U.S. patent application number 09/254783 was filed with the patent office on 2002-03-21 for g-coupled receptor showing selective affinity for atp.
Invention is credited to BOEYNAEMS, JEAN-MARIE, COMMUNI, DIDIER.
Application Number | 20020035734 09/254783 |
Document ID | / |
Family ID | 8231018 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020035734 |
Kind Code |
A1 |
COMMUNI, DIDIER ; et
al. |
March 21, 2002 |
G-COUPLED RECEPTOR SHOWING SELECTIVE AFFINITY FOR ATP
Abstract
The present invention concerns a G-coupled receptor which has an
amino acid sequence having more than 50% homology with the amino
acid sequence shown in FIG. 1.
Inventors: |
COMMUNI, DIDIER; (VILVOORDE,
BE) ; BOEYNAEMS, JEAN-MARIE; (WEMMEL, BE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
8231018 |
Appl. No.: |
09/254783 |
Filed: |
August 16, 1999 |
PCT Filed: |
July 9, 1998 |
PCT NO: |
PCT/BE98/00108 |
Current U.S.
Class: |
800/8 ;
435/320.1; 435/325; 435/69.1; 436/6; 536/23.1; 536/24.33 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 37/04 20180101; A01K 2217/075 20130101; C12N 2799/021
20130101; C07K 14/705 20130101; A01K 2217/05 20130101; A01K 67/0275
20130101; A61P 35/00 20180101 |
Class at
Publication: |
800/8 ;
435/320.1; 435/325; 435/69.1; 436/6; 536/23.1; 536/24.33 |
International
Class: |
A01K 067/00; C12Q
001/68; C12P 021/02; C12N 005/06; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 9, 1997 |
EP |
97870101.9 |
Claims
1. Receptor which has an amino acid sequence having more than 50%
homology with the amino acid sequence shown in FIG. 1.
2. Receptor according to claim 1, which has at least the amino acid
sequence shown in FIG. 1 or a portion thereof.
3. Receptor according to claim 1 or 2 having a selective affinity
ADP.
4. Receptor according to any of the preceding claims, belonging to
the P2Y receptor family.
5. Receptor according to any of the preceding claims, being a G
protein-coupled receptor.
6. Receptor according to any of the preceding claims, being a human
receptor.
7. Nucleic acid molecule encoding the receptor according to any of
the preceding claims.
8. Nucleic acid molecule according to claim 7, wherein the nucleic
acid molecule is DNA or RNA molecule.
9. DNA molecule according to claim 8, which is a cDNA molecule or a
genomic DNA molecule.
10. Nucleic acid molecule according to any of the claims 7 to 9,
having more than 50% homology to the DNA sequence shown in FIG.
1.
11. DNA molecule according to claim 10, which has at least the DNA
sequence as shown in FIG. 1 or a portion thereof.
12. Vector comprising the nucleic acid molecule according to any of
the claims 7 to 11.
13. Vector according to claim 12, adapted for expression in a cell,
which comprises the regulatory elements necessary for expression of
the nucleic acid molecule in said cell operatively linked to the
nucleic acid molecule according to any of the claims 7 to 11 as to
permit expression thereof.
14. Vector of claim 13, wherein the cell is selected from the group
consisting of bacterial cells, yeast cells, insect cells or
mammalian cells.
15. Vector according to any of the claims 12 to 14, wherein the
vector is a plasmid or a virus, preferably a baculovirus, an
adenovirus or a Semliki Forest virus.
16. Cell comprising the vector according to any of the claims 12 to
15.
17. Cell of claim 16, wherein the cell is a mammalian cell,
preferably non neuronal in origin.
18. Cell of claim 16, wherein the cell is selected from the group
consisting of COS-7 cells, CHO cells, LM(tk-) cells, NIH-3T3 cells
or 1321N1 astrocytoma cells.
19. Nucleic acid probe comprising a nucleic acid molecule of at
least 15 nucleotides capable of specifically hybridising with a
unique sequence included within the nucleic acid molecule according
to any of the claims 11 to 15.
20. Nucleic acid probe of claim 19, wherein the nucleic acid is DNA
or RNA.
21. Antisense oligonucleotide having a sequence capable of
specifically hybridising to a mRNA molecule of claim 8, so as to
prevent translation of the mRNA molecule.
22. Antisense oligonucleotide having a sequence capable of
specifically hybridising to the DNA molecule of claim 9.
23. Antisense oligonucleotide according to claim 21 or 22,
comprising chemical analogs of nucleotides.
24. Ligand other than purine and pyridine nucleotides capable of
binding to a receptor according to any of the claims 1 to 6.
25. Anti-ligand capable of competitively inhibiting the binding of
the ligand according to claim 24 to the receptor according to any
of the claims 1 to 6.
26. Ligand according to claim 24, which is an antibody.
27. Anti-ligand according to claim 25, which is an antibody.
28. Antibody according to claim 26 or 27, which is a monoclonal
antibody.
29. Monoclonal antibody according to claim 28, directed to an
epitope of the receptor according to any of the claims 1 to 6,
present on the surface of a cell expressing said receptor.
30. Pharmaceutical composition comprising an amount of the
oligonucleotide according to claim 21, effective to decrease
activity of the receptor according to any of the claims 1 to 6 by
passing through a cell membrane and binding specifically with mRNA
encoding said receptor in the cell so as to prevent its
translation, and a pharmaceutically acceptable carrier capable of
passing through a cell membrane.
31. Pharmaceutical composition of claim 30, wherein the
oligonucleotide is coupled to a substance which inactivates
mRNA.
32. Pharmaceutical composition of claim 31, wherein the substance
which inactivates mRNA is a ribozyme.
33. Pharmaceutical composition according to any of the claims 30 to
32, wherein the pharmaceutically acceptable carrier comprises a
structure which binds to a receptor on a cell capable of being
taken up by cell after binding to the structure.
34. Pharmaceutical composition of claim 33, wherein the structure
of the pharmaceutically acceptable carrier is capable of binding to
a receptor which is specific for a selected cell type.
35. Pharmaceutical composition which comprises an effective amount
of the anti-ligand of claim 30, effective to block binding of a
ligand to the receptor according to any of the claims 1 to 6 and a
pharmaceutically acceptable carrier.
36. Transgenic non human mammal expressing the nucleic acid
molecule according to any of the claims 7 to 11.
37. Transgenic non human mammal comprising a homologous
recombination knockout of the native receptor according to any of
the claims 1 to 6.
38. Transgenic non human mammal whose genome comprises antisense
nucleic acid complementary to the nucleic acid molecule according
to any of the claims 7 to 11 so placed as to be transcripted into
antisense mRNA which is complementary to the mRNA of claim 8 and
which hybridises to said mRNA thereby reducing its translation.
39. Transgenic non human mammal according to any of the claims 36
to 38, wherein the nucleic acid according to any of the claims 7 to
11 additionally comprises an inducible promoter.
40. Transgenic non human mammal according to any of the claims 36
to 39, wherein the nucleic acid according to claim 7 to 11
additionally comprises tissue specific regulatory elements.
41. Transgenic non human mammal according to any of the claims 36
to 40, which is a mouse.
42. Method for determining whether a ligand can specifically bind
to a receptor according to any of the claims 1 to 6, possibly as an
agonist or an antagonist of said receptor; said method comprising
the steps of contacting a cell or cell extract from cells
transfected with a vector expressing the nucleic acid molecule
encoding said receptor, possibly isolating a membrane fraction from
the cell extract, with the ligand under conditions permitting
binding of said ligand to said receptor, possibly by the activation
of a functional response, and detecting the presence of any such
ligand bound specifically to said receptor, possibly by means of a
bioassay such as a modification of the production of a second
messenger or an increasing in the receptor activity, thereby
determining whether the ligand binds specifically to said receptor,
possibly as an agonist or an antagonist of said receptor.
43. A method according to claim 42, wherein the second messenger
assay comprises measurement of intracellular cAMP, intracellular
Inositol phosphate, intracellular diacylglycerol concentration or
intracellular calcium mobilisation.
44. Method according to claim 42 or 43, wherein the cell is a
mammalian cell, preferably non neuronal in origin, and selected
from the group consisting of COS-7 cells, CHO cells, LM(tk-) cells,
NIH-3T3 cells or 1321N1 cells.
45. Method according to any of the preceding claims 42 to 44,
wherein the ligand is not previously known.
46. Ligand detected by the method according to any of the preceding
claims 42 to 45.
47. Pharmaceutical composition which comprises the ligand according
to claim 46 and a pharmaceutically acceptable carrier.
48. Method of screening drugs to identify drugs which specifically
bind to the receptor according to any of the claims 1 to 6 on the
surface of the cell, which comprises contacting a cell transfected
with a vector expressing the nucleic acid molecule encoding said
receptor with a plurality of drugs under conditions permitting
binding of said drugs to the receptor, and determining those drugs
which specifically bind to the transfected cell, thereby
identifying drugs which specifically bind to the receptor.
49. Method of screening drugs to identify drugs which specifically
bind to the receptor according to any of the claims 1 to 6 on the
surface of the cell, which comprises preparing a cell extract from
cells transfected with a vector expressing the nucleic acid
molecule encoding said receptor, isolating a membrane fraction from
the cells extract, contacting the membrane fraction with a
plurality of drugs and determining those drugs which bind to the
transfected cell, thereby identifying drugs which specifically bind
to said receptor.
50. Method of screening drugs to identify drugs which act as
agonists of the receptor according to any of the claims 1 to 6,
which comprises contacting a cell transfected with a vector
expressing the nucleic acid molecule encoding said receptor with a
plurality of drugs under conditions permitting the activation of a
functional receptor response, and determining those drugs which
activate such receptor using a bio-assay, such as a modification in
a second messenger concentration or modification in the cellular
metabolism, thereby identifying drugs which act as receptor
agonists.
51. Method of screening drugs to identify drugs which act as
agonists of the receptor according to any of the claims 1 to 6,
which comprises preparing a cell extract from cells transfected
with a vector expressing the nucleic acid molecule encoding said
receptor, isolating a membrane fraction from the cell extract,
contacting the membrane fraction with a plurality of drugs under
conditions permitting the activation of a functional receptor
response, and determining those drugs which activate such receptor
using a bio-assay, such as a modification in a second messenger
concentration, thereby identifying drugs which act as receptor
agonists.
52. Method of screening drugs to identify drugs which act as
antagonists of the receptor according to any of the claims 1 to 6,
which comprises contacting a cell transfected with a vector
expressing the nucleic acid molecule encoding said receptor with a
plurality of drugs in the presence of a known receptor agonist,
under conditions permitting the activation of a functional receptor
response, and determining those drugs which inhibit the activation
of the receptor using a bio-assay, such as a modification in a
second messenger concentration or modification in the cellular
metabolism, thereby identifying drugs which act as receptor
antagonists.
53. Method of screening drugs to identify drugs which act as
antagonists of the receptor according to any of the claims 1 to 6,
which comprises preparing a cell extract from cells transfected
with a vector expressing the nucleic acid molecule encoding said
receptor, isolating a membrane fraction from the cell extract,
contacting the membrane fraction with a plurality of drugs in
presence of a known receptor agonist, under conditions permitting
the activation of a functional receptor response, and determining
those drugs which inhibit the activation of the receptor using a
bio-assay, such as a modification in a second messenger
concentration, thereby identifying drugs which act as receptor
antagonists.
54. Drug detected by any of the methods according to claims 48 to
53.
55. Pharmaceutical composition comprising a drug according to claim
54.
56. Method of detecting the expression of the receptor according to
any of the claims 1 to 6, by detecting the presence of -mRNA coding
said receptor, which comprises obtaining total RNA or total mRNA
from the cell and contacting the RNA or mRNA so obtained with the
nucleic acid probe according to claim 19 under hybridising
conditions, and detecting the presence of mRNA hybridised to the
probe, thereby detecting the expression of the receptor by the
cell.
57. Method of detecting the presence of the receptor according to
any of the claims 1 to 6 on the surface of a cell, which comprises
contacting the cell with the antibody of claim 26 under conditions
permitting binding of the antibody to the receptor, and detecting
the presence of the antibody bound to the cell, thereby detecting
the presence of the receptor on the surface of the cell.
58. Method of determining the physiological effects of expressing
varying levels of the receptor according to any of the claims 1 to
6, which comprises producing a transgenic non human mammal
according to any of the claims 36 to 41 whose levels of receptor
expression are varied by use of an inducible promoter which
regulates the receptor expression.
59. Method of determining the physiological effects of expressing
varying levels of the receptor according to any of the claims 1 to
6, which comprises producing a panel of transgenic non human
mammals according to any of the claims 36 to 41, each expressing a
different amount of said receptor.
60. Method for identifying an antagonist of the receptor according
to any of the claims 1 to 6 capable of alleviating an abnormality
in a subject wherein the abnormality is alleviated by decreasing
the activity of the receptor, which comprises administering the
antagonist to a transgenic non human mammal according to any of the
claims 36 to 41 and determining whether the antagonist alleviates
the physical and behavioural abnormalities displayed by the
transgenic non human mammal as a result of receptor activity,
thereby identifying the antagonist.
61. Antagonist identified by the method of claim 60.
62. Pharmaceutical composition comprising an antagonist according
to claim 61 and a pharmaceutically acceptable carrier.
63. Method for identifying an agonist of the receptor according to
any of the claims 1 to 6 capable of alleviating an abnormality in a
subject wherein the abnormality is alleviated by activation of said
receptor, which comprises administering the agonist to a transgenic
non human mammal according to any of the claims 36 to 41 and
determining whether the antagonist alleviates the physical and
behavioural abnormalities displayed by the transgenic non human
mammal, the alleviation of the abnormalities indicating the
identification of the agonist.
64. Agonist identified by the method of claim 63.
65. Pharmaceutical composition comprising an agonist according to
claim 64 and a pharmaceutically acceptable carrier.
66. Method for diagnosing a predisposition to a disorder associated
with the activity of a specific allele of the receptor according to
any of the claims 1 to 6, which comprises: a) obtaining nucleic
acid molecules of subjects suffering from said disorder; b)
performing a restriction digest of said nucleic acid molecules with
a panel of restriction enzymes; c) electrophoretically separating
the resulting nucleic acid fragments on a sized gel; d) contacting
the resulting gel with a nucleic acid probe capable of specifically
hybridising to said nucleic acid molecule and labelled with a
detectable marker; e) detecting labelled bands which have
hybridised to the said nucleic acid molecule labelled with a
detectable marker to create a unique band pattern specific to
subjects suffering from said disorder; f) preparing nucleic acid
molecules obtained for diagnosis by step a-e; and g) comparing the
unique band pattern specific to the nucleic acid molecule of
subjects suffering from the disorder from step e and the nucleic
acid molecule obtained for diagnosis from step f to determine
whether the patterns are the same or different and to diagnose
thereby predisposition to the disorder if the patterns are the
same.
67. Method of preparing the purified receptor according to any of
the claims 1 to 6, which comprises: a) constructing a vector
adapted for expression in a cell which comprises the regulatory
elements necessary for the expression of nucleic acid molecules in
the cell operatively linked to nucleic acid molecule encoding said
receptor so as to permit expression thereof, wherein the cell is
selected from the group consisting of bacterial cells, yeast cells,
insect cells and mammalian cells; b) inserting the vector of step a
in a suitable host cell; c) incubating the cell of step b under
conditions allowing the expression of the receptor according to the
invention; d) recovering the receptor so obtained; and e) purifying
the receptor so recovered, thereby preparing an isolated receptor
according to the invention.
68. Use of the pharmaceutical composition according to claim 47,
55, 62 or 65, in the preparation of a medicament for the treatment
and/or the prevention of the neutropenie, agranulocytose infections
or cancer.
Description
OBJECT OF THE PRESENT INVENTION
[0001] The present invention concerns a new G protein-coupled
receptor having selective affinity for ATP and the nucleic acid
molecule encoding said receptor, vectors comprising said nucleic
acid molecule, cells transformed by said vector, antibodies
directed against said receptor, nucleic acid probes directed
against said nucleic acid molecule, pharmaceutical compositions
comprising said products and non human transgenic animals
expressing the receptor according to the invention or the nucleic
acid molecule according to said receptor.
BACKGROUND OF THE INVENTION
[0002] An impressive number of P2 receptors subtypes has been
cloned since 1993. A new molecular nomenclature has then been
created in which G protein-coupled P2 receptors have been named P2Y
while P2 receptors having an intrinsic ion channel activity have
been named P2X. The P2Y family encompasses selective purinoceptors
(the P2Y.sub.1 receptor activated by ATP and ADP), nucleotide
receptors responsive to both adenine and uracil nucleotides
(P2Y.sub.2 receptor: activated equipotentally by ATP and UTP) and
pyrimidinoceptors (the P2Y.sub.3 and P2Y.sub.6 receptors activated
by UDP; the P2Y.sub.4.sup.- receptor: activated by UTP). The
P2Y.sub.5 and P2Y.sub.7 receptors display limited homologies with
the other members of the P2Y family. They have been included in
this family especially on the basis of radioligand binding studies
showing affinities for adenine nucleotides (1-18).
SUMMARY OF THE INVENTION
[0003] The present invention concerns a new receptor having the
amino acids sequence of FIG. 1 or any receptor which presents more
than 50%, preferably more than 70%, more preferably more than 85%,
more specifically more than 95% homology with the amino acids
sequence of FIG. 1.
[0004] The present invention concerns also the receptor having at
least the amino acids sequence of FIG. 1 or a portion thereof,
preferably an amino acids sequence wherein the large extracellular
part (the NH.sub.2 portion of 450 amino acids sequence the end of
which (.diamond-solid.) is represented on FIG. 1) has been
truncated or active parts of said portion such as the sixth and
seventh transmembrane domains comprising the amino acids :
His.sup.686, Arg.sup.689 and Arg.sup.728.
[0005] The present invention is also related to said NH.sub.2
portion of 450 amino acids sequence, including peptides reproducing
or mimicking a portion of this sequence or of organic molecules
sharing the effects of these peptides.
[0006] Indeed, the inventors have discovered that either the whole
receptor having the amino acids sequence of FIG. 1 or its portion
(preferably the amino acids sequence wherein the large
extracellular part of 450 amino acids has been truncated and
starting from (.diamond-solid.) in FIG. 1) seems to have the same
industrial application (said portion will be identified hereafter
as the P2Y11 receptor or sequence).
[0007] The first industrial application of this receptor or its
portions is the screening of agonists and antagonists of said
receptor which may have advantageous pharmaceutical or diagnostical
properties. The second industrial application of the receptor
according to the invention or of its portions or of active parts of
its portions is the identification of patients who may present
genetic disorders induced by an inactive receptor or by an inactive
portion of said receptor.
[0008] According to a preferred embodiment of the present
invention, said receptor is a human receptor.
[0009] ATP seems to be the preferential natural agonist of this
receptor : UTP, UDP, AP.sub.4A, AP.sub.6A, AMP and adenosine seem
to be unable to stimulate the phosphoinositide pathway or were much
less potent that ATP.
[0010] Therefore, the invention is also related to a new G-coupled
receptor, its portions or active parts of its portions having a
selective affinity for ATP. "A selective affinity for ATP" means
that ATP is able to induce the formation of a functional response
(preferably the accumulation of Inositol triphosphate IP.sub.3 and
a rise of intracellular Ca.sup.2+) in a short time of incubation
with said agonist (preferably in less than 5 min, more preferably
less than 1 min) while the other known agonists of P2Y (UTP, UDP,
AP.sub.4A, AP.sub.6A, AMP and adenosine were unable to stimulate
said receptor or were much less potent than ATP and induce a
detectable functional response by said receptor.
[0011] The present invention is also related to a nucleic acid
molecule, such as a DNA molecule or an RNA molecule, encoding the
receptor, its portions or active parts of its portions according to
the invention.
[0012] Preferably, said DNA molecule is a cDNA molecule or a
genomic DNA molecule.
[0013] Preferably, said nucleic acid molecule has more than 50l%,
preferably more than 70%, more preferably more than 85%, more
specifically more than 95% homology with the DNA sequence shown in
FIG. 1.
[0014] Preferably, the invention is related to a nucleic acid
molecule which has more than 50%, preferably more than 70%, more
preferably more than 85%, more specifically more than 95% homology
with this DNA sequence (shown in the FIG. 1), wherein the DNA
sequence encoding the 450 amino acids of the NH.sub.2 portion were
truncated.
[0015] The present invention is also related to the vector
comprising the nucleic acid molecule according to the invention.
Preferably, said vector is adapted for expression in a cell and
comprises the regulatory elements necessary for expressing the
amino acid molecule in said cell operatively linked to the nucleic
acid sequence according to the invention as to permit expression
thereof.
[0016] Preferably, said cell is selected from the group consisting
of bacterial cells, yeast cells, insect cells or mammalian cells.
The vector according to the invention is a plasmid or a virus,
preferably a baculovirus, an adenovirus or a semliki forest
virus.
[0017] The present invention concerns also the cell transformed by
the vector according to the invention, said cell is preferably
non-neuronal in origin and is selected from the group consisting of
a COS-7 cell, a CHO cell, an LM(tk-) cell, an NIH-3T3 cell or a
1321N1 astrocytoma cell.
[0018] The present invention is also related to a nucleic acid
probe comprising the nucleic acid molecule according to the
invention, of at least 15 nucleotides capable of specifically
hybridising with a unique sequence included in the sequence of the
nucleic acid molecule encoding the receptor according to the
invention. Said nucleic acid probe may be a DNA or an RNA
molecule.
[0019] The invention concerns also an antisense oligonucleotide
having a sequence capable of specifically hybridising to an mRNA
molecule encoding the receptor according to-the invention so as to
prevent translation of said mRNA molecule or an antisense
oligonucleotide having a sequence capable of specifically
hybridising to the CDNA molecule encoding the receptor according to
the invention.
[0020] Said antisense oligonucleotide may comprise chemical analogs
of nucleotide or substances which inactivate MRNA, or be included
in an RNA molecule endowed with ribozyme activity.
[0021] Another aspect of the present invention concerns a ligand
(preferably an antibody) other than known molecules, especially the
ATP, capable of binding to the receptor according to the invention
and an anti-ligand (preferably also an antibody) capable of
competitively inhibiting the binding of said ligand to the receptor
according to the invention.
[0022] Preferably, said antibody is a monoclonal antibody directed
to an epitope of the receptor according to the invention and
present on the surface of a cell expressing said receptor.
[0023] The invention concerns also the pharmaceutical composition
comprising an effective amount of oligonucleotide according to the
invention, effective to decrease the activity of said receptor by
passing through a cell membrane and binding specifically with MRNA
encoding the receptor according to the invention in the cell so as
to prevent its translation. The pharmaceutical composition
comprises also a pharmaceutically acceptable carrier capable of
passing through said cell membrane.
[0024] Preferably, in said pharmaceutical composition, the
oligonucleotide is coupled to a substance, such as a ribozyme,
which inactivates mRNA.
[0025] Preferably, the pharmaceutically acceptable carrier
comprises a structure which binds to a receptor on a cell capable
of being taken up by cell after binding to the structure. The
structure of the pharmaceutically acceptable carrier in said
pharmaceutical composition is capable of binding to a receptor
which is specific for a selected cell type.
[0026] Preferably, said pharmaceutical composition comprises an
amount of the antibody according to the invention effective to
block the binding of a ligand to the receptor according to the
invention and a pharmaceutically acceptable carrier.
[0027] The present invention concerns also a transgenic non human
mammal overexpressing (or expressing ectopically) the nucleic acid
molecule encoding the receptor according to the invention.
[0028] The present invention also concerns a transgenic non human
mammal comprising a homologous recombination knockout of the native
receptor according to the invention.
[0029] According to a preferred embodiment of the invention, the
transgenic non human mammal whose genome comprises antisense
nucleic acid complementary to the nucleic acid according to the
invention is so placed as to be transcripted into antisense mRNA
which is complementary to the mRNA encoding the receptor according
to the invention and which hybridises to mRNA encoding said
receptor, thereby reducing its translation. Preferably, the
transgenic non human mammal according to the invention comprises a
nucleic acid molecule encoding the receptor according to the
invention and comprises additionally an inducible promoter or a
tissue specific regulatory element.
[0030] Preferably, the transgenic non human mammal is a mouse.
[0031] The invention relates also to a method for determining
whether a ligand as an agonist or an antagonist of the receptor
according to the invention can be specifically bound to said
receptor; said method comprising the steps of contacting a cell or
a cell extract from cells transfected with a vector according to
the invention and expressing the nucleic acid molecule encoding
said receptor, possibly isolating a membrane fraction from the cell
extract, contacting the ligand with the membrane fraction or with
the cell under conditions permitting binding of said ligand to the
receptor and detecting, possibly by means of a bioassay such as a
modification in the second messenger concentration or a
modification in the cellular metabolism (preferably determined by
the acidification rate of the culture medium), an increase in the
receptor activity), thereby determining whether the ligand binds to
the receptor, possibly as an agonist or as an antagonist of said
receptor.
[0032] Preferably, the second messenger assay comprises measurement
of intracellular cAMP, intracellular inositol phosphate (IP3),
intracellular diacylglycerol (DAG) concentration or intracellular
calcium mobilisation.
[0033] Preferably, the cell used in said method is a mammalian cell
non neuronal in origin, such as a COS-7 cell, a CHO cell, a LM(tk-)
cell an NIH-3T3 cell or 1321N1 cell. In said method, the ligand is
not previously known.
[0034] The invention is also related to the ligand isolated and
detected by any of the preceding methods.
[0035] The present invention concerns also the pharmaceutical
composition which comprises an effective amount of an agonist or an
antagonist of the receptor according to the invention, effective to
reduce the activity of said receptor and a pharmaceutically
acceptable carrier.
[0036] The P2Y11 transcripts (obtained from the nucleotidic
sequence starting from (.diamond-solid.) in FIG. 1) are detectable
in HL-60 human leukaemia cells. Expression of P2Y11 receptor mRNA
is increased by agents (ripnoic acid, DMSO) known to induce the
granulocytic differenciations of HL-60 cells. However, the P2Y11
transcripts could not be detected in mature neutrophils. Therefore,
a first industrial application of the product according to the
invention is the diagnostic of leukaemia, preferably by Northern
blot analysis using the nucleotidic sequence encoding the P2Y11
receptor according to the invention.
[0037] The present invention is also related to a diagnostic device
or kit comprising the elements for the diagnostic of specific
leukaemia, preferably HL-60 human leukaemia, comprising the
receptor according to the invention, the nucleic acid sequence
encoding said receptor, a nucleic acid probe comprising the nucleic
acid molecule according to the invention of at least 15 nucleotides
capable of specifically hybridising with a unique sequence included
in the sequence of the nucleic acid molecule encoding the receptor
according to the invention, such as an antisense oligonucleotide or
a ligand such as an antibody, preferably a monoclonal antibody,
capable of binding or competitively inhibiting the binding of a
ligand to the receptor according to the invention. Said diagnostic
device or kit could be used for the specific diagnostic or for the
monitoring of the evolution of tumoral cells, especially leukaemia
HL-60 cells.
[0038] Therefore, the previously described methods may be used for
the screening of drugs (having advantageously anti-tumoral
properties) which specifically bind to the receptor according to
the invention.
[0039] Another industrial application of the present invention is
related to the use of said drugs, preferably ligands or
anti-ligands according to the invention, for the prevention and/or
the treatment of specific diseases such as neutropenie or
agranulocytose infections or cancer.
[0040] The invention is also related to the drugs isolated and
detected by any of these methods.
[0041] The present invention concerns also a pharmaceutical
composition comprising said drugs and a pharmaceutically acceptable
carrier.
[0042] The invention is also related to a method of detecting
expression of a receptor according to the invention by detecting
the presence of mRNA coding for a receptor, which comprises
obtaining total RNA or total MRNA from the cell and contacting the
RNA or mRNA so obtained with the nucleic acid probe according to
the invention under hybridising conditions and detecting the
presence of mRNA hybridised to the probe, thereby detecting the
expression of the receptor by the cell.
[0043] The hybridisation conditions above-described are preferably
standard stringent conditions as described by Sambrook et al.
(.sctn.9.47-9.51 in Molecular Cloning Laboratory Manual, Cold
Spring Harbour, Laboratory Press, New York (1989)).
[0044] The present invention concerns also a method for diagnosing
a predisposition to a disorder associated with the activity of the
receptor according to the invention. Said method comprises:
[0045] a) obtaining nucleic acid molecules of subjects suffering
from said disorder;
[0046] b) performing a restriction digest of said nucleic acid
molecules with a panel of restriction enzymes;
[0047] c) electrophoretically separating the resulting nucleic acid
fragments on a sized gel;
[0048] d) contacting the resulting gel with a nucleic acid probe
capable of specifically hybridising to said nucleic acid molecule
and labelled with a detectable marker;
[0049] e) detecting labelled bands which have hybridised to the
said nucleic acid molecule labelled with a detectable marker to
create a unique band pattern specific to subjects suffering from
said disorder;
[0050] f) preparing nucleic acid molecules obtained for diagnosis
by step a-e; and
[0051] g) comparing the unique band pattern specific to the nucleic
acid molecule of subjects suffering from the disorder from step e
and the nucleic acid molecule obtained for diagnosis from step f to
determine whether the patterns are the same or different and to
diagnose thereby predisposition to the disorder if the patterns are
the same.
[0052] A last aspect of the present invention concerns a method of
preparing the receptor according to the invention, which
comprises:
[0053] a) constructing a vector adapted for expression in a cell
which comprises the regulatory elements necessary for the
expression of nucleic acid molecules in the cell operatively linked
to nucleic acid molecule encoding said receptor so as to permit
expression thereof, wherein the cell is selected from the group
consisting of bacterial cells, yeast cells, insect cells and
mammalian cells;
[0054] b) inserting the vector of step a in a suitable host
cell;
[0055] c) incubating the cell of step b under conditions allowing
the expression of the receptor according to the invention;
[0056] d) recovering the receptor so obtained; and
[0057] e) purifying the receptor so recovered, thereby preparing an
isolated receptor according to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] FIG. 1 represents the nucleotide and deduced amino acid
sequence of the new human P2Y receptor. The putative
phosphorylation sites by protein kinase C or by
calmodulin-dependent protein kinases are indicated respectively by
a black circle (.circle-solid.) or a black diamond
(.diamond-solid.). The potential N-glycosylation site is indicated
by a black square (.box-solid.).
[0059] FIG. 2 represents dendrogram of the structural relatedness
of the P2Y.sub.11 receptor with the other P2Y subtypes. The plot
was constructed using the multiple sequence alignment program
Pileup of the GCG package The P2Y.sub.5-like published sequence
(18) is identical to the P.sup.2Y.sub.9 sequence submitted to the
GenBank/EMBL Data Bank.
[0060] FIG. 3 represents Northern blot analysis of P2Y.sub.11
messenger expression. Each lane of the MTN blot contains 2 of
polyA.sup.+ RNA from several human tissues. Each lane of the HL-60
blot contains 10 .mu.g of total RNA from differentiated or
undifferentiated HL-60 cells. Hybridization with the probe was
performed as described under Materials and Methods. The pictures of
the MTNII blot and the HL-60 blot were obtained respectively, from
an autoradiography and from a PhosphorImager SI (Molecular
Dynamics). The 2 kb-length P2Y.sub.11 transcripts are indicated by
a black arrow.
[0061] FIG. 4 represents concentration-action curves of several
nucleotides on IP3 and cAMP accumulation in cells transfected with
the P2Y.sub.11 receptor. 1321N1 and CHO-K1 transfected cells were
assayed for the accumulation of, respectively, IP.sub.3 (A) or cAMP
(B) in response to various concentrations of the following
nucleotides: ATP, 2MeSATP, ADP and 2MeSADP. Incubation times were
30 s for IP.sub.3 measurements and 15 min for cAMP assays. The data
represent the means.+-.S.D. of triplicate experimental points and
are representative of two independent experiments.
DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION
Experimental Procedures
[0062] Materials
[0063] Trypsin was from Flow Laboratories (Bioggio, Switzerland) .
Culture media, G418, fetal calf serum (FCS), restriction enzymes
and Taq polymerase were purchased from GIBCO BRL (Grand Island,
N.Y.). The radioactive products myo-D-[2-.sup.3H]inositol (17.7
Ci/mmol) and [(.alpha..sup.32P]ATP (800 Ci/mmol) were from Amersham
(Ghent, Belgium) . Dowex AG1X8 (formate form) was from Bio-Rad
Laboratories (Richmond, Calif.) ATP, ADP, AMP, adenosine, UTP, UDP,
AP.sub.4A, AP.sub.6A, all-trans retinoic acid (RA) and
12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma
Chemical Co. (St. Louis, Mo.). 2-methylthio-ATP (2MeSATP),
2-methylthio-ADP (2MeSADP) and 8 (p-sulfophenyl) theophylline were
from Research Biochemicals International (Natick, Mass.). Forskolin
was purchased from Calbiochem (Bierges, Belgium). Indomethacin and
dimethyl sulfoxide (DMSO) were from Merck (Netherlands). Rolipram
was obtained from the Laboratoires Jacques Logeais (Trappes,
France). The HL-60 human cell line was obtained from the American
Type Culture Collection (Rockville, USA). The human genomic DNA
library was from Stratagene (La Jolla, Calif.). pEFIN3 is an
expression vector obtained from Euroscreen (Brussels, Belgium).
Multiple Human Tissues Northern blot (MTN) were from Clontech (Palo
Alto, Calif.).
[0064] Cloning and sequencing
[0065] A human placenta cDNA library was screened at moderate
stringency with an [.alpha..sup.32P] DATP labelled P2Y.sub.4
receptor probe corresponding to a partial sequence covering the
third to the seventh transmembrane segments. Three overlapping
clones encoding a new G protein-coupled receptor were isolated, but
did not contain the 3' end of the coding region. A human genomic
DNA library was then screened with this partial sequence to obtain
the complete sequence of this new receptor. The hybridization
conditions for screening the two libraries were 6.times.SSC
(1.times.SSC: 0.15 M NaCl, 0.015 M sodium citrate) and 40%
formamide at 42.degree. C. for 14 hours and the final washing
conditions were 0.5.times.SSC, 0.1% SDS at 60.degree. C. Four
genomic clones were purified and shown to contain the 3' end of the
open reading frame missing in the cDNA clones. The sequence was
obtained on both strands after subcloning of overlapping
restriction fragments in M13mp18 and M13mp19 using the Sanger
dideoxy nucleotide chain termination method.
[0066] Northern blot analysis
[0067] Two blots of human organs (MTN I and MTN II: 2 .mu.g
polyA.sup.+ RNA/lane) and a blot containing total RNA from
differentiated and undifferentiated HL-60 cells (10 .mu.g of total
RNA/lane) were hybridized with a probe corresponding to the new
receptor in order to characterize its tissue distribution. The
HL-60 cells were maintained in RPMI 1640 supplemented with 10% FCS,
5 mM L-glutamine, 50 U/ml penicillin and 50 .mu.g/ml streptomycin
at 37.degree. C. with 5% CO.sub.2. The HL-60 cells were incubated
during six days with or without 1 .mu.M retinoic acid or 1.25% DMSO
or during eight hours with 25 nM TPA. The RNA from the
differentiated or undifferentiated HL-60 cells was prepared with
the RNeasy kit (Quiagen) The blots were prehybridized 8 hours at
42.degree. C. in a 50% formamide, 2% SDS solution and hybridized
for 18 hours in the same solution supplemented with the
[.alpha..sup.32P] labelled probe. The final washing conditions were
0.1.times.SSC and 0.1% SDS at 55.degree. C. The blots were exposed
during twelve days and visualized as an autoradiography or using
the PhosphorImager SI (Molecular Dynamics).
[0068] Cell culture and transfection
[0069] The complete sequence of the new receptor according to the
invention was subcloned between the Hind III and Nhe I sites of the
bicistronic pEFIN3 expression vector. 1321N1 and CHO-K1 cells were
transfected with the recombinant pEFIN3 plasmid or with the plasmid
alone using the calcium phosphate precipitation method as described
(19). The transfected cells were selected with 400 .mu.g/ml G418 in
complete medium (10% FCS, 100 units/ml penicillin, 100 .mu.g/ml
streptomycin and 2.5 .mu.g/ml amphotericin B in Dulbecco's modified
Eagle's medium (DMEM)) two days after transfection and maintained
in the same medium (10).
[0070] Inositol phosphates (IP) measurement
[0071] 1321N1 cells were labelled for 24 hours with 10 mCi/ml
[.sup.3H] inositol in inositol free DMEM containing 5% FCS,
antibiotics, amphotericin, sodium pyruvate and 400 .mu.g/ml G418.
Cells were washed twice with Krebs-Ringer Hepes (KRH) buffer of the
following composition (124 mM NaCl, 5 mM KCl, 1.25 mM MgSO.sub.4,
1.45 mM CaCl.sub.2, 1.25 mM KH.sub.2PO.sub.4, 25 mM Hepes (pH:7.4)
and 8 mM glucose) and incubated in the same medium for 30 min. The
cells were then challenged by various nucleotides for 30 s. The
incubation was stopped by the addition of an ice cold 3% perchloric
acid solution. IP were extracted and separated on Dowex columns as
previously described (20).
[0072] Cyclic AMP measurements
[0073] Stably transfected CHO-K1 or 1321N1 cell lines were spread
on Petri dishes (150.000 cells per dish) and cultured in Ham's F12
or DMEM medium containing 10% FCS, antibiotics, amphotericin,
sodium pyruvate and 400 .mu.g/ml G418. Cells were preincubated for
30 min in KRH buffer with rolipram (25 .mu.M) and incubated for
different times in the presence of the agonists (15 min in most
experiments). The incubation was stopped by the addition of 1 ml
HCl 0.1 M. The incubation medium was dried up, resuspended in water
and diluted as required. Cyclic AMP was quantified by
radioimnunoassay after acetylation as previously described
(21).
Results
[0074] Cloning and Sequencing
[0075] A human cDNA placenta library was screened at moderate
stringency with a human P2Y.sub.4 probe. Nine clones which
hybridized weakly with the P2Y.sub.4 probe were obtained, purified
and analyzed. Six of them corresponded to the sequence of the
P2Y.sub.6 receptor (10) while three overlapping clones corresponded
to a partial sequence encoding a new G protein-coupled receptor,
displaying about 30% identity with the other P2Y receptors. The
partial open reading frame started with an ATG-codon in a Kozak
consensus but the 3' end was missing in all three cDNA clones. The
Inventors screened a human genomic DNA library using this partial
sequence as a probe. Four overlapping genomic clones were obtained.
Mapping of the coding sequence and partial sequencing allowed to
determine that the gene encoding the new receptor contains an
intron interrupting the coding sequence at the 5' end of the gene.
This intron separates the three first codons from the rest of the
coding sequence. Beside these first codons, the four genomic clones
contained the complete open reading frame including the 3' end
missing in the cDNA clones. The full open reading frame appeared as
1113 base pairs (bp) long and encoded a protein of 371 amino acids
containing one potential site for N-linked glycosylation and two
potential sites for phosphorylation by protein kinase C or
calmodulin-dependent protein kinases (FIG. 1). The new receptor,
provisionally named P2Y.sub.11, displays significant homologies
with the other P2Y receptors (FIG. 2). In particular, 33% and 28%
amino acid identity were observed respectively with the human
P2Y.sub.1 and P2Y.sub.2 receptors.
[0076] Tissue distribution of the new receptor
[0077] The tissue distribution of the new receptor transcripts was
investigated by Northern blotting (FIG. 3) by using a probe
corresponding to a partial sequence encoding transmembrane segments
3 to 7. The strongest signal was observed for human spleen and
corresponded to a 2 kilobase (kb)-length messenger RNA (MTN II). A
weaker signal was observed in small intestine (MTN II) All the
lanes in MTN I (heart, - brain, placenta, lung, liver, skeletal
muscle, kidney, pancreas) were negative. The Inventors also
detected specific 2 kb-length transcripts in HL-60 cells. The
signal was very weak in the undifferentiated HL-60 cells but
increased when the cells had been treated with retinoic acid or
DMSO. No increase was observed when the HL-60 cells were stimulated
with TPA. A weak non-specific hybridization with 18S rRNA was
observed. These data were confirmed with a non-overlapping probe
corresponding to the first 300 bp of the coding region, presenting
limited homologies with the other P2Y subtypes.
[0078] Functional Expression of the new receptor in 1321N1
astrocytoma cells
[0079] The complete sequence of the new receptor was introduced in
the pEFIN3 expression vector in order to transfect the 1321N1
astrocytoma cell line, used previously to characterize several P2Y
subtypes (6, 10, 12) The pool of G418-resistant clones was tested
for its functional response to several nucleotides. ATP (100 .mu.M)
induced a strong inositol trisphosphate (IP.sub.3) accumulation in
cells transfected with the recombinant plasmid, whereas ADP, AMP,
adenosine, UTP, UDP, AP.sub.4A and AP.sub.6A were inactive at the
same concentration. All nucleotides were totally inactive on the
cells transfected with the vector alone. We then tested ATP,
2MeSATP, ADP and 2MeSADP in a large range of concentrations. As
shown in FIG. 4A, ATP was the most potent agonist (EC.sub.50
ATP=38.+-.7 .mu.M; EC.sub.50 2MeSATP=118.+-.15 .mu.M;
means.+-.range of two independent experiments). The effect of ADP
and 2MeSADP were minimal. Pertussis toxin (50 ng/ml; 24 h
pretreatment) had no effect on the ATP response, whereas a lower
concentration of pertussis toxin was previously shown to abolish
the response to UTP in P2Y.sub.4 transfected 1321N1 astrocytoma
cells (22). A response to ATP (10 .mu.M) was also obtained
following [Ca.sup.2+].sub.i measurements performed on the 1321N1
transfected cells while ADP was inactive at this concentration.
[0080] Functional expression of the new receptor in CHO-K1
cells
[0081] The 1321N1 cells transfected with the new receptor displayed
a strong cAMP increase in response to ATP. A much lower but
significant endogeneous response due to the degradation of adenine
nucleotides into adenosine was also obtained in the 1321N1 cells
transfected with the vector alone. The CHO-K1 cells express an
endogeneous P2Y.sub.2 receptor coupled to the phosphoinositide
pathway (23) but do not possess adenosine receptors coupled to
adenylyl cyclase. We therefore used CHO-K1 cells in order to
characterize the coupling of the new receptor to the cAMP pathway.
A pool of G418 resistant CHO-K1 clones was first tested for its
response to several nucleotides at a concentration of 100 .mu.M.
ATP was able to induce a strong increase in the cAMP content,
whereas it was inactive on cells transfected with the vector alone.
ADP, AMP, adenosine, UTP and UDP were completely inactive.
Concentration-action curves were established for ATP, 2MeSATP, ADP
and 2MeSADP (FIG. 4B). The rank order of potency was the same as in
the inositol phosphate study on 1321N1 cells. The curves were
obtained after 15 min of stimulation by the agonists; however a
significant cAMP response to ATP was already obtained after 2 min
of stimulation. The response to ATP (30 .mu.M) was inhibited
neither by indomethacin (10 .mu.g/ml, present from 30 minutes
before the stimulation and readded in the stimulation medium) nor
by 8 (p-sulfophenyl) theophylline (100 .mu.M).
[0082] The receptor according to the invention presents some
structural peculiarities which differentiate it from some other P2Y
subtypes. Concerning its gene structure, the coding sequence is
interrupted by an intron. Comparison between the cDNA and the
genomic DNA sequences has clearly demonstrated the absence of
intron in the coding region of the human P2Y.sub.1 receptor (24,
25), the rat P2Y.sub.2 receptor (26) and the rat P2Y.sub.6 receptor
(11). In terms of protein structure, the second and third
extracellular loops are significantly longer than those of the
other P2Y receptors. The homology with the other subtypes is
relatively weak (about 30%). The closest G-coupled receptor is the
human P2Y.sub.1 receptor (33%) which is also a receptor responsive
to adenine nucleotides (3, 4). Mutagenesis experiments with the
P2Y.sub.2 receptor have identified three positively charged amino
acids in the sixth and seventh transmembrane domains (His.sup.262,
Arg.sup.265 and Arg.sup.292), which play a crucial role in
nucleotide binding (presumably by neutralizing the negative charge
of the phosphate groups) (27). These three residues are conserved
in this new receptor.
[0083] So far, eight P2Y receptor subtypes are described in the
literature (P2Y.sub.1-P2Y.sub.8) . In addition, two sequences
related to the P2Y.sub.5 receptor and named P2Y.sub.9 and
P2Y.sub.10, have been recently submitted to the GenBank/EMBL Data
Bank. The P2Y.sub.9 sequence is identical to that recently
published under the name "P2Y.sub.5-like" (18). Therefore the new
receptor described in this paper might be called P2Y.sub.11.
However, it is already clear that the nomenclature needs a
revision. It was recently demonstrated that the P2Y.sub.7 receptor
is actually a receptor for leukotriene B.sub.4 (16) and there is no
functional evidence that the P2Y.sub.5 and related receptors
(P2Y.sub.5-like or P2Y.sub.9, P2Y.sub.10) are nucleotide receptors
(17, 18).
[0084] Among the sixteen human organs tested by Northern blotting,
P2Y.sub.11 transcripts of 2 kb size were only detectable in spleen,
and with lower intensity in small intestine. This distribution is
reminiscent of that of the human P2Y.sub.6 1.7 kb-messenger. The
observation of the expression of the P2Y.sub.11 receptor in the
HL-60 cell line shows that this expression was strongly increased
following treatment by DMSO or retinoic acid, two agents known to
induce the differentiation of these cells into granulocytes (28) .
On the contrary, TPA, which is known to induce the monocytic
differentiation of the HL-60 cells (29), did not stimulate the
expression of the P2Y11 receptor. The confirmation of these data
with a second probe of the P2Y.sub.11 cDNA, that shares little
similarity with other P2Y sequences, excludes possible
cross-hybridization with another P2Y receptor transcript. In view
of the Northern blots results, it is tempting to speculate that the
P2Y.sub.11 receptor is involved in the recently described
accumulation of cAMP in ATP-stimulated HL-60 cells (30).
[0085] Among the P2Y receptors, the P2Y11 subtype has the unique
property to activate both the phosphoinositide and the cAMP
pathways. Other cloned P2Y receptors are coupled to phospholipase C
exclusively. The rank order of potency of agonists was the same for
the two pathways. ATP was clearly much more potent than ADP. This
difference may be even underestimated as a result of low level ATP
contamination in ADP preparation or conversion of ADP into ATP
during assays (4, 11). On the other hand, 2MeSATP had the same
maximal effect than ATP but presented a lower potency, while
2MeSADP, a potent activator of the P2Y.sub.1 and P2T subtypes (4),
was almost inactive. The EC.sub.50 values were comparable to those
obtained in the study concerning the effects of extracellular
nucleotides on the cAMP accumulation in the HL-60 cells (30).
[0086] Stimulatory effects of adenine nucleotides on the cAMP
pathway have been described in different cell types (31, 32). In
most cases, the stimulatory effect of nucleotides was inhibited by
xanthines. These data suffer from the fact that it is difficult to
exclude that the effect of adenine nucleotides is mediated by their
degradation into adenosine due to the ubiquitous presence of
ectonucleotidases expressed at the cell surface. The cAMP study has
been performed with CHO-K1 cells to avoid the endogeneous cAMP
response to adenosine in the astrocytoma cell line. Neither in
untransfected CHO-K1 cells nor in P2Y.sub.11-transfected CHO-K1
cells did adenosine increase cAMP accumulation. Furthermore the
cAMP response to ATP was insensitive to xanthine inhibition. It was
also insensitive to indomethacin, indicating that is not mediated
by the release of prostaglandins. It is unlikely that the cAMP
response would be an indirect consequence of the calcium response
since the use of ATP, which activates the phosphoinositide pathway
by the activation of P2Y.sub.2 endogeneous receptors, or the use of
calcium ionophores in the CHO-K1 cells failed to stimulate cAMP
accumulation (33). Therefore these data constitute the first strong
evidence that a P2 receptor can be coupled to the stimulation of
adenylyl cyclase.
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Sequence CWU 1
1
2 1 805 PRT Homo sapien 1 Met Gly Gln Ser Gly Arg Ser Arg His Gln
Lys Arg Ala Arg Ala Gln 1 5 10 15 Ala Gln Leu Arg Asn Leu Glu Ala
Tyr Ala Ala Asn Pro His Ser Phe 20 25 30 Val Phe Thr Arg Gly Cys
Thr Gly Arg Asn Ile Arg Gln Leu Ser Leu 35 40 45 Asp Val Arg Arg
Val Met Glu Pro Leu Thr Ala Ser Arg Leu Gln Val 50 55 60 Arg Lys
Lys Asn Ser Leu Lys Asp Cys Val Ala Val Ala Gly Pro Leu 65 70 75 80
Gly Val Thr His Phe Leu Ile Leu Ser Lys Thr Glu Thr Asn Val Tyr 85
90 95 Phe Lys Leu Met Arg Leu Pro Gly Gly Pro Thr Leu Thr Phe Gln
Val 100 105 110 Lys Lys Tyr Ser Leu Val Arg Asp Val Val Ser Ser Leu
Arg Arg His 115 120 125 Arg Met His Glu Gln Gln Phe Ala His Pro Pro
Leu Leu Val Leu Asn 130 135 140 Ser Phe Gly Pro His Gly Met His Val
Lys Leu Met Ala Thr Met Phe 145 150 155 160 Gln Asn Leu Phe Pro Ser
Ile Asn Val His Lys Val Asn Leu Asn Thr 165 170 175 Ile Lys Arg Cys
Leu Leu Ile Asp Tyr Asn Pro Asp Ser Gln Glu Leu 180 185 190 Asp Phe
Arg His Tyr Ser Ile Lys Val Val Pro Val Gly Ala Ser Arg 195 200 205
Gly Met Lys Lys Leu Leu Gln Glu Lys Phe Pro Asn Met Ser Arg Leu 210
215 220 Gln Asp Ile Ser Glu Leu Leu Ala Thr Gly Ala Gly Leu Ser Glu
Ser 225 230 235 240 Glu Ala Glu Pro Asp Gly Asp His Asn Ile Thr Glu
Leu Pro Gln Ala 245 250 255 Val Ala Gly Arg Gly Asn Met Arg Ala Gln
Gln Ser Ala Val Arg Leu 260 265 270 Thr Glu Ile Gly Pro Arg Met Thr
Leu Gln Leu Ile Lys Val Gln Glu 275 280 285 Gly Val Gly Glu Gly Lys
Val Met Phe His Ser Phe Val Ser Lys Thr 290 295 300 Glu Glu Glu Leu
Gln Ala Ile Leu Glu Ala Lys Glu Lys Lys Leu Arg 305 310 315 320 Leu
Lys Ala Gln Arg Gln Ala Gln Gln Ala Gln Asn Tyr Gln Arg Lys 325 330
335 Gln Glu Gln Arg Glu Ala His Arg Lys Lys Ser Leu Glu Gly Met Lys
340 345 350 Lys Ala Arg Val Gly Gly Ser Asp Glu Glu Ala Ser Gly Ile
Pro Ser 355 360 365 Arg Thr Ala Ser Leu Glu Leu Gly Glu Asp Asp Asp
Glu Gln Glu Asp 370 375 380 Asp Asp Ile Glu Tyr Phe Cys Gln Ala Val
Gly Glu Ala Pro Ser Glu 385 390 395 400 Asp Leu Phe Pro Glu Ala Lys
Gln Lys Arg Leu Ala Lys Ser Pro Gly 405 410 415 Arg Lys Arg Lys Arg
Trp Glu Met Asp Arg Gly Ala Lys Ser Cys Pro 420 425 430 Ala Asn Phe
Leu Ala Ala Ala Asp Asp Lys Leu Ser Gly Phe Gln Gly 435 440 445 Asp
Phe Leu Trp Pro Ile Leu Val Val Glu Phe Leu Val Ala Val Ala 450 455
460 Ser Asn Gly Leu Ala Leu Tyr Arg Phe Ser Ile Arg Lys Gln Arg Pro
465 470 475 480 Trp His Pro Ala Val Val Phe Ser Val Gln Leu Ala Val
Ser Asp Leu 485 490 495 Leu Cys Ala Leu Thr Leu Pro Pro Leu Ala Ala
Tyr Leu Tyr Pro Pro 500 505 510 Lys His Trp Arg Tyr Gly Glu Ala Ala
Cys Arg Leu Glu Arg Phe Leu 515 520 525 Phe Thr Cys Asn Leu Leu Gly
Ser Val Ile Phe Ile Thr Cys Ile Ser 530 535 540 Leu Asn Arg Tyr Leu
Gly Ile Val His Pro Phe Phe Ala Arg Ser His 545 550 555 560 Leu Arg
Pro Lys His Ala Trp Ala Val Ser Ala Ala Gly Trp Val Leu 565 570 575
Ala Ala Leu Leu Ala Met Pro Thr Leu Ser Phe Ser His Leu Lys Arg 580
585 590 Pro Pro Gln Gln Gly Ala Gly Asn Cys Ser Val Ala Arg Pro Glu
Ala 595 600 605 Cys Ile Lys Cys Leu Gly Thr Ala Asp His Gly Leu Ala
Ala Tyr Arg 610 615 620 Ala Tyr Ser Leu Val Leu Ala Gly Leu Gly Cys
Gly Leu Pro Leu Leu 625 630 635 640 Leu Thr Leu Ala Ala Tyr Gly Ala
Leu Gly Arg Ala Val Leu Arg Ser 645 650 655 Pro Gly Met Thr Val Ala
Glu Lys Leu Arg Val Ala Ala Leu Val Ala 660 665 670 Ser Gly Val Ala
Leu Tyr Ala Ser Ser Tyr Val Pro Tyr His Ile Met 675 680 685 Arg Val
Leu Asn Val Asp Ala Arg Arg Arg Trp Ser Thr Arg Cys Pro 690 695 700
Ser Phe Ala Asp Ile Ala Gln Ala Thr Ala Ala Leu Glu Leu Gly Pro 705
710 715 720 Tyr Val Gly Tyr Gln Val Met Arg Gly Leu Met Pro Leu Ala
Phe Cys 725 730 735 Val His Pro Leu Leu Tyr Met Ala Ala Val Pro Ser
Leu Gly Cys Cys 740 745 750 Cys Arg His Cys Pro Gly Tyr Arg Asp Ser
Trp Asn Pro Glu Asp Ala 755 760 765 Lys Ser Thr Gly Gln Ala Leu Pro
Leu Asn Ala Thr Ala Ala Pro Lys 770 775 780 Pro Ser Glu Pro Gln Ser
Arg Glu Leu Ser Gln Glu Asp Cys Ser Ala 785 790 795 800 His Ala His
Asp Cys 805 2 2427 DNA Homo sapien 2 gaattcggca cgaggaggcc
tcgtggagga cacagcagca tgggacagtc agggaggtcc 60 cggcaccaga
agcgcgcccg cgcccaggcg cagctccgca acctcgaggc ctatgccgcg 120
aacccgcact cgttcgtgtt cacgcgaggc tgcacgggtc gcaacatccg gcagctcagc
180 ctggacgtgc ggcgggtcat ggagccgctc actgccagcc gtctgcaggt
tcgtaagaag 240 aactcgctga aggactgcgt ggcagtggct gggcccctcg
gggtcacaca ctttctgatc 300 ctgagcaaaa cagagaccaa tgtctacttt
aagctgatgc gcctcccagg aggccccacc 360 ttgaccttcc aggtgaagaa
gtactcgctg gtgcgtgatg tggtctcctc actgcgccgg 420 caccgcatgc
acgagcagca gtttgcccac ccacccctcc tggtactcaa cagctttggc 480
ccccatggta tgcatgtgaa gctcatggcc accatgttcc agaacctgtt cccctccatc
540 aacgtgcaca aggtgaacct gaacaccatc aagcgctgcc tcctcatcga
ctacaacccc 600 gactcccagg agctggactt ccgccactat agcatcaaag
ttgttcctgt gggcgcgagt 660 cgcgggatga agaagctgct ccaggagaag
ttccccaaca tgagccgcct gcaggacatc 720 agcgagctgc tggccacggg
cgcggggctg tcggagagcg aggcagagcc tgacggcgac 780 cacaacatca
cagagctgcc tcaggctgtc gctggccgtg gcaacatgcg ggcccagcag 840
agtgcagtgc ggctcaccga gatcggcccg cggatgacac tgcagctcat caaggtccag
900 gagggcgtcg gggagggcaa agtgatgttc cacagttttg tgagcaagac
ggaggaggag 960 ctgcaggcca tcctggaagc caaggagaag aagctgcggc
tgaaggcgca gaggcaggcc 020 cagcaggccc agaatgtgca gcgcaagcag
gagcagcggg aggcccacag aaagaagagc 080 ctggagggca tgaagaaggc
acgggtcggg ggtagtgatg aagaggcctc tgggatccct 140 tcaaggacgg
cgagcctgga gttgggtgag gacgatgatg aacaggaaga tgatgacatc 200
gagtatttct gccaggcggt gggcgaggcg cccagtgagg acctgttccc cgaggccaag
260 cagaaacggc ttgccaagtc tccagggcgg aagcggaagc ggtgggaaat
ggatcgaggt 320 gccaagtcct gccctgccaa cttcttggca gctgccgacg
acaaactcag tgggttccag 380 ggggacttcc tgtggcccat actggtggtt
gagttcctgg tggccgtggc cagcaatggc 440 ctggccctgt accgcttcag
catccggaag cagcgcccat ggcaccccgc cgtggtcttc 500 tctgtccagc
tggcagtcag cgacctgctc tgcgctctga cgctgccccc gctggccgcc 560
tacctctatc cccccaagca ctggcgctat ggggaggccg cgtgccgcct ggagcgcttc
620 ctcttcacct gcaacctgct gggcagcgtc atcttcatca cctgcatcag
cctcaaccgc 680 tacctgggca tcgtgcaccc cttcttcgcc cgaagccacc
tgcgacccaa gcacgcctgg 740 gccgtgagcg ctgccggctg ggtcctggcc
gccctgctgg ccatgcccac actcagcttc 800 tcccacctga agaggccgcc
gcagcagggg gcgggcaact gcagcgtggc caggcccgag 860 gcctgcatca
agtgtctggg gacagcagac cacgggctgg cggcctacag agcgtatagc 920
ctggtgctgg cggggttggg ctgcggcctg ccgctgctgc tcacgctggc agcctacggc
980 gccctcgggc gggccgtgct acgcagccca ggcatgactg tggccgagaa
gctgcgtgtg 040 gcagcgttgg tggccagtgg tgtggccctc tacgccagct
cctatgtgcc ctaccacatc 100 atgcgggtgc tcaacgtgga tgctcggcgg
cgctggagca cccgctgccc gagctttgca 160 gacatagccc aggccacagc
agccctggag ctggggccct acgtgggcta ccaggtgatg 220 cggggcctca
tgcccctggc cttctgtgtc caccctctac tctacatggc cgcagtgccc 280
agcctgggct gctgctgccg acactgcccc ggctacaggg acagctggaa cccagaggac
340 gccaagagca ctggccaagc cctgcccctc aatgccacag ccgcccctaa
accgtcagag 400 ccccagtccc gtgagctgag ccaatga 427
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