or agent can be packaged in a suitable container. The kit
can further comprise instructions for using the kit to detect
mACHR-6 mRNA or polypeptide.
[0148] The methods of the invention can also be used to detect
naturally occurring genetic mutations in an mACHR-6 gene, thereby
determining if a subject with the mutated gene is at risk for a
disorder characterized by aberrant or abnormal mACHR-6 nucleic acid
expression or mACHR-6 polypeptide activity as described herein. In
preferred embodiments, the methods include detecting, in a sample
of cells from the subject, the presence or absence of a genetic
mutation characterized by at least one of an alteration affecting
the integrity of a gene encoding an mACHR-6 polypeptide, or the
misexpression of the mACHR-6 gene. For example, such genetic
mutations can be detected by ascertaining the existence of at least
one of 1) a deletion of one or more nucleotides from an mACHR-6
gene; 2) an addition of one or more nucleotides to an mACHR-6 gene;
3) a substitution of one or more nucleotides of an mACHR-6 gene, 4)
a chromosomal rearrangement of an mACHR-6 gene; 5) an alteration in
the level of a messenger RNA transcript of an mACHR-6 gene, 6)
aberrant modification of an mACHR-6 gene, such as of the
methylation pattern of the genomic DNA, 7) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of an
mACHR-6 gene, 8) a non-wild type level of an mACHR-6-polypeptide,
9) allelic loss of an mACHR-6 gene, and 10) inappropriate
post-translational modification of an mACHR-6-polypeptide. As
described herein, there are a large number of assay techniques
known in the art which can be used for detecting mutations in an
mACHR-6 gene.
[0149] In certain embodiments, detection of the mutation involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
mACHR-6-gene (see Abravaya et al. (1995) Nucleic Acids Res
0.23:675-682). This method can include the steps of collecting a
sample of cells from a patient, isolating nucleic acid (e.g.,
genomic, mRNA or both) from the cells of the sample, contacting the
nucleic acid sample with one or more primers which specifically
hybridize to an mACHR-6 gene under conditions such that
hybridization and amplification of the mACHR-6-gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample.
[0150] In an alternative embodiment, mutations in an mACHR-6 gene
from a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0151] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
mACHR-6 gene and detect mutations by comparing the sequence of the
sample mACHR-6 with the corresponding wild-type (control) sequence.
Examples of sequencing reactions include those based on techniques
developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger
((1977) PNAS 74:5463). A variety of automated sequencing procedures
can be utilized when performing the diagnostic assays ((1995)
Biotechniques 19:448), including sequencing by mass spectrometry
(see, e.g., PCT International Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147-159).
[0152] Other methods for detecting mutations in the mACHR-6 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et
al. (1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton (1993) Mutat Res 285:125-144; and
Hayashi (1992) Genet Anal Tech Appl 9:73-79), and movement of
mutant or wild-type fragments in polyacrylamide gels containing a
gradient of denaturant is assayed using denaturing gradient gel
electrophoresis (Myers et al (1985) Nature 313:495). Examples of
other techniques for detecting point mutations include, selective
oligonucleotide hybridization, selective amplification, and
selective primer extension.
[0153] c. Methods of Treatment
[0154] Another aspect of the invention pertains to methods for
treating a subject, e.g., a human, having a disease or disorder
characterized by (or associated with) aberrant or abnormal mACHR-6
nucleic acid expression and/or mACHR-6 polypeptide activity. These
methods include the step of administering an mACHR-6 modulator
(agonist or antagonist) to the subject such that treatment occurs.
The language "aberrant or abnormal mACHR-6 expression" refers to
expression of a non-wild-type mACHR-6 polypeptide or a
non-wild-type level of expression of an mACHR-6 polypeptide.
Aberrant or abnormal mACHR-6 activity refers to a neon-wild-type
mACHR-6 activity or a non-wild-type level of mACHR-6 activity. As
the mACHR-6 polypeptide is involved in a pathway involving
modulation of neurotransmitter, e.g., acetylcholine, release;
modulation of smooth muscle contraction; modulation of cardiac
muscle contraction; and modulation of gland, e.g., exocrine gland
function, aberrant or abnormal mACHR-6 activity or expression
interferes with the normal neurotransmitter, e.g., acetylcholine,
release; normal smooth muscle; and cardiac muscle contraction; and
normal gland, e.g., exocrine gland function. Non-limiting examples
of disorders or diseases characterized by or associated with
abnormal or aberrant mACHR-6 activity or expression include nervous
system related disorders, e.g., central nervous system related
disorders. Examples of nervous system related disorders include
cognitive disorders, e.g., memory and learning disorders, such as
amnesia, apraxia, agnosia, amnestic dysnomia, amnestic spatial
disorientation, Kluver-Bucy syndrome, Alzheimer's related memory
loss (Eglen R. M. (1996) Pharmacol. and Toxicol. 78(2):59-68; Perry
E. K. (1995) Brain and Cognition 28(3):240-58) and learning
disability; disorders affecting consciousness, e.g., visual
hallucinations, perceptual disturbances, or delerium associated
with Lewy body dementia; schitzo-effective disorders (Dean B.
(1996) Mol. Psychiatry 1(1):54-8), schizophrenia with mood swings
(Bymaster F. P. (1997) J. Clin. Psychiatry 58 (suppl. 10):28 36;
Yeomans J. S. (1995) Neuropharmacol. 12(1):3-16; Reimann D. (1994)
J. Psychiatric Res. 28(3):195-210), depressive illness (primary or
secondary); affective disorders (Janowsky D. S. (1994) Am. J. Med.
Genetics 54(4):335-44); sleep disorders (Kimura F. (1997) J.
Neurophysiol. 77(2):709-16), e.g., REM sleep abnormalities in
patients suffering from, for example, depression (Riemann D. (1994)
J. Psychosomatic Res. 38 Suppl. 1:15-25; Bourgin P. (1995)
Neuroreport 6(3): 532-6), paradoxical sleep abnormalities (Sakai K.
(1997) Eur. J. Neuroscience 9(3):415-23), sleep-wakefulness, and
body temperature or respiratory depression abnormalities during
sleep (Shuman S. L. (1995) Am. J. Physiol. 269(2 Pt 2):R308-17;
Mallick B. N. (1997) Brain Res. 750(1-2):311-7). Other examples of
nervous system related disorders include disorders affecting pain
generation mechanisms, e.g., pain related to irritable bowel
syndrome (Mitch C. H. (1997) J. Med. Chem. 40(4):538-46; Shannon H.
E. (1997) J. Pharmac. and Exp. Therapeutics 281(2):884-94; Bouaziz
H. (1995) Anesthesia and Analgesia 80(6):1140-4; or Guimaraes A. P.
(1994) Brain Res. 647(2):220-30) or chest pain; movement disorders
(Monassi C. R. (1997) Physiol. and Behav. 62(1):53-9), e.g.,
Parkinson's disease related movement disorders (Finn M. (1997)
Pharmacol. Biochem. & Behavior 57(1-2):243-9; Mayorga A. J.
(1997) Pharmacol. Biochem. & Behavior 56(2):273-9); eating
disorders, e.g., insulin hypersecretion related obesity (Maccario
M. (1997) J. Endocrinol. Invest. 20(1):8-12; Premawardhana L. D.
(1994) Clin. Endocrinol. 40(5): 617-21); or drinking disorders,
e.g., diabetic polydipsia (Murzi E. (1997) Brain Res.
752(1-2):184-8; Yang X. (1994) Pharmacol. Biochem. & Behavior
49(1):1-6). Yet further examples of disorders or diseases
characterized by or associated with abnormal or aberrant mACHR-6
activity or expression include smooth muscle related disorders such
as irritable bowel syndrome, diverticular disease, urinary
incontinence, oesophageal achalasia, or chronic obstructive airways
disease; heart muscle related disorders such as pathologic
bradycardia or tachycardia, arrhythmia, flutter or fibrillation; or
gland related disorders such as xerostomia, or diabetes mellitus.
The terms "treating" or "treatment", as used herein, refer to
reduction or alleviation of at least one adverse effect or symptom
of a disorder or disease, e.g., a disorder or disease characterized
by or associated with abnormal or aberrant mACHR-6 polypeptide
activity or mACHR-6 nucleic acid expression.
[0155] As used herein, an mACHR-6 modulator is a molecule which can
modulate mACHR-6 nucleic acid expression and/or mACHR-6 polypeptide
activity. For example, an mACHR-6 modulator can modulate, e.g.,
upregulate (activate/agonize) or downregulate
(suppress/antagonize), mACHR-6 nucleic acid expression. In another
example, an mACHR-6 modulator can modulate (e.g., stimulate/agonize
or inhibit/antagonize) mACHR-6 polypeptide activity. If it is
desirable to treat a disorder or disease characterized by (or
associated with) aberrant or abnormal (non-wild-type) mACHR-6
nucleic acid expression and/or mACHR-6 polypeptide activity by
inhibiting mACHR-6 nucleic acid expression, an mACHR-6 modulator
can be an antisense molecule, e.g., a ribozyme, as described
herein. Examples of antisense molecules which can be used to
inhibit mACHR-6 nucleic acid expression include antisense molecules
which are complementary to a portion of the 5' untranslated region
of SEQ ID NO:1 which also includes the start codon and antisense
molecules which are complementary to a portion of the 3'
untranslated region of SEQ ID NO:1, 4, or 31. An example of an
antisense molecule which is complementary to a portion of the 5'
untranslated region of SEQ ID NO:1 and which also includes the
start codon is a nucleic acid molecule which includes nucleotides
which are complementary to nucleotides 280 to 296 of SEQ ID NO:1.
This antisense molecule has the following nucleotide sequence:
5'CCTGCGGGGCCATGGAG 3' (SEQ ID NO:21). An example of an antisense
molecule which is complementary to a portion of the 3' untranslated
region of SEQ ID NO:1 is a nucleic acid molecule which includes
nucleotides which are complementary to nucleotides 1629 to 1645 of
SEQ ID NO:1. This antisense molecule has the following sequence: 5'
GTGGCCCACCAGAGCCT 3' (SEQ ID NO:22). An additional example of an
antisense molecule which is complementary to a portion of the 3'
untranslated region of SEQ ID NO:1 is a nucleic acid molecule which
includes nucleotides which are complementary to nucleotides 1650 to
1666 of SEQ ID NO:1. This antisense molecule has the following
sequence: 5'CAGCCACGCCTCTCTCA 3' (SEQ ID NO:23). An example of an
antisense molecule which is complementary to a portion of the 5'
untranslated region of SEQ ID NO:4 and which also includes the
start codon, is a nucleic acid molecule which includes nucleotides
which are complementary to nucleotides 766 to 783 of SEQ ID NO:4.
This antisense molecule has the following nucleotide sequence: 5'
GCCTGCTGGGCCATGGAG 3' (SEQ ID NO:24). An example of an antisense
molecule which is complementary to a portion of the 3' untranslated
region of SEQ ID NO:4 is a nucleic acid molecule which includes
nucleotides which are complementary to nucleotides 2113 to 2128 of
SEQ ID NO:4. This antisense molecule has the following sequence: 5'
TGAGCAGCTGCCCCAC 3' (SEQ ID NO:25). An additional example of an
antisense molecule which is complementary to a portion of the 3'
untranslated region of SEQ ID NO:4 is a nucleic acid molecule which
includes nucleotides which are complementary to nucleotides 2133 to
2148 of SEQ ID NO:4. This antisense molecule has the following
sequence: 5'CTGAGGCCAGGCCCTT 3' (SEQ ID NO:26).
[0156] An mACHR-6 modulator which inhibits mACHR-6 nucleic acid
expression can also be a small molecule or other drug, e.g., a
small molecule or drug identified using the screening assays
described herein, which inhibits mACHR-6 nucleic acid expression.
If it is desirable to treat a disease or disorder characterized by
(or associated with) aberrant or abnormal (non-wild-type) mACHR-6
nucleic acid expression and/or mACHR-6 polypeptide activity by
stimulating mACHR-6 nucleic acid expression, an mACHR-6 modulator
can be, for example, a nucleic acid molecule encoding mACHR-6
(e.g., a nucleic acid molecule comprising a nucleotide sequence
homologous to the nucleotide sequence of SEQ ID NO:1, 4, or 31) or
a small molecule or other drug, e.g., a small molecule (peptide) or
drug identified using the screening assays described herein, which
stimulates mACHR-6 nucleic acid expression.
[0157] Alternatively, if it is desirable to treat a disease or
disorder characterized by (or associated with) aberrant or abnormal
(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6
polypeptide activity by inhibiting mACHR-6 polypeptide activity, an
mACHR-6 modulator can be an anti-mACHR-6 antibody or a small
molecule or other drug, e.g., a small molecule or drug identified
using the screening assays described herein, which inhibits mACHR-6
polypeptide activity. If it is desirable to treat a disease or
disorder characterized by (or associated with) aberrant or abnormal
(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6
polypeptide activity by stimulating mACHR-6 polypeptide activity,
an mACHR-6 modulator can be an active mACHR-6 polypeptide or
portion thereof (e.g., an mACHR-6 polypeptide or portion thereof
having an amino acid sequence which is homologous to the amino acid
sequence of SEQ ID NO:2, 5, or 32 or a portion thereof) or a small
molecule or other drug, e.g., a small molecule or drug identified
using the screening assays described herein, which stimulates
mACHR-6 polypeptide activity.
[0158] Other aspects of the invention pertain to methods for
modulating a cell associated activity. These methods include
contacting the cell with an agent (or a composition which includes
an effective amount of an agent) which modulates mACHR-6
polypeptide activity or mACHR-6 nucleic acid expression such that a
cell associated activity is altered relative to a cell associated
activity (for example, phosphatidylinositol metabolism) of the cell
in the absence of the agent. As used herein, "a cell associated
activity" refers to a normal or abnormal activity or function of a
cell. Examples of cell associated activities include
phosphatidylinositol turnover, production or secretion of
molecules, such as proteins, contraction, proliferation, migration,
differentiation, and cell survival. In a preferred embodiment, the
cell is neural cell of the brain, e.g., a hippocampal cell. The
term "altered" as used herein refers to a change, e.g., an increase
or decrease, of a cell associated activity particularly
phosphatidylinositol turnover and phospholipase C activation. In
one embodiment, the agent stimulates mACHR-6 polypeptide activity
or mACHR-6 nucleic acid expression. Examples of such stimulatory
agents include an active mACHR-6 polypeptide, a nucleic acid
molecule encoding mACHR-6 that has been introduced into the cell,
and a modulatory agent which stimulates mACHR-6 polypeptide
activity or mACHR-6 nucleic acid expression and which is identified
using the drug screening assays described herein. In another
embodiment, the agent inhibits mACHR-6 polypeptide activity or
mACHR-6 nucleic acid expression. Examples of such inhibitory agents
include an antisense mACHR-6 nucleic acid molecule, an anti-mACHR-6
antibody, and a modulatory agent which inhibits mACHR-6 polypeptide
activity or mACHR-6 nucleic acid expression and which is identified
using the drug screening assays described herein. These modulatory
methods can be performed in vitro (e.g., by culturing the cell with
the agent) or, alternatively, in vivo (e.g., by administering the
agent to a subject). In a preferred embodiment, the modulatory
methods are performed in vivo, i.e., the cell is present within a
subject, e.g., a mammal, e.g., a human, and the subject has a
disorder or disease characterized by or associated with abnormal or
aberrant mACHR-6 polypeptide activity or mACHR-6 nucleic acid
expression.
[0159] A nucleic acid molecule, a polypeptide, an mACHR-6
modulator, a compound etc. used in the methods of treatment can be
incorporated into an appropriate pharmaceutical composition
described herein and administered to the subject through a route
which allows the molecule, polypeptide, modulator, or compound etc.
to perform its intended function. Examples of routes of
administration are also described herein under subsection IV.
[0160] d. Pharmacogenomics
[0161] Test/candidate compounds, or modulators which have a
stimulatory or inhibitory effect on mACHR-6 activity (e.g., mACHR-6
gene expression) as identified by a screening assay described
herein can be administered to individuals to treat
(prophylactically or therapeutically) disorders (e.g., CNS
disorders) associated with aberrant mACHR-6 activity. In
conjunction with such treatment, the pharmacogenomics (i.e., the
study of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) of the
individual may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds (e.g.,
drugs) for prophylactic or therapeutic treatments based on a
consideration of the individual's genotype. Such pharmacogenomics
can further be used to determine appropriate dosages and
therapeutic regimens. Accordingly, the activity of mACHR-6
polypeptide, expression of mACHR-6 nucleic acid, or mutation
content of mACHR-6 genes in an individual can be determined to
thereby select appropriate compound(s) for therapeutic or
prophylactic treatment of the individual.
[0162] Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder,
M. W. (1997) Clin. Chem. 43(2):254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body (altered drug action) or genetic conditions
transmitted as single factors altering the way the body acts on
drugs (altered drug metabolism). These pharmacogenetic conditions
can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0163] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C
19 quite frequently experience exaggerated drug response and side
effects when they receive standard doses. If a metabolite is the
active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0164] Thus, the activity of mACHR-6 polypeptide, expression of
mACHR-6 nucleic acid, or mutation content of mACHR-6 genes in an
individual can be determined to thereby select appropriate agent(s)
for therapeutic or prophylactic treatment of a subject. In
addition, pharmacogenetic studies can be used to apply genotyping
of polymorphic alleles encoding drug-metabolizing enzymes to the
identification of a subject's drug responsiveness phenotype. This
knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
an mACHR-6 modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0165] e. Monitoring of Effects During Clinical Trials
[0166] Monitoring the influence of compounds (e.g., drugs) on the
expression or activity of mACHR-6 (e.g., the ability to modulate
the effects of acetylcholine on acetylcholine responsive cells) can
be applied not only in basic drug screening, but also in clinical
trials. For example, the effectiveness of an agent determined by a
screening assay, as described herein, to increase mACHR-6 gene
expression, polypeptide levels, or up-regulate mACHR-6 activity,
can be monitored in clinical trails of subjects exhibiting
decreased mACHR-6 gene expression, polypeptide levels, or
down-regulated mACHR-6 activity. Alternatively, the effectiveness
of an agent, determined by a screening assay, to decrease mACHR-6
gene expression, polypeptide levels, or down-regulate mACHR-6
activity, can be monitored in clinical trails of subjects
exhibiting increased mACHR-6 gene expression, polypeptide levels,
or up-regulated mACHR-6 activity. In such clinical trials, the
expression or activity of mACHR-6 and, preferably, other genes
which have been implicated in, for example, a nervous system
related disorder can be used as a "read out" or markers of the
acetylcholine responsiveness of a particular cell.
[0167] For example, and not by way of limitation, genes, including
mACHR-6, which are modulated in cells by treatment with a compound
(e.g., drug or small molecule) which modulates mACHR-6 activity
(e.g., identified in a screening assay as described herein) can be
identified. Thus, to study the effect of compounds on CNS
disorders, for example, in a clinical trial, cells can be isolated
and RNA prepared and analyzed for the levels of expression of
mACHR-6 and other genes implicated in the disorder. The levels of
gene expression (i.e., a gene expression pattern) can be quantified
by Northern blot analysis or RT-PCR, as described herein, or
alternatively by measuring the amount of polypeptide produced, by
one of the methods described herein, or by measuring the levels of
activity of mACHR-6 or other genes. In this way, the gene
expression pattern can serve as a marker, indicative of the
physiological response of the cells to the compound. Accordingly,
this response state may be determined before, and at various points
during, treatment of the individual with the compound.
[0168] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with a compound (e.g., an agonist, antagonist, peptidomimetic,
polypeptide, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the compound; (ii)
detecting the level of expression of an mACHR-6 polypeptide, mRNA,
or genomic DNA in the preadministration sample; (iii) obtaining one
or more post-administration samples from the subject; (iv)
detecting the level of expression or activity of the mACHR-6
polypeptide, mRNA, or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mACHR-6 polypeptide, mRNA, or genomic DNA in the pre-administration
sample with the mACHR-6 polypeptide, mRNA, or genomic DNA in the
post administration sample or samples; and (vi) altering the
administration of the compound to the subject accordingly. For
example, increased administration of the compound may be desirable
to increase the expression or activity of mACHR-6 to higher levels
than detected, i.e., to increase the effectiveness of the agent.
Alternatively, decreased administration of the agent may be
desirable to decrease expression or activity of mACHR-6 to lower
levels than detected, i.e. to decrease the effectiveness of the
compound.
[0169] VI. Uses of Partial mACHR-6 Sequences
[0170] Portions or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (a) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (b) identify an individual from a minute biological sample
(tissue typing); and (c) aid in forensic identification of a
biological sample. These applications are described in the
subsections below.
[0171] a. Chromosome Mapping
[0172] Once the sequence (or a portion of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, portions or fragments of the mACHR-6,
sequences, described herein, can be used to map the location of the
mACHR-6 gene, respectively, on a chromosome. The mapping of the
mACHR-6 sequence to chromosomes is an important first step in
correlating these sequence with genes associated with disease.
[0173] Briefly, the mACHR-6 gene can be mapped to a chromosome by
preparing PCR primers (preferably)15-25 bp in length) from the
mACHR-6 sequence. Computer analysis of the mACHR-6, sequence can be
used to rapidly select primers that do not span more than one exon
in the genomic DNA, thus complicating the amplification process.
These primers can then be used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the mACHR-6 sequence
will yield an amplified fragment.
[0174] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio P. et al. (1983)
Science 220:919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[0175] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the mACHR-6 sequence to design oligonucleotide
primers, sublocalization can be achieved with panels of fragments
from specific chromosomes. Other mapping strategies which can
similarly be used to map a mACHR-6 sequence to its chromosome
include in situ hybridization (described in Fan, Y. et al. (1990)
PNAS, 87:6223-27), pre-screening with labeled flow-sorted
chromosomes, and pre-selection by hybridization to chromosome
specific cDNA libraries.
[0176] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical like colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence-as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual of Basic
Techniques (Pergamon Press, New York, 1988).
[0177] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0178] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data (such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[0179] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the mACHR-6 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0180] b. Tissue Typing
[0181] The mACHR-6 sequences of the present invention can also be
used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0182] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected portions of an
individual's genome. Thus, the mACHR-6 sequences described herein
can be used to prepare two PCR primers from the 5' and 3' ends of
the sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0183] Panels of corresponding DNA sequences from individuals,
prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The mACHR-6 sequences
of the invention uniquely represent portions of the human genome.
Allelic variation occurs to some degree in the coding regions of
these sequences, and to a greater degree in the noncoding regions.
It is estimated that allelic variation between individual humans
occurs with a frequency of about once per each 500 bases. Each of
the sequences described herein can, to some degree, be used as a
standard against which DNA from an individual can be compared for
identification purposes. Because greater numbers of polymorphisms
occur in the noncoding regions, fewer sequences are necessary to
differentiate individuals. The noncoding sequences of SEQ ID NOs:1,
4, and 31, can comfortably provide positive individual
identification with a panel of perhaps 10 to 1,000 primers which
each yield a noncoding amplified sequence of 100 bases. If
predicted coding sequences, such as those in SEQ ID NOs:3, 6, and
33, are used, a more appropriate number of primers for positive
individual identification would be 500-2,000.
[0184] If a panel of reagents from mACHR-6 sequences described
herein is used to generate a unique identification database for an
individual, those same reagents can later be used to identify
tissue from that individual. Using the unique identification
database, positive identification of the individual, living or
dead, can be made from extremely small tissue samples.
[0185] c. Use of Partial mACHR-6 Sequences in Forensic Biology
[0186] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0187] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As described
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to
noncoding regions of SEQ ID NOs:1, 4, and 31 are particularly
appropriate for this use as greater numbers of polymorphisms occur
in the noncoding regions, making it easier to differentiate
individuals using this technique. Examples of polynucleotide
reagents include the mACHR-6 sequences or portions thereof, e.g.,
fragments derived from the noncoding regions of SEQ ID NOs:1, 4,
and 31, having a length of at least 20 bases, preferably at least
30 bases.
[0188] The mACHR-6 sequences described herein can further be used
to provide polynucleotide reagents, e.g., labeled or labelable
probes which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue, e.g., brain tissue. This
can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such mACHR-6
probes can be used to identify tissue by species and/or by organ
type.
[0189] In a similar fashion, these reagents, e.g., mACHR-6 primers
or probes can be used to screen tissue culture for contamination
(i.e. screen for the presence of a mixture of different types of
cells in a culture).
[0190] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patent applications, patents, and published patent
applications cited throughout this application are hereby
incorporated by reference.
EXAMPLES
Example 1
Identification of Rat And Human mACHR-6 cDNA
[0191] In this example, mACHR-6 nucleic acid molecules were
identified by screening appropriate cDNA libraries. More
specifically, a rat frontal cortex oligo dT-primed cDNA library was
plated out and colonies picked into 96 well plates. The colonies
were cultured, plasmids were prepared from each well, and the 5'
end of each insert sequenced. After automated "trimming" of
non-insert sequences, the nucleotide sequences were compared
against the public protein databases using the BLAST sequence
comparison program (BLASTN1.3MP, Altschul et al. (1990) J. Mol.
Biol. 215:403). Upon review of the results from this sequence
comparison, a single clone was identified, designated 84g5, whose
highest similarity was with the rat muscarinic acetylcholine
receptor M1 (mACHR M1; GenBank.TM. Accession Number P08482). The
clone containing this sequence was recovered from the 96 well
plate, plasmid was prepared using standard methods and the insert
fully sequenced using standard "contigging" techniques. A repeat
BLAST analysis using the entire insert sequence once again showed
that the sequence in the protein database with the greatest
similarity corresponded to GenBank.TM. Accession Number P08482.
This sequence and the insert sequence were compared using the GAP
program in the GCG software package using a gap weight of 5.000 and
a length weight of 0.100. The results showed a 27.97% identity and
49.01% similarity between the two sequences with the insertion of 4
gaps for optimized sequence alignment. The alignment indicated that
the 84g5 clone does not extend fully across the P08482 sequence,
apparently missing approximately 30 amino acid residues at the
N-terminal region of the molecule. A probe spanning residues
143-249 of SEQ ID NO:31 was then used to re-screen the same frontal
cortex library. This resulted in the indentification of the full
length rat mACHR-6 sequence shown in SEQ ID NO:4 BLAST analysis of
public nucleotide databases revealed no equivalent human sequences.
Only a single mouse EST was identified (GenBank.TM. Accession
Number AA118949) which is similar to the 84g5 clone between
residues 1101 and 1650.
[0192] The human mACHR-6 nucleic acid molecule was identified by
screening a human cerebellum cDNA library using a Nci I/Not I
restriction fragment of the rat cDNA as a probe. BLAST analysis of
protein and nucleic acid databases in the public domain again
showed that the mACHR-6 nucleic acid molecule is most similar to
mACHR M1 sequences. The alignments also revealed that mAChR-6
nucleic acid molecule encodes a full length mACHR polypeptide.
Example 2
Tissue Expression of the mACHR-6 Gene
[0193] Northern Analysis Using RNA from Human and Rat Tissue
[0194] Human brain multiple tissue northern (MTN) blots, human MTN
I, II, and III blots, and rat MTN blots (Clontech, Palo Alto,
Calif.), containing 2 .mu.g of poly A+ RNA per lane were probed
with the rat mACHR-6 nucleotide sequence (Nci I/Not I restriction
fragment). The filters were prehybridized in 10 ml of Express Hyb
hybridization solution (Clontech, Palo Alto, Calif.) at 68.degree.
C. for 1 hour, after which 100 ng of .sup.32P labeled probe was
added. The probe was generated using the Stratagene Prime-It kit,
Catalog Number 300392 (Clontech, Palo Alto, Calif.). Hybridization
was allowed to proceed at 68.degree. C. for approximately 2 hours.
The filters were washed in a 0.05% SDS/2.times.SSC solution for 15
minutes at room temperature and then twice with a 0.1%
SDS/0.1.times.SSC solution for 20 minutes at 50.degree. C. and then
exposed to autoradiography film overnight at -80.degree. C. with
one screen. The human tissues tested included: heart, brain
(regions of the brain tested included cerebellum, corpus callosum,
cerebral cortex, medulla, occipital pole, frontal lobe, temporal
lobe, putamen, amygdala, caudate nucleus, hippocampus, substantia
nigra, subthalamic nucleus and thalamus), placenta, lung, liver,
skeletal muscle, kidney, pancreas, spleen, thymus, prostate,
testis, ovary, small intestine, colon, peripheral blood leukocyte,
stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland
and bone marrow. The rat tissues tested included: heart, brain,
spleen, lung, liver, skeletal muscle, kidney, and testis.
[0195] There was a strong hybridization to human whole brain, the
following human brain regions: cerebellum, corpus callosum,
cerebral cortex, medulla, occipital pole, frontal lobe, temporal
lobe, putamen, amygdala, caudate nucleus, hippocampus, substantia
nigra, subthalamic nucleus and thalamus; and rat brain indicating
that the approximately 3 kb mACHR-6 gene transcript is expressed in
these tissues. There was also hybridization to human spinal
cord.
[0196] In Situ Hybridization
[0197] For in situ analysis, the brain of an adult Sprague-Dawley
rat was removed and frozen on dry ice. Ten-micrometer-thick coronal
sections of the brain were postfixed with 4% formaldehyde in DEPC
treated 1.times.phosphate-buffered saline at room temperature for
10 minutes before being rinsed twice in DEPC
1.times.phosphate-buffered saline and once in 0.1 M
triethanolamine-HCl (pH 8.0). Following incubation in 0.25% acetic
anhydride-0.1 M triethanolamine-HCl for 10 minutes, sections were
rinsed in DEPC 2.times.SSC (1.times.SSC is 0.15 M NaCl plus 0.015 M
sodium citrate). Tissue was then dehydrated through a series of
ethanol washes, incubated in 100% chloroform for 5 minutes, and
then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1
minute and allowed to air dry.
[0198] Hybridizations were performed with .sup.35S-radiolabeled
(5.times.10.sup.7 cpm/ml) cRNA probes encoding a 474-bp fragment of
the rat gene (generated with PCR primers F, 5'-CAAGAACCCMTTAAGCCAAG
(SEQ ID NO:27), and R, 5'-GAAGAAGGTAACGCTGAGGA (SEQ ID NO:28)) and
a 529-bp fragment of the rat gene (generated with PCR primers F,
5'-CAGAACCCCCACCAGATGCC (SEQ ID NO:29), and R,
5'-TAGTGGCACAGTGGGTAGAG (SEQ ID NO:30)). Probes were incubated in
the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH
7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA,
0.05% yeast total RNA type X1, 1.times. Denhardt's solution, 50%
formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium
dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at
55.degree. C.
[0199] After hybridization, slides were washed with 2.times.SSC.
Sections were then sequentially incubated at 37.degree. C. in TNE
(a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1
mM EDTA), for 10 minutes, in TNE with 10 .mu.g of RNase A per ml
for 30 minutes, and finally in TNE for 10 minutes. Slides were then
rinsed with 2.times.SSC at room temperature, washed with
2.times.SSC at 50.degree. C. for 1 hour, washed with 0.2.times.SSC
at 55.degree. C. for 1 hour, and 0.2.times.SSC at 60.degree. C. for
1 hour. Sections were then dehydrated rapidly through serial
ethanol-0.3 M sodium acetate concentrations before being air dried
and exposed to Kodak Biomax MR scientific imaging film for 24 hours
and subsequently dipped in NB-2 photoemulsion and exposed at
4.degree. C. for 7 days before being developed and counter
stained.
[0200] Significant hybridization was seen in a number of brain
regions. These included the cortex, caudate putamen, hippocampus,
thalamus and cerebellum. Analysis of these regions at high
magnification showed that significant labeling was seen over the
cell bodies of neurons.
Example 3
Expression of Recombinant mACHR-6 Polypeptide in Bacterial
Cells
[0201] In this example, mACHR-6 is expressed as a recombinant
glutathione-S-transferase (GST) fusion polypeptide in E. coli and
the fusion polypeptide is isolated and characterized. Specifically,
mACHR-6 is fused to GST and this fusion polypeptide is expressed in
E. coli, e.g., strain PEB199. As the human and rat mACHR-6
polypeptides are predicted to be approximately 51.3 kDa, and 51.2
kDa, respectively, and GST is predicted to be 26 kDa, the fusion
polypeptides are predicted to be approximately 77.3 kDa and 77.2
kDa, respectively, in molecular weight. Expression of the
GST-mACHR-6 fusion polypeptide in PEB199 is induced with IPTG. The
recombinant fusion polypeptide is purified from crude bacterial
lysates of the induced PEB199 strain by affinity chromatography on
glutathione beads. Using polyacrylamide gel electrophoretic
analysis of the polypeptide purified from the bacterial lysates,
the molecular weight of the resultant fusion polypeptide is
determined.
Example 4
Expression of Recombinant mACHR-6 Polypeptide in COS Cells
[0202] To express the mACHR-6 gene in COS cells, the pcDNA/Amp
vector by Invitrogen Corporation (San Diego, Calif.) is used. This
vector contains an SV40 origin of replication, an ampicillin
resistance gene, an E. coli replication origin, a CMV promoter
followed by a polylinker region, and an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire mACHR-6
polypeptide and a HA tag (Wilson et al. (1984) Cell 37:767) fused
in-frame to its 3' end of the fragment is cloned into the
polylinker region of the vector, thereby placing the expression of
the recombinant polypeptide under the control of the CMV
promoter.
[0203] To construct the plasmid, the mACHR-6 DNA sequence is
amplified by PCR using two primers. The 5' primer contains the
restriction site of interest followed by approximately twenty
nucleotides of the mACHR-6 coding sequence starting from the
initiation codon; the 3' end sequence contains complementary
sequences to the other restriction site of interest, a translation
stop codon, the HA tag and the last 20 nucleotides of the mACHR-6
coding sequence. The PCR amplified fragment and the pcDNA/Amp
vector are digested with the appropriate restriction enzymes and
the vector is dephosphorylated using the CIAP enzyme (New England
Biolabs, Beverly, Mass.). Preferably the two restriction sites
chosen are different so that the mACHR-6 gene is inserted in the
correct orientation. The ligation mixture is transformed into E.
coli cells (strains HB101, DH5a, SURE, available from Stratagene
Cloning Systems, La Jolla, Calif., can be used), the transformed
culture is plated on ampicillin media plates, and resistant
colonies are selected. Plasmid DNA is isolated from transformants
and examined by restriction analysis for the presence of the
correct fragment.
[0204] COS cells are subsequently transfected with the
mACHR-6-pcDNA/Amp plasmid DNA using the calcium phosphate or
calcium chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of
the mACHR-6 polypeptide is detected by radiolabelling
(.sup.35S-methionine or .sup.35S-cysteine available from NEN,
Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and
Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory-Press, Cold Spring Harbor, N.Y., 1988) using an HA
specific monoclonal antibody. Briefly, the cells are labelled for 8
hours with .sup.35S-methionine (or .sup.35S-cysteine). The culture
media are then collected and the cells are lysed using detergents
(RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM
Tris, pH 7.5). Both the cell lysate and the culture media are
precipitated with an HA specific monoclonal antibody. Precipitated
polypeptides are then analyzed by SDS-PAGE.
[0205] Alternatively, DNA containing the mACHR-6 coding sequence is
cloned directly into the polylinker of the pCDNA/Amp vector using
the appropriate restriction sites. The resulting plasmid is
transfected into COS cells in the manner described above, and the
expression of the mACHR-6 polypeptide is detected by radiolabelling
and immunoprecipitation using an mACHR-6 specific monoclonal
antibody.
Example 5
Characterization of the Human and Rat mACHR-6 Polypeptides
[0206] In this example, the amino acid sequences of the human and
the rat mACHR-6 polypeptides were compared to amino acid sequences
of known polypeptides and various motifs were identified.
[0207] The human mACHR-6 polypeptide, the amino acid sequence of
which is shown in FIG. 1 (SEQ ID NO:2), is a novel polypeptide
which includes 445 amino acid residues. The human mACHR-6
polypeptide contains seven transmembrane domains between amino acid
residues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID NO:8), 109-130 (SEQ ID
NO:9), 152-174 (SEQ ID NO:10), 197-219 (SEQ ID NO:11), 360-380 (SEQ
ID NO:12), and 396-416 (SEQ ID NO:13). The nucleotide sequence of
the human mACHR-6 was used as a database query using the BLASTN
program (BLASTN1.3 MP, Altschul et al. (1990) J. Mol. Biol.
215:403). The closest hits were human, rat, mouse and pig mACHR M1
(GenBank.TM. Accession Numbers P11229, P08482, P12657, and P04761,
respectively). The highest similarity is 32/70 amino acid
identities.
[0208] The rat mACHR-6 polypeptide, the amino acid sequence of
which is shown in FIG. 2 (SEQ ID NO:5), is a novel polypeptide
which includes 445 amino acid residues. The rat mACHR-6 polypeptide
contains seven transmembrane domains between amino acid residues
34-59 (SEQ ID NO:14), 73-91 (SEQ ID NO:15), 109-130 (SEQ ID NO:16),
152-174 (SEQ ID NO:17), 197-219 (SEQ ID NO:18), 360-380 (SEQ ID
NO:19) and 396416 (SEQ ID NO:20), which correspond to the human
mACHR-6 polypeptide transmembrane domains 1-7 (SEQ ID NOs:7-13).
The nucleotide sequence of the rat mACHR-6 was used as a database
query using the BLASTN program (BLASTN1.3 MP, Altschul et al.
(1990) J. Mol. Biol. 215:403) The closest hits were human, rat,
mouse and pig mACHR M1 (GenBank.TM. Accession Numbers P11229,
P08482, P12657, and P04761, respectively). The highest similarity
is 33/70 amino acid identities. Hydropathy plots indicated that the
transmembrane domains of the rat mACHR-6 polypeptide are similar to
those of the rat mACHR M1. The cysteines (residues 63 and 44 of SEQ
ID NO:5) that give rise to intramolecular disulfide bonds are also
conserved.
[0209] Equivalents
[0210] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *
References