U.S. patent application number 13/445358 was filed with the patent office on 2013-06-13 for g-protein-conjugated receptor having altered ligand affinity, and use thereof.
This patent application is currently assigned to Nippon Chemiphar Co., Ltd.. The applicant listed for this patent is Ikunobu Muramatsu. Invention is credited to Ikunobu Muramatsu.
Application Number | 20130149715 13/445358 |
Document ID | / |
Family ID | 40074960 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130149715 |
Kind Code |
A1 |
Muramatsu; Ikunobu |
June 13, 2013 |
G-PROTEIN-CONJUGATED RECEPTOR HAVING ALTERED LIGAND AFFINITY, AND
USE THEREOF
Abstract
A modified G-protein-coupled receptor (GPCR), having modified
ligand affinity is provided by binding a G-protein-coupled receptor
to a polypeptide consisting of an amino acid sequence of SEQ ID NO:
1. Furthermore, agonists for or antagonists against the modified
GPCR are screened using a transformant in which the modified GPCR
has been expressed. This makes it possible to provide a technique
for analyzing the function of many putative GPCRs whose entities
have not been clarified.
Inventors: |
Muramatsu; Ikunobu; (Fukui,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Muramatsu; Ikunobu |
Fukui |
|
JP |
|
|
Assignee: |
Nippon Chemiphar Co., Ltd.
Chiyoda-ku
JP
Pharmacome LLC
Fukui
JP
|
Family ID: |
40074960 |
Appl. No.: |
13/445358 |
Filed: |
April 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12451558 |
Nov 18, 2009 |
8173378 |
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PCT/JP2008/059459 |
May 22, 2008 |
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13445358 |
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Current U.S.
Class: |
435/7.21 ;
435/361; 435/375; 435/455; 530/409 |
Current CPC
Class: |
G01N 33/566 20130101;
C07K 14/723 20130101; C07K 14/705 20130101; G01N 2500/00 20130101;
C12N 15/85 20130101; G01N 2333/726 20130101 |
Class at
Publication: |
435/7.21 ;
530/409; 435/361; 435/455; 435/375 |
International
Class: |
G01N 33/566 20060101
G01N033/566; C12N 15/85 20060101 C12N015/85; C07K 14/72 20060101
C07K014/72 |
Foreign Application Data
Date |
Code |
Application Number |
May 23, 2007 |
JP |
137275/2007 |
Claims
1. A protein complex of a GPCR and a polypeptide, the polypeptide
being: (A) a polypeptide consisting of an amino-acid sequence of
SEQ ID NO: 1; (B) a polypeptide (i) consisting of an amino-acid
sequence of SEQ ID NO: 1 with a deletion, insertion, substitution,
or addition of one or several amino acids, and (ii) having activity
to modify ligand affinity of a GPCR with which the polypeptide has
formed a complex; (C) a polypeptide encoded by a polynucleotide
consisting of a nucleotide sequence of SEQ ID NO: 2; (D) a
polypeptide (i) encoded by a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,
substitution, or addition of one or several nucleotides, and (ii)
having activity to modify ligand affinity of a GPCR with which the
polypeptide has formed a complex; (E) a polypeptide (i) encoded by
a polynucleotide capable of hybridizing under stringent conditions
with a polynucleotide consisting of a sequence complementary to a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex; or (F) a polypeptide (i) coded for by a
polynucleotide having a sequence identity of 70% or higher with a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2
and (ii) having activity to modify ligand affinity of a GPCR with
which the polypeptide has formed a complex.
2. The protein complex as set forth in claim 1, wherein the GPCR is
an adrenergic receptor, a dopamine receptor, a muscarinic receptor,
or an endothelin receptor.
3. The protein complex as set forth in claim 2, wherein the
adrenergic receptor is .alpha..sub.1-receptor or a
.beta..sub.1-receptor.
4. The protein complex as set forth in claim 3, wherein the
.alpha..sub.1-receptor is an .alpha..sub.1A-receptor.
5. The protein complex as set forth in claim 2, wherein the
dopamine receptor is a D2 receptor.
6. A lipid membrane containing a protein complex as set forth in
claim 1.
7. A method for producing a lipid membrane as set forth in claim 6,
comprising the step of causing a GPCR and a polypeptide to coexist
on a lipid membrane, the polypeptide being: (A) a polypeptide
consisting of an amino-acid sequence of SEQ ID NO: 1; (B) a
polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO:
1 with a deletion, insertion, substitution, or addition of one or
several amino acids, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(C) a polypeptide encoded by a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2; (D) a polypeptide (i) encoded
by a polynucleotide consisting of a nucleotide sequence of SEQ ID
NO: 2 with a deletion, insertion, substitution, or addition of one
or several nucleotides, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(E) a polypeptide (i) encoded by a polynucleotide capable of
hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (F) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex.
8. A transformant expressing a protein complex as set forth in
claim 1.
9. A method for producing a transformant as set forth in claim 8,
comprising the step of coexpressing a GPCR and a polypeptide in a
cell, the polypeptide being: (A) a polypeptide consisting of an
amino-acid sequence of SEQ ID NO: 1; (B) a polypeptide (i)
consisting of an amino-acid sequence of SEQ ID NO: 1 with a
deletion, insertion, substitution, or addition of one or several
amino acids, and (ii) having activity to modify ligand affinity of
a G-protein-coupled receptor with which the polypeptide has formed
a complex; (C) a polypeptide encoded by a polynucleotide consisting
of a nucleotide sequence of SEQ ID NO: 2; (D) a polypeptide (i)
encoded by a polynucleotide consisting of a nucleotide sequence of
SEQ ID NO: 2 with a deletion, insertion, substitution, or addition
of one or several nucleotides, and (ii) having activity to modify
ligand affinity of a GPCR with which the polypeptide has formed a
complex; (E) a polypeptide (i) encoded by a polynucleotide capable
of hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (F) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex.
10.-12. (canceled)
13. A method for producing a transformant expressing a
G-protein-coupled receptor having modified ligand affinity,
comprising the step of inhibiting expression of a polypeptide in a
cell in which a GPCR has been expressed, the polypeptide being: (A)
a polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;
(B) a polypeptide (i) consisting of an amino-acid sequence of SEQ
ID NO: 1 with a deletion, insertion, substitution, or addition of
one or several amino acids, and (ii) having activity to modify
ligand affinity of a GPCR with which the polypeptide has formed a
complex; (C) a polypeptide encoded by a polynucleotide consisting
of a nucleotide sequence of SEQ ID NO: 2; (D) a polypeptide (i)
encoded by a polynucleotide consisting of a nucleotide sequence of
SEQ ID NO: 2 with a deletion, insertion, substitution, or addition
of one or several nucleotides, and (ii) having activity to modify
ligand affinity of a GPCR with which the polypeptide has formed a
complex; (E) a polypeptide (i) encoded by a polynucleotide capable
of hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (F) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex.
14. The method as set forth in claim 13, the polypeptide is an
endogenous protein.
15. The method as set forth in claim 13, wherein the step of
inhibiting the expression of the polypeptide is performed according
to an RNAi method.
16. The method as set forth in claim 15, wherein the RNAi method is
performed by inserting an oligonucleotide consisting of a
nucleotide sequence of SEQ ID NO: 5.
17. The method as set forth in claim 13, wherein the cell is a
transformant expressing an exogenous GPCR.
18. A method for modifying ligand affinity of a GPCR, comprising
the step of inhibiting expression of a polypeptide in a cell in
which the GPCR has been expressed, the polypeptide being: (A) a
polypeptide consisting of an amino-acid sequence of SEQ ID NO: 1;
(B) a polypeptide (i) consisting of an amino-acid sequence of SEQ
ID NO: 1 with a deletion, insertion, substitution, or addition of
one or several amino acids, and (ii) having activity to modify
ligand affinity of a GPCR with which the polypeptide has formed a
complex; (C) a polypeptide encoded by a polynucleotide consisting
of a nucleotide sequence of SEQ ID NO: 2; (D) a polypeptide (i)
encoded by a polynucleotide consisting of a nucleotide sequence of
SEQ ID NO: 2 with a deletion, insertion, substitution, or addition
of one or several nucleotides, and (ii) having activity to modify
ligand affinity of a GPCR with which the polypeptide has formed a
complex; (E) a polypeptide (i) encoded by a polynucleotide capable
of hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (F) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex.
19. A method for screening an agonist for or antagonist against a
GPCR having modified ligand affinity, comprising the steps of:
inhibiting expression of a polypeptide in a cell in which a GPCR
has been expressed; and incubating the cell together with a
candidate factor, the polypeptide being: (A) a polypeptide
consisting of an amino-acid sequence of SEQ ID NO: 1; (B) a
polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO:
1 with a deletion, insertion, substitution, or addition of one or
several amino acids, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(C) a polypeptide encoded by a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2; (D) a polypeptide (i) encoded
by a polynucleotide consisting of a nucleotide sequence of SEQ ID
NO: 2 with a deletion, insertion, substitution, or addition of one
or several nucleotides, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(E) a polypeptide (i) encoded by a polynucleotide capable of
hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (F) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex.
20. The method as set forth in claim 19, further comprising the
step of measuring an intracellular Ca.sup.2+ concentration or the
step of measuring metabolism of intracellular inositol phosphate.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
Ser. No. 12/451,558, filed Nov. 18, 2009, now U.S. Pat. No.
8,173,378, which is the 35 U.S.C. .sctn.371 national stage of PCT
application PCT/JP2008/059459, filed May 22, 2008, which claims
benefit of Japanese Application 2007-137275, filed May 23, 2007,
the disclosures of all of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to G-protein-coupled receptors
(GPCRs) having modified ligand affinity and use thereof and, more
particularly, the present invention relates to a GPCR having ligand
affinity modified by forming a complex with a particular protein
and use thereof.
BACKGROUND ART
[0003] Many reactions in living organisms are caused by entrance of
extracellular information into cells and by propagation of the
information in the cells. Membrane receptors serve as mediators
through which extracellular information is transmitted into cells.
Among them, GPCRs having seven transmembrane domains is well known
as a major category of membrane receptors.
[0004] When a ligand (such as amino acids, peptides or amines)
binds to a GPCR, the GPCR transmits its information into a cell via
a trimeric G protein. G proteins coupled to GPCRs are classified
into Gs, Gi, Gq, and the like, which activate/inactivate different
effector pathways (e.g., cAMP pathway, cGMP pathway, and
phospholipase C pathway), respectively. For example,
.alpha..sub.1-adrenergic receptor is mainly coupled to Gq protein
to promote phospholipase C system, which produces diacylglycerol
and inositol trisphosphate, thus increasing intracellular
Ca.sup.2+. .alpha..sub.2-adrenergic receptor is mainly coupled to
Gi protein to suppress adenylate cyclase system, thus decreasing
cAMP. Further, .beta.-adrenergic receptor is mainly coupled to Gs
protein to promote adenylate cyclase system, which thus increases
cAMP.
[0005] GPCRs widely occur and function in our body. For example,
.alpha..sub.1-adrenergic receptors exist peripherally in blood
vessel, prostate, and produce the contractions. Further,
.alpha..sub.1-adrenergic receptors are known to function in central
nervous system. .beta.-adrenergic receptors in heart and fat tissue
play important roles in heart rate and lipolysis.
[0006] GPCRs and their signal transduction systems are known not
only to control the physiological homeostasis in our body but also
to be involved in pathophysiological status of various diseases.
Therefore, in order to treat the diseases it will be very
significant to identify the GPCRs which are related to the diseases
and then to develop their specific drugs (such as agonists or
antagonists). [0007] Non Patent Literature 1 [0008] Muramatsu, I.
et al. Br. J. Pharmacol., 99: 197 (1990) [0009] Non Patent
Literature 2 [0010] Muramatsu, I. et al. Pharmacol. Commun., 6: 23
(1995) [0011] Non Patent Literature 3 [0012] Morishima, S. et al.
J. Urol. 177: 377-381 (2007) [0013] Non Patent Literature 4 [0014]
Molenaar, P. and Parsonage, W. A. Trends Pharmacol. Sci., 26:
368-375 (2005) [0015] Non Patent Literature 5 [0016] Samaha, A. N.
et al., J. Neurosci. 27: 2979-2986 (2007)
SUMMARY OF INVENTION
[0017] There has been significant progress in GPCR research,
whereby a large number of GPCRs have been identified. However,
there still exist several putative GPCRs whose phenotypes are
identified but whose entities are yet unknown. For example,
.alpha..sub.1-adrenergic receptors are now classified into three
subtypes (.alpha..sub.1A, .alpha..sub.1B and .alpha..sub.1D) based
on their distinct genes. However, in addition to the classical
.alpha..sub.1-adrenergic receptors, the presence of an additional
subtype (.alpha..sub.1L) which shows different pharmacological
profile (phenotype) from the classical subtypes has been proposed.
The .alpha..sub.1L-subtype has significantly lower affinity for a
representative .alpha..sub.1 blocker (prazosin) than
.alpha..sub.1A-, .alpha..sub.1B- and .alpha..sub.1D-subtypes. The
.alpha..sub.1L-subtype can be detected only in intact strips or
segments of native tissues but not be identified in their tissue
homogenates (see Non Patent Literatures 1 to 3). Further, subtypes
of .beta..sub.1 adrenaline receptor (.beta..sub.1H and
.beta..sub.1L) that differ in phenotype are known to be expressed
from the same genes (see Non Patent Literature 4). Such a subtype
only has its phenotype known, and has its entity unknown. The
similar cases may be also pointed out for dopamine receptors,
muscarinic receptors, or endothelin receptors (see Non Patent
Literature 5 and the like). However, the underlying mechanisms for
different phenotype formation of the same gene product have not yet
known.
[0018] The present invention has been made in view of this problem,
and it is an object of the present invention to provide a technique
for analyzing the function of a G-protein-coupled receptor whose
entity has not been clarified.
[0019] A protein complex according to the present invention is
characterized by binding of a GPCR to (1) a polypeptide consisting
of an amino-acid sequence of SEQ ID NO: 1; (2) a polypeptide (i)
consisting of an amino-acid sequence of SEQ ID NO: 1 with a
deletion, insertion, substitution, or addition of one or several
amino acids, and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; (3) a
polypeptide encoded by a polynucleotide consisting of a nucleotide
sequence of SEQ ID NO: 2; (4) a polypeptide (i) encoded by a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2
with a deletion, insertion, substitution, or addition of one or
several nucleotides, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(5) a polypeptide (i) encoded by a polynucleotide capable of
hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (6) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex.
[0020] A method according to the present invention for producing a
protein complex is characterized by including the step of causing a
GPCR and a polypeptide to coexist on a lipid membrane, the
polypeptide being (1) a polypeptide consisting of an amino-acid
sequence of SEQ ID NO: 1; (2) a polypeptide (i) consisting of an
amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion,
substitution, or addition of one or several amino acids, and (ii)
having activity to modify ligand affinity of a GPCR with which the
polypeptide has formed a complex; (3) a polypeptide encoded by a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;
(4) a polypeptide (i) encoded by a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,
substitution, or addition of one or several nucleotides, and (ii)
having activity to modify ligand affinity of a GPCR with which the
polypeptide has formed a complex; (5) a polypeptide (i) encoded by
a polynucleotide capable of hybridizing under stringent conditions
with a polynucleotide consisting of a sequence complementary to a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex; or (6) a polypeptide (i) coded for by a
polynucleotide having a sequence identity of 70% or higher with a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2
and (ii) having activity to modify ligand affinity of a GPCR with
which the polypeptide has formed a complex.
[0021] With this feature, the present invention can modify the
ligand affinity of a GPCR. That is, the method according to the
present invention for producing a protein complex can also be a
method for modifying the affinity of a GPCR for its ligands.
[0022] A lipid membrane according to the present invention is
characterized by containing the protein complex. A method according
to the present invention for producing the lipid membrane is
characterized by including the step of causing a GPCR and a
polypeptide to coexist on the lipid membrane, the polypeptide being
(1) a polypeptide consisting of an amino-acid sequence of SEQ ID
NO: 1; (2) a polypeptide (i) consisting of an amino-acid sequence
of SEQ ID NO: 1 with a deletion, insertion, substitution, or
addition of one or several amino acids, and (ii) having activity to
modify ligand affinity of a G-protein-coupled receptor with which
the polypeptide has formed a complex; (3) a polypeptide encoded by
a polynucleotide consisting of a nucleotide sequence of SEQ ID NO:
2; (4) a polypeptide (i) encoded by a polynucleotide consisting of
a nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,
substitution, or addition of one or several nucleotides, and (ii)
having activity to modify ligand affinity of a GPCR with which the
polypeptide has formed a complex; (5) a polypeptide (i) encoded by
a polynucleotide capable of hybridizing under stringent conditions
with a polynucleotide consisting of a sequence complementary to a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex; or (6) a polypeptide (i) coded for by a
polynucleotide having a sequence identity of 70% or higher with a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2
and (ii) having activity to modify ligand affinity of a GPCR with
which the polypeptide has formed a complex.
[0023] Furthermore, a transformant according to the present
invention is characterized by containing the protein complex. A
method according to the present invention for producing the
transformant is characterized by including the step of expressing
the protein complex, and preferably includes the step of
introducing, into a cell, a gene encoding a GPCR and a gene
encoding the polypeptide.
[0024] With this feature, the present invention makes it easy to
analyze the function of a GPCR having modified ligand affinity.
[0025] A method according to the present invention for screening
agonists or antagonists of a GPCR having modified ligand affinity
is characterized by including the steps of: [I] generating a
protein complex by causing a GPCR and a polypeptide to coexist on a
lipid membrane; and [II] incubating the protein complex together
with a candidate factor, the polypeptide being (1) a polypeptide
consisting of an amino-acid sequence of SEQ ID NO: 1; (2) a
polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO:
1 with a deletion, insertion, substitution, or addition of one or
several amino acids, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(3) a polypeptide encoded by a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2; (4) a polypeptide (i) encoded
by a polynucleotide consisting of a nucleotide sequence of SEQ ID
NO: 2 with a deletion, insertion, substitution, or addition of one
or several nucleotides, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(5) a polypeptide (i) encoded by a polynucleotide capable of
hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (6) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex.
[0026] A method according to the present invention for producing a
transformant expressing a GPCR having modified ligand affinity is
characterized by including the step of inhibiting expression of a
polypeptide in the cell in which a GPCR has been expressed, the
polypeptide being (1) a polypeptide consisting of an amino-acid
sequence of SEQ ID NO: 1; (2) a polypeptide (i) consisting of an
amino-acid sequence of SEQ ID NO: 1 with a deletion, insertion,
substitution, or addition of one or several amino acids, and (ii)
having activity to modify ligand affinity of a GPCR with which the
polypeptide has formed a complex; (3) a polypeptide encoded by a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2;
(4) a polypeptide (i) encoded by a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,
substitution, or addition of one or several nucleotides, and (ii)
having activity to modify ligand affinity of a GPCR with which the
polypeptide has formed a complex; (5) a polypeptide (i) encoded by
a polynucleotide capable of hybridizing under stringent conditions
with a polynucleotide consisting of a sequence complementary to a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex; or (6) a polypeptide (i) coded for by a
polynucleotide having a sequence identity of 70% or higher with a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2
and (ii) having activity to modify ligand affinity of a GPCR with
which the polypeptide has formed a complex.
[0027] With this feature, the present invention makes it easy to
analyze the function of a GPCR having modified ligand affinity, and
can modify the ligand affinity of a GPCR. That is, the present
invention can also be a method for modifying the affinity of a GPCR
for its ligands.
[0028] The producing method according to the present invention is
preferably such that the polypeptide is an endogenous protein, and
that the step of inhibiting the expression of the polypeptide is
performed according to an RNAi method. Further, the cell may be a
transformant expressing an exogenous GPCR.
[0029] A method according to the present invention for screening
agonists or antagonists of a GPCR having modified ligand affinity
is characterized by including the steps of: [I] inhibiting
expression of a polypeptide in the cell in which a GPCR has been
expressed; and [II] incubating the cell together with a candidate
factor, the polypeptide being (1) a polypeptide consisting of an
amino-acid sequence of SEQ ID NO: 1; (2) a polypeptide (i)
consisting of an amino-acid sequence of SEQ ID NO: 1 with a
deletion, insertion, substitution, or addition of one or several
amino acids, and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; (3) a
polypeptide encoded by a polynucleotide consisting of a nucleotide
sequence of SEQ ID NO: 2; (4) a polypeptide (i) encoded by a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2
with a deletion, insertion, substitution, or addition of one or
several nucleotides, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(5) a polypeptide (i) encoded by a polynucleotide capable of
hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (6) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex. It is preferable that the screening method
according to the present invention further include the step of
measuring an intracellular Ca.sup.2+ concentration or the step of
measuring metabolism of intracellular inositol phosphate.
[0030] In the present invention, it is preferable that the GPCR
constituting the protein complex be an adrenergic receptor, a
dopamine receptor, a muscarinic receptor, or an endothelin
receptors, and the adrenergic receptors may be .alpha.-receptor or
.beta.-receptor. Further, it is preferable that the dopamine
receptor be a D2 receptor. It is preferable that the
.alpha.-receptor be an .alpha..sub.1-receptor, and it is more
preferable that the .alpha..sub.1-receptor be an
.alpha..sub.1A-subtype. In a preferred embodiment, a protein
complex according to the present invention is
.alpha..sub.1L-subtype or .beta..sub.1L-subtype of adrenergic
receptors.
[0031] For a fuller understanding of the nature and advantages of
the invention, reference should be made to the ensuing detailed
description taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0032] FIG. 1
[0033] FIG. 1 shows saturation binding curves for
[.sup.3H]-silodosin in .alpha..sub.1L cells (whole cell binding
experiment).
[0034] FIGS. 2(a) and (b)
[0035] (a) of FIG. 2 shows competition-binding curves for prazosin
in .alpha..sub.1L cells and .alpha..sub.1A cells (whole cell
binding experiment). (b) of FIG. 2 shows competition-binding curves
for prazosin in homogenates of .alpha..sub.1L cells and
.alpha..sub.1A cells. Dashed line with closed squares: A
competition-binding curve for prazosin in whole .alpha..sub.1L
cells was described for comparison against the curves in
homogenates.
DESCRIPTION OF EMBODIMENTS
[0036] [1: Protein Complex]
[0037] The present invention provides a protein complex of a GPCR
and a particular polypeptide. When used in the present
specification, the term "complex" means an integrated combination
of a plurality of substances, and "complex formation" and
"integration" are used interchangeably. It should be noted that a
plurality of substances forming a complex only need to interact
with each other in close proximity and may or may not bind to each
other. In a preferred embodiment, a protein complex according to
the present invention is such a state that a GPCR and a particular
polypeptide interact with each other in close proximity and, as a
result, functions as a GPCR having modified ligand affinity.
[0038] The present specification describes the present invention by
taking an adrenergic receptor (in particular, of
.alpha..sub.1A-subtype) as an example of a GPCR whose ligand
affinity is modified by interacting with a particular polypeptide.
However, a person skilled in the art who has read the present
specification would easily understand that a GPCR constituting the
present invention is not limited to an adrenergic receptor.
Further, a person skilled in the art could easily obtain
information on the sequence of a GPCR constituting the present
invention.
[0039] Adrenergic receptors are well known to mediate the functions
of the autonomic nervous system (sympathetic nervous system).
Binding of adrenaline, noradrenaline, or the like to the adrenergic
receptor causes various physiological responses such as
contractions of vascular smooth muscle, increases in blood pressure
and heart rate, dilation of the pupil, and an increase in blood
glucose level.
[0040] The progress in research and the development of a new
variety of drugs have made clear that adrenergic receptors are
classified into various types. In the past, adrenergic receptors
were classified into .alpha.-receptor and .beta.-receptor according
to their difference in reactivity to drugs such as isoproterenol
and phentolamine, and further classified into pharmacological
subtypes (.alpha..sub.1-receptor, .alpha..sub.2-receptor, and
.beta.-receptor). These receptors are known to exhibit different
distributions in each tissue and to play different functions. For
example, the .alpha..sub.1-adrenergic receptors are known to play
as an important mediator causing contractions of vascular smooth
muscle, prostate, and the like, and also known to be involved in
the regulation of consciousness and emotion in central nervous
system.
[0041] Now that the mapping of the human genome has been finished,
the structures and functions of many proteins can be shown on a
genetic level. In .alpha..sub.1-adrenergic receptors, three
subtypes (.alpha..sub.1A, .alpha..sub.1B, and .alpha..sub.1D) were
identified according to their distinct genes. These classical
subtypes are known to coincide well with pharmacologically
identified subtypes. In this way, it may be considered that one
receptor subtype is basically originated from one distinct
gene.
[0042] However, it has been long pointed out that a unique
.alpha..sub.1-adrenergic receptor occurs and functions in some
tissues of our body. Because of its low affinity for a
representative .alpha..sub.1 blocker (prazosin), the unique subtype
has been called ".alpha..sub.1L-subtype", although the
corresponding gene has not been yet cloned.
[0043] Table 1 shows the classification and drug selectivity of
.alpha..sub.1-adrenergic receptors.
TABLE-US-00001 TABLE 1 Classification and Drug Selectivity of
.alpha..sub.1-adrenergic Receptors Affinity (pKb) Subtypes
silodosin.sup.1, 3, 4, 5 tamsulosin.sup.1, 3, 4 prazosin.sup.1, 2,
3, 4, 5 RS- 17053.sup.2, 4 BMY 7378.sup.4, 5 .alpha.1A 10.7-9.5
10.4-9.9 10.6-9.3 9.1-8.4 6.9-5.6 .alpha.1L 10.7-9.5 10.4-9.9
8.3-7.6 6.3 6.9-5.6 .alpha.1B 8.1 9.3 10.6-10.1 7.8 7.4 .alpha.1D
8.6 9.9 10.1-9.9 7.8 9.1
[0044] The effects of the compounds shown in Table 1 are based on
the following literatures: [0045] 1. Muramatsu, I. et al.
Pharmacol. Commun., 6: 23 (1995) [0046] 2. Ford, A P. et al. Mol.
Pharmacol., 49: 209 (1996) [0047] 3. Murata, S. et al. J. Urol.,
164: 578 (2000) [0048] 4. Hiraizumi-Hiraoka, Y. et al. J.
Pharmacol. Exp. Ther., 310: 995 (2004) [0049] 5. Murata, S. et al.
Br. J. Pharmacol., 127: 19 (1999)
[0050] In bioassay studies, the .alpha..sub.1L-subtype has been
clearly demonstrated as a functional receptor in the lower urinary
tract systems of human and other mammals. The
.alpha..sub.1L-subtype was also identified, if the intact segments
of native tissues (e.g. human prostate). However, the
.alpha..sub.1L-subtype was not detected by a conventional binding
experiment conducted with homogenized tissue. The fact that
.alpha..sub.1L-subtype is not detected in homogenized tissue means
that purification of the .alpha..sub.1L-subtype from tissue is very
difficult. Unless the entity of the .alpha..sub.1L-subtype is
clarified, it will be very difficult to analyze the function of
.alpha..sub.1L-adrenergic receptor, in particular, to develop
.alpha..sub.1L-selective drugs (such as agonists or
antagonists).
[0051] The inventors assumed that the .alpha..sub.1L-subtype, whose
entity is unknown, is constituted by binding of some sort of
ancillary molecule to an already known subtype (probably,
.alpha..sub.1A-subtype). That is, the inventors assumed that the
interaction between the subtype molecule and the molecule ancillary
thereto, which constitute the .alpha..sub.1L-subtype together, is
dissolved by homogenizing tissue and, as a result, the
.alpha..sub.1L-subtype changes its properties into those of the
.alpha..sub.1A-subtype. In the result, the inventors found that the
pharmacological profile of .alpha..sub.1L-subtype was converted to
that of .alpha..sub.1A-subtype upon tissue homogenization.
Furthermore, as a result of their diligent studies, the inventors
confirmed that a particular protein binds to the
.alpha..sub.1A-subtype, and that coexpression of the protein with
the .alpha..sub.1A-subtype in a cultured cell leads to expression
of a phenotype of .alpha..sub.1L-subtype. That is, the inventors
found that the .alpha..sub.1L-subtype consists of a protein
complex, identified a protein constituting the complex, and thereby
accomplished the present invention.
[0052] In one embodiment, the present invention provides a protein
complex that forms an .alpha..sub.1L-subtype of adrenergic
receptor. That is, a protein complex according to the present
embodiment is an .alpha..sub.1L-subtype of adrenergic receptor. The
present embodiment makes it possible to provide treatment for any
disease associated with an .alpha..sub.1L-subtype whose entity has
been clarified.
[0053] A polypeptide constituting the protein complex according to
the present embodiment is already publicly known as a CRELD
(cysteine-rich with EGF-like domains) 1.alpha. protein, whose
missense mutation is pointed out as being associated with an
atrioventricular septal defect (Gene 293: 47-57 (2002), Am. J. Hum.
Genet. 72: 1047-1052 (2003)). Information on the sequence of the
polypeptide is provided as NCBI Accession No. NM.sub.--015513 and,
in the present specification, represented as SEQ ID NO: 1
(amino-acid sequence) and SEQ ID NO: 2 (nucleotide sequence). That
is, the polypeptide constituting the protein complex according to
the present embodiment may be a polypeptide consisting of an
amino-acid sequence of SEQ ID NO: 1, or may be a polypeptide coded
for by a polynucleotide consisting of a nucleotide sequence of SEQ
ID NO: 2.
[0054] The polypeptide constituting the protein complex according
to the present embodiment is not limited to a polypeptide
consisting of an amino-acid sequence of SEQ ID NO: 1, and may be a
mutant polypeptide retaining the activity of the original
polypeptide. An example of such a mutant polypeptide is a
polypeptide (i) consisting of an amino-acid sequence of SEQ ID NO:
1 with a deletion, insertion, substitution, or addition of one or
several amino acids, and (ii) having the activity to form a complex
with a GPCR and modify the ligand affinity of the GPCR.
[0055] With technical common sense in the field, a person skilled
in the art could easily produce a mutant polypeptide, in the
amino-acid sequence of a particular polypeptide with a deletion,
insertion, substitution, or addition of one or several amino acids.
Further, based on the descriptions in the present specification and
the technical common sense, a person skilled in the art could
easily confirm whether or not the mutant polypeptide retains the
same activity as the original polypeptide.
[0056] Further, the polynucleotide encoding the polypeptide
constituting the protein complex according to the present
embodiment is not limited to a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2, and only needs to be a mutant
polynucleotide encoding a polypeptide retaining the activity to
modify the ligand affinity of a GPCR with which the polypeptide has
formed a complex. An example of such a mutant polynucleotide is,
but is not limited to, (1) a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 with a deletion, insertion,
substitution, or addition of one or several nucleotides, (2) a
polynucleotide capable of hybridizing under stringent conditions
with a polynucleotide consisting of a sequence complementary to a
nucleotide sequence of SEQ ID NO: 2, or (3) a polynucleotide having
a sequence identity of 70% or higher, preferably 80% or higher, or
more preferably 85% or higher, with a polynucleotide consisting of
a nucleotide sequence of SEQ ID NO: 2.
[0057] With technical common sense in the field, a person skilled
in the art could easily produce: a mutant polypeptide, in the
amino-acid sequence of a particular polynucleotide with a deletion,
insertion, substitution, or addition of one or several amino acids;
a mutant polynucleotide capable of hybridizing under stringent
conditions with a particular polynucleotide; or a mutant
polynucleotide having a sequence identity of 70% or higher with a
particular polynucleotide. Further, based on the descriptions in
the present specification and the technical common sense, a person
skilled in the art could easily confirm whether or not a
polypeptide coded for by the mutant polynucleotide retains the same
activity as a polypeptide coded for by the original
polynucleotide.
[0058] When used in the present specification, the term "stringent
hybridization conditions" means overnight incubation at 42.degree.
C. in a hybridization solution (containing 50% formamide,
5.times.SSC [150 mM of NaCl, 15 mM of trisodium citrate], 50 mM of
sodium phosphate (with a pH of 7.6), 5.times.Denhardt's solution,
10% dextran sulfate, and 20 .mu.g/ml of sheared and denatured
salmon sperm), followed by washing of a filter at approximately
65.degree. C. in 0.1.times.SSC.
[0059] A specific procedure for hybridization only needs to be
performed according to a method well known in the field (e.g., a
method described in "Molecular Cloning: A Laboratory Manual, 3rd
ed., J. Sambrook and D. W. Russell ed., Cold Spring Harbor
Laboratory, NY (2001)" [which is incorporated by a reference to the
present specification]).
[0060] The present invention thus far has been described by taking
an adrenergic receptor as an example of a GPCR constituting a
protein complex according to the present invention. However, a
protein complex according to the present invention only needs to be
a complex of a GPCR and (1) a polypeptide consisting of an
amino-acid sequence of SEQ ID NO: 1; (2) a polypeptide (i)
consisting of an amino-acid sequence of SEQ ID NO: 1 with a
deletion, insertion, substitution, or addition of one or several
amino acids, and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; (3) a
polypeptide encoded by a polynucleotide consisting of a nucleotide
sequence of SEQ ID NO: 2; (4) a polypeptide (i) encoded by a
polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 2
with a deletion, insertion, substitution, or addition of one or
several nucleotides, and (ii) having activity to modify ligand
affinity of a GPCR with which the polypeptide has formed a complex;
(5) a polypeptide (i) encoded by a polynucleotide capable of
hybridizing under stringent conditions with a polynucleotide
consisting of a sequence complementary to a nucleotide sequence of
SEQ ID NO: 2 and (ii) having activity to modify ligand affinity of
a GPCR with which the polypeptide has formed a complex; or (6) a
polypeptide (i) coded for by a polynucleotide having a sequence
identity of 70% or higher with a polynucleotide consisting of a
nucleotide sequence of SEQ ID NO: 2 and (ii) having activity to
modify ligand affinity of a GPCR with which the polypeptide has
formed a complex. That is, the GPCR constituting the protein
complex is not limited to an adrenergic receptor, but may be
dopamine receptors, muscarinic receptors, or endothelin receptors.
Further, a person skilled in the art could easily obtain, from a
publicly-known database, information on the sequence of a GPCR
constituting the present invention, and therefore could easily
produce a desired protein complex, based on the descriptions in the
present specification and the technical common sense.
[0061] [2. Lipid Membrane Containing a Protein Complex]
[0062] The present invention also provides a method for producing a
protein complex of a GPCR and a particular polypeptide. The method
according to the present invention for producing a protein complex
is characterized by including the step of causing a GPCR and a
particular polypeptide to coexist on a lipid membrane. That is, the
present invention provides a lipid membrane containing a protein
complex and a method for producing the same.
[0063] A lipid membrane according to the present invention is
characterized by containing such a protein complex as described
above. The lipid membrane according to the present invention may be
a naturally-occurring lipid membrane, or may be an artificial lipid
membrane. In cases where the lipid membrane is a
naturally-occurring lipid membrane, the lipid membrane is intended
to be a biological membrane. In cases where the lipid membrane is
an artificial lipid membrane, the lipid membrane is intended to be
a lipid planar membrane or liposome.
[0064] In one aspect of the present invention, a lipid membrane can
be a biological membrane of a transformant having introduced
thereinto a polynucletoide encoding a polypeptide constituting a
protein complex according to the present invention. Such a
transformant can be obtained by introducing an expression vector
containing the polynucleotide into a living organism so that the
polypeptide is expressed in the form of a biological membrane. It
should be noted that the living organism for use as the
transformant may be a prokaryotic organism or a eucaryotic
organism.
[0065] In another aspect of the present invention, a lipid membrane
can be a lipid bilayer containing a polypeptide constituting a
protein complex of the present invention. The lipid bilayer is a
membranous structure composed of two layers of polar lipid (in
particular, phospholipid). The lipid bilayer structure is
stabilized as a two-dimensional structure when it takes the form of
a sphere, but can be a planar structure if its end is isolated from
a water molecule. When used in the present specification, the term
"liposome" means a spherical lipid bilayer that is made
artificially, and the term "lipid planar membrane" means a planar
lipid bilayer that is made artificially. In the field, an
artificial lipid bilayer is used in in vitro measurement of the
activity of a membrane protein (e.g., a channel protein). In this
way, a person skilled in the art could easily produce a lipid
planar membrane and cause the lipid planar membrane to retain a
protein (polypeptide) of interest. Further, liposome is a lipid
artificial membrane that is referred to also as "vesicle", and can
be produced by beating up a suspension of lipid (e.g.,
phospholipid) and then subjecting it to ultrasonication. In the
field, researches have been widely conducted with liposome as a
cell membrane model or as a means of drug delivery system (DDS). In
this way, a person skilled in the art could easily produce liposome
and cause the liposome to retain a protein (polypeptide) of
interest.
[0066] In one embodiment, the present invention provides a lipid
membrane containing an .alpha..sub.1L-subtype of adrenergic
receptor. As described above, the .alpha..sub.1L-subtype of
adrenergic receptor is a protein complex of an
.alpha..sub.1A-subtype molecule and a CRELD1.alpha. protein. The
.alpha..sub.1A-subtype of adrenergic receptor and the CRELD1.alpha.
are both membrane-bound proteins, and, as described above, their
interaction with each other is dissolved by homogenization
(membrane disruption). That is, their interaction with each other
is expressed by placing and retaining both of them on a lipid
membrane.
[0067] In cases where the lipid membrane according to the present
embodiment is a biological membrane, a method according to the
present embodiment for producing a lipid membrane can also be a
method for producing a transformant that expresses an
.alpha..sub.1L-subtype of adrenergic receptor, and only needs to
include the step of coexpressing an .alpha..sub.1A-adrenergic
receptor protein and a CRELD1.alpha. protein. Information on the
sequence of the .alpha..sub.1A-subtype of adrenergic receptor is
provided as NCBI Accession No. U03866 or NM.sub.--000680 and, in
the present specification, represented as SEQ ID NO: 3 (amino-acid
sequence) and SEQ ID NO: 4 (nucleotide sequence). That is, a method
according to the present embodiment for producing a protein complex
only needs to include the step of transforming a host with a vector
containing a polynucleotide consisting of a nucleotide sequence of
SEQ ID NO: 2 and a vector containing a polynucleotide consisting of
a nucleotide sequence of SEQ ID NO: 4.
[0068] It is preferable that the vectors be each an expression
vector having a polynucleotide of interest operably linked
therewith. When used in the present specification, the term
"operably linked" means that a polynucleotide encoding a peptide
(or protein) of interest is under control of a control region such
as a promoter and is in such a form that the peptide (or protein)
can be expressed in a host. A procedure for establishing a desired
vector by "operably linking" a polynucleotide encoding a peptide of
interest with an expression vector is well known in the field.
Further, a method for introducing an expression vector into a host
is well known, too, in the field. Therefore, a person skilled in
the art could easily generate a desired peptide in a host.
[0069] When used in the present specification, the term
"transformant" means not only a cell, tissue, or an organ, but also
an individual living organism. Examples of a living organism
serving as an object of transformation include, but are not limited
to, various microorganisms, plants, and animals. It should be noted
that a transformant according to the present invention only needs
to have introduced thereinto at least a polynucleotide encoding a
polypeptide constituting a protein complex according to the present
invention and express such polypeptides. That is, it should be
noted that a transformant generated by means other than an
expression vector is encompassed, too, in the technical scope of
the present invention. Further, although it is preferable that a
transformant according to the present invention be stably
expressing a polypeptide constituting a protein complex according
to the present invention, the polypeptide of interest may be
transiently expressed. It should be noted that use of a
transformant coexistent with a protein complex according to the
present embodiment makes it possible to screen an agonist for or
antagonist against the complex by observing the behavior of a
second messenger in a host cell.
[0070] In cases where the lipid membrane according to the present
embodiment is an artificial membrane, the method according to the
present embodiment for producing a lipid membrane can also be a
method for producing a lipid planer membrane or liposome containing
an .alpha..sub.1L-subtype of adrenergic receptor and, more
specifically, only needs to include the step of reconstituting an
.alpha..sub.1A-adreneregic receptor protein and a CRELD1.alpha.
protein on an artificial planer membrane. With common sense in the
field, a person skilled in the art could easily reconstitute a
membrane protein on an artificial lipid membrane. Use of an
artificial lipid membrane coexistent with a protein complex
according to the present embodiment makes it possible to measure
the ligand affinity of the complex.
[0071] [3. Use of a Lipid Membrane]
[0072] A person skilled in the art would easily understand that the
above-described method for producing a lipid membrane can be both a
method for producing a protein complex and a method for modifying
the ligand affinity of a GPCR. That is, the present invention
provides a method for modifying the affinity of a GPCR for its
ligands.
[0073] Further, use of the lipid membrane according to the present
invention makes it possible to screen an agonist for or antagonist
against a GPCR having modified ligand affinity. That is, the
present invention provides a method for screening agonists for or
antagonists against a GPCR having modified ligand affinity.
[0074] The screening method according to the present invention is
characterized by including the steps of generating such a protein
complex as described above on a lipid membrane; and incubating the
protein complex together with a candidate factor. When used in the
present specification, "incubating" means causing a plurality of
substances to coexist and putting them in such a state that they
make sufficient contact with each other.
[0075] In a preferred embodiment, the screening method according to
the present invention includes the step of incubating, together
with a candidate factor, a transformant containing a GPCR of
interest (e.g., a cell having .alpha..sub.1L-subtype of adrenergic
receptor expressed therein). The screening method according to the
present embodiment makes it possible to screen agonists for or
antagonists against a GPCR of interest (e.g., an
.alpha..sub.1L-subtype of adrenergic receptor) by measuring the
fluctuation of a second messenger in a cell (e.g., the amount of
cAMP in cases where the G protein is Gs protein or Gi protein or
the concentrations of inosine triphosphate and Ca.sup.2+ in cases
where the G protein is Gq protein).
[0076] The agonists for .alpha..sub.1L-subtype of adrenergic
receptor can be used as a drug for urinary incontinence, a
mydriatic drug, a drug for glaucoma, and a drug for central
stimulation. The antagonists against .alpha..sub.1L-subtype of
adrenergic receptor can be used as a drug for urinary disturbance,
a drug for Raynaud's disease, a drug for microcirculatory failure,
an antihypertensive drug, a central depressant, a drug for
improvement of renal blood flow, and a diuretic drug. Further,
since .beta..sub.1L-subtype of adrenergic receptor is known to be
associated with heart disease and dopamine D2 receptor and
muscarinic receptor are known to be associated with central nervous
system disease, agonists or antagonists thereto are useful,
too.
[0077] [4. Knockdown Cell and Use Thereof]
[0078] The present invention also provides a transformant in which
a polypeptide that forms a protein complex with a GPCR has been
knocked down and a method for producing the same. A method
according to the present invention for producing a transformant is
characterized by including the step of inhibiting expression of an
endogenous polypeptide.
[0079] For example, the CRELD1.alpha. protein, which is a
polypeptide that forms a complex with a GPCR, is expressed in
various cells. Therefore, a transformant in which expression of an
endogenous CRELD1.alpha. protein has been inhibited is very useful
as a control cell that is used in analyzing the function of the
protein complex or screening a target compound (e.g., an agonist or
antagonist). The knockdown cell, described in this section, may be
used alone, but is preferably used in combination with the
above-described protein complex or the above-described lipid
membrane containing the protein complex. It should be noted that
the GPCR expressed in the object cell may be endogenous or
exogenous.
[0080] In one aspect, the present invention can include the step of
inhibiting expression of an endogenous CRELD protein in a cell in
which a GPCR has been expressed. This makes it possible to modify
the ligand affinity of the GPCR expressed in the cell. That is, the
method according to the present invention for producing a
transformant can be both a method for producing a transformant
expressing a GPCR having modified ligand affinity and a method for
modifying the affinity of a GPCR for its ligand. In one embodiment,
the present invention can include the step of inhibiting expression
of an endogenous CRELD1.alpha. protein in a cell expressing an
.alpha..sub.1L-subtype of adrenergic receptor. This makes it
possible to modify a cell indicative of a phenotype of
.alpha..sub.1L-subtype to be a cell indicative of a phenotype of
.alpha..sub.1A-subtype.
[0081] It is preferable that an RNAi method be used as a technique
for inhibiting expression of an endogenous CRELD1.alpha. protein.
The RNAi method is a technique well known in the field; for
example, expression of an endogenous CRELD1.alpha. protein can be
successfully inhibited by introducing, into a cell of interest, an
oligonucleotide consisting of a nucleotide sequence of SEQ ID NO:
5. In the case of use of a vector for introducing, into a cell of
interest, an oligonucleotide consisting of a nucleotide sequence of
SEQ ID NO: 5, it is preferable that an oligonucleotide consisting
of a nucleotide sequence of SEQ ID NO: 6 be used as an antisense,
without implying any limitation.
[0082] The present invention further provides a method,
characterized by using the above-described transformant, for
screening an agonist for or antagonist against a GPCR having
modified ligand affinity.
[0083] The present invention is not limited to the description of
the embodiments above, but may be altered by a skilled person
within the scope of the claims. An embodiment based on a proper
combination of technical means disclosed in different embodiments
is encompassed in the technical scope of the present invention.
[0084] All the academic and patent literatures cited herein are
incorporated by references to the present specification.
EXAMPLE
1. Structures
[0085] A human .alpha..sub.1A-adrenergic receptor gene (ADRA1A) was
cloned from a human prostate library with a PCR technique. The
cloned gene had a whole length of 1465 bp, and had an ORF sequence
perfectly matching that of the human alpha-1A gene (NCBI Accession
No. U03866 or NM.sub.--000680), which has conventionally been
reported. Further, sequences preceding and following the ORF
sequence matched those reported in Hirasawa et al. (1993).
[0086] A CRELD1.alpha. gene was cloned from a human prostate cDNA
library. As a result of sequence determination, the cloned gene
matched that reported in Rupp et al. (2002) (NCBI Accession No.
NM.sub.--015513).
[0087] Next, the coding region of the ADRA1A gene was subcloned
into the EcoRI restriction enzyme site of the multicloning site A
of the pIRES (Clonetech, Catalog No. PT3266-5). Further, the coding
region of the CRELD1.alpha. was subcloned into the XbaI restriction
enzyme site of the multicloning site B of the pIRES. For the
purpose of subcloning, each of the genes had a restriction enzyme
adapter added to each end.
2. Production of a Transformant
[0088] The vector was amplified/purified in a conventional method,
and then transfected into CHO-K1 cells, which are cells of Chinese
Hamster ovarian origin, with Lipofectamine 2000 (Invitrogen
Corporation) according to the manufacturer's protocol. Two days
after the transfection, 1200 .mu.g/ml of the antibiotic Geneticin
(G418) were added to the DMEM culture medium. Cells into which the
vector had not been transfected, i.e., cells subjected to the same
operation with Lipofectamine 2000 and the like expect that the
vector had not been added, were completely annihilated at a G418
concentration of 1000 .mu.g/ml. The G418-resistant cells were
diluted, suspended, and injected dividedly into a 96-well plate so
that an average of 0.5 cells was put in per well, and a single
colony was chosen. This operation was repeated three times, whereby
a stable clone (.alpha..sub.1L cells) having coexpressed the ADRA1A
gene and the CREDL1.alpha. gene was produced. Further, as control
groups, the cells (.alpha..sub.1A cells) having stably expressed
only the ADRA1A gene and the cells (CREDL1.alpha. cells) having
stably expressed only the CREDL1.alpha. gene were produced.
3. Binding Experiment
[0089] The drugs used in the binding experiment and their proper
chemical names are as follows: silodosin, prazosin, tamsulosin,
RS-17053
(N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-.alpha.,.alpha.-dimeth-
yl-1H-indole-3-ethanamine hydrochloride), BMY 7378
(8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]8-azaspiro[4,5]decane7,9-d-
ione dihydrochloride).
[0090] The stable clone was studied by a pharmacological binding
method with [.sup.3H]-silodosin, which is a radioligand. An
experiment was conducted using a whole-cell binding method. That
is, cells were scattered two to three days before the experiment so
that they are semi-confluent, washed twice with ice-cold PBS, and
then scraped by a scraper. The cells thus collected were suspended
again in a Krebs-HEPES solution, and then incubated at 4.degree. C.
for four hours together with [.sup.3H]-silodosin and another drug
as needed. Thereafter, the cells were filtered/washed by a Whatmann
GC/F filter pretreated with polyethylene imine, and the
radioactivity was measured by a liquid scintillation counter. The
non-specific binding was evaluated as binding in the presence of 30
.mu.M phenotolamine.
[0091] [A] Saturation Binding Experiment According to the
Whole-Cell Method
[0092] The binding of [.sup.3H]-silodosin of various concentrations
(30 to 1000 pM) to the .alpha..sub.1L cells was examined (FIG. 1).
FIG. 1 shows a saturation binding curve obtained by the whole-cell
method. The specific binding of [.sup.3H]-silodosin to the
.alpha..sub.1L cells exhibited a maximum binding amount (Bmax) of
134 fmol/mg protein and a Kd value of 843 pM (pK.sub.D=9.1).
[0093] [B] Competitive Binding Experiment
[0094] Silodosin is known to bind selectively to the
.alpha..sub.1A- and .alpha..sub.1L-subtypes with a high affinity.
The term "high affinity" here means a Kd value of 1 nM or less. In
order to identify the pharmacologic properties of receptors
expressed in the .alpha..sub.1L cells, a competitive binding
experiments with prazosin at 300 pM [.sup.3H]-silodosin binding
sites were conducted using the whole-cell binding experiment
method. The .alpha..sub.1A cells were used as control cell line.
[.sup.3H]-silodosin is known as a selective antagonist of
.alpha..sub.1A- and .alpha..sub.1L-subtypes both.
[0095] As shown by the competitive curve in (a) of FIG. 2, most
(88% or higher) of receptors in the .alpha..sub.1L cells exhibit
low sensitivity to prazosin (pK.sub.i=7.6). Further, the receptors
are similarly low in sensitivity to RS-17053, and have high
sensitivity to tamsulosin. Further, the receptors in the
.alpha..sub.1L cells exhibit only low sensitivity to BMY 7378
(Table 2). On the other hand, the sensitivities (pK.sub.i values)
of the .alpha..sub.1A cells, which were used as a control group, to
prazosin (FIG. 2), tamsulosin, and RS-17053 are as high as 9.5,
9.9, and 8.8, respectively, which correspond to the .alpha..sub.1A
properties hitherto reported (Table 2).
TABLE-US-00002 TABLE 2 Pharmacologic Properties of
.alpha..sub.1L-adrenergic Receptors in Two Cell Lines and Human
Prostate Human Drug .alpha..sub.1L Cells .alpha..sub.1A Cells
Prostate [.sup.3H]-silodosin (pK.sub.D) 9.1 9.7 9.5 Prazosin 7.6
9.5 8.3 Tamsulosin 9.3 9.9 10.0 RS-17053 <6.0 8.8 6.6 BMY 7378
<6.0 6.5 5.9
[0096] In this table, the inhibition constants (Kr) of competitive
drugs at [.sup.3H]-silodosin binding sites in the .alpha..sub.1L
cells, the .alpha..sub.1A cells, and the human prostate tissue
segments are shown as -log K.sub.i (pK.sub.i). However, the value
of [.sup.3H]-silodosin exhibits a pK.sub.D value (dissociation
equilibrium constant) calculated from the saturation binding curve.
The value is an average of three to four examples.
[0097] Further, (b) of FIG. 2 shows results obtained by examining
sensitivity to prazosin in the homogenates of au. cells and the
homogenates of .alpha..sub.1A cells. In the homogenates, the
.alpha..sub.1L cells exhibited as high sensitivity to prazosin as
the .alpha..sub.1A cells. This fact coincides well with the results
that the .alpha..sub.1L receptors in prostate and brain disappeared
upon tissue homogenization and that the pharmacological profile was
converted to .alpha..sub.1A phenotype.
[0098] Binding affinities for various drugs of
.alpha..sub.1-adrenergic receptors in .alpha..sub.1L and a 1A cells
and human prostate are summarized in Table 2. The .alpha..sub.1L
cells, in which the ADRA1A gene and the CRELD1.alpha. gene were
co-expressed, differ in receptor properties from a 1A receptors,
and coincides well in pharmacologic properties with .alpha..sub.1L
receptors reported in human prostates and the like. Therefore,
unlike the .alpha..sub.1A cells, the .alpha..sub.1L cells are
considered as a cell line having expressed .alpha..sub.1L receptors
predominantly.
4. Intracellular Ca.sup.2+ Measurement Experiment
[0099] Furthermore, in order to completely get rid of the influence
of CRELD1.alpha., which might have been endogenously expressed in
the .alpha..sub.1A cells, the RNAi method was used to produce cells
in which CRELD1.alpha. expression had been knocked down (such cells
being hereinafter referred to as "KD cells". In order to inhibit
gene expression with the RNAi method, a vector was produced by
inserting an oligonucleotide (sense sequence;
GATCCAGGCGACTTAGTGTTCACCTTCAAGAGAGGTGAACACTAAGTCGCCTTTA: SEQ ID NO:
5, antisense sequence;
AGCTTAAAGGCGACTTAGTGTTCACCTCTCTTGAAGGTGAACACTAAGTCGCCTG: SEQ ID NO:
6) containing a coding region of mRNA of chinese hamster
CRELD1.alpha. into a commercially-available pSilencer.TM.4.1-CMV
hygro (Ambion, Inc.) according to the instruction manual therefor,
and then transfected into the .alpha..sub.1A cells in the same
procedure as described above. The Ca.sup.2+ response to
noradrenaline in the KD cells was examined according to a
fluorescent photometric method with Fura-2, which is a fluorescent
dye. The pEC.sub.50 value of the Ca.sup.2+ response to
noradrenaline in the KD cells was 7.9. On the other hand, when the
cells were treated in advance with prazosin 10.sup.-8 M for two
minutes, the dose-response curve of Ca.sup.2+ response to
noradrenaline shifted rightward, and the pK.sub.B value of prazosin
was calculated to be 9.3. This shows that the Ca.sup.2+ response to
noradrenaline in the KD cells coincides with the properties of the
.alpha..sub.1A-subtype.
[0100] Thus, when the expression of endogenous CRELD1.alpha. in a
CHO cell having a gene of .alpha..sub.1A-subtype expressed therein
was inhibited by the RNAi method, .alpha..sub.1A receptor
properties were perfectly exhibited. It should be noted that there
have been some experiments where the receptor functions, such as
Ca.sup.2+ response, of .alpha..sub.1A-subtype were examined by
using CHO cells in which a gene of the receptor had been expressed.
However, there has not necessarily been an agreement in the
receptor properties obtained as a result of those experiments.
[0101] Use of the present invention makes it possible to clarify a
GPCR whose entity has been unknown and thereby provide treatment
for any disease associated with the receptor, and also makes it
possible to modify the ligand affinity of a GPCR.
[0102] The embodiments and concrete examples of implementation
discussed in the foregoing detailed explanation serve solely to
illustrate the technical details of the present invention, which
should not be narrowly interpreted within the limits of such
embodiments and concrete examples, but rather may be applied in
many variations within the spirit of the present invention,
provided such variations do not exceed the scope of the patent
claims set forth below.
INDUSTRIAL APPLICABILITY
[0103] It is now clear that .alpha..sub.1L-AR is a functional
receptor and a main target for therapeutic drugs in lower urinary
tract system. Thus, the present .alpha..sub.1L-AR expression cells,
developed by the inventors, should be considered to be extremely
useful in drug development for urinary disturbance in patients with
benign prostatic hyperplasia and for urinary incontinence. Further,
the .alpha..sub.1L-AR expression cells are considered to be
extremely useful as a method for screening therapeutic drugs for
any disease associated with .alpha..sub.1L-AR.
Sequence CWU 1
1
61420PRThuman 1Met Ala Pro Trp Pro Pro Lys Gly Leu Val Pro Ala Val
Leu Trp Gly1 5 10 15Leu Ser Leu Phe Leu Asn Leu Pro Gly Pro Ile Trp
Leu Gln Pro Ser 20 25 30Pro Pro Pro Gln Ser Ser Pro Pro Pro Gln Pro
His Pro Cys His Thr 35 40 45Cys Arg Gly Leu Val Asp Ser Phe Asn Lys
Gly Leu Glu Arg Thr Ile 50 55 60Arg Asp Asn Phe Gly Gly Gly Asn Thr
Ala Trp Glu Glu Glu Asn Leu65 70 75 80Ser Lys Tyr Lys Asp Ser Glu
Thr Arg Leu Val Glu Val Leu Glu Gly 85 90 95Val Cys Ser Lys Ser Asp
Phe Glu Cys His Arg Leu Leu Glu Leu Ser 100 105 110Glu Glu Leu Val
Glu Ser Trp Trp Phe His Lys Gln Gln Glu Ala Pro 115 120 125Asp Leu
Phe Gln Trp Leu Cys Ser Asp Ser Leu Lys Leu Cys Cys Pro 130 135
140Ala Gly Thr Phe Gly Pro Ser Cys Leu Pro Cys Pro Gly Gly Thr
Glu145 150 155 160Arg Pro Cys Gly Gly Tyr Gly Gln Cys Glu Gly Glu
Gly Thr Arg Gly 165 170 175Gly Ser Gly His Cys Asp Cys Gln Ala Gly
Tyr Gly Gly Glu Ala Cys 180 185 190Gly Gln Cys Gly Leu Gly Tyr Phe
Glu Ala Glu Arg Asn Ala Ser His 195 200 205Leu Val Cys Ser Ala Cys
Phe Gly Pro Cys Ala Arg Cys Ser Gly Pro 210 215 220Glu Glu Ser Asn
Cys Leu Gln Cys Lys Lys Gly Trp Ala Leu His His225 230 235 240Leu
Lys Cys Val Asp Ile Asp Glu Cys Gly Thr Glu Gly Ala Asn Cys 245 250
255Gly Ala Asp Gln Phe Cys Val Asn Thr Glu Gly Ser Tyr Glu Cys Arg
260 265 270Asp Cys Ala Lys Ala Cys Leu Gly Cys Met Gly Ala Gly Pro
Gly Arg 275 280 285Cys Lys Lys Cys Ser Pro Gly Tyr Gln Gln Val Gly
Ser Lys Cys Leu 290 295 300Asp Val Asp Glu Cys Glu Thr Glu Val Cys
Pro Gly Glu Asn Lys Gln305 310 315 320Cys Glu Asn Thr Glu Gly Gly
Tyr Arg Cys Ile Cys Ala Glu Gly Tyr 325 330 335Lys Gln Met Glu Gly
Ile Cys Val Lys Glu Gln Ile Pro Glu Ser Ala 340 345 350Gly Phe Phe
Ser Glu Met Thr Glu Asp Glu Leu Val Val Leu Gln Gln 355 360 365Met
Phe Phe Gly Ile Ile Ile Cys Ala Leu Ala Thr Leu Ala Ala Lys 370 375
380Gly Asp Leu Val Phe Thr Ala Ile Phe Ile Gly Ala Val Ala Ala
Met385 390 395 400Thr Gly Tyr Trp Leu Ser Glu Arg Ser Asp Arg Val
Leu Glu Gly Phe 405 410 415Ile Lys Gly Arg 42022723DNAhuman
2tgcgttttac gcaggctgtg gcagcgacgc ggtgaggaga cggcccacgg cgcccgcggg
60ctggggcggt cgcttcttcc ttctccgtgg cctacgaggg tctggatcct tctctgccgg
120ctcgtgggcc gtgcctttgc ccttctgcga ggccctgaat ctgatccctt
cccttcatat 180ccggatccgg gctcctccct ccaagcccgg ggttccggac
acctccccca agacaacccc 240tctggcctcc tctccttcag tacttggaat
ctgatctctt ctccctaatt ctgcggatcc 300ggcccctaat attctttatc
agaccctcag acaagaggct gacttctgcc cccttgtcaa 360ggagcgaggc
cactttcctc tccaccccat gctagcgagg ataacttatt tctcttctgg
420aattgcatct tatgcgcctt tccccaccca tccccacagc ccctgcaata
cccagtttgg 480cctcttttgc ttgtaataac gcagatccca gcgccacggc
accttagaac agaccttttt 540ctttctcgcg tggggcctga ctccttcagt
gaagcctctc cacgccctct atctgcaggt 600ccccagcctg ggtaaagatg
gccccatggc ccccgaaggg cctagtccca gctgtgctct 660ggggcctcag
cctcttcctc aacctcccag gacctatctg gctccagccc tctccacctc
720cccagtcttc tcccccgcct cagccccatc cgtgtcatac ctgccgggga
ctggttgaca 780gctttaacaa gggcctggag agaaccatcc gggacaactt
tggaggtgga aacactgcct 840gggaggaaga gaatttgtcc aaatacaaag
acagtgagac ccgcctggta gaggtgctgg 900agggtgtgtg cagcaagtca
gacttcgagt gccaccgcct gctggagctg agtgaggagc 960tggtggagag
ctggtggttt cacaagcagc aggaggcccc ggacctcttc cagtggctgt
1020gctcagattc cctgaagctc tgctgccccg caggcacctt cgggccctcc
tgccttccct 1080gtcctggggg aacagagagg ccctgcggtg gctacgggca
gtgtgaagga gaagggacac 1140gagggggcag cgggcactgt gactgccaag
ccggctacgg gggtgaggcc tgtggccagt 1200gtggccttgg ctactttgag
gcagaacgca acgccagcca tctggtatgt tcggcttgtt 1260ttggcccctg
tgcccgatgc tcaggacctg aggaatcaaa ctgtttgcaa tgcaagaagg
1320gctgggccct gcatcacctc aagtgtgtag acattgatga gtgtggcaca
gagggagcca 1380actgtggagc tgaccaattc tgcgtgaaca ctgagggctc
ctatgagtgc cgagactgtg 1440ccaaggcctg cctaggctgc atgggggcag
ggccaggtcg ctgtaagaag tgtagccctg 1500gctatcagca ggtgggctcc
aagtgtctcg atgtggatga gtgtgagaca gaggtgtgtc 1560cgggagagaa
caagcagtgt gaaaacaccg agggcggtta tcgctgcatc tgtgccgagg
1620gctacaagca gatggaaggc atctgtgtga aggagcagat cccagagtca
gcaggcttct 1680tctcagagat gacagaagac gagttggtgg tgctgcagca
gatgttcttt ggcatcatca 1740tctgtgcact ggccacgctg gctgctaagg
gcgacttggt gttcaccgcc atcttcattg 1800gggctgtggc ggccatgact
ggctactggt tgtcagagcg cagtgaccgt gtgctggagg 1860gcttcatcaa
gggcagataa tcgcggccac cacctgtagg acctcctccc acccacgctg
1920cccccagagc ttgggctgcc ctcctgctgg acactcagga cagcttggtt
tatttttgag 1980agtggggtaa gcacccctac ctgccttaca gagcagccca
ggtacccagg cccgggcaga 2040caaggcccct ggggtaaaaa gtagccctga
aggtggatac catgagctct tcacctggcg 2100gggactggca ggcttcacaa
tgtgtgaatt tcaaaagttt ttccttaatg gtggctgcta 2160gagctttggc
ccctgcttag gattaggtgg tcctcacagg ggtggggcca tcacagctcc
2220ctcctgccag ctgcatgctg ccagttcctg ttctgtgttc accacatccc
cacaccccat 2280tgccacttat ttattcatct caggaaataa agaaaggtct
tggaaagtta aaaggcatca 2340gtcttactac ctgtcccacc acccccacct
tagggaaatg tcctagaatc ctgggaaatt 2400gagggcttct ttgatggtga
gtggagaaaa gatagaggag aaggttgccc ctgaagtgct 2460gttaggagaa
ggaggataga ggaatcagcc ttaggagggt tccatgccag ctgtcatttg
2520gcaaaggacc ctggacagat gacttttgcc tctgaacttc actcttctct
ttcctcaaat 2580gggcttcata atgctttcca ctcaggctta acatgagaat
taaatgaggt gacaaatgtg 2640aagacctgga cagtacacaa cagatattca
ataaaagtgt ggtcgccatt atgaccagag 2700cctccaaaaa aaaaaaaaaa aaa
27233466PRThuman 3Met Val Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser
Asn Cys Thr Gln1 5 10 15Pro Pro Ala Pro Val Asn Ile Ser Lys Ala Ile
Leu Leu Gly Val Ile 20 25 30Leu Gly Gly Leu Ile Leu Phe Gly Val Leu
Gly Asn Ile Leu Val Ile 35 40 45Leu Ser Val Ala Cys His Arg His Leu
His Ser Val Thr His Tyr Tyr 50 55 60Ile Val Asn Leu Ala Val Ala Asp
Leu Leu Leu Thr Ser Thr Val Leu65 70 75 80Pro Phe Ser Ala Ile Phe
Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95Val Phe Cys Asn Ile
Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110Ser Ile Met
Gly Leu Cys Ile Ile Ser Ile Asp Arg Tyr Ile Gly Val 115 120 125Ser
Tyr Pro Leu Arg Tyr Pro Thr Ile Val Thr Gln Arg Arg Gly Leu 130 135
140Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val Ile Ser Ile
Gly145 150 155 160Pro Leu Phe Gly Trp Arg Gln Pro Ala Pro Glu Asp
Glu Thr Ile Cys 165 170 175Gln Ile Asn Glu Glu Pro Gly Tyr Val Leu
Phe Ser Ala Leu Gly Ser 180 185 190Phe Tyr Leu Pro Leu Ala Ile Ile
Leu Val Met Tyr Cys Arg Val Tyr 195 200 205Val Val Ala Lys Arg Glu
Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr 210 215 220Asp Lys Ser Asp
Ser Glu Gln Val Thr Leu Arg Ile His Arg Lys Asn225 230 235 240Ala
Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys Thr His 245 250
255Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys
260 265 270Thr Leu Gly Ile Val Val Gly Cys Phe Val Leu Cys Trp Leu
Pro Phe 275 280 285Phe Leu Val Met Pro Ile Gly Ser Phe Phe Pro Asp
Phe Lys Pro Ser 290 295 300Glu Thr Val Phe Lys Ile Val Phe Trp Leu
Gly Tyr Leu Asn Ser Cys305 310 315 320Ile Asn Pro Ile Ile Tyr Pro
Cys Ser Ser Gln Glu Phe Lys Lys Ala 325 330 335Phe Gln Asn Val Leu
Arg Ile Gln Cys Leu Cys Arg Lys Gln Ser Ser 340 345 350Lys His Ala
Leu Gly Tyr Thr Leu His Pro Pro Ser Gln Ala Val Glu 355 360 365Gly
Gln His Lys Asp Met Val Arg Ile Pro Val Gly Ser Arg Glu Thr 370 375
380Phe Tyr Arg Ile Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe
Phe385 390 395 400Ser Ser Met Pro Arg Gly Ser Ala Arg Ile Thr Val
Ser Lys Asp Gln 405 410 415Ser Ser Cys Thr Thr Ala Arg Val Arg Ser
Lys Ser Phe Leu Gln Val 420 425 430Cys Cys Cys Val Gly Pro Ser Thr
Pro Ser Leu Asp Lys Asn His Gln 435 440 445Val Pro Thr Ile Lys Val
His Thr Ile Ser Leu Ser Glu Asn Gly Glu 450 455 460Glu
Val46541500DNAhuman 4ccgcctccgc gccagcccgg gaggtggccc tgacagccgg
acctcgcccg gccccggctg 60ggaccatggt gtttctctcg ggaaatgctt ccgacagctc
caactgcacc caaccgccgg 120caccggtgaa catttccaag gccattctgc
tcggggtgat cttggggggc ctcattcttt 180tcggggtgct gggtaacatc
ctagtgatcc tctccgtagc ctgtcaccga cacctgcact 240cagtcacgca
ctactacatc gtcaacctgg cggtggccga cctcctgctc acctccacgg
300tgctgccctt ctccgccatc ttcgaggtcc taggctactg ggccttcggc
agggtcttct 360gcaacatctg ggcggcagtg gatgtgctgt gctgcaccgc
gtccatcatg ggcctctgca 420tcatctccat cgaccgctac atcggcgtga
gctacccgct gcgctaccca accatcgtca 480cccagaggag gggtctcatg
gctctgctct gcgtctgggc actctccctg gtcatatcca 540ttggacccct
gttcggctgg aggcagccgg cccccgagga cgagaccatc tgccagatca
600acgaggagcc gggctacgtg ctcttctcag cgctgggctc cttctacctg
cctctggcca 660tcatcctggt catgtactgc cgcgtctacg tggtggccaa
gagggagagc cggggcctca 720agtctggcct caagaccgac aagtcggact
cggagcaagt gacgctccgc atccatcgga 780aaaacgcccc ggcaggaggc
agcgggatgg ccagcgccaa gaccaagacg cacttctcag 840tgaggctcct
caagttctcc cgggagaaga aagcggccaa aacgctgggc atcgtggtcg
900gctgcttcgt cctctgctgg ctgccttttt tcttagtcat gcccattggg
tctttcttcc 960ctgatttcaa gccctctgaa acagttttta aaatagtatt
ttggctcgga tatctaaaca 1020gctgcatcaa ccccatcata tacccatgct
ccagccaaga gttcaaaaag gcctttcaga 1080atgtcttgag aatccagtgt
ctctgcagaa agcagtcttc caaacatgcc ctgggctaca 1140ccctgcaccc
gcccagccag gccgtggaag ggcaacacaa ggacatggtg cgcatccccg
1200tgggatcaag agagaccttc tacaggatct ccaagacgga tggcgtttgt
gaatggaaat 1260ttttctcttc catgccccgt ggatctgcca ggattacagt
gtccaaagac caatcctcct 1320gtaccacagc ccgggtgaga agtaaaagct
ttttgcaggt ctgctgctgt gtagggccct 1380caacccccag ccttgacaag
aaccatcaag ttccaaccat taaggtccac accatctccc 1440tcagtgagaa
cggggaggaa gtctaggaca ggaaagatgc agaggaaagg ggaatatctt
1500555DNAArtificial SequenceDescription of Artificial Sequence
Synthesized Oligo Nucleotide 5gatccaggcg acttagtgtt caccttcaag
agaggtgaac actaagtcgc cttta 55655DNAArtificial SequenceDescription
of Artificial Sequence Synthesized Oligo Nucleotide 6agcttaaagg
cgacttagtg ttcacctctc ttgaaggtga acactaagtc gcctg 55
* * * * *