U.S. patent application number 09/771287 was filed with the patent office on 2003-05-01 for a method of treating depression using a galr3 receptor antagonist.
This patent application is currently assigned to Synaptic Pharmaceutical Corporation. Invention is credited to Bard, Jonathan A., Borowsky, Beth, Branchek, Theresa A., Gerald, Christophe P.G., Jones, Kenneth A., Smith, Kelli E..
Application Number | 20030082641 09/771287 |
Document ID | / |
Family ID | 27505578 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030082641 |
Kind Code |
A1 |
Bard, Jonathan A. ; et
al. |
May 1, 2003 |
A METHOD OF TREATING DEPRESSION USING A GALR3 RECEPTOR
ANTAGONIST
Abstract
This invention provides an isolated nucleic acid encoding a
mammalian galanin receptor, an isolated galanin receptor protein,
vectors comprising isolated nucleic acid encoding a mammalian
galanin receptor, cells comprising such vectors, antibodies
directed to a mammalian galanin receptor, nucleic acid probes
useful for detecting nucleic acid encoding a mammalian galanin
receptor, antisense oligonucleotides complementary to unique
sequences of nucleic acid encoding a mammalian galanin receptor,
nonhuman transgenic animals which express DNA encoding a normal or
a mutant mammalian galanin receptor, as well as methods of
determining binding of compounds to mammalian galanin
receptors.
Inventors: |
Bard, Jonathan A.;
(Doylestown, PA) ; Borowsky, Beth; (Montclair,
NJ) ; Smith, Kelli E.; (Wayne, NJ) ; Branchek,
Theresa A.; (Teaneck, NJ) ; Gerald, Christophe
P.G.; (Ridgewood, NJ) ; Jones, Kenneth A.;
(Bergenfield, NJ) |
Correspondence
Address: |
John P. White, Esq.
Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
Synaptic Pharmaceutical
Corporation
|
Family ID: |
27505578 |
Appl. No.: |
09/771287 |
Filed: |
January 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09771287 |
Jan 26, 2001 |
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09058333 |
Apr 9, 1998 |
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6368812 |
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09058333 |
Apr 9, 1998 |
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PCT/US97/18222 |
Oct 9, 1997 |
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PCT/US97/18222 |
Oct 9, 1997 |
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08900230 |
Jul 23, 1997 |
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08900230 |
Jul 23, 1997 |
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08787261 |
Jan 24, 1997 |
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08787261 |
Jan 24, 1997 |
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08767964 |
Dec 17, 1996 |
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08767964 |
Dec 17, 1996 |
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08728139 |
Oct 9, 1996 |
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Current U.S.
Class: |
435/7.21 ;
435/320.1; 435/325; 435/69.1; 530/350 |
Current CPC
Class: |
C07K 14/72 20130101;
G01N 33/74 20130101; C07K 16/2869 20130101; A01K 2217/05 20130101;
A61K 38/00 20130101; G01N 2500/10 20130101; G01N 2333/72 20130101;
G01N 33/5088 20130101 |
Class at
Publication: |
435/7.21 ;
435/69.1; 435/325; 435/320.1; 530/350 |
International
Class: |
G01N 033/567; C12P
021/02; C07K 014/715 |
Claims
What is claimed is:
1. An isolated nucleic acid encoding a GALR3 receptor.
2. The nucleic acid of claim 1, wherein the nucleic acid is
DNA.
3. The DNA of claim 2, wherein the DNA is cDNA.
4. The DNA of claim 2, wherein the DNA is genomic DNA.
5. The nucleic acid of claim 1, wherein the nucleic acid is
RNA.
6. The nucleic acid of claim 1, wherein the nucleic acid encodes a
vertebrate GALR3 receptor.
7. The nucleic acid of claim 1, wherein the nucleic acid encodes a
mammalian GALR3 receptor.
8. The nucleic acid of claim 1, wherein the nucleic acid encodes a
rat GALR3 receptor.
9. The nucleic acid of claim 1, wherein the nucleic acid encodes a
human GALR3 receptor.
10. The nucleic acid of claim 7, wherein the nucleic acid encodes a
receptor characterized by an amino acid sequence in the
transmembrane region which has a homology of 70% or higher to the
amino acid sequence in the transmembrane region of the rat GALR3
receptor and a homology of less than 70% to the amino acid sequence
in the transmembrane region of any GALR1 receptor.
11. The nucleic acid of claim 7, wherein the nucleic acid encodes a
mammalian GALR3 receptor which has substantially the same amino
acid sequence as does the GALR3 receptor encoded by the plasmid
K1086 (ATCC Accession No. 97747).
12. The nucleic acid of claim 8, wherein the nucleic acid encodes a
rat GALR3 receptor which has an amino acid sequence encoded by the
plasmid K1086 (ATCC Accession No. 97747).
13. The nucleic acid of claim 7, wherein the nucleic acid encodes a
mammalian GALR3 receptor which has substantially the same amino
acid sequence as does the GALR3 receptor encoded by the plasmid
pEXJ-RGalR3T (ATCC Accession No. 97826).
14. The nucleic acid of claim 8, wherein the nucleic acid encodes a
rat GALR3 receptor which has an amino acid sequence encoded by the
plasmid pEXJ-RGalR3T (ATCC Accession No. 97826).
15. The nucleic acid of claim 8, wherein the nucleic acid encodes a
rat GALR3 receptor having substantially the same amino acid
sequence as the amino acid sequence shown in FIG. 2 (Seq. ID No.
2).
16. The nucleic acid of claim 8, wherein the nucleic acid encodes a
rat GALR3 receptor having the amino acid sequence shown in FIG. 2
(Seq. ID No. 2).
17. The nucleic acid of claim 7, wherein the nucleic acid encodes a
mammalian galanin receptor which has substantially the same amino
acid sequence as does the GALR3 receptor encoded by the plasmid
pEXJ-hGalR3 (ATCC Accession No. 97827).
18. The nucleic acid of claim 9, wherein the nucleic acid encodes a
human galanin receptor which has an amino acid sequence encoded by
the plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
19. The nucleic acid of claim 9, wherein the human GALR3 receptor
has a sequence, which sequence comprises substantially the same
amino acid sequence as the sequence shown in FIG. 4 (Seq. I.D. No.
4) from amino acid 60 through amino acid 427.
20. The nucleic acid of claim 19, wherein the human GALR3 receptor
has a sequence, which sequence comprises the sequence shown in FIG.
4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
21. An isolated nucleic acid encoding a modified GALR3 receptor,
which differs from a GALR3 receptor by having an amino acid(s)
deletion, replacement or addition in the third intracellular
domain.
22. The nucleic acid of claim 21, wherein the modified GALR3
receptor differs by having a deletion in the third intracellular
domain.
23. The nucleic acid of claim 21, wherein the modified GALR3
receptor differs by having a replacement or addition to the third
intracellular domain.
24. A purified GALR3 receptor protein.
25. A vector comprising the nucleic acid of claim 1.
26. A vector comprising the nucleic acid of claim 9.
27. A vector of claim 25 adapted for expression in a bacterial cell
which comprises the regulatory elements necessary for expression of
the nucleic acid in the bacterial cell operatively linked to the
nucleic acid encoding a GALR3 receptor as to permit expression
thereof.
28. A vector of claim 25 adapted for expression in an amphibian
cell which comprises the regulatory elements necessary for
expression of the nucleic acid in the amphibian cell operatively
linked to the nucleic acid encoding a GALR3 receptor as to permit
expression thereof.
29. A vector of claim 25 adapted for expression in a yeast cell
which comprises the regulatory elements necessary for expression of
the nucleic acid in the yeast cell operatively linked to the
nucleic acid encoding a GALR3 receptor as to permit expression
thereof.
30. A vector of claim 25 adapted for expression in an insect cell
which comprises the regulatory elements necessary for expression of
the nucleic acid in the insect cell operatively linked to the
nucleic acid encoding the GALR3 receptor as to permit expression
thereof.
31. A vector of claim 30 which is a baculovirus.
32. A vector of claim 25 adapted for expression in a mammalian cell
which comprises the regulatory elements necessary for expression of
the nucleic acid in the mammalian cell operatively linked to the
nucleic acid encoding a GALR3 receptor as to permit expression
thereof.
33. A vector of claim 25 wherein the vector is a plasmid.
34. The plasmid of claim 33 designated K1086 (ATCC Accession No.
97747).
35. The plasmid of claim 33 designated pEXJ-hGalR3 (ATCC Accession
No. 97827).
36. The plasmid of claim 33 designated pEXJ-RGalR3T (ATCC Accession
No. 97826).
37. The plasmid of claim 33 designated M54 (ATCC Accession No.
209312).
38. The plasmid of claim 33 designated M67 (ATCC Accession No.
______).
39. A cell comprising the vector of claim 25.
40. A cell of claim 39, wherein the cell is a non-mammalian
cell.
41. A cell of claim 40, wherein the non-mammalian cell is a Xenopus
oocyte cell or a Xenopus melanophore cell.
42. A cell of claim 39, wherein the cell is a mammalian cell.
43. A mammalian cell of claim 42, wherein the cell is a COS-7 cell,
a 293 human embryonic kidney cell, a NIH-3T3 cell, a mouse Y1 cell,
a LM(tk-) cell or a CHO cell.
44. The 293 human embryonic kidney cell of claim 43 designated
293-rGalR3-105 (ATCC Accession No. CRL-12287).
45. The LM(tk-) cell of claim 43 designated L-hGalR3-228 (ATCC
Accession No. CRL-12373).
46. An insect cell comprising the vector of claim 32.
47. An insect cell of claim 46, wherein the insect cell is an Sf9
cell.
48. An insect cell of claim 46, wherein the insect cell is an Sf21
cell.
49. A membrane preparation isolated from the cell of claim 39 or
46.
50. A nucleic acid probe comprising at least 15 nucleotides, which
probe specifically hybridizes with a nucleic acid encoding a GALR3
receptor, wherein the probe has a unique sequence corresponding to
a sequence present within one of the two strands of the nucleic
acid encoding the GALR3 receptor contained in plasmid K1086.
51. A nucleic acid probe comprising at least 15 nucleotides, which
probe specifically hybridizes with a nucleic acid encoding a GALR3
receptor, wherein the probe has a unique sequence corresponding to
a sequence present within (a) the nucleic acid sequence shown in
FIG. 1 (Seq. ID No. 1) or (b) the reverse complement to the nucleic
acid sequence shown in FIG. 1 (Seq. ID No. 1).
52. A nucleic acid probe comprising at least 15 nucleotides, which
probe specifically hybridizes with a nucleic acid encoding a GALR3
receptor, wherein the probe has a unique sequence corresponding to
a sequence present within one of the two strands of the nucleic
acid encoding the GALR3 receptor contained in plasmid
pEXJ-hGalR3.
53. A nucleic acid probe comprising at least 15 nucleotides, which
probe specifically hybridizes with a nucleic acid encoding a GALR3
receptor, wherein the probe has a unique sequence corresponding to
a sequence present within (a) the nucleic acid sequence shown in
FIG. 3 (Seq. ID No. 3) or (b) the reverse complement to the nucleic
acid sequence shown in FIG. 3 (Seq. ID No. 3).
54. The nucleic acid probe of claim 52 or 53, wherein the nucleic
acid is DNA.
55. The nucleic acid probe of claim 52 or 53, wherein the nucleic
acid is RNA.
56. A nucleic acid probe comprising a nucleic acid molecule of at
least 15 nucleotides which is complementary to a unique fragment of
the sequence of a nucleic acid molecule encoding a GALR3
receptor.
57. A nucleic acid probe comprising a nucleic acid molecule of at
least 15 nucleotides which is complementary to the antisense
sequence of a unique fragment of the sequence of a nucleic acid
molecule encoding a GALR3 receptor.
58. An antisense oligonucleotide having a sequence capable of
specifically hybridizing to the RNA of claim 5, so as to prevent
translation of the RNA.
59. An antisense oligonucleotide having a sequence capable of
specifically hybridizing to the genomic DNA of claim 4.
60. An antisense oligonucleotide of either of claims 58 or 59,
wherein the oligonucleotide comprises chemically modified
nucleotides or nucleotide analogues.
61. An antibody capable of binding to a GALR3 receptor encoded by
the nucleic acid of claim 1.
62. The antibody of claim 61, wherein the GALR3 receptor is a human
GALR3 receptor.
63. An antibody capable of competitively inhibiting the binding of
the antibody of claim 61 to a GALR3 receptor.
64. An antibody of claim 61, wherein the antibody is a monoclonal
antibody.
65. A monoclonal antibody of claim 64 directed to an epitope of a
GALR3 receptor present on the surface of a GALR3 receptor
expressing cell.
66. A pharmaceutical composition comprising an amount of the
oligonucleotide of claim 58 capable of passing through a cell
membrane effective to reduce expression of a GALR3 receptor and a
pharmaceutically acceptable carrier capable of passing through a
cell membrane.
67. A pharmaceutical composition of claim 66, wherein the
oligonucleotide is coupled to a substance which inactivates
mRNA.
68. A pharmaceutical composition of claim 67, wherein the substance
which inactivates mRNA is a ribozyme.
69. A pharmaceutical composition of claim 66, wherein the
pharmaceutically acceptable carrier comprises a structure which
binds to a receptor on a cell capable of being taken up by the
cells after binding to the structure.
70. A pharmaceutical composition of claim 69 wherein the
pharmaceutically acceptable carrier is capable of binding to a
receptor which is specific for a selected cell type.
71. A pharmaceutical composition which comprises an amount of the
antibody of claim 61 effective to block binding of a ligand to the
GALR3 receptor and a pharmaceutically acceptable carrier.
72. A transgenic nonhuman mammal expressing DNA encoding a GALR3
receptor of claim 1.
73. A transgenic nonhuman mammal comprising a homologous
recombination knockout of the native GALR3 receptor.
74. A transgenic nonhuman mammal whose genome comprises antisense
DNA complementary to DNA encoding a GALR3 receptor of claim 1 so
placed as to be transcribed into antisense mRNA which is
complementary to mRNA encoding a GALR3 receptor and which
hybridizes to mRNA encoding a GALR3 receptor, thereby reducing its
translation.
75. The transgenic nonhuman mammal of either of claims 72 or 73,
wherein the DNA encoding a GALR3 receptor additionally comprises an
inducible promoter.
76. The transgenic nonhuman mammal of either of claims 72 or 73,
wherein the DNA encoding a GALR3 receptor additionally comprises
tissue specific regulatory elements.
77. A transgenic nonhuman mammal of any one of claims 72, 73 or 74,
wherein the transgenic nonhuman mammal is a mouse.
78. A process for identifying a chemical compound which
specifically binds to a GALR3 receptor which comprises contacting
cells containing DNA encoding and expressing on their cell surface
the GALR3 receptor, wherein such cells do not normally express the
GALR3 receptor, with the compound under conditions suitable for
binding, and detecting specific binding of the chemical compound to
the GALR3 receptor.
79. A process for identifying a chemical compound which
specifically binds to a GALR3 receptor which comprises contacting a
membrane fraction from a cell extract of cells containing DNA
encoding and expressing on their cell surface the GALR3 receptor,
wherein such cells do not normally express the GALR3 receptor, with
the compound under conditions suitable for binding, and detecting
specific binding of the chemical compound to the GALR3
receptor.
80. The process of claim 78 or 79, wherein the GALR3 receptor is a
mammalian GALR3 receptor.
81. The process of claim 78 or 79, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid K1086 (ATCC Accession No. 97747).
82. The process of claim 78 or 79, wherein the GALR3 receptor has
substantially the same sequence as the amino acid sequence shown in
FIG. 2 (Seq. ID No. 2).
83. The process of claim 78 or 79, wherein the GALR3 receptor has
the amino acid sequence shown in FIG. 2 (Seq. ID No. 2).
84. The process of claims 78 or 79, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid pEXJ-hGalR3 (ATTC Accession No. 97827).
85. The process of claim 78 or 79, wherein the GALR3 receptor has a
sequence, which sequence comprises substantially the same amino
acid sequence as a sequence shown in FIG. 4 (Seq. ID No. 4) from
amino acid 60 through amino acid 427.
86. The process of claim 78 or 79, wherein the GALR3 receptor has a
sequence, which sequence comprises a sequence shown in FIG. 4 (Seq.
ID No. 4) from amino acid 60 through amino acid 427.
87. The process of claim 78 or 79, wherein the cells are
transfected with plasmid pEXJ-RGalR3T (ATCC Accession No.
97826).
88. The process of claim 85, wherein the compound is not previously
known to bind to a GALR3 receptor.
89. A compound determined by the process of claim 88.
90. A process of claim 85, wherein the cell is an insect cell.
91. A process of claim 85, wherein the cell is a mammalian
cell.
92. A process of claim 91, wherein the cell is nonneuronal in
origin.
93. A process of claim 92, wherein the nonneuronal cell is a COS-7
cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell a
mouse Y1 cell or LM(tk-) cell.
94. A process of claim 91, wherein the compound is not previously
known to bind to a GALR3 receptor.
95. A compound determined by the process of claim 94.
96. A process involving competitive binding for identifying a
chemical compound which specifically binds to a GALR3 receptor
which comprises separately contacting cells expressing on their
cell surface the GALR3 receptor, wherein such cells do not normally
express the GALR3 receptor, with both the chemical compound and a
second chemical compound known to bind to the receptor, and with
only the second chemical compound, under conditions suitable for
binding of both compounds, and detecting specific binding of the
chemical compound to the GALR3 receptor, a decrease in the binding
of the second chemical compound to the GALR3 receptor in the
presence of the chemical compound indicating that the chemical
compound binds to the GALR3 receptor.
97. A process involving competitive binding for identifying a
chemical compound which specifically binds to a human GALR3
receptor which comprises separately contacting a membrane fraction
from a cell extract of cells expressing on their cell surface the
GALR3 receptor, wherein such cells do not normally express the
GALR3 receptor, with both the chemical compound and a second
chemical compound known to bind to the receptor, and with only the
second chemical compound, under conditions suitable for binding of
both compounds, and detecting specific binding of the chemical
compound to the GALR3 receptor, a decrease in the binding of the
second chemical compound to the GALR3 receptor in the presence of
the chemical compound indicating that the chemical compound binds
to the GALR3 receptor.
98. A process of claim 96 or 97, wherein the GALR3 receptor is a
mammalian GALR3 receptor.
99. The process of claim 98, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by plasmid
K1086 (ATCC Accession No. 97747).
100. The process of claim 96 or 97, wherein the GALR3 receptor has
substantially the same amino acid sequence as shown in FIG. 2 (Seq.
ID No. 2).
101. The process of either of claims 96 or 97, wherein the GALR3
receptor has the amino acid sequence shown in FIG. 2 (Seq. ID No.
2).
102. The process of claim 96 or 97, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by plasmid
pEXJ-hGalR3 (ATCC Accession No. 97827).
103. The process of claim 96 or 97, wherein the GALR3 receptor has
a sequence, which sequence comprises substantially the same amino
acid sequence as the sequence shown in FIG. 4 (Seq. ID No. 4) from
amino acid 60 through amino acid 427.
104. The process of claim 96 or 97, wherein the GALR3 receptor has
a sequence, which sequence comprises a sequence shown in FIG. 4
(Seq. ID No. 4) from amino acid 60 through amino acid 427.
105. The process of claim 96 or 97, wherein the cells are
transfected with plasmid pEXJ-RGalR3T (ATCC Accession No.
97826).
106. The process of claim 104, wherein the cell is an insect
cell.
107. The process of claim 104, wherein the cell is a mammalian
cell.
108. The process of claim 107, wherein the cell is nonneuronal in
origin.
109. The process of claim 107, wherein the nonneuronal cell is a
COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3
cell a mouse Y1 cell or LM(tk-) cell.
110. The process of claim 106, wherein the compound is not
previously known to bind to a GALR3 receptor.
111. A compound determined by the process of claim 110.
112. A method of screening a plurality of chemical compounds not
known to bind to a GALR3 receptor to identify a compound which
specifically binds to the GALR3 receptor, which comprises (a)
contacting cells transfected with and expressing DNA encoding the
GALR3 receptor with a compound known to bind specifically to the
GALR3 receptor; (b) contacting the preparation of step (a) with the
plurality of compounds not known to bind specifically to the GALR3
receptor, under conditions permitting binding of compounds known to
bind the GALR3 receptor; (c) determining whether the binding of the
compound known to bind to the GALR3 receptor is reduced in the
presence of the compounds, relative to the binding of the compound
in the absence of the plurality of compounds; and if so (d)
separately determining the binding to the GALR3 receptor of each
compound included in the plurality of compounds, so as to thereby
identify the compound which specifically binds to the GALR3
receptor.
113. A method of screening a plurality of chemical compounds not
known to bind to a GALR3 receptor to identify a compound which
specifically binds to the GALR3 receptor, which comprises (a)
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, contacting the membrane fraction with a compound
known to bind specifically to the GALR3 receptor; (b) contacting
the preparation of step (a) with the plurality of compounds not
known to bind specifically to the GALR3 receptor, under conditions
permitting binding of compounds known to bind the GALR3 receptor;
(c) determining whether the binding of the compound known to bind
to the GALR3 receptor is reduced in the presence of the compounds,
relative to the binding of the compound in the absence of the
plurality of compounds; and if so (d) separately determining the
binding to the GALR3 receptor of each compound included in the
plurality of compounds, so as to thereby identify the compound
which specifically binds to the GALR3 receptor.
114. A method of either of claims 112 or 113, wherein the GALR3
receptor is a mammalian GALR3 receptor.
115. A method of either of claims 112 or 113, wherein the cell is a
mammalian cell.
116. A method of claim 115, wherein the mammalian cell is
non-neuronal in origin.
117. The method of claim 116, wherein the non-neuronal cell is a
COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell, a
CHO cell, a mouse Y1 cell or an NIH-3T3 cell.
118. A method of detecting expression of a GALR3 receptor by
detecting the presence of mRNA coding for the GALR3 receptor which
comprises obtaining total mRNA from the cell and contacting the
mRNA so obtained with the nucleic acid probe of claim 52 under
hybridizing conditions, detecting the presence of mRNA hybridized
to the probe, and thereby detecting the expression of the GALR3
receptor by the cell.
119. A method of detecting the presence of a GALR3 receptor on the
surface of a cell which comprises contacting the cell with the
antibody of claim 61 under conditions permitting binding of the
antibody to the receptor, detecting the presence of the antibody
bound to the cell, and thereby detecting the presence of a GALR3
receptor on the surface of the cell.
120. A method of determining the physiological effects of varying
levels of activity of GALR3 receptors which comprises producing a
transgenic nonhuman mammal of claim 75 whose levels of GALR3
receptor activity are varied by use of an inducible promoter which
regulates GALR3 receptor expression.
121. A method of determining the physiological effects of varying
levels of activity of GALR3 receptors which comprises producing a
panel of transgenic nonhuman mammals of claim 75 each expressing a
different amount of GALR3 receptor.
122. A method for identifying an antagonist capable of alleviating
an abnormality wherein the abnormality is alleviated by decreasing
the activity of a GALR3 receptor comprising administering a
compound to the transgenic nonhuman mammal of any one of claims 72,
75, 76, or 77, and determining whether the compound alleviates the
physical and behavioral abnormalities displayed by the transgenic
nonhuman mammal as a result of overactivity of a GALR3 receptor,
the alleviation of the abnormality identifying the compound as an
antagonist.
123. An antagonist identified by the method of claim 122.
124. A pharmaceutical composition comprising an antagonist
identified by the method of claim 123 and a pharmaceutically
acceptable carrier.
125. A method of treating an abnormality in a subject wherein the
abnormality is alleviated by decreasing the activity of a GALR3
receptor which comprises administering to a subject an effective
amount of the pharmaceutical composition of claim 124, thereby
treating the abnormality.
126. A method for identifying an agonist capable of alleviating an
abnormality in a subject wherein the abnormality is alleviated by
increasing the activity of a GALR3 receptor comprising
administering a compound to the transgenic nonhuman mammal of any
one of claims 72, 75, 76, or 77, and determining whether the
compound alleviates the physical and behavioral abnormalities
displayed by the transgenic nonhuman mammal, the alleviation of the
abnormality identifying the compound as an agonist.
127. An agonist identified by the method of claim 126.
128. A pharmaceutical composition comprising an agonist identified
by the method of claim 126 and a pharmaceutically acceptable
carrier.
129. A method for treating an abnormality in a subject wherein the
abnormality is alleviated by increasing the activity of a GALR3
receptor which comprises administering to a subject an effective
amount of the pharmaceutical composition of claim 128, thereby
treating the abnormality.
130. A method for diagnosing a predisposition to a disorder
associated with the activity of a specific human GALR3 receptor
allele which comprises: a. obtaining DNA of subjects suffering from
the disorder; b. performing a restriction digest of the DNA with a
panel of restriction enzymes; c. electrophoretically separating the
resulting DNA fragments on a sizing gel; d. contacting the
resulting gel with a nucleic acid probe capable of specifically
hybridizing with a unique sequence included within the sequence of
a nucleic acid molecule encoding a human GALR3 receptor and
labelled with a detectable marker; e. detecting labelled bands
which have hybridized to the DNA encoding a human GALR3 receptor of
claim 9 labelled with a detectable marker to create a unique band
pattern specific to the DNA of subjects suffering from the
disorder; f. preparing DNA obtained for diagnosis by steps a-e; and
g. comparing the unique band pattern specific to the DNA of
subjects suffering from the disorder from step e and the DNA
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.
131. The method of claim 130, wherein a disorder associated with
the activity of a specific human GALR3 receptor allele is
diagnosed.
132. A method of preparing the purified GALR3 receptor of claim 24
which comprises: a. inducing cells to express GALR3 receptor; b.
recovering the receptor from the induced cells; and c. purifying
the receptor so recovered.
133. A method of preparing the purified GALR3 receptor of claim 24
which comprises: a. inserting nucleic acid encoding the GALR3
receptor in a suitable vector; b. introducing the resulting vector
in a suitable host cell; c. placing the resulting cell in suitable
condition permitting the production of the isolated GALR3 receptor;
d. recovering the receptor produced by the resulting cell; and e.
purifying the receptor so recovered.
134. A method of modifying feeding behavior of a subject which
comprises administering to the subject an amount of a compound
which is a GALR3 receptor agonist or antagonist effective to
increase or decrease the consumption of food by the subject so as
to thereby modify feeding behavior of the subject.
135. The method of claim 134, wherein the compound is a GALR3
receptor antagonist and the amount is effective to decrease the
consumption of food by the subject.
136. The method of either of claims 134 or 135, wherein the
compound is administered in combination with food.
137. The method of claim 134, wherein the compound is a GALR3
receptor agonist and the amount is effective to increase the
consumption of food by the subject.
138. The method of either of claims 134 or 135, wherein the
compound is administered in combination with food.
139. The method of claim 134, wherein the subject is a vertebrate,
a mammal, a human or a canine.
140. The method of claim 135 or 137, wherein the compound binds
selectively to a GALR3 receptor.
141. The method of claim 140, wherein the compound binds to the
GALR3 receptor with an affinity greater than ten-fold higher than
the affinity with which the compound binds to a GALR1 receptor.
142. The method of claim 140, wherein the compound binds to the
GALR3 receptor with an affinity greater than ten-fold higher than
the affinity with which the compound binds to a GALR2 receptor.
143. A method for determining whether a compound is a GALR3
antagonist which comprises: (a) administering to an animal a GALR3
agonist and measuring the amount of food intake in the animal; (b)
administering to a second animal both the GALR3 agonist and the
compound, and measuring the amount of food intake in the second
animal; and (c) determining whether the amount of food intake is
reduced in the presence of the compound relative to the amount of
food intake in the absence of the compound, so as to thereby
determine whether the compound is a GALR3 antagonist.
144. A method of screening a plurality of compounds to identify a
compound which is a GALR3 antagonist which comprises: (a)
administering to an animal a GALR3 agonist and measuring the amount
of food intake in the animal; (b) administering to a second animal
the GALR3 agonist and at least one compound of the plurality of
compounds and measuring the amount of food intake in the animal;
(c) determining whether the amount of food intake is reduced in the
presence of at least one compound of the plurality relative to the
amount of food intake in the absence of at least one compound of
the plurality, and if so; (d) separately determining whether each
compound is a GALR3 antagonist according to the method of claim
122, so as to thereby identify a compound which is a GALR3
antagonist.
145. The method of either of claims 143 or 144, wherein the GALR3
agonist is galanin.
146. The method of either of claims 143 or 144, wherein the animal
is a non-human mammal.
147. The method of claim 146, wherein the mammal is a rodent.
148. A process of claim 78 or 79, which further comprises
determining whether the compound selectively binds to the GALR3
receptor relative to another galanin receptor.
149. The process of claim 148, wherein the determination whether
the compound selectively binds to the GALR3 receptor comprises: (a)
determining the binding affinity of the compound for the GALR3
receptor and for such other galanin receptor; and (b) comparing the
binding affinities so determined, the presence of a higher binding
affinity for the GALR3 receptor than for such other galanin
receptor indicating that the compound selectively binds to the
GALR3 receptor.
150. A process of claim 148, wherein the other galanin receptor is
a GALR1 receptor.
151. A process of claim 148, wherein the other galanin receptor is
a GALR2 receptor.
152. A method of decreasing feeding behavior of a subject which
comprises administering a compound which is a GALR3 receptor
antagonist and a compound which is a Y5 receptor antagonist, the
amount of such antagonists being effective to decrease the feeding
behavior of the subject.
153. The method of claim 152, wherein the GALR3 antagonist and the
Y5 antagonist are administered in combination.
154. The method of claim 152, wherein the GALR3 antagonist and the
Y5 antagonist are administered once.
155. The method of claim 152, wherein the GALR3 antagonist and the
Y5 antagonist are administered separately.
156. The method of claim 155, wherein the GALR3 antagonist and the
Y5 antagonist are administered once.
157. The method of claim 155, wherein the galanin receptor
antagonist is administered for about 1 week to 2 weeks.
158. The method of claim 155, wherein the Y5 receptor antagonist is
administered for about 1 week to 2 weeks.
159. The method of claim 155, wherein the GALR3 antagonist and the
Y5 antagonist are administered alternately.
160. The method of claim 159, wherein the GALR3 antagonist and the
Y5 antagonist are administered repeatedly.
161. A method of claim 159 or claim 160, wherein the galanin
receptor antagonist is administered for about 1 week to 2
weeks.
162. A method of claim 159 or claim 160, wherein the Y5 receptor
antagonist is administered for about 1 week to 2 weeks.
163. A method of any one of claims 152, 153, 154, or 155, wherein
the compound is administered in a pharmaceutical composition
comprising a sustained release formulation.
164. A method of decreasing nociception in a subject which
comprises administering to the subject an amount of a compound
which is a GALR3 receptor agonist effective to decrease nociception
in the subject.
165. A method of treating pain in a subject which comprises
administering to the subject an amount of a compound which is a
GALR3 receptor agonist effective to treat pain in the subject.
166. A method of treating diabetes in a subject which comprises
administering to the subject an amount of a compound which is a
GALR3 receptor antagonist effective to treat diabetes in the
subject.
167. A method of enhancing cognition in a subject which comprises
administering to the subject an amount of a compound which is a
GALR3 receptor antagonist effective to enhance cognition in the
subject.
168. A process for determining whether a chemical compound is a
GALR3 receptor agonist which comprises contacting cells which
express the GALR3 receptor with the compound under conditions
permitting the activation of the GALR3 receptor, and detecting an
increase in GALR3 receptor activity, so as to thereby determine
whether the compound is a GALR3 receptor agonist, wherein the cells
do not normally express the GALR3 receptor.
169. A process for determining whether a chemical compound is a
GALR3 receptor antagonist which comprises contacting cells which
express the GALR3 receptor with the compound in the presence of a
known GALR3 receptor agonist, under conditions permitting the
activation of the GALR3 receptor, and detecting a decrease in GALR3
receptor activity, so as to thereby determine whether the compound
is a GALR3 receptor antagonist, wherein the cells do not normally
express the GALR3 receptor.
170. A process of claim 168 or 169, wherein the cells are
transfected with and express DNA encoding the GALR3 receptor.
171. A process of claim 168 or 169, wherein RNA encoding and
expressing the GALR3 receptor has been injected into the cells.
172. A process of any one of claims 168, 169, 170, or 171, wherein
the cells also express GIRK1 and GIRK4.
173. A process of any one of claims 168, 169, 170, or 172, wherein
the GALR3 receptor is a mammalian GALR3 receptor.
174. A process of claim 171, wherein the cells are injected with
RNA synthesized in vitro from the plasmid of claim 37 designated
M54 (ATCC Accession No. 209312).
175. A process of claim 171, wherein the cells are injected with
RNA synthesized in vitro from the plasmid of claim 37 designated
M67 (ATCC Accession No. ______).
176. A pharmaceutical composition which comprises an amount of a
GALR3 receptor agonist determined by the process of claim 168
effective to increase activity of a GALR3 receptor and a
pharmaceutically acceptable carrier.
177. A pharmaceutical composition of claim 176, wherein the GALR3
receptor agonist is not previously known.
178. A pharmaceutical composition which comprises an amount of a
GALR3 receptor antagonist determined by the process of claim 169
effective to reduce activity of a GALR3 receptor and a
pharmaceutically acceptable carrier.
179. A pharmaceutical composition of claim 178, wherein the GALR3
receptor antagonist is not previously known.
180. A process for determining whether a chemical compound
specifically binds to and activates a GALR3 receptor, which
comprises contacting cells producing a second messenger response
and expressing on their cell surface the GALR3 receptor, wherein
such cells do not normally express the GALR3 receptor, with the
chemical compound under conditions suitable for activation of the
GALR3 receptor, and measuring the second messenger response in the
presence and in the absence of the chemical compound, a change in
the second messenger response in the presence of the chemical
compound indicating that the compound activates the GALR3
receptor.
181. The process of claim 180, wherein the second messenger
response comprises potassium channel activation and the change in
second messenger is an increase in the level of potassium
current.
182. A process for determining whether a chemical compound
specifically binds to and inhibits activation of a GALR3 receptor,
which comprises separately contacting cells producing a second
messenger response and expressing on their cell surface the GALR3
receptor, wherein such cells do not normally express the GALR3
receptor, with both the chemical compound and a second chemical
compound known to activate the GALR3 receptor, and with only the
second chemical compound, under conditions suitable for activation
of the GALR3 receptor, and measuring the second messenger response
in the presence of only the second chemical compound and in the
presence of both the second chemical compound and the chemical
compound, a smaller change in the second messenger response in the
presence of both the chemical compound and the second chemical
compound than in the presence of only the second chemical compound
indicating that the chemical compound inhibits activation of the
GALR3 receptor.
183. The process of claim 182, wherein the second messenger
response comprises potassium channel activation and the change in
second messenger response is a smaller increase in the level of
potassium current in the presence of both the chemical compound and
the second chemical compound than in the presence of only the
second chemical compound.
184. A process of any one of claims 180, 181, 182 or 183, wherein
the GALR3 receptor is a mammalian GALR3 receptor.
185. The process of claim 184, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid K1086 (ATCC Accession No. 97747).
186. The process of claim 184, wherein the GALR3 receptor has
substantially the same amino acid sequence as that shown in FIG. 2
(Seq. ID No. 2).
187. The process of claim 184, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
188. The process of claim 184, wherein the GALR3 receptor has a
sequence, which sequence comprises substantially the same amino
acid sequence as that shown in FIG. 4 (Seq. ID No. 4) from amino
acid 60 through amino acid 427.
189. The process of claim 184, wherein the GALR3 receptor has a
sequence, which sequence comprises a sequence shown in FIG. 4 (Seq.
ID No. 4) from amino acid 60 through amino acid 427.
190. The process of claim 184, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid pEXJ-RGalR3T (ATCC Accession No. 97826).
191. The process of claim 184, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid M54 (ATCC Accession No. 209312).
192. The process of claim 184, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid M67 (ATCC Accession No. ______).
193. The process of any one of claims 180, 181, 182, 183, 184, 185,
186, 187, 188, 189, 190, 191, or 192, wherein the cell is an insect
cell.
194. The process of any one of claims 180, 181, 182, 183, 184, 185,
186, 187, 188, 189, 190, 191, or 192, wherein the cell is a
mammalian cell.
195. The process of claim 194, wherein the mammalian cell is
nonneuronal in origin.
196. The process of claim 195, wherein the nonneuronal cell is a
COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell
or LM(tk-) cell.
197. The process of claim 196, wherein the nonneuronal cell is the
293 human embryonic kidney cell designated 293-rGALR3-105 (ATCC
Accession No. CRL-12287).
198. The process of claim 196, wherein the nonneuronal cell is the
LM(tk-) cell designated L-hGALR3-228 (ATCC Accession No.
CRL-12373).
199. The process of claim 194, wherein the compound is not
previously known to bind to a GALR3 receptor.
200. A compound determined by the process of claim 199.
201. A pharmaceutical composition which comprises an amount of a
GALR3 receptor agonist determined by the process of claim 180 or
181 effective to increase activity of a GALR3 receptor and a
pharmaceutically acceptable carrier.
202. A pharmaceutical composition of claim 201, wherein the GALR3
receptor agonist is not previously known.
203. A pharmaceutical composition which comprises an amount of a
GALR3 receptor antagonist determined by the process of any one of
claims 182 or 183 effective to reduce activity of a GALR3 receptor
and a pharmaceutically acceptable carrier.
204. A pharmaceutical composition of claim 203, wherein the GALR3
receptor antagonist is not previously known.
205. A method of screening a plurality of chemical compounds not
known to activate a GALR3 receptor to identify a compound which
activates the GALR3 receptor which comprises: (a) contacting cells
which express the GALR3 receptor with the plurality of compounds
not known to activate the GALR3 receptor, under conditions
permitting activation of the GALR3 receptor, wherein the cells do
not normally express the GALR3 receptor; (b) determining whether
the activity of the GALR3 receptor is increased in the presence of
the compounds; and if so (c) separately determining whether the
activation of the GALR3 receptor is increased by each compound
included in the plurality of compounds, so as to thereby identify
the compound which activates the GALR3 receptor.
206. The process of claim 205, wherein the cells also express GIRK1
and GIRK4.
207. A method of claim 205, wherein the GALR3 receptor is a
mammalian GALR3 receptor.
208. A method of screening a plurality of chemical compounds not
known to inhibit the activation of a GALR3 receptor to identify a
compound which inhibits the activation of the GALR3 receptor, which
comprises: (a) contacting cells which express the GALR3 receptor
with the plurality of compounds in the presence of a known GALR3
receptor agonist, under conditions permitting activation of the
GALR3 receptor, wherein the cells do not normally express the GALR3
receptor; (b) determining whether the activation of the GALR3
receptor is reduced in the presence of the plurality of compounds,
relative to the activation of the GALR3 receptor in the absence of
the plurality of compounds; and if so (c) separately determining
the inhibition of activation of the GALR3 receptor for each
compound included in the plurality of compounds, so as to thereby
identify the compound which inhibits the activation of the GALR3
receptor.
209. The process of claim 208, wherein the cells also express GIRK1
and GIRK4.
210. A method of claim 208 or 209, wherein the GALR3 receptor is a
mammalian GALR3 receptor.
211. A method of any one of claims 205, 206, 207, 208, 209, or 210
wherein the cell is a mammalian cell.
212. A method of claim 211, wherein the mammalian cell is
non-neuronal in origin.
213. The method of claim 212, wherein the non-neuronal cell is a
COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell or an
NIH-3T3 cell.
214. A pharmaceutical composition comprising a compound identified
by the method of any one of claims 205, 206, or 207 effective to
increase GALR3 receptor activity and a pharmaceutically acceptable
carrier.
215. A pharmaceutical composition comprising a compound identified
by the method of any one of claims 208, 209 or 210 effective to
decrease GALR3 receptor activity and a pharmaceutically acceptable
carrier.
216. A process of any one of claims 168, 180 or 181, which further
comprises determining whether the compound selectively activates
the GALR3 receptor relative to another galanin receptor.
217. The process of claim 216, wherein the determination whether
the compound selectively activates the GALR3 receptor comprises:
(a) determining the potency of the compound for the GALR3 receptor
and for such other galanin receptor; and (b) comparing the
potencies so determined, the presence of a higher potency for the
GALR3 receptor than for such other galanin receptor indicating that
the compound selectively activates the GALR3 receptor.
218. A process of claim 217, wherein such other galanin receptor is
a GALR1 receptor.
219. A process of claim 217, wherein such other galanin receptor is
a GALR2 receptor.
220. A process of any one of claims 169, 182 or 183, which further
comprises determining whether the compound selectively inhibits the
activation of the GALR3 receptor relative to another galanin
receptor.
221. The process of claim 210, wherein the determination whether
the compound selectively inhibits the activation of the GALR3
receptor comprises: (a) determining the decrease in the potency of
a known galanin receptor agonist for the GALR3 receptor in the
presence of the compound, relative to the potency of the agonist in
the absence of the compound; (b) determining the decrease in the
potency of the agonist for such other galanin receptor in the
presence of the compound, relative to the potency of the agonist in
the absence of the compound; and (c) comparing the decrease in
potencies so determined, the presence of a greater decrease in
potency for the GALR3 receptor than for such other galanin receptor
indicating that the compound selectively inhibits the activation of
the GALR3 receptor.
222. A process of claim 221, wherein such other galanin receptor is
a GALR1 receptor.
223. A process of claim 221, wherein such other galanin receptor is
a GALR2 receptor.
224. A process for determining whether a chemical compound is a
GALR3 receptor agonist, which comprises preparing a cell extract
from cells transfected with and expressing DNA encoding the GALR3
receptor, isolating a membrane fraction from the cell extract,
separately contacting the membrane fraction with both the chemical
compound and GTP.gamma.S, and with only GTP.gamma.S, under
conditions permitting the activation of the GALR3 receptor, and
detecting GTP.gamma.S binding to the membrane fraction, an increase
in GTP.gamma.S binding in the presence of the compound indicating
that the chemical compound activates the GALR3 receptor.
225. A process for determining whether a chemical compound is a
GALR3 receptor antagonist, which comprises preparing a cell extract
from cells transfected with and expressing DNA encoding the GALR3
receptor, isolating a membrane fraction from the cell extract,
separately contacting the membrane fraction with the chemical
compound, GTP.gamma.S and a second chemical compound known to
activate the GALR3 receptor, with GTP.gamma.S and only the second
compound, and with GTP.gamma.S alone, under conditions permitting
the activation of the GALR3 receptor, detecting GTP.gamma.S binding
to each membrane fraction, and comparing the increase in
GTP.gamma.S binding in the presence of the compound and the second
compound relative to the binding of GTP.gamma.S alone, to the
increase in GTP.gamma.S binding in the presence of the second
chemical compound relative to the binding of GTP.gamma.S alone, a
smaller increase in GTP.gamma.S binding in the presence of the
compound and the second compound indicating that the compound is a
GALR3 receptor antagonist.
226. A process of claim 224 or 225, wherein the GALR3 receptor is a
mammalian GALR3 receptor.
227. The process of claim 226, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid K1086 (ATCC Accession No. 97747).
228. The process of claim 226, wherein the GALR3 receptor has
substantially the same amino acid sequence as that shown in FIG. 2
(Seq. ID No. 2).
229. The process of claim 226, wherein the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
230. The process of claim 226, wherein the GALR3 receptor has a
sequence, which sequence comprises substantially the same amino
acid sequence as that shown in FIG. 4 (Seq. ID No. 4) from amino
acid 60 through amino acid 427.
231. The process of claim 226, wherein the GALR3 receptor has a
sequence, which sequence comprises a sequence shown in FIG. 4 (Seq.
ID No. 4) from amino acid 60 through amino acid 427.
232. The process of any one of claims 224, 225, 226, 227, 228, 229,
230, or 231 wherein the cell is an insect cell.
233. The process of any one of claims 224, 225, 226, 227, 228, 229,
230, or 231, wherein the cell is a mammalian cell.
234. The process of claim 233, wherein the mammalian cell is
nonneuronal in origin.
235. The process of claim 234, wherein the nonneuronal cell is a
COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell
or LM(tk-) cell.
236. The process of claim 235, wherein the nonneuronal cell is the
293 human embryonic kidney cell designated 293-rGALR3-105 (ATCC
Accession No. CRL-12287).
237. The process of claim 235, wherein the nonneuronal cell is the
LM(tk-) cell designated L-hGALR3-228 (ATCC Accession No.
CRL-12373).
238. The process of claim 233, wherein the compound is not
previously known to bind to a GALR3 receptor.
239. A compound determined by the process of claim 238.
Description
[0001] This application is a continuation-in-part of
PCT/US97/18222, filed Oct. 9, 1997, which is a continuation-in-part
in the U.S. of U.S. Ser. No. 08/900,230, filed Jul. 23, 1997, which
is a continuation-in-part of U.S. Ser. No. 08/787,261, filed Jan.
24, 1997, which is a continuation-in-part of U.S. Ser. No.
08/767,964, filed Dec. 17, 1996, which is a continuation-in-part of
U.S. Ser. No. 08/728,139, filed Oct. 9, 1996, the contents of which
are incorporated by reference. Throughout this application, various
references are referred to within parentheses. Disclosures of these
publications in their entireties are hereby incorporated by
reference into this application to more fully describe the state of
the art to which this invention pertains. Full bibliographic
citation for these references may be found at the end of this
application, preceding the sequence listing and the claims.
BACKGROUND OF THE INVENTION
[0002] The neuropeptide galanin and its receptors hold great
promise as targets for the development of novel therapeutic agents.
Galanin is widely distributed throughout the peripheral and central
nervous systems and is associated with the regulation of processes
such as somatosensory transmission, smooth muscle contractility,
hormone release, and feeding (for review, see Bartfai et al.,
1993). In the periphery galanin is found in the adrenal medulla,
uterus, gastrointestinal tract, dorsal root ganglia (DRG), and
sympathetic neurons. Galanin released from sympathetic nerve
terminals in the pancreas is a potent regulator of insulin release
in several species (Ahrn and Lindskog, 1992; Boyle et al., 1994),
suggesting a potential role for galanin in the etiology or
treatment of diabetes. High levels of galanin are observed in human
and rat anterior pituitary where galanin MRNA levels are potently
upregulated by estrogen (Vrontakis et al., 1987; Kaplan et al.,
1988) The presence of galanin in the hypothalamic-pituitary-adrenal
axis coupled with its potent hormonal effects has led to the
suggestion that galanin may play an integral role in the hormonal
response to stress (Bartfai et, al., 1993).
[0003] Within the CNS galanin-containing cell bodies are found in
the hypothalamus, hippocampus, amygdala, basal forebrain, brainstem
nuclei, and spinal cord, with highest concentrations of galanin in
the hypothalamus and pituitary (Skofitsch and Jacobowitz, 1985;
Bennet et al., 1991; Merchenthaler et al., 1993). The distribution
of galanin receptors in the CNS generally complements that of
galanin peptide, with high levels of galanin binding observed in
the hypothalamus, amygdala, hippocampus, brainstem and dorsal
spinal cord (Skofitsch et al., 1986; Merchenthaler et al., 1993;
see Bartfai et al., 1993). Accordingly, agents modulating the
activity of galanin receptors would have multiple potential
therapeutic applications in the CNS. One of the most important of
these is the regulation of food intake. Galanin injected into the
paraventricular nucleus (PVN) of the hypothalamus stimulates
feeding in satiated rats (Kyrkouli et al., 1990), an effect which
is blocked by the peptide galanin antagonist M40 (Crawley et al.,
1993). In freely feeding rats, PVN injection of galanin
preferentially stimulates fat-preferring feeding (Tempel et al.,
1988); importantly, the galanin antagonist M40 administered alone
decreases overall fat intake (Leibowitz and Kim, 1992). These data
indicate that specific receptors in the hypothalamus mediate the
effects of galanin on feeding behavior, and further suggest that
agents acting at hypothalamic galanin receptors may be
therapeutically useful in the treatment of human eating
disorders.
[0004] Galanin receptors elsewhere in the CNS may also serve as
therapeutic targets. In the spinal cord galanin is released from
the terminals of sensory neurons as well as spinal interneurons and
appears to play a role in the regulation of pain threshold
(Wiesenfeld-Hallin et al., 1992). Intrathecal galanin potentiates
the anti-nociceptive effects of morphine in rats and produces
analgesia when administered alone (Wiesenfeld-Hallin et al., 1993;
Post et al., 1988); galanin receptor agonists may therefore be
useful as analgesic agents in the spinal cord. Galanin may also
play a role in the development of Alzheimer's disease. In the
hippocampus galanin inhibits both the release (Fisone et al., 1987)
and efficacy (Palazzi et al., 1988) of acetylcholine, causing an
impairment of cognitive functions (Sundstrom et al., 1988). Autopsy
samples from humans afflicted with Alzheimer's disease reveal a
galaninergic hyperinnervation of the nucleus basalis (Chan-Palay,
1988), suggesting a role for galanin in the impaired cognition
characterizing Alzheimer's disease. Together these data suggest
that a galanin antagonist may be effective in ameliorating the
symptoms of Alzheimer's disease (see Crawley, 1993). This
hypothesis is supported by the report that intraventricular
administration of the peptide galanin antagonist M35 improves
cognitive performance in rats (gren et al., 1992). Human galanin
receptors thus provide targets for therapeutic intervention in
multiple CNS disorders.
[0005] High-affinity galanin binding sites have been characterized
in brain, spinal cord, pancreatic islets and cell lines, and
gastrointestinal smooth muscle in several mammalian species, and
all show similar affinity for .sup.125I-porcine galanin
(.about.0.5-1 nM). Nevertheless, recent in vitro and in vivo
pharmacological studies in which fragments and analogues of galanin
were used suggest the existence of multiple galanin receptor
subtypes. For example, a galanin binding site in guinea pig stomach
has been reported that exhibits high affinity for porcine galanin
(3-29) (Gu, et al. 1995), which is inactive at CNS galanin
receptors. The chimeric galanin analogue M15 (galantide) acts as
antagonist at CNS galanin receptors (Bartfai et al., 1991) but as a
full agonist in gastrointestinal smooth muscle (Gu et al., 1993)
Similarly, the galanin-receptor ligand M40 acts as a weak agonist
in RINm5F insulinoma cells and a full antagonist in brain (Bartfai
et al, 1993a). The pharmacological profile of galanin receptors in
RINm5F cells can be further distinguished from those in brain by
the differential affinities of [D-Tyr.sup.2]- and
[D-Phe.sup.2]-galanin analogues (Lagny-Pourmir et al., 1989). The
chimeric galanin analogue M35 displaces .sup.125I-galanin binding
to RINm5F membranes in a biphasic manner, suggesting the presence
of multiple galanin receptor subtypes, in this cell line (Gregersen
et al., 1993).
[0006] Multiple galanin receptor subtypes may also co-exist within
the CNS. Galanin receptors in the dorsal hippocampus exhibit high
affinity for Gal (1-15) but not for Gal (1-29) (Hedlund et al.,
1992), suggesting that endogenous croteolytic processing may
release bioactive fragments of galanin to act at distinct
receptors. The rat pituitary exhibits high-affinity binding for
.sup.125I-Bolton and Hunter (N-terminus)-labeled galanin (1-29) but
not for [.sup.125I]Tyr.sup.26-por- cine galanin (Wynick et al.,
1993), suggesting that the pituitary galanin receptor is a
C-terminus-preferring subtype. Spinal cord galanin binding sites,
while similar to those in brain, show an affinity for the chimeric
peptide antagonist M35 intermediate between the brain and smooth
muscle (Bartfai et al., 1991), raising the possibility of further
heterogeneity.
[0007] A galanin receptor cDNA was recently isolated by expression
cloning from a human Bowes melanoma cell line (Habert-Ortoli et
al., 1994). The pharmacological profile exhibited by this receptor
is similar to that observed in brain and pancreas, and on that
basis the receptor has been termed GALR1. The cloned human GALR1
receptor ("hGALR1") binds native human, porcine and rat galanin
with .about.1 nM affinity (K.sub.i vs. .sup.125I-galanin) and
porcine galanin 1-16 at a slightly lower affinity (.about.5 nm)
Porcine galanin 3-29 does not bind to the receptor. The GALR1
receptor appears to couple to inhibition of adenylate cyclase, with
half-maximal inhibition of forskolin-stimulated cAMP production by
1 nM galanin, and maximal inhibition occurring at about 1
.mu.M.
[0008] Recently the rat homologue of GALR1 ("GALR1") was cloned
from the RIN14B pancreatic cell line (Burgevin, et al., (1995),
Parker et al., 1995. The pharmacologic data reported to date do not
suggest substantial differences between the pharmacologic
properties of the rat and human GALR1 receptors. Localization
studies reveal GALR1 mRNA in rat hypothalamus, ventral hippocampus,
brainstem, and spinal cord (Gustafson et al., 1996), regions
consistent with roles for galanin in feeding, cognition, and pain
transmission. However, GALR1 appears to be distinct from the
pituitary and hippocampal receptor subtypes described above.
[0009] The indication of multiple galanin receptor subtypes within
the brain underscores the importance of defining galanin receptor
heterogeneity at the molecular level in order to develop specific
therapeutic agents for CNS disorders. Pharmacological tools capable
of distinguishing galanin receptor subtypes in tissue preparations
are only beginning to appear. Several high-affinity peptide-based
galanin antagonists have been developed and are proving useful in
probing the functions of galanin receptors (see Bartfai et al.,
1993), but their peptide character precludes practical use as
therapeutic agents. In light of galanin's multiple neuroendocrine
roles, therapeutic agents targeting a specific disorder must be
selective for the appropriate receptor subtype to minimize side
effects.
[0010] Accordingly, applicants have endeavored to clone the entire
family of galanin receptors for use in target-based drug design
programs. The identification of non-peptide agents acting
selectively only at specific galanin receptors will be greatly
facilitated by the cloning, expression, and characterization of the
galanin receptor family.
[0011] Applicants have recently isolated by expression cloning from
a rat hypothalamic cDNA library a novel galanin receptor, termed
"GALR2," not described herein, which is distinguishable from GALR1
both by its unique sequence and distinct pharmacologic properties.
The GALR2 receptor is the subject of PCT International Application
PCT/US97/01301, published on Jul. 31, 1997, as WO 97/26853.
[0012] Applicants now report the isolation of a novel galanin
receptor subtype, referred to herein as "GALR3," from a rat
hypothalamic cDNA library. This discovery provides a novel
approach, through the use of heterologous expression systems, to
develop subtype selective, high-affinity non-peptide compounds that
could serve as therapeutic agents for eating disorders, diabetes,
pain, depression, ischemia, Alzheimer's disease, neuroendocrine
disorders. The distribution of mRNA encoding the rat GALR3 receptor
in multiple CNS regions as well as other organs supports the notion
that the GALR3 is involved in these disorders. Pathophysiological
disorders proposed to be linked to galanin receptor activation
include eating disorders, diabetes, pain, depression, ischemia,
Alzheimer's disease and reproductive disorders.
[0013] Accordingly, treatment of such disorders may be effected by
the administration of GALR3 receptor-selective compounds. The
presence of galanin binding sites in multiple CNS areas suggests
that GALR3 receptors may also play a role in cognition, analgesia,
sensory processing (olfactory, visual), processing of visceral
information, motor coordination, modulation of dopaminergic
activity, neuroendocrine function, sleep disorders, migraine, and
anxiety.
SUMMARY OF THE INVENTION
[0014] This invention provides an isolated nucleic acid encoding a
GALR3 galanin receptor. This invention also provides an isolated
GALR3 receptor protein. This invention also provides a purified
GALR3 receptor protein. This invention further provides DNA, cDNA,
genomic DNA, RNA, and MRNA encoding the GALR3 receptor.
[0015] This invention further provides a vector comprising the
GALR3 receptor. Such a vector may be adapted for expression of the
GALR3 receptor in mammalian or non-mammalian cells. This invention
also provides a plasmid which comprises the regulatory elements
necessary for expression of GALR3 nucleic acid in a mammalian cell
operatively linked to a nucleic acid encoding the GALR3 receptor so
as to permit expression thereof, designated K1086 (ATCC Accession
No. 97747). This invention also provides a plasmid which comprises
the regulatory elements necessary for expression of GALR3 nucleic
acid in a mammalian cell operatively linked to a nucleic acid
encoding a human GALR3 receptor so as to permit expression thereof,
designated pEXJ-hGalR3 (ATCC Accession No. 97827). This invention
provides mammalian cells comprising the above-described plasmid or
vector. This invention also provides a membrane preparation
isolated from the cells.
[0016] This invention provides an isolated nucleic acid encoding a
modified GALR3 receptor, which differs from a GALR3 receptor by
having an amino acid(s) deletion, replacement or addition in the
third intracellular domain.
[0017] This invention provides a nucleic acid probe comprising at
least 15 nucleotides, which probe specifically hybridizes with a
nucleic acid encoding a GALR3 receptor, wherein the probe has a
unique sequence corresponding to a sequence present within one of
the two strands of the nucleic acid encoding the GALR3 receptor
contained in plasmid K1086. This invention still further provides a
nucleic acid probe comprising at least 15 nucleotides, which probe
specifically hybridizes with a nucleic acid encoding a GALR3
receptor, wherein the probe has a unique sequence corresponding to
a sequence present within (a) the nucleic acid sequence described
in FIG. 1 (Seq. ID No. 1) or (b) the reverse complement to the
nucleic acid sequence shown in FIG. 1 (Seq. ID No. 1).
[0018] In yet another embodiment, the GALR3 receptor is the rat
GALR3 receptor having substantially the same amino acid sequence as
the amino acid sequence shown in FIG. 2. In another embodiment, the
GALR3 receptor is the rat GALR3 receptor having the amino acid
sequence shown in FIG. 2. In another embodiment, the GALR3 receptor
is the human GALR3 receptor. In another embodiment, the GALR3
receptor is the human GALR3 receptor encoded by the coding sequence
of plasmid pEXJ-hGalR3. This invention also provides a nucleic acid
probe comprising at least 15 nucleotides, which probe specifically
hybridizes with a nucleic acid encoding a GALR3 receptor, wherein
the probe has a unique sequence corresponding to a sequence present
within one of the two strands of the nucleic acid encoding the
GALR3 receptor contained in plasmid pEXJ-hGalR3. This invention
provides a nucleic acid probe comprising at least 15 nucleotides,
which probe specifically hybridizes with a nucleic acid encoding a
GALR3 receptor, wherein the probe has a unique sequence
corresponding to a sequence present within (a) the nucleic acid
sequence described in FIG. 3 (Seq. ID No. 3) or (b) the reverse
complement to the nucleic acid sequence shown in FIG. 3 (Seq. ID
No. 3).
[0019] This invention further provides a nucleic acid probe
comprising a nucleic acid molecule of at least 15 nucleotides which
is complementary to a unique fragment of the sequence of a nucleic
acid molecule encoding a GALR3 receptor.
[0020] This invention also provides a nucleic acid probe comprising
a nucleic acid molecule of at least 15 nucleotides which is
complementary to the antisense sequence of a unique fragment of the
sequence of a nucleic acid molecule encoding a GALR3 receptor.
[0021] This invention provides an antisense oligonucleotide having
a sequence capable of specifically hybridizing to mRNA encoding a
GALR3 galanin receptor, so as to prevent translation of the mRNA.
This invention also provides an antisense oligonucleotide having a
sequence capable of specifically hybridizing to the genomic DNA
molecule encoding a GALR3 receptor.
[0022] This invention provides an antibody directed to a GALR3
receptor. This invention also provides a monoclonal antibody
directed to an epitope of a GALR3 receptor, which epitope is
present on the surface of a cell expressing a GALR3 receptor.
[0023] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide effective to reduce
activity of a GALR3 receptor by passing through a cell membrane and
binding specifically with mRNA encoding a GALR3 receptor in the
cell so as to prevent its translation and a pharmaceutically
acceptable carrier capable of passing through a cell membrane. In
an embodiment, the oligonucleotide is coupled to a substance which
inactivates mRNA. In another embodiment, the substance which
inactivates mRNA is a ribozyme.
[0024] This invention provides a pharmaceutical composition
comprising an amount of an antagonist effective to reduce the
activity of a GALR3 receptor and a pharmaceutically acceptable
carrier.
[0025] This invention provides a pharmaceutical composition
comprising an amount of an agonist effective to increase activity
of a GALR3 receptor and a pharmaceutically acceptable carrier.
[0026] This invention provides a transgenic nonhuman mammal
expressing DNA encoding a GALR3 receptor. This invention provides a
transgenic nonhuman mammal comprising a homologous recombination
knockout of the native GALR3 receptor. This invention provides a
transgenic nonhuman mammal whose genome comprises antisense DNA
complementary to DNA encoding a GALR3 receptor so placed as to be
transcribed into antisense mRNA which is complementary to mRNA
encoding a GALR3 receptor and which hybridizes to mRNA encoding a
GALR3 receptor thereby reducing its translation.
[0027] This invention also provides a process for determining
whether a compound can specifically bind to a GALR3 receptor which
comprises contacting a cell transfected with and expressing DNA
encoding the GALR3 receptor with the compound under conditions
permitting binding of compounds to such receptor, and detecting the
presence of any such compound specifically bound to the GALR3
receptor, so as to thereby determine whether the ligand
specifically binds to the GALR3 receptor.
[0028] This invention provides a process for determining whether a
compound can specifically bind to a GALR3 receptor which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, contacting the membrane fraction with the
compound under conditions permitting binding of compounds to such
receptor, and detecting the presence of the compound specifically
bound to the GALR3 receptor, so as to thereby determine whether the
compound specifically binds to the GALR3 receptor.
[0029] In one embodiment, the GALR3 receptor is a mammalian GALR3
receptor. In another embodiment, the GALR3 receptor is a rat GALR3
receptor. In still another embodiment, the GALR3 receptor has
substantially the same amino acid sequence encoded by the plasmid
K1086. In a still further embodiment, the GALR3 receptor has the
amino acid sequence encoded by the plasmid K1086. In another
embodiment, the GALR3 receptor is a human GALR3 receptor.
[0030] This invention provides a process for determining whether a
compound is a GALR3 receptor agonist which comprises contacting a
cell transfected with and expressing DNA encoding the GALR3
receptor with the compound under conditions permitting the
activation of the GALR3 receptor, and detecting an increase in
GALR3 receptor activity, so as to thereby determine whether the
compound is a GALR3 receptor agonist.
[0031] This invention provides a process for determining whether a
compound is a GALR3 receptor antagonist which comprises contacting
a cell transfected with and expressing DNA encoding the GALR3
receptor with the compound in the presence of a known GALR3
receptor agonist, such as galanin, under conditions permitting the
activation of the GALR3 receptor, and detecting a decrease in GALR3
receptor activity, so as to thereby determine whether the compound
is a GALR3 receptor antagonist.
[0032] This invention provides a compound determined by the
above-described processes. In one embodiment of the above-described
processes, the compound is not previously known. In another
embodiment, the compound is not known to bind a GALR3 receptor.
[0033] This invention provides a method of screening a plurality of
chemical compounds not known to bind to a GALR3 receptor to
identify a compound which specifically binds to the GALR3 receptor,
which comprises (a) contacting cells transfected with and
expressing DNA encoding the GALR3 receptor with a compound known to
bind specifically to the GALR3 receptor; (b) contacting the
preparation of step (a) with the plurality of compounds not known
to bind specifically to the GALR3 receptor, under conditions
permitting binding of compounds known to bind the GALR3 receptor;
(c) determining whether the binding of the compound known to bind
to the GALR3 receptor is reduced in the presence of the compounds,
relative to the binding of the compound in the absence of the
plurality of compounds; and if so (d) separately determining the
binding to the GALR3 receptor of each compound included in the
plurality of compounds, so as to thereby identify the compound
which specifically binds to the GALR3 receptor.
[0034] This invention provides a method of screening a plurality of
chemical compounds not known to activate a GALR3 receptor to
identify a compound which activates the GALR3 receptor which
comprises (a) contacting cells transfected with and expressing the
GALR3 receptor with the plurality of compounds not known to
activate the GALR3 receptor, under conditions permitting activation
of the GALR3 receptor; (b) determining whether the activity of the
GALR3 receptor is increased in the presence of the compounds; and
if so (c) separately determining whether the activation of the
GALR3 receptor is increased by each compound included in the
plurality of compounds, so as to thereby identify the compound
which activates the GALR3 receptor.
[0035] This invention provides a method of screening a plurality of
chemical compounds not known to inhibit the activation of a GALR3
receptor to identify a compound which inhibits the activation of
the GALR3 receptor, which comprises (a) preparing a cell extract
from cells transfected with and expressing DNA encoding the GALR3
receptor, isolating a membrane fraction from the cell extract,
contacting the membrane fraction with the plurality of compounds in
the presence of a known GALR3 receptor agonist, under conditions
permitting activation of the GALR3 receptor; (b) determining
whether the activation of the GALR3 receptor is reduced in the
presence of the plurality of compounds, relative to the activation
of the GALR3 receptor in the absence of the plurality of compounds:
and if so (c) separately determining the inhibition of activation
of the GALR3 receptor for each compound included in the plurality
of compounds, so as to thereby identify the compound which inhibits
the activation of the GALR3 receptor.
[0036] This invention provides a method of detecting expression of
a GALR3 receptor by detecting the presence of mPNA coding for the
GALR3 receptor which comprises obtaining total mRNA from the cell
and contacting the mRNA so obtained with the above-described
nucleic acid probe under hybridizing conditions, detecting the
presence of mRNA hybridized to the probe, and thereby detecting the
expression of the GALR3 receptor by the cell.
[0037] This invention provides a method of treating an abnormality
in a subject, wherein the abnormality is alleviated by the
inhibition of a GALR3 receptor which comprises administering to a
subject an effective amount of the above-described pharmaceutical
composition effective to decrease the activity of the GALR3
receptor in the subject, thereby treating the abnormality in the
subject. In an embodiment, the abnormality is obesity.
[0038] In another embodiment, the abnormality is bulimia.
[0039] This invention provides a method of treating an abnormality
in a subject wherein the abnormality is alleviated by the
activation of a GALR3 receptor which comprises administering to a
subject an effective amount of the above-described pharmaceutical
composition effective to activate the GALR3 receptor in the
subject. In an embodiment, the abnormal condition is anorexia.
[0040] This invention provides a method for diagnosing a
predisposition to a disorder associated with the activity of a
specific human GALR3 receptor allele which comprises: (a) obtaining
DNA of subjects suffering from the disorder; (b) performing a
restriction digest of the DNA with a panel of restriction enzymes;
(c) electrophoretically separating the resulting DNA fragments on a
sizing gel; (d) contacting the resulting gel with a nucleic acid
probe capable of specifically hybridizing with a unique sequence
included within the sequence of a nucleic acid molecule encoding a
human GALR3 receptor and labeled with a detectable marker; (e)
detecting labeled bands which have hybridized to DNA encoding a
human GALR3 receptor labeled with a detectable marker to create a
unique band pattern specific to the DNA of subjects suffering from
the disorder; (f) preparing DNA obtained for diagnosis by steps
a-e; and (g) comparing the unique band pattern specific to the DNA
of subjects suffering from the disorder from step e and the DNA
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.
[0041] This invention provides a method of modifying feeding
behavior of a subject which comprises administering to the subject
an amount of a compound which is a galanin receptor agonist or
antagonist effective to increase or decrease the consumption of
food by the subject so as to thereby modify feeding behavior of the
subject. In an embodiment, the compound is a GALR3 receptor
antagonist and the amount is effective to decrease the consumption
of food by the subject. In another embodiment the compound is
administered in combination with food.
[0042] In yet another embodiment the compound is a GALR3 receptor
agonist and the amount is effective to increase the consumption of
food by the subject. In a still further embodiment, the compound is
administered in combination with food. In other embodiments the
subject is a vertebrate, a mammal, a human or a canine.
[0043] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor agonist, which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, separately contacting the membrane fraction with
both the chemical compound and GTP.gamma.S, and with only
GTP.gamma.S, under conditions permitting the activation of the
GALR3 receptor, and detecting GTP.gamma.S binding to the membrane
fraction, an increase in GTP.gamma.S binding in the presence of the
compound indicating that the chemical compound activates the GALR3
receptor.
[0044] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor antagonist, which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, separately contacting the membrane fraction with
the chemical compound, GTP.gamma.S and a second chemical compound
known to activate the GALR3 receptor, with GTP.gamma.S and only the
second compound, and with GTP.gamma.S alone, under conditions
permitting the activation of the GALR3 receptor, detecting
GTP.gamma.S binding to each membrane fraction, and comparing the
increase in GTP.gamma.S binding in the presence of the compound and
the second compound relative to the binding of GTP.gamma.S alone,
to the Increase in GTP.gamma.S binding in the presence of the
second chemical compound relative to the binding of GTP.gamma.S
alone, a smaller increase in GTP.gamma.S binding in the presence of
the compound and the second compound indicating that the compound
is a GALR3 receptor antagonist.
[0045] This invention further provides a process for identifying a
chemical compound which specifically binds to a GALR3 receptor
which comprises contacting cells containing DNA encoding and
expressing on their cell surface the GALR3 receptor, wherein such
cells do not normally express the GALR3 receptor, with the compound
under conditions suitable for binding, and detecting specific
binding of the chemical compound to the GALR3 receptor.
[0046] This invention also provides a process for identifying a
chemical compound which specifically binds to a GALR3 receptor
which comprises contacting a membrane fraction from a cell extract
of cells containing DNA encoding and expressing on their cell
surface the GALR3 receptor, wherein such cells do not normally
express the GALR3 receptor, with the compound under conditions
suitable for binding, and detecting specific binding of the
chemical compound to the GALR3 receptor.
[0047] This invention provides a process involving competitive
binding for identifying a chemical compound which specifically
binds to a GALR3 receptor which comprises separately contacting
cells expressing on their cell surface the GALR3 receptor, wherein
such cells do not normally express the GALR3 receptor, with both
the chemical compound and a second chemical compound known to bind
to the receptor, and with only the second chemical compound, under
conditions suitable for binding of both compounds, and detecting
specific binding of the chemical compound to the GALR3 receptor, a
decrease in the binding of the second chemical compound to the
GALR3 receptor in the presence of the chemical compound indicating
that the chemical compound binds to the GALR3 receptor.
[0048] This invention further provides a process involving
competitive binding for identifying a chemical compound which
specifically binds to a human GALR3 receptor which comprises
separately contacting a membrane fraction from a cell extract of
cells expressing on their cell surface the GALR3 receptor, wherein
such cells do not normally express the GALR3 receptor, with both
the chemical compound and a second chemical compound known to bind
to the receptor, and with only the second chemical compound, under
conditions suitable for binding of both compounds, and detecting
specific binding of the chemical compound to the GALR3 receptor, a
decrease in the binding of the second chemical compound to the
GALR3 receptor in the presence of the chemical compound indicating
that the chemical compound binds to the GALR3 receptor.
[0049] This invention provides a method of screening a plurality of
chemical compounds not known to bind to a GALR3 receptor to
identify a compound which specifically binds to the GALR3 receptor,
which comprises (a) preparing a cell extract from cells transfected
with and expressing DNA encoding the GALR3 receptor, isolating a
membrane fraction from the cell extract, contacting the membrane
fraction with a compound known to bind specifically to the GALR3
receptor; (b)contacting the preparation of step (a) with the
plurality of compounds not known to bind specifically to the GALR3
receptor, under conditions permitting binding of compounds known to
bind the GALR3 receptor; (c) determining whether the binding of the
compound known to bind to the GALR3 receptor is reduced in the
presence of the compounds, relative to the binding of the compound
in the absence of the plurality of compounds; and if so (d)
separately determining the binding to the GALR3 receptor of each
compound included in the plurality of compounds, so as to thereby
identify the compound which specifically binds to the GALR3
receptor.
[0050] This invention provides a method for determining whether a
compound is a GALR3 antagonist which comprises: (a) administering
to an animal a GALR3 agonist and measuring the amount of food
intake in the animal; (b) administering to a second animal both the
GALR3 agonist and the compound, and measuring the amount of food
intake in the second animal; and (c) determining whether the amount
of food intake is reduced in the presence of the compound relative
to the amount of food intake in the absence of the compound, so as
to thereby determine whether the compound is a GALR3
antagonist.
[0051] This invention provides a method of screening a plurality of
compounds to identify a compound which is a GALR3 antagonist which
comprises: (a) administering to an animal a GALR3 agonist and
measuring the amount of food intake in the animal; (b)
administering to a second animal the GALR3 agonist and at least one
compound of the plurality of compounds and measuring the amount of
food intake in the animal; (c) determining whether the amount of
food intake is reduced in the presence of at least one compound of
the plurality relative to the amount of food intake in the absence
of at least one compound of the plurality, and if so; (d)
separately determining whether each compound is a GALR3 antagonist
according to the method of claim 118, so as to thereby identify a
compound which is a GALR3 antagonist.
[0052] This invention further provides a method of decreasing
feeding behavior of a subject which comprises administering a
compound which is a GALR3 receptor antagonist and a compound which
is a Y5 receptor antagonist, the amount of such antagonists being
effective to decrease the feeding behavior of the subject.
[0053] This invention provides a method of decreasing nociception
in a subject which comprises administering to the subject an amount
of a compound which is a GALR3 receptor agonist effective to
decrease nociception in the subject.
[0054] This invention also provides a method of treating pain in a
subject which comprises administering to the subject an amount of a
compound which is a GALR3 receptor agonist effective to treat pain
in the subject.
[0055] This invention further provides a method of treating
diabetes in a subject which comprises administering to the subject
an amount of a compound which is a GALR3 receptor antagonist
effective to treat diabetes in the subject.
[0056] This invention also provides a process for determining
whether a chemical compound specifically binds to and activates a
GALR3 receptor, which comprises contacting cells producing a second
messenger response and expressing on their cell surface the GALR3
receptor, wherein such cells do not normally express the GALR3
receptor, with the chemical compound under conditions suitable for
activation of the GALR3 receptor, and measuring the second
messenger response in the presence and in the absence of the
chemical compound, a change in the second messenger response in the
presence of the chemical compound indicating that the compound
activates the GALR3 receptor.
[0057] This invention provides a process for determining whether a
chemical compound specifically binds to and inhibits activation of
a GALR3 receptor, which comprises separately contacting cells
producing a second messenger response and expressing on their cell
surface the GALR3 receptor, wherein such cells do not normally
express the GALR3 receptor, with both the chemical compound and a
second chemical compound known to activate the GALR3 receptor, and
with only the second chemical compound, under conditions suitable
for activation of the GALR3 receptor, and measuring the second
messenger response in the presence of only the second chemical
compound and in the presence of both the second chemical compound
and the chemical compound, a smaller change in the second messenger
response in the presence of both the chemical compound and the
second chemical compound than in the presence of only the second
chemical compound indicating that the chemical compound inhibits
activation of the GALR3 receptor.
[0058] This invention provides a method of screening a plurality of
chemical compounds not known to activate a GALR3 receptor to
identify a compound which activates the GALR3 receptor which
comprises: (a) contacting cells transfected with and expressing the
GALR3 receptor with the plurality of compounds not known to
activate the GALR3 receptor, under conditions permitting activation
of the GALR3 receptor; (b) determining whether the activity of the
GALR3 receptor is increased in the presence of the compounds; and
if so (c) separately determining whether the activation of the
GALR3 receptor is increased by each compound included in the
plurality of compounds, so as to thereby identify the compound
which activates the GALR3 receptor.
[0059] This invention provides a method of screening a plurality of
chemical compounds not known to inhibit the activation of a GALR3
receptor to identify a compound which inhibits the activation of
the GALR3 receptor, which comprises: (a) contacting cells
transfected with and expressing the GALR3 receptor with the
plurality of compounds in the presence of a known GALR3 receptor
agonist, under conditions permitting activation of the GALR3
receptor; (b) determining whether the activation of the GALR3
receptor is reduced in the presence of the plurality of compounds,
relative to the activation of the GALR3 receptor in the absence of
the plurality of compounds; and if so (c) separately determining
the inhibition of activation of the GALR3 receptor for each
compound included in the plurality of compounds, so as to thereby
identify the compound which inhibits the activation of the GALR3
receptor.
[0060] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor antagonist, which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, separately contacting the membrane fraction with
the chemical compound, GTP.gamma.S and a second chemical compound
known to activate the GALR3 receptor, with GTP.gamma.S and only the
second compound, and with GTP.gamma.S alone, under conditions
permitting the activation of the GALR3 receptor, detecting
GTP.gamma.S binding to each membrane fraction, and comparing the
increase in GTP.gamma.S binding in the presence of the compound and
the second compound relative to the binding of GTP.gamma.S alone,
to the increase in GTP.gamma.S binding in the presence of the
second chemical compound relative to the binding of GTP.gamma.S
alone, a smaller increase in GTP.gamma.S binding in the presence of
the compound and the second compound indicating that the compound
is a GALR3 receptor antagonist.
BRIEF DESCRIPTION OF THE FIGURES
[0061] FIG. 1 Nucleotide coding sequence of the rat hypothalamic
galanin GALR3 receptor (Seq. I.D. No. 1), with partial 5' and 3'
untranslated sequences. Start and stop codons are underlined.
[0062] FIG. 2 Deduced amino acid sequence of the rat hypothalamic
galanin GALR3 receptor (Seq. I.D. No. 2) encoded by the rat
nucleotide sequence shown in FIG. 1.
[0063] FIG. 3 Nucleotide coding sequence of the human galanin GALR3
receptor (Seq. I.D. No. 3), with partial 5' and 3' untranslated
sequences. Start and stop codons are underlined.
[0064] FIG. 4 Deduced amino acid sequence of the human galanin
GALR3 receptor (Seq. I.D. No. 4) encoded by the human nucleotide
sequence shown in FIG. 3. The nucleotide sequence shown in FIG. 3
is translated from nucleotide 1 to the stop codon. Two possible
starting methionines are underlined.
[0065] FIGS. 5A-5D Amino acid sequence alignment of the rat GALR3
receptor (top row) (Seq. ID No. 2), human GALR3 receptor (middle
row) (Seq. ID No. 4) and rat GALR1 receptor (bottom row) (Seq. ID
No. 5). Transmembrane domains (TM 1-7) are indicated by brackets
above the sequence.
[0066] FIGS. 6A-6B FIG. 6A: Long continuous trace (3 segments)
demonstrates galanin responsivity and sensitivity to Ba.sup.++
block in an oocyte expressing hGalR3 and GIRK1 and GIRK4. Switching
from ND96 to 1/2hK solution causes the appearance of a large
resting (inward) K.sup.+ current that increases further upon
transient addition of 3 .mu.M galanin. Subsequent addition of 300
.mu.M Ba.sup.++ largely blocks both the resting and
galanin-stimulated K.sup.+ currents. After removal of Ba.sup.++
galanin responsivity is partially restored. FIG. 6B:
Concentration-response characteristic of a second oocyte expressing
both hGalR3 and GIRKs. Stepwise increases in the concentration of
porcine galanin from 10 to 10,000 nM result in a saturable increase
in inward current.
[0067] FIG. 7 Pertussis toxin sensitivity of GalR3 and GalR1
stimulation of GIRK currents. Normalized mean currents elicited by
0.1 .mu.M (GalR1) and 1 .mu.M (GalR3) galanin in oocytes injected 3
h prior with 2 ng of pertussis toxin compared to water-injected
oocytes. For oocytes expressing GalR2 and .alpha.1a receptors, the
response amplitude was measured as the peak of the Cl.sup.- current
stimulated by 1 .mu.M galanin or epinephrine, respectively. Number
of observations appears in parenthesis below the x-axis. Apparent
absence of a bar indicates an amplitude of 0 (no response above
baseline).
[0068] FIGS. 8A-8F Concentration-response relations for 6 peptides
at GalR3 receptors expressed in oocytes. FIG. 8A: M32; FIG. 8B:
porcine galanin; FIG. 8C: C7; FIG. 8D: Gal -7-29; FIG. 8E: Gal
1-16; FIG. 8F: M40. Measurements of GIRK currents were made as
shown for galanin in FIG. 6B. For all peptides, responses from 3-6
oocytes were averaged for each data point. Curves were fitted with
the logistic equation I=Imax/(1+(EC.sub.50/[Agonist]).sup.n), where
EC.sub.50 is the concentration of agonist that produced
half-maximal activation, and n the Hill coefficient. Fits were made
with a Marquardt-Levenberg non-linear least-squares curve fitting
algorithm.
[0069] FIGS. 9A-9B FIG. 9A: Current-voltage relation for responses
generated by galanin in oocytes expressing hGalR3, GIRK1 and GIRK4.
Voltage ramps from -100 to +20 mV were applied at a rate of 50
mV/s. Ramps were generated in 1/2hK, 1/2hK+1 .mu.M galanin, and
1/2hK+galanin+300 .mu.M Ba.sup.++. FIG. 9B: the galanin-sensitive
current (I.sub.gal) was derived by subtracting the background
current (1/2hK) from the galanin current (+gal); the total inward
rectifier current (I.sub.tot) was similarly obtained by subtracting
the current in the presence of Ba.sup.++ from the galanin current.
Both I.sub.gal and I.sub.tot display steep inward rectification and
reverse at approximately -24 mV.
[0070] FIG. 10 Auto radiograph demonstrating hybridization of
radiolabeled rat GALR3 probe to RNA extracted from rat tissue in a
solution hybridization/nuclease protection assay using .sup.32P
labeled riboprobe. 2 .mu.g of mRNA was used in each assay. The
single band represents mRNA coding for the rat GALR3 receptor
extracted from tissue indicated at the bottom of the gel. mRNA
coding for the rGalR3 is present in: kidney, stomach, pancreas,
pituitary, adrenal medulla, whole brain, hypothalamus, spinal cord,
and medulla. Integrity of RNA was assessed using hybridization to
mRNA coding to GAPDH. Biomax Film; 18 hr exposure, -70.degree.
C.
[0071] FIG. 11 Localization of rGALR3 mRNA by solution
hybridization/ RNAse protection assay. Auto radiograph
demonstrating protection of radiolabeled rat GALR3 RNA probe by
poly A+RNA (2 .mu.g) from various rat tissues in a solution
hybridization/nuclease protection assay. The single band (arrow)
represents levels of rat GALR3 receptor mRNA in the tissues
indicated: (sc, spinal cord; ad ctx, adrenal cortex; cpu, caudate
putamen; cblm, cerebellum; choroid, choroid plexus; ctx, cerebral
cortex; drg, dorsal root ganglia; hif, hippocampal formation;
medulla, medulla oblongata; olf bulb, olfactory bulb; sn,
substantia nigra; pit, pituitary; duod, duodenum; vas def, vas
deferens.)
[0072] FIG. 12 Functional activation of rat GALR3 receptors
expressed in Xenopus oocytes. Under voltage clamp an inward current
develops following application of porcine galanin (1 .mu.M) during
the period indicated by the bar. Oocyte was continuously bathed in
elevated K.sup.+ (hK); holding potential was -80 mV. Oocyte was
previously injected with mRNAs encoding rat GALR3 and the potassium
channel subunits GIRK1 and GIRK4.
[0073] FIG. 13 Coupling of hGALR3 to G protein in LMTK-. Membranes
were prepared from hGALR3-LMTK-cells which had been grown for 16
hours in the presence (open circle) or absence (closed circle)
pertussis toxin (100 ng/ml) Membranes were distributed into 96 well
plates (40 .mu.g membrane protein/250 .mu.l) together with porcine
.sup.125I-galanin (375,000 dpm/250 .mu.l) and guanine nucleotides
(GTP.gamma.S, GDP or GMP). Nonspecific binding was defined by 1
.mu.M porcine galanin. Pertussis toxin-treated membranes appear to
have a reduced population of hGALR3/G protein-coupled receptors, as
indicated by 1) a reduction in specific binding vs. control
membranes, and 2) the absence of guanine nucleotide sensitivity in
the remaining fraction of .sup.125I-galanin binding sites. Each
symbol is the average of duplicate data points from a single
assay.
DETAILED DESCRIPTION OF THE INVENTION
[0074] Throughout this application, the following standard
abbreviations are used to indicate specific nucleotide bases:
1 C = cytosine A = adenine T = thymine G = guanine
[0075] Furthermore, the term "agonist" is used throughout this
application to indicate any peptide or non-peptidyl compound which
increases the activity of any of the receptors of the subject
invention. The term "antagonist" is used throughout this
application to indicate any peptide or non-peptidyl compound which
decreases the activity of any of the receptors of the subject
invention.
[0076] The activity of a G-protein coupled receptor such as a
galanin receptor may be measured using any of a variety of
functional assays which are well-known in the art, in which
activation of the receptor in question results in an observable
change in the level of some second messenger, including but not
limited to adenylate cyclase, calcium mobilization, arachidonic
acid release, ion channel activity, inositol phospholipid
hydrolysis or guanylyl cyclase. Heterologous expression systems
utilizing appropriate host cells to express the nucleic acid of the
subject invention are used to obtain the desired second messenger
coupling. Receptor activity may also be assayed in an oocyte
expression system, using methods well known in the art.
[0077] This invention provides an isolated nucleic acid encoding a
GALR3 galanin receptor. This invention further provides a
recombinant nucleic acid encoding a GALR3 galanin receptor. In an
embodiment, the galanin receptor is a vertebrate or a mammalian
GALR3 receptor. In another embodiment, the galanin receptor is a
rat GALR3 receptor. In another embodiment, the galanin receptor is
a human GALR3 receptor. In an embodiment, the isolated nucleic acid
encodes a receptor characterized by an amino acid sequence in the
transmembrane region, which has a homology of 70% or higher to the
amino acid sequence in the transmembrane region of the rat galanin
GALR3 receptor and a homology of less than 70% to the amino acid
sequence in the transmembrane region of any GALR1 receptor. In an
embodiment, the GALR3 receptor is a rat GALR3 receptor. In another
embodiment, the GALR3 receptor is a human GALR3 receptor.
[0078] This invention provides an isolated nucleic acid encoding a
GALR3 receptor having the same or substantially the same amino acid
sequence as the amino acid sequence encoded by the plasmid K1086
(ATCC Accession No. 97747). In an embodiment, the nucleic acid is
DNA. This invention further provides an isolated nucleic acid
encoding a rat GALR3 receptor having the amino acid sequence
encoded by the plasmid K1086. This invention provides an isolated
nucleic acid encoding a GALR3 receptor having the same or
substantially the same amino acid sequence as the amino acid
sequence encoded by the plasmid pEXJ-RGalR3T (ATCC Accession No.
97826). In an embodiment, the nucleic acid is DNA. This invention
further provides an isolated nucleic acid encoding a rat GALR3
receptor having the amino acid sequence encoded by the plasmid
pEXJ-RGalR3T (ATCC Accession No. 97826). This invention provides an
isolated nucleic acid encoding a GALR3 receptor having
substantially the same amino acid sequence as the amino acid
sequence shown in FIG. 2 (Seq. I.D. No. 2). In another embodiment,
the GALR3 receptor is the rat GALR3 receptor having the amino acid
sequence shown in FIG. 2 (Seq. ID NO. 2). In another embodiment,
the nucleic acid comprises at least an intron. In still another
embodiment, the nucleic acid comprises alternately spliced nucleic
acid transcribed from the nucleic acid contained in plasmid K1086.
In an embodiment, the alternately spliced nucleic acid is mRNA
transcribed from DNA encoding a galanin receptor.
[0079] In an embodiment, the GALR3 receptor is a human GALR3
receptor. This invention provides an isolated nucleic acid encoding
a human GALR3 receptor having the same or substantially the same
amino acid sequence as the amino acid sequence encoded by plasmid
pEXJ-hGalR3 (ATCC Accession No. 97827). This invention provides an
isolated nucleic acid encoding a human GALR3 receptor, wherein the
human GALR3 receptor has a sequence, which sequence comprises
substantially the same amino acid sequence as the sequence shown in
FIG. 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
In another embodiment, the GALR3 receptor has a sequence, which
sequence comprises the sequence shown in FIG. 4 (Seq. ID NO. 4)
from amino acid 60 through amino acid 427.
[0080] In another embodiment, the nucleic acid encoding the human
GALR3 receptor comprises an intron. In still another embodiment,
the nucleic acid encoding the human GALR3 receptor comprises
alternately spliced nucleic acid.
[0081] The fact that introns are found in many G protein coupled
receptors raises the possibility that introns could exist in coding
or non-coding regions of GALR3; if so, a spliced form of mRNA may
encode additional amino acids either upstream of the currently
defined starting methionine or within the coding region. Further,
the existence and use of alternative exons is possible, whereby the
mRNA may encode different amino acids within the region comprising
the exon. In addition, single amino acid substitutions may arise
via the mechanism of RNA editing such that the amino acid sequence
of the expressed protein is different than that encoded by the
original gene (Burns et al., 1996; Chu et al., 1996). Such variants
may exhibit pharmacologic properties differing from the receptor
encoded by the original gene.
[0082] This invention provides a splice variant of the GALR3
receptors disclosed herein. This invention further provides for
alternate translation initiation sites and alternately spliced or
edited variants of nucleic acids encoding rat and human GALR3
receptors.
[0083] This invention provides the above-described isolated nucleic
acid, wherein the nucleic acid is DNA. In one embodiment, the DNA
is cDNA. In another embodiment, the DNA is genomic DNA. In still
another embodiment, the nucleic acid molecule is RNA. Methods for
production and manipulation of nucleic acid molecules are well
known in the art.
[0084] This invention provides a vector encoding the nucleic acid
of human GALR3 receptor.
[0085] In another embodiment, the nucleic acid encodes a vertebrate
GALR3 receptor. In a separate embodiment, the nucleic acid encodes
a mammalian GALR3 receptor. In another embodiment, the nucleic acid
encodes a rat GALR3 receptor. In still another embodiment, the
nucleic acid encodes a human GALR3 receptor.
[0086] This invention further provides nucleic acid which is
degenerate with respect to the DNA comprising the coding sequence
of the plasmid K1086 (ATCC Accession No. 97747). This invention
further provides nucleic acid which is degenerate with respect to
any DNA encoding a GALR3 receptor. In an embodiment, the nucleic
acid comprises a nucleotide sequence which is degenerate with
respect to the nucleotide sequence of plasmid K1086, that is, a
nucleotide sequence which is translated into the same amino acid
sequence. In an embodiment, the nucleic acid comprises a nucleotide
sequence which is degenerate with respect to the nucleotide
sequence of plasmid pEXJ-rGalR3T (ATCC Accession No. 97826). In
another embodiment, the nucleic acid comprises a nucleotide
sequence which is degenerate with respect to the nucleotide
sequence of plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
[0087] This invention also encompasses DNAs and cDNAs which encode
amino acid sequences which differ from those of the GALR3 galanin
receptor, but which should not produce phenotypic changes.
Alternatively, this invention also encompasses DNAs, cDNAs, and
RNAs which hybridize to the DNA, cDNA, and RNA of the subject
invention. Hybridization methods are well known to those of skill
in the art.
[0088] The nucleic acids of the subject invention also include
nucleic acid molecules coding for polypeptide analogs, fragments or
derivatives of antigenic polypeptides which differ from
naturally-occurring forms in terms of the identity or location of
one or more amino acid residues (deletion analogs containing less
than all of the residues specified for the protein, substitution
analogs wherein one or more residues specified are replaced by
other residues and addition analogs where in one or more amino acid
residues is added to a terminal or medial portion of the
polypeptides) and which share some or all properties of
naturally-occurring forms. These molecules include: the
incorporation of codons "preferred" for expression by selected
non-mammalian hosts; the provision of sites for cleavage by
restriction endonuclease enzymes; and the provision of additional
initial, terminal or intermediate DNA sequences that facilitate
construction of readily expressed vectors.
[0089] G-protein coupled receptors such as the GALR3 receptors of
the present invention are characterized by the ability of an
agonist to promote the formation of a high-affinity ternary complex
between the agonist, the receptor, and an intracellular G-protein.
This complex is formed in the presence of physiological
concentrations of GTP, and results in the dissociation of the alpha
subunit of the G protein from the beta and gamma subunits of the G
protein, which further results in a functional response, i.e.,
activation of downstream effectors such as adenylyl cyclase or
phospholipase C. This high-affinity complex is transient even in
the presence of GTP, so that if the complex is destablized, the
affinity of the receptor for agonists is reduced. Thus, if a
receptor is not optimally coupled to G protein under the conditions
of an assay, an agonist will bind to the receptor with low
affinity. In contrast, the affinity of the receptor for an
antagonist is normally not significantly affected by the presence
or absence of G protein. Functional assays may be used to determine
whether a compound binds to the receptor, but may be more
time-consuming or difficult to perform than a binding assay.
Therefore, it may desirable to produce a receptor which will bind
to agonists with high affinity in a binding assay. Examples of
modified receptors which bind agonists with high affinity are
disclosed in WO 96/14331, which describes neuropeptide Y receptors
modified in the third intracellular domain. The modifications may
include deletions of 6-13 amino acids in the third intracellular
loop. Such deletions preferably end immediately before the polar or
charged residue at the beginning of helix six. In an embodiment,
the deleted amino acids are at the carboxy terminus of the third
intracellular domain. Such modified receptors may be produced using
methods well-known in the art such as site-directed mutagenesis or
recombinant techniques using restriction enzymes.
[0090] This invention provides an isolated nucleic acid encoding a
modified GALR3 receptor, which differs from a GALR3 receptor by
having an amino acid(s) deletion, replacement or addition in the
third intracellular domain. In one embodiment, the modified GALR3
receptor differs by having a deletion in the third intracellular
domain. In another embodiment, the modified GALR3 receptor differs
by having an amino acid replacement or addition to the third
intracellular domain.
[0091] The modified receptors of this invention may be transfected
into cells either transiently or stably using methods well-known in
the art, examples of which are disclosed herein. This invention
also provides for binding assays using the modified receptors, in
which the receptor is expressed either transiently or in stable
cell lines. This invention further provides for a compound
identified using a modified receptor in a binding assay such as the
binding assays described herein.
[0092] The nucleic acids described and claimed herein are useful
for the information which they provide concerning the amino acid
sequence of the polypeptide and as products for the large scale
synthesis of the polypeptide by a variety of recombinant
techniques. The nucleic acid molecule is useful for generating new
cloning and expression vectors, transformed and transfected
prokaryotic and eukaryotic host cells, and new and useful methods
for cultured growth of such host cells capable of expression of the
polypeptide and related products.
[0093] This invention also provides an isolated galanin GALR3
receptor protein. In one embodiment, the GALR3 receptor protein has
the same or substantially the same amino acid sequence as the amino
acid sequence encoded by plasmid K1086. In another embodiment, the
GALR3 receptor protein has the amino acid sequence encoded by
plasmid K1086. In another embodiment, the protein has the amino
acid sequence encoded by the plasmid pEXJ-hGalR3. In an embodiment,
the GALR3 receptor protein has the same or substantially the same
amino acid sequence as the amino acid sequence shown in FIG. 2
(Seq. I.D. No. 2). In an embodiment, the GALR3 receptor comprises
the same or substantially the same amino acid sequence as the amino
acid sequence shown in FIG. 4 (Seq. I.D. No. 4) from amino acid 60
through amino acid 427.
[0094] This invention provides a vector comprising the
above-described nucleic acid molecule.
[0095] Vectors which comprise the isolated nucleic acid molecule
described hereinabove also are provided. Suitable vectors comprise,
but are not limited to, a plasmid or a virus. Some such vectors may
be transformed into a suitable host cell to form a host cell
expression system for the production of a polypeptide having the
biological activity of a galanin GALR3 receptor. Suitable host
cells include, for example, neuronal cells such as the glial cell
line C6, a Xenopus cell such as an oocyte or melanophore cell, as
well as numerous mammalian cells and non-neuronal cells. Other such
vectors may be used for in vitro transcription to produce RNA
encoding GALR3, which RNA is introduced, e.g., by injection, into
oocytes.
[0096] This invention provides the above-described vector adapted
for expression in a bacterial cell which further comprises the
regulatory elements necessary for expression of the nucleic acid in
the bacterial cell operatively linked to the nucleic acid encoding
the GALR3 receptor as to permit expression thereof.
[0097] This invention provides the above-described vector adapted
for expression in a yeast cell which comprises the regulatory
elements necessary for expression of the nucleic acid in the yeast
cell operatively linked to the nucleic acid encoding the GALR3
receptor as to permit expression thereof.
[0098] This invention provides the above-described vector adapted
for expression in an insect cell which comprises the regulatory
elements necessary for expression of the nucleic acid in the insect
cell operatively linked to the nucleic acid encoding the GALR3
receptor as to permit expression thereof. In a still further
embodiment, the vector is a baculovirus.
[0099] This invention provides the above-described vector adapted
for expression in a amphibian cell which further comprises the
regulatory elements necessary for expression of the nucleic acid in
the amphibian cell operatively linked to the nucleic acid encoding
the GALR3 receptor as to permit expression thereof.
[0100] In an embodiment, the vector is adapted for expression in a
mammalian cell which comprises the regulatory elements necessary
for expression of the nucleic acid in the mammalian cell
operatively linked to the nucleic acid encoding the mammalian GALR3
receptor as to permit expression thereof.
[0101] In a further embodiment, the vector is adapted for
expression in a mammalian cell which comprises the regulatory
elements necessary for expression of the nucleic acid in the
mammalian cell operatively linked to the nucleic acid encoding the
rat GALR3 receptor as to permit expression thereof.
[0102] In a still further embodiment, the vector is a plasmid.
[0103] In another embodiment, the plasmid is adapted for expression
in a mammalian cell which comprises the regulatory elements
necessary for expression of the nucleic acid in the mammalian cell
operatively linked to the nucleic acid encoding the human GALR3
receptor as to permit expression thereof.
[0104] This invention provides the above-described plasmid adapted
for expression in a mammalian cell which comprises the regulatory
elements necessary for expression of nucleic acid in a mammalian
cell operatively linked to the nucleic acid encoding the mammalian
GALR3 receptor as to permit expression thereof.
[0105] This invention provides a plasmid designated K1086 (ATCC
Accession No. 97747) which comprises the regulatory elements
necessary for expression of DNA in a mammalian cell operatively
linked to DNA encoding the GALR3 galanin receptor so as to permit
expression thereof.
[0106] This plasmid (K1086) was deposited on Oct. 8, 1996, with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852, U.S.A. under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure and was
accorded ATCC Accession No. 97747.
[0107] This invention provides a plasmid designated pEXJ-hGalR3
(ATCC Accession No. 97827) which comprises the regulatory elements
necessary for expression of DNA in a mammalian cell operatively
linked to DNA encoding the human GALR3 galanin receptor so as to
permit expression thereof. This plasmid was deposited Dec. 17,
1996, with the ATCC, 12301 Parklawn Drive, Rockville, Md., 20852,
U.S.A. under the provisions of the Budapest Treaty forth
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure and was accorded ATCC Accession No.
97827.
[0108] This invention provides a plasmid designated pEXJ-rGalR3T
(ATCC Accession No. 97826) which comprises the regulatory elements
necessary for expression of DNA in a mammalian cell operatively
linked to DNA encoding the rat GALR3 galanin receptor so as to
permit expression thereof.
[0109] This plasmid was deposited Dec. 17, 1996, with the ATCC,
12301 Parklawn Drive, Rockville, Md., 20852, U.S.A. under the
provisions of the Budapest Treaty for the International Recognition
of the Deposit of Microorganisms for the Purposes of Patent
Procedure and was accorded ATCC Accession No. 97826.
[0110] This invention provides a plasmid designated M54 (ATCC
Accession No. 209312).
[0111] This plasmid (M54) was deposited on Sep. 30, 1997, with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852, U.S.A. under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure and was
accorded ATCC Accession No. 209312.
[0112] This invention provides a plasmid designated M67 (ATCC
Accession No. ).
[0113] This plasmid (M67) was deposited on Mar. 27, 1998, with the
American Type Culture Collection (ATCC), 12301 Parklawn Drive,
Rockville, Md. 20852, U.S.A. under the provisions of the Budapest
Treaty for the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure and was
accorded ATCC Accession No xxxxx.
[0114] This invention further provides for any vector or plasmid
which comprises modified untranslated sequences, which are
beneficial for expression in desired host cells or for use in
binding or functional assays. For example, a vector or plasmid with
untranslated sequences of varying lengths may express differing
amounts of the receptor depending upon the host cell used. In one
embodiment, the vector or plasmid comprises the coding sequence of
the GALR3 receptor and the regulatory elements necessary for
expression in the host cell.
[0115] This invention provides a eukaryotic cell comprising the
above-described plasmid or vector. This invention provides a
mammalian cell comprising the above-described plasmid or vector. In
an embodiment the cell is a Xenopus oocyte or melanophore cell. In
an embodiment, the cell is a neuronal cell such as the glial cell
line C6. In an embodiment, the mammalian cell is non-neuronal in
origin. In an embodiment, the mammalian cell is a COS-7 cell. In
another embodiment the mammalian cell is a Chinese hamster ovary
(CHO) cell. In another embodiment, the cell is a mouse Y1 cell.
[0116] In still another embodiment, the mammalian cell is a 293
human embryonic kidney cell. In still another embodiment, the
mammalian cell is a NIH-3T3 cell. In another embodiment, the
mammalian cell is an LM(tk-) cell.
[0117] In an embodiment, the mammalian cell is the 293 cell
designated 293-rGALR3-105, which comprises the "trimmed" plasmid
pEXJ-rGalR3T. This cell line was deposited with the ATCC on Feb.
19, 1997, under the provisions of the Budapest Treaty for the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure, and was accorded ATCC Accession No.
CRL-12287.
[0118] In an embodiment, the mammalian cell is the LM(tk-) cell
designated L-hGALR3-228, which comprises the plasmid pEXJ-hGalR3.
This cell line was deposited with the ATCC on Jun. 25, 1997, under
the provisions of the Budapest Treaty for the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure, and was accorded ATCC Accession No.
CRL-12373.
[0119] This invention also provides an insect cell comprising the
above-described vector. In an embodiment, the insect cell is an Sf9
cell. In another embodiment, the insect cell is an Sf21 cell.
[0120] This invention provides a membrane preparation isolated from
any of the above-described cells.
[0121] This invention provides a nucleic acid probe comprising at
least 15 nucleotides, which probe specifically hybridizes with a
nucleic acid encoding a GALR3 receptor, wherein the probe has a
unique sequence corresponding to a sequence present within one of
the two strands of the nucleic acid encoding the GALR3 receptor
contained in plasmid K1086.
[0122] This invention further provides a nucleic acid probe
comprising at least 15 nucleotides, which probe specifically
hybridizes with a nucleic acid encoding a GALR3 receptor, wherein
the probe has a unique sequence corresponding to a sequence present
within one of the two strands of the nucleic acid encoding the
GALR3 receptor contained in plasmid pEXJ-rGalR3T.
[0123] This invention still further provides a nucleic acid probe
comprising at least 15 nucleotides, which probe specifically
hybridizes with a nucleic acid encoding a GALR3 receptor, wherein
the probe has a unique sequence corresponding to a sequence present
within (a) the nucleic acid sequence shown in FIG. 1 (Seq. ID NO.
1) or (b) the reverse complement to the nucleic acid sequence shown
in FIG. 1 (Seq. ID No. 1).
[0124] This invention also provides a nucleic acid probe comprising
at least 15 nucleotides, which probe specifically hybridizes with a
nucleic acid encoding a GALR3 receptor, wherein the probe has a
unique sequence corresponding to a sequence present within one of
the two strands of the nucleic acid encoding the GALR3 receptor
contained in plasmid pEXJ-hGalR3. This invention provides a nucleic
acid probe comprising at least 15 nucleotides, which probe
specifically hybridizes with a nucleic acid encoding a GALR3
receptor, wherein the probe has a unique sequence corresponding to
a sequence present within (a) the nucleic acid sequence shown in
FIG. 3 (Seq. ID No. 3) or (b) the reverse complement to the nucleic
acid sequence shown in FIG. 3 (Seq. ID NO. 3).
[0125] This invention provides a nucleic acid probe comprising a
nucleic acid which specifically hybridizes with a nucleic acid
encoding a GALR3 receptor, wherein the probe comprises a unique
sequence of at least 15 nucleotides within a fragment of (a) the
nucleic acid sequence contained in plasmid K1086 or (b) the
antisense nucleic acid sequence capable of specifically hybridizing
to the nucleic acid sequence contained in plasmid K1086. In one
embodiment the GALR3 receptor is encoded by the coding sequence of
the plasmid K1086, or the reverse complement (antisense sequence)
of the coding sequence of plasmid K1086. In an embodiment, the
nucleic acid encoding a GALR3 receptor comprises an intron.
[0126] This invention further provides a nucleic acid probe
comprising a nucleic acid molecule of at least 15 nucleotides which
is complementary to a unique fragment of the sequence of a nucleic
acid molecule encoding a GALR3 receptor. This invention also
provides a nucleic acid probe comprising a nucleic acid molecule of
at least 15 nucleotides which is complementary to the antisense
sequence of a unique fragment of the sequence of a nucleic acid
molecule encoding a GALR3 receptor.
[0127] In an embodiment, the nucleic acid probe is DNA. In another
embodiment the nucleic acid probe is RNA. As used herein, the
phrase "specifically hybridizing" means the ability of a nucleic
acid molecule to recognize a nucleic acid sequence complementary to
its own and to form double-helical segments through hydrogen
bonding between complementary base pairs.
[0128] This nucleic acid of at least 15 nucleotides capable of
specifically hybridizing with a sequence of a nucleic acid encoding
the GALR3 galanin receptors can be used as a probe. Nucleic acid
probe technology is well known to those skilled in the art who will
readily appreciate that such probes may vary greatly in length and
may be labeled with a detectable label, such as a radioisotope or
fluorescent dye, to facilitate detection of the probe. DNA probe
molecules may be produced by insertion of a DNA molecule which
encodes the GALR3 receptor into suitable vectors, such as plasmids
or bacteriophages, followed by transforming into suitable bacterial
host cells, replication in the transformed bacterial host cells and
harvesting of the DNA probes, using methods well known in the art.
Alternatively, probes may be generated chemically from DNA
synthesizers.
[0129] RNA probes may be generated by inserting the DNA molecule
which encodes the GALR3 galanin receptor downstream of a
bacteriophage promoter such as T3, T7 or SP6. Large amounts of RNA
probe may be produced by incubating the labeled nucleotides with
the linearized fragment where it contains an upstream promoter in
the presence of the appropriate RNA polymerase.
[0130] This invention provides an antisense oligonucleotide having
a sequence capable of specifically hybridizing to mRNA encoding a
GALR3 galanin receptor, so as to prevent translation of the
mRNA.
[0131] This invention provides an antisense oligonucleotide having
a sequence capable of specifically hybridizing to the genomic DNA
molecule encoding a GALR3 receptor.
[0132] This invention provides an antisense oligonucleotide
comprising chemical analogues of nucleotides.
[0133] This invention provides an antibody directed to a GALR3
receptor. This invention also provides an antibody directed to a
rat GALR3 receptor. This invention also provides an antibody
directed to a human GALR3 receptor. In an embodiment, the rat GALR3
has an amino acid sequence substantially the same as an amino acid
sequence encoded by plasmid K1086. In an embodiment, the human
GALR3 receptor has a sequence, which sequence comprises
substantially the same sequence as the sequence shown in FIG. 4
(Seq. I.D. No. 4) from amino acid 60 through amino acid 427. This
invention further provides an antibody capable of competitively
inhibiting the binding of a second antibody to a GALR3
receptor.
[0134] This invention provides a monoclonal antibody directed to an
epitope of a GALR3 receptor, which epitope is present on the
surface of a cell expressing a GALR3 receptor.
[0135] This invention provides a pharmaceutical composition
comprising an amount of the oligonucleotide effective to reduce
activity of a GALR3 receptor by passing through a cell membrane and
binding specifically with mRNA encoding a GALR3 receptor in the
cell so as to prevent its translation and a pharmaceutically
acceptable carrier capable of passing through a cell membrane. In
an embodiment, the oligonucleotide is coupled to a substance which
inactivates mRNA. In another embodiment, the substance which
inactivates mRNA is a ribozyme.
[0136] This invention provides the above-described pharmaceutical
composition, wherein the pharmaceutically acceptable carrier
capable of passing through a cell membrane comprises a structure
which binds to a receptor specific for a selected cell type and is
thereby taken up by cells of the selected cell type. In an
emdodiment, the pharmaceutically acceptable carrier is capable of
binding to a receptor which is specific for a selected cell
type.
[0137] This invention provides a pharmaceutical composition
comprising an amount of an antagonist effective to reduce the
activity of a GALR3 receptor and a pharmaceutically acceptable
carrier.
[0138] This invention provides a pharmaceutical composition
comprising an amount of an agonist effective to increase activity
of a GALR3 receptor and a pharmaceutically acceptable carrier.
[0139] This invention provides the above-described pharmaceutical
composition which comprises an amount of the antibody effective to
block binding of a ligand to the GALR3 receptor and a
pharmaceutically acceptable carrier.
[0140] As used herein, "pharmaceutically acceptable carriers" means
any of the standard pharmaceutically acceptable carriers. Examples
include, but are not limited to, phosphate buffered saline,
physiological saline, water and emulsions, such as oil/water
emulsions.
[0141] This invention provides a transgenic nonhuman mammal
expressing DNA encoding a GALR3 receptor.
[0142] This invention provides a transgenic nonhuman mammal
comprising a homologous recombination knockout of the native GALR3
receptor.
[0143] This invention provides a transgenic nonhuman mammal whose
genome comprises antisense DNA complementary to DNA encoding a
GALR3 receptor so placed as to be transcribed into antisense mRNA
which is complementary to mRNA encoding a GALR3 receptor and which
hybridizes to mRNA encoding a GALR3 receptor thereby reducing its
translation.
[0144] This invention provides the above-described transgenic
nonhuman mammal, wherein the DNA encoding a GALR3 receptor
additionally comprises an inducible promoter.
[0145] This invention provides the transgenic nonhuman mammal,
wherein the DNA encoding a GALR3 receptor additionally comprises
tissue specific regulatory elements.
[0146] In an embodiment, the transgenic nonhuman mammal is a
mouse.
[0147] Animal model systems which elucidate the physiological and
behavioral roles of GALR3 receptor are produced by creating
transgenic animals in which the activity of the GALR3 receptor is
either increased or decreased, or the amino acid sequence of the
expressed GALR3 receptor is altered, by a variety of techniques.
Examples of these techniques include, but are not limited to: 1)
Insertion of normal or mutant versions of DNA encoding a GALR3
receptor, by microinjection, electroporation, retroviral
transfection or other means well known to those skilled in the art,
into appropriate fertilized embryos in order to produce a
transgenic animal or 2) Homologous recombination of mutant or
normal, human or animal versions of these genes with the native
gene locus in transgenic animals to alter the regulation of
expression or the structure of these GALR3 receptor sequences. The
technique of homologous recombination is well known in the art. It
replaces the native gene with the inserted gene and so is useful
for producing an animal that cannot express native GALR3 receptors
but does express, for example, an inserted mutant GALR3 receptor,
which has replaced the native GALR3 receptor in the animal's genome
by recombination, resulting in underexpression of the transporter.
Microinjection adds genes to the genome, but does not remove them,
and so is useful for producing an animal which expresses its own
and added GALR3 receptors, resulting in overexpression of the GALR3
receptors.
[0148] One means available for producing a transgenic animal, with
a mouse as an example, is as follows: Female mice are mated, and
the resulting fertilized eggs are dissected out of their oviducts.
The eggs are stored in an appropriate medium such as M2 medium. DNA
or cDNA encoding a GALR3 receptor is purified from a vector by
methods well known in the art. Inducible promoters may be fused
with the coding region of the DNA to provide an experimental means
to regulate expression of the trans-gene. Alternatively or in
addition, tissue specific regulatory elements may be fused with the
coding region to permit tissue-specific expression of the
trans-gene. The DNA, in an appropriately buffered solution, is put
into a microinjection needle (which may be made from capillary
tubing using a pipet puller) and the egg to be injected is put in a
depression slide. The needle is inserted into the pronucleus of the
egg, and the DNA solution is injected. The injected egg is then
transferred into the oviduct of a pseudopregnant mouse (a mouse
stimulated by the appropriate hormones to maintain pregnancy but
which is not actually pregnant), where it proceeds to the uterus,
implants, and develops to term. As noted above, microinjection is
not the only method for inserting DNA into the egg cell, and is
used here only for exemplary purposes.
[0149] This invention provides a process for identifying a chemical
compound which specifically binds to a GALR3 receptor which
comprises contacting cells containing DNA encoding and expressing
on their cell surface the GALR3 receptor, wherein such cells do not
normally express the GALR3 receptor, with the compound under
conditions suitable for binding, and detecting specific binding of
the chemical compound to the GALR3 receptor.
[0150] This invention further provides a process for identifying a
chemical compound which specifically binds to a GALR3 receptor
which comprises contacting a membrane fraction from a cell extract
of cells containing DNA encoding and expressing on their cell
surface the GALR3 receptor, wherein such cells do not normally
express the GALR3 receptor, with the compound under conditions
suitable for binding, and detecting specific binding of the
chemical compound to the GALR3 receptor.
[0151] This invention also provides a process for determining
whether a chemical compound can specifically bind to a GALR3
receptor which comprises contacting cells transfected with and
expressing DNA encoding the GALR3 receptor with the compound under
conditions permitting binding of compounds to such receptor, and
detecting the presence of any such compound specifically bound to
the GALR3 receptor, so as to thereby determine whether the ligand
specifically binds to the GALR3 receptor.
[0152] This invention provides a process for determining whether a
chemical compound can specifically bind to a GALR3 receptor which
comprises preparing a cell extract from cells transfected with and
expressing DNA encoding the GALR3 receptor, isolating a membrane
fraction from the cell extract, contacting the membrane fraction
with the compound under conditions permitting binding of compounds
to such receptor, and detecting the presence of the compound
specifically bound to the GALR3 receptor, so as to thereby
determine whether the compound specifically binds to the GALR3
receptor.
[0153] In one embodiment, the GALR3 receptor is a mammalian GALR3
receptor. In another embodiment, the GALR3 receptor is a rat GALR3
receptor. In still another embodiment, the GALR3 receptor has the
same or substantially the same amino acid sequence as that encoded
by plasmid K1086. In still another embodiment, the GALR3 receptor
has the amino acid sequence encoded by plasmid K1086. In another
embodiment, the GALR3 receptor has substantially the same amino
acid sequence as the amino acid sequence shown in FIG. 2 (Seq. ID
NO. 2). In another embodiment, the GALR3 receptor has the amino
acid sequence shown in FIG. 2 (Seq. ID NO. 2). In still another
embodiment, the cells are transfected with the plasmid pEXJ-RGALR3T
(ATCC Accession No. 97826), encoding the rat GALR3 receptor.
Plasmid pEXJ-RGalR3T comprises the entire coding region of rat
GALR3, but in which the 5' initiating ATG is joined directly to the
vector, and which comprises only 100 nucleotides from the 3'
untranslated region after the stop codon (i.e., up to and including
nucleotide 1275 in FIG. 1 (Seq. ID NO. 1)). Transfection of cells
with the "trimmed" plasmid results in a higher level of expression
of the rat GALR3 receptor than the level of expression when plasmid
K1086 is used. The use of the "trimmed" plasmid provides for
greater convenience and accuracy in binding assays. In another
embodiment the GALR3 receptor is a human GALR3 receptor. In still
another embodiment, the GALR3 receptor has the same or
substantially the same amino acid sequence as that encoded by
plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In an embodiment,
the human GALR3 receptor has a sequence, which sequence comprises
substantially the same amino acid sequence as the sequence shown in
FIG. 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
In another embodiment, the GALR3 receptor has a sequence, which
sequence comprises the sequence shown in FIG. 4 (Seq. ID NO. 4)
from amino acid 60 through amino acid 427.
[0154] In an embodiment, the above process further comprises
determining whether the compound selectively binds to the GALR3
receptor relative to another galanin receptor. In another
embodiment, the determination whether the compound selectively
binds to the GALR3 receptor comprises: (a) determining the binding
affinity of the compound for the GALR3 receptor and for such other
galanin receptor; and (b) comparing the binding affinities so
determined, the presence of a higher binding affinity for the GALR3
receptor than for such other galanin receptor indicating that the
compound selectively binds to the GALR3 receptor. In one
embodiment, the other galanin receptor is a GALR1 receptor. In
another embodiment, the other galanin receptor is a GALR2
receptor.
[0155] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor agonist which comprises
contacting cells which express the GALR3 receptor with the compound
under conditions permitting the activation of the GALR3 receptor,
and detecting an increase in GALR3 receptor activity, so as to
thereby determine whether the compound is a GALR3 receptor agonist,
wherein the cells do not normally express the GALR3 receptor.
[0156] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor agonist which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, contacting the membrane fraction with the
compound under conditions permitting the activation of the GALR3
receptor, and detecting an increase in GALR3 receptor activity, so
as to thereby determine whether the compound is a GALR3 receptor
agonist.
[0157] In one embodiment, the GALR3 receptor is a rat GALR3
receptor. In another embodiment, the GALR3 receptor has the same or
substantially the same amino acid sequence as that encoded by the
plasmid K1086. In yet another embodiment, the GALR3 receptor has
the amino acid sequence encoded by the plasmid K1086. In another
embodiment, the GALR3 receptor has substantially the same amino
acid sequence as the amino acid sequence shown in FIG. 2 (Seq. ID
No. 2). In another embodiment, the GALR3 receptor has the amino
acid sequence shown in FIG. 2 (Seq. ID No. 2). In another
embodiment, the GALR3 receptor is a human GALR3 receptor. In still
another embodiment, the GALR3 receptor has the same or
substantially the same amino acid sequence as that encoded by
plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In another
embodiment, the human GALR3 receptor has a sequence, which sequence
comprises substantially the same amino acid sequence as the
sequence shown in FIG. 4 (Seq. I.D. No. 4) from amino acid 60
through amino acid 427. In another embodiment, the GALR3 receptor
has a sequence, which sequence comprises the sequence shown in FIG.
4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427. In
another embodiment of this invention the cells are transfected with
plasmid pEXJ-RGalR3T (ATCC Accession No. 97826).
[0158] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor antagonist which comprises
contacting cells which express the GALR3 receptor with the compound
in the presence of a known GALR3 receptor agonist, such as galanin,
under conditions permitting the activation of the GALR3 receptor,
and detecting a decrease in GALR3 receptor activity, so as to
thereby determine whether the compound is a GALR3 receptor
antagonist, wherein the cells do not normally express the GALR3
receptor.
[0159] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor antagonist which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, contacting the membrane fraction with the ligand
in the presence of a known GALR3 receptor agonist, such as galanin,
under conditions permitting the activation of the GALR3 receptor,
and detecting a decrease in GALR3 receptor activity, so as to
thereby determine whether the compound is a GALR3 receptor
antagonist.
[0160] In an embodiment, the GALR3 receptor is a mammalian GALR3
receptor. In one embodiment of the invention, the GALR3 receptor is
a rat GALR3 receptor. In another embodiment, the GALR3 receptor has
the same or substantially the same amino acid sequence as that
encoded by the plasmid K1086.
[0161] In still another embodiment, the GALR3 receptor has the
amino acid sequence encoded by the plasmid K1086. In another
embodiment, the GALR3 receptor has substantially the same amino
acid sequence as the amino acid sequence shown in FIG. 2 (Seq. ID
No. 2). In another embodiment, the GALR3 receptor has the amino
acid sequence shown in FIG. 2 (Seq. ID No. 2). In another
embodiment, the GALR3 receptor is a human GALR3 receptor. In still
another embodiment, the GALR3 receptor has the same or
substantially the same amino acid sequence as that encoded by
plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In another
embodiment, the human GALR3 receptor has a sequence, which sequence
comprises substantially the same amino acid sequence as the
sequence shown in FIG. 4 (Seq. I.D. No. 4) from amino acid 60
through amino acid 427. In another embodiment, the GALR3 receptor
has a sequence, which sequence comprises the sequence shown in FIG.
4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
[0162] In an embodiment of the above-described methods, the cell is
a non-mammalian cell such as an insect cell or a Xenopus cell. In
another embodiment, the cell is a mammalian cell. In a further
embodiment, the cell is non-neuronal in origin. In still further
embodiments, the non-neuronal cell is a COS-7 cell, 293 human
embryonic kidney cell, NIH-3T3 cell, a CHO cell, or LM(tk-) cell.
In another embodiment, the cell is a mouse Y1 cell.
[0163] This invention provides a compound determined by the
above-described methods. In one embodiment of the above-described
methods, the compound is not previously known to bind to a GALR3
receptor.
[0164] This invention provides a GALR3 agonist determined by the
above-described methods. This invention also provides a GALR3
antagonist determined by the above-described methods.
[0165] In an embodiment of any of the above processes, the cells
are transfected with and express DNA encoding the GALR3
receptor.
[0166] In an embodiment of any of the above processes, RNA encoding
and expressing the GALR3 receptor has been injected into the
cells.
[0167] In an embodiment of any of the above processes, the cells
also express GIRK1 and GIRK4.
[0168] In an embodiment of any of the above processes, the GALR3
receptor is a mammalian GALR3 receptor.
[0169] In an embodiment of any of the above processes, the cells
are injected with RNA synthesized in vitro from the plasmid
designated M54 (ATCC Accession No. 209312).
[0170] In an embodiment of any of the above processes, the cells
are injected with RNA synthesized in vitro from the plasmid
designated M67 (ATCC Accession No.
[0171] This invention provides a pharmaceutical composition which
comprises an amount of a GALR3 receptor agonist determined by the
above-described processes effective to increase activity of a GALR3
receptor and a pharmaceutically acceptable carrier. In an
embodiment, the GALR3 receptor agonist is not previously known.
[0172] This invention provides a pharmaceutical composition which
comprises an amount of a GALR3 receptor antagonist determined by
the above-described processes effective to reduce activity of a
GALR3 receptor and a pharmaceutically acceptable carrier. In an
embodiment, the GALR3 receptor antagonist is not previously
known.
[0173] This invention provides a pharmaceutical composition which
comprises an amount of a GALR3 receptor agonist effective to
increase activity of a GALR3 receptor and a pharmaceutically
acceptable carrier.
[0174] This invention provides a pharmaceutical composition which
comprises an amount of a GALR3 receptor antagonist effective to
reduce activity of a GALR3 receptor and a pharmaceutically
acceptable carrier.
[0175] In further embodiments of the above-described processes, the
agonist or antagonist is not previously known to bind to a GALR3
receptor.
[0176] This invention provides a process involving competitive
binding for identifying a chemical compound which specifically
binds to a GALR3 receptor which comprises separately contacting
cells expressing on their cell surface the GALR3 receptor, wherein
such cells do not normally express the GALR3 receptor, with both
the chemical compound and a second chemical compound known to bind
to the receptor, and with only the second chemical compound, under
conditions suitable for binding of both compounds, and detecting
specific binding of the chemical compound to the GALR3 receptor, a
decrease in the binding of the second chemical compound to the
GALR3 receptor in the presence of the chemical compound indicating
that the chemical compound binds to the GALR3 receptor.
[0177] This invention further provides a process involving
competitive binding for identifying a chemical compound which
specifically binds to a human GALR3 receptor which comprises
separately contacting a membrane fraction from a cell extract of
cells expressing on their cell surface the GALR3 receptor, wherein
such cells do not normally express the GALR3 receptor, with both
the chemical compound and a second chemical compound known to bind
to the receptor, and with only the second chemical compound, under
conditions suitable for binding of both compounds, and detecting
specific binding of the chemical compound to the GALR3 receptor, a
decrease in the binding of the second chemical compound to the
GALR3 receptor in the presence of the chemical compound indicating
that the chemical compound binds to the GALR3 receptor.
[0178] This invention further provides a process for determining
whether a chemical compound specifically binds to and activates a
GALR3 receptor, which comprises contacting cells producing a second
messenger response and expressing on their cell surface the GALR3
receptor, wherein such cells do not normally express the GALR3
receptor, with the chemical compound under conditions suitable for
activation of the GALR3 receptor, and measuring the second
messenger response in the presence and in the absence of the
chemical compound, a change in the second messenger response in the
presence of the chemical compound indicating that the compound
activates the GALR3 receptor.
[0179] This invention further provides a process for determining
whether a chemical compound specifically binds to and activates a
GALR3 receptor, which comprises contacting a membrane fraction from
a cell extract of cells producing a second messenger response and
expressing on their cell surface the GALR3 receptor, wherein such
cells do not normally express the GALR3 receptor, with the chemical
compound under conditions suitable for activation of the GALR3
receptor, and measuring the second messenger response in the
presence and in the absence of the chemical compound, a change in
the second messenger response in the presence of the chemical
compound indicating that the compound activates the GALR3
receptor.
[0180] In an embodiment of the above processes, the second
messenger response comprises potassium channel activation and the
change in second messenger is an increase in the level of potassium
current.
[0181] In one embodiment of the above processes, the second
messenger response comprises adenylate cyclase activity and the
change in second messenger response is a decrease in adenylate
cyclase activity. In an embodiment, adenylate cyclase activity is
determined by measurement of cyclic AMP levels.
[0182] In another embodiment of the above processes, the second
messenger response comprises arachidonic acid release and the
change in second messenger response is an increase in arachidonic
acid levels.
[0183] In another embodiment of the above processes, the second
messenger response comprises intracellular calcium levels and the
change in second messenger response is an increase in intracellular
calcium levels.
[0184] In a still further embodiment of the above processes, the
second messenger response comprises inositol phospholipid
hydrolysis and the change in second messenger response is an
increase in inositol phospholipid hydrolysis.
[0185] This invention further provides a process for determining
whether a chemical compound specifically binds to and inhibits
activation of a GALR3 receptor, which comprises separately
contacting cells producing a second messenger response and
expressing on their cell surface the GALR3 receptor, wherein such
cells do not normally express the GALR3 receptor, with both the
chemical compound and a second chemical compound known to activate
the GALR3 receptor, and with only the second compound, under
conditions suitable for activation of the GALR3 receptor, and
measuring the second messenger response in the presence of only the
second chemical compound and in the presence of both the second
chemical compound and the chemical compound, a smaller change in
the second messenger response in the presence of both the chemical
compound and the second chemical compound than in the presence of
only the second chemical compound indicating that the chemical
compound inhibits activation of the GALR3 receptor.
[0186] This invention further provides a process for determining
whether a chemical compound specifically binds to and inhibits
activation of a GALR3 receptor, which comprises separately
contacting a membrane fraction from a cell extract of cells
producing a second messenger response and expressing on their cell
surface the GALR3 receptor, wherein such cells do not normally
express the GALR3 receptor, with both the chemical compound and a
second chemical compound known to activate the GALR3 receptor, and
with only the second chemical compound, under conditions suitable
for activation of the GALR3 receptor, and measuring the second
messenger response in the presence of only the second chemical
compound and in the presence of both the second chemical compound
and the chemical compound, a smaller change in the second messenger
response in the presence of both the chemical compound and the
second chemical compound than in the presence of only the second
chemical compound indicating that the chemical compound inhibits
activation of the GALR3 receptor.
[0187] In an embodiment of the above processes, the second
messenger response comprises potassium channel activation and the
change in second messenger response is a smaller increase in the
level of potassium current in the presence of both the chemical
compound and the second chemical compound than in the presence of
only the second chemical compound.
[0188] In one embodiment of the above processes, the second
messenger response comprises adenylate cyclase activity and the
change in second messenger response is a smaller decrease in the
level of adenylate cyclase activity in the presence of both the
chemical compound and the second chemical compound than in the
presence of only the second chemical compound. In an embodiment,
adenylate cyclase activity is determined by measurement of cyclic
AMP levels.
[0189] In another embodiment of the above processes the second
messenger response comprises arachidonic acid release, and the
change in second messenger response is a smaller increase in
arachidonic acid levels in the presence of both the chemical
compound and the second chemical compound than in the presence of
only the second chemical compound.
[0190] In another embodiment of the above processes the second
messenger response comprises intracellular calcium levels, and the
change in second messenger response is a smaller increase in
intracellular calcium levels in the presence of both the chemical
compound and the second chemical compound than in the presence of
only the second chemical compound.
[0191] In yet another embodiment of the above processes, the second
messenger response comprises inositol phospholipid hydrolysis, and
the change in second messenger response is a smaller increase in
inositol phospholipid hydrolysis in the presence of both the
chemical compound and the second chemical compound than in the
presence of only the second chemical compound.
[0192] In an embodiment of any of the above processes, the GALR3
receptor is a mammalian GALR3 receptor. In another embodiment of
the above processes, the GALR3 receptor is a rat GALR3 receptor or
a human GALR3 receptor. In still another embodiment of the above
processes, the GALR3 receptor has the same or substantially the
same amino acid sequence as encoded by the plasmid K1086 (ATCC
Accession No. 97747). In another embodiment, the GALR3 receptor has
substantially the same amino acid sequence as the amino acid
sequence shown in FIG. 2 (Seq. ID No. 2). In another embodiment,
the GALR3 receptor has the amino acid sequence shown in FIG. 2
(Seq. ID No. 2). In still another embodiment, the GALR3 receptor
has the same or substantially the same amino acid sequence as that
encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In
another embodiment, the human GALR3 receptor has a sequence, which
sequence comprises substantially the same amino acid sequence as
the sequence shown in FIG. 4 (Seq. I.D. No. 4) from amino acid 60
through amino acid 427. In another embodiment, the GALR3 receptor
has a sequence, which sequence comprises the sequence shown in FIG.
4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427. In
another embodiment of this invention the cells are transfected with
plasmid pEXJ-RGalR3T (ATCC Accession No. 97826).
[0193] In one embodiment of the above-described processes, the cell
is a non-mammalian cell such as an insect cell or a Xenopus cell.
In another embodiment of any of the above processes, the cell is a
mammalian cell. In still further embodiments, the cell is
nonneuronal in origin. In another embodiment of the above
processes, the nonneuronal cell is a COS-7 cell, 293 human
embryonic kidney cell, CHO cell, mouse Y1 cell, NIH-3T3 cell or
LM(tk-) cell.
[0194] This invention further provides a compound determined by any
of the above processes. In another embodiment, the compound is not
previously known to bind to a GALR3 receptor.
[0195] This invention provides a pharmaceutical composition which
comprises an amount of a GALR3 receptor agonist determined by any
of the above processes effective to increase activity of a GALR3
receptor and a pharmaceutically acceptable carrier. In an
embodiment, the GALR3 receptor agonist is not previously known.
[0196] This invention provides a pharmaceutical composition which
comprises an amount of a GALR3 receptor antagonist determined by
any of the above processes effective to reduce activity of a GALR3
receptor and a pharmaceutically acceptable carrier. In an
embodiment, the GALR3 receptor antagonist is not previously
known.
[0197] This invention provides a method of screening a plurality of
chemical compounds not known to bind to a GALR3 receptor to
identify a compound which specifically binds to the GALR3 receptor,
which comprises (a) contacting cells transfected with and
expressing DNA encoding the GALR3 receptor with a compound known to
bind specifically to the GALR3 receptor; (b) contacting the
preparation of step (a) with the plurality of compounds not known
to bind specifically to the GALR3 receptor, under conditions
permitting binding of compounds known to bind the GALR3 receptor;
(c) determining whether the binding of the compound known to bind
to the GALR3 receptor is reduced in the presence of the compounds,
relative to the binding of the compound in the absence of the
plurality of compounds; and if so (d) separately determining the
binding to the GALR3 receptor of each compound included in the
plurality of compounds, so as to thereby identify the compound
which specifically binds to the GALR3 receptor.
[0198] This invention provides a method of screening a plurality of
chemical compounds not known to bind to a GALR3 receptor to
identify a compound which specifically binds to the GALR3 receptor,
which comprises (a) preparing a cell extract from cells transfected
with and expressing DNA encoding the GALR3 receptor, isolating a
membrane fraction from the cell extract, contacting the membrane
fraction with a compound known to bind specifically to the GALR3
receptor; (b) contacting the preparation of step (a) with the
plurality of compounds not known to bind specifically to the GALR3
receptor, under conditions permitting binding of compounds known to
bind the GALR3 receptor; (c) determining whether the binding of the
compound known to bind to the GALR3 receptor is reduced in the
presence of the compounds, relative to the binding of the compound
in the absence of the plurality of compounds; and if so (d)
separately determining the binding to the GALR3 receptor of each
compound included in the plurality of compounds, so as to thereby
identify the compound which specifically binds to the GALR3
receptor.
[0199] In an embodiment of any of the above processes, the GALR3
receptor is a mammalian GALR3 receptor. In an embodiment of the
above-described methods, the GALR3 receptor is a rat GALR3
receptor. In another embodiment, the GALR3 receptor has the same or
substantially the same amino acid sequence as the amino acid
sequence encoded by plasmid K1086. In another embodiment, the GALR3
receptor has substantially the same amino acid sequence as the
amino acid sequence shown in FIG. 2 (Seq. ID NO. 2). In another
embodiment, the GALR3 receptor has the amino acid sequence shown in
FIG. 2 (Seq. ID No. 2). In another embodiment, the GALR3 receptor
is a human GALR3 receptor. In still another embodiment, the GALR3
receptor has the same or substantially the same amino acid sequence
as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
In another embodiment, the human GALR3 receptor has a sequence,
which sequence comprises substantially the same amino acid sequence
as the sequence shown in FIG. 4 (Seq. I.D. No. 4) from amino acid
60 through amino acid 427. In another embodiment, the GALR3
receptor has a sequence, which sequence comprises the sequence
shown in FIG. 4 (Seq. ID NO. 4) from amino acid 60 through amino
acid 427.
[0200] This invention provides a method of screening a plurality of
chemical compounds not known to activate a GALR3 receptor to
identify a compound which activates the GALR3 receptor which
comprises (a) contacting cells expressing the GALR3 receptor with
the plurality of compounds not known to activate the GALR3
receptor, under conditions permitting activation of the GALR3
receptor, wherein the cells do not normally express the GALR3
receptor; (b) determining whether the activity of the GALR3
receptor is increased in the presence of the compounds; and if so
(c) separately determining whether the activation of the GALR3
receptor is increased by each compound included in the plurality of
compounds, so as to thereby identify the compound which activates
the GALR3 receptor.
[0201] This invention provides a method of screening a plurality of
chemical compounds not known to activate a GALR3 receptor to
identify a compound which activates the GALR3 receptor which
comprises (a) preparing a cell extract from cells transfected with
and expressing DNA encoding the GALR3 receptor, isolating a
membrane fraction from the cell extract, contacting the membrane
fraction with the plurality of compounds not known to activate the
GALR3 receptor, under conditions permitting activation of the GALR3
receptor; (b) determining whether the activity of the GALR3
receptor is increased in the presence of the compounds; and if so
(c) separately determining whether the activation of the GALR3
receptor is increased by each compound included in the plurality of
compounds, so as to thereby identify the compound which activates
the GALR3 receptor.
[0202] In an embodiment of the above processes, the cells also
express GIRK1 and GIRK4. In another embodiment, the GALR3 receptor
is a mammalian GALR3 receptor.
[0203] In an embodiment of any of the above-described methods, the
GALR3 receptor is a rat GALR3 receptor. In still another
embodiment, the GALR3 receptor has the same or substantially the
same amino acid sequence as the amino acid sequence encoded by
plasmid K1086. In another embodiment, the GALR3 receptor has
substantially the same amino acid sequence as the amino acid
sequence shown in FIG. 2 (Seq. ID No. 2). In another embodiment,
the GALR3 receptor has the amino acid sequence shown in FIG. 2
(Seq. ID No. 2). In another embodiment, the GALR3 receptor is a
human GALR3 receptor. In still another embodiment, the GALR3
receptor has the same or substantially the same amino acid sequence
as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
In another embodiment, the human GALR3 receptor has a sequence,
which sequence comprises substantially the same amino acid sequence
as the sequence shown in FIG. 4 (Seq. I.D. No. 4) from amino acid
60 through amino acid 427. In another embodiment, the GALR3
receptor has a sequence, which sequence comprises the sequence
shown in FIG. 4 (Seq. ID NO. 4) from amino acid 60 through amino
acid 427.
[0204] This invention provides a method of screening a plurality of
chemical compounds not known to inhibit the activation of a GALR3
receptor to identify a compound which inhibits the activation of
the GALR3 receptor, which comprises (a) contacting cells which
express the GALR3 receptor with the plurality of compounds in the
presence of a known GALR3 receptor agonist, under conditions
permitting activation of the GALR3 receptor, wherein the cells do
not normally express the GALR3 receptor; (b) determining whether
the activation of the GALR3 receptor is reduced in the presence of
the plurality of compounds, relative to the activation of the GALR3
receptor in the absence of the plurality of compounds; and if so
(c) separately determining the inhibition of activation of the
GALR3 receptor for each compound included in the plurality of
compounds, so as to thereby identify the compound which inhibits
the activation of the GALR3 receptor.
[0205] This invention provides a method of screening a plurality of
chemical compounds not known to inhibit the activation of a GALR3
receptor to identify a compound which inhibits the activation of
the GALR3 receptor, which comprises (a) preparing a cell extract
from cells transfected with and expressing DNA encoding the GALR3
receptor, isolating a membrane fraction from the cell extract,
contacting the membrane fraction with the plurality of compounds in
the presence of a known GALR3 receptor agonist, under conditions
permitting activation of the GALR3 receptor; (b) determining
whether the activation of the GALR3 receptor is reduced in the
presence of the plurality of compounds, relative to the activation
of the GALR3 receptor in the absence of the plurality of compounds;
and if so (c) separately determining the inhibition of activation
of the GALR3 receptor for each compound included in the plurality
of compounds, so as to thereby identify the compound which inhibits
the activation of the GALR3 receptor.
[0206] In an embodiment of the above processes, the cells also
express GIRK1 and GIRK4. In another embodiment, the GALR3 receptor
is a mammalian GALR3 receptor.
[0207] In an embodiment of any of the above-described methods, the
GALR3 receptor is a rat GALR3 receptor. In another embodiment, the
GALR3 receptor has the same or substantially the same amino acid
sequence as the amino acid sequence encoded by plasmid K1086. In
another embodiment, the GALR3 receptor has substantially the same
amino acid sequence as the amino acid sequence shown in FIG. 2
(Seq. ID No. 2). In another embodiment, the GALR3 receptor has the
amino acid sequence shown in FIG. 2 (Seq. ID No. 2). In another
embodiment, the GALR3 receptor is a human GALR3 receptor. In still
another embodiment, the GALR3 receptor has the same or
substantially the same amino acid sequence as that encoded by
plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In still another
embodiment, the GALR3 receptor has the same or substantially the
same amino acid sequence as that encoded by plasmid pEXJ-RGalR3T
(ATCC Accession No. 97826). In still another embodiment, the GALR3
receptor has the same or substantially the same amino acid sequence
as that encoded by plasmid M54 (ATCC Accession No. 209312). In
still another embodiment, the GALR3 receptor has the same or
substantially the same amino acid sequence as that encoded by
plasmid M67 (ATCC Accession No. ). In another embodiment, the human
GALR3 receptor has a sequence, which sequence comprises
substantially the same amino acid sequence as the sequence shown in
FIG. 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
In another embodiment, the GALR3 receptor has a sequence, which
sequence comprises the sequence shown in FIG. 4 (Seq. ID NO. 4)
from amino acid 60 through amino acid 427.
[0208] In an embodiment of the above processes, the cells are
transfected with and expressing GIRK1 and GIRK4. In an embodiment
of the above processes, receptor activation is determined by
measurement of potassium channel activation. In an embodiment,
receptor activation is determined by measurement of an increase in
potassium current. In another embodiment, inhibition of receptor
activation is determined by a smaller increase in potassium current
in the presence of the compound and a galanin receptor agonist than
in the presence of only the galanin receptor agonist. In an
embodiment, the galanin receptor agonist is galanin.
[0209] This invention provides a pharmaceutical composition
comprising a compound identified by any of the above-described
methods effective to increase GALR3 receptor activity and a
pharmaceutically acceptable carrier.
[0210] This invention provides a pharmaceutical composition
comprising a compound identified by any of the above-described
methods effective to decrease GALR3 receptor activity and a
pharmaceutically acceptable carrier.
[0211] This invention provides any of the above processes, which
further comprises a process for determining whether the compound
selectively activates the GALR3 receptor relative to another
galanin receptor.
[0212] This invention provides a process for determining whether a
compound selectively activates the GALR3 receptor relative to
another galanin receptor which comprises: (a) determining the
potency of the compound for the GALR3 receptor and for such other
galanin receptor; and (b) comparing the potencies so determined,
the presence of a higher potency for the GALR3 receptor than for
such other galanin receptor indicating that the compound
selectively activates the GALR3 receptor. In an embodiment of the
above process such other galanin receptor is a GALR1 receptor. In
another embodiment, such other galanin receptor is a GALR2
receptor.
[0213] This invention further provides any of the above processes,
which further comprises a process for determining whether the
compound selectively inhibits the activation of the GALR3 receptor
relative to another galanin receptor.
[0214] This invention provides a process for determining whether a
compound selectively inhibits the activation of the GALR3 receptor
relative to another galanin receptor, which comprises: (a)
determining the decrease in the potency of a known galanin receptor
agonist for the GALR3 receptor in the presence of the compound,
relative to the potency of the agonist in the absence of the
compound; (b) determining the decrease in the potency of the
agonist for such other galanin receptor in the presence of the
compound, relative to the potency of the agonist in the absence of
the compound; and (c) comparing the decrease in potencies so
determined, the presence of a greater decrease in potency for the
GALR3 receptor than for such other galanin receptor indicating that
the compound selectively inhibits the activation of the GALR3
receptor. In an embodiment of the above processes, such other
galanin receptor is a GALR1 receptor. In another embodiment, such
other galanin receptor is a GALR2 receptor.
[0215] In an embodiment of any of the above-described methods, the
activation of the GALR3 receptor is determined by a second
messenger assay. In an embodiment, the second messenger assay
measures adenylate cyclase activity. In other embodiments, the
second messenger is cyclic AMP, intracellular calcium, or
arachidonic acid or a phosphoinositol lipid metabolite. Receptor
activation may also be measured by assaying the binding of
GTP.gamma.S (gamma thiol GTP) to membranes, which precedes and is
therefore independent of second messenger coupling.
[0216] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor agonist, which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, separately contacting the membrane fraction with
both the chemical compound and GTP.gamma.S, and with only
GTP.gamma.S, under conditions permitting the activation of the
GALR3 receptor, and detecting GTP.gamma.S binding to the membrane
fraction, an increase in GTP.gamma.S binding in the presence of the
compound indicating that the chemical compound activates the GALR3
receptor.
[0217] This invention provides a process for determining whether a
chemical compound is a GALR3 receptor antagonist, which comprises
preparing a cell extract from cells transfected with and expressing
DNA encoding the GALR3 receptor, isolating a membrane fraction from
the cell extract, separately contacting the membrane fraction with
the chemical compound, GTP.gamma.S and a second chemical compound
known to activate the GALR3 receptor, with GTP.gamma.S and only the
second compound, and with GTP.gamma.S alone, under conditions
permitting the activation of the GALR3 receptor, detecting
GTP.gamma.S binding to each membrane fraction, and comparing the
increase in GTP.gamma.S binding in the presence of the compound and
the second compound relative to the binding of GTP.gamma.S alone,
to the increase in GTP.gamma.S binding in the presence of the
second chemical compound relative to the binding of GTP.gamma.S
alone, a smaller increase in GTP.gamma.S binding in the presence of
the compound and the second compound indicating that the compound
is a GALR3 receptor antagonist.
[0218] In an embodiment of any of the above-described processes,
the second chemical compound is a labeled compound. In another
embodiment, the second chemical compound is a radiolabeled
compound.
[0219] In an embodiment of any of the above-described processes,
the GALR3 receptor is a mammalian GALR3 receptor. In another
embodiment of any of the above-described processes, the GALR3
receptor has substantially the same amino acid sequence as encoded
by the plasmid K1086 (ATCC Accession No. 97747). In another
embodiment of any of the above-described processes, the GALR3
receptor has substantially the same amino acid sequence as that
shown in FIG. 2 (Seq. ID No. 2). In still another embodiment of any
of the above-described processes, the GALR3 receptor has
substantially the same amino acid sequence as encoded by the
plasmid pEXJ-hGalR3 (ATCC Accession No. 97827). In an embodiment of
any of the above-described processes, the GALR3 receptor has a
sequence, which sequence comprises substantially the same amino
acid sequence as that shown in FIG. 4 (Seq. ID No. 4) from amino
acid 60 through amino acid 427. In still another embodiment of any
of the above-described processes, the GALR3 receptor has a
sequence, which sequence comprises a sequence shown in FIG. 4 (Seq.
ID No. 4) from amino acid 60 through amino acid 427.
[0220] In an embodiment of any of the above-described processes,
the cell is an insect cell.
[0221] In an embodiment of any of the above-described processes,
the cell is a mammalian cell. In another embodiment of any of the
above-described processes, the mammalian cell is nonneuronal in
origin. In another embodiment of any of the above-described
processes, the nonneuronal cell is a COS-7 cell, CHO cell, 293
human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell. In
another embodiment, the nonneuronal cell is the 293 human embryonic
kidney cell designated 293-rGALR3-105 (ATCC Accession No.
CRL-12287). In still another embodiment, the nonneuronal cell is
the LM(tk-) cell designated L-hGALR3-228 (ATCC Accession No.
CRL-12373).
[0222] GTP.gamma.S assays are well-known in the art, and it is
expected that variations on the method described above, such as are
described by e.g., Tian et al. (1994) or Lazareno and Birdsall
(1993), may be used by one of ordinary skill in the art. In an
embodiment of any of the above-described processes, the compound is
not previously known to bind to a GALR3 receptor. This invention
also provides a compound determined by any of the above-described
processes.
[0223] This invention further provides a method of measuring GALR3
receptor activation in an oocyte expression system such as a
Xenopus oocyte or melanophore. In an embodiment, receptor
activation is determined by measurement of ion channel activity,
e.g., using the voltage clamp technique (Stuhmer, 1992). In an
embodiment, receptor activation is determined by the measurement of
potassium current. In the experiments described hereinbelow,
receptor activation was determined by measurement of inward
potassium current in the presence of elevated external potassium
levels. However, this invention also provides a method of
determining GALR3 receptor activation by measurement of outward
potassium current in the presence of low (i.e., physiologic)
external potassium levels, using similar methods, which are
well-known in the art.
[0224] Expression of genes in Xenopus oocytes is well known in the
art (A. Coleman, Transcription and Translation: A Practical
Approach (B. D. Hanes, S. J. Higgins, eds., pp 271-302, IRL Press,
Oxford, 1984; Y. Masu et al., Nature 329:21583-21586, 1994) and is
performed using microinjection of native mRNA or in vitro
synthesized mRNA into frog oocytes. The preparation of in vitro
synthesized mRNA can be performed by various standard techniques
(J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
1989) including using T7 polymerase with the mCAP RNA capping kit
(Stratagene). The use of DNA vectors that include 5' and 3'
untranslated (UT) regions of Xenopus .beta.-globin gene flanking
the coding region of the gene of interest has been found to
increase the level of expression in Xenopus oocytes (Linman, et
al., 1992).
[0225] In an embodiment of any of the above-described processes or
methods, the cell is a non-mammalian cell such as an insect cell or
Xenopus cell. In a further embodiment of the invention, the cell is
a mammalian cell. In another embodiment of the invention, the
mammalian cell is non-neuronal in origin. In still further
embodiments of the invention, the non-neuronal cell is a COS-7
cell, a 293 human embryonic kidney cell, a LM(tk-) cell, a mouse Y1
cell, a CHO cell, or an NIH-3T3 cell.
[0226] This invention provides a pharmaceutical composition
comprising a compound identified by the above-described methods and
a pharmaceutically acceptable carrier.
[0227] In an embodiment of the above-described methods, the cell is
non-neuronal in origin. In a further embodiment, the non-neuronal
cell is a COS-7 cell, 293 human embryonic kidney cell, CHO cell,
NIH-3T3 cell or LM(tk-) cell.
[0228] In one embodiment of the above-described methods, the
compound is not previously known to bind to a GALR3 receptor.
[0229] This invention provides a GALR3 receptor agonist detected by
the above-described methods. This invention provides a GALR3
receptor antagonist detected by the above-described methods. In an
embodiment the cell is a non-mammalian cell, for example, a Xenopus
oocyte or melanophore. In another embodiment the cell is a neuronal
cell, for example, a glial cell line such as C6. In an embodiment,
the cell is non-neuronal in origin. In a further embodiment, the
cell is a Cos-7 or a CHO cell, a 293 human embryonic kidney cell,
an LM(tk-) cell or an NIH-3T3 cell.
[0230] This invention provides a pharmaceutical composition
comprising a drug candidate identified by the above-described
methods and a pharmaceutically acceptable carrier.
[0231] This invention provides a method for determining whether a
chemical compound is a GALR3 antagonist which comprises: (a)
administering to an animal a GALR3 agonist and measuring the amount
of food intake in the animal; (b) administering to a second animal
both the GALR3 agonist and the chemical compound, and measuring the
amount of food intake in the second animal; and (c) determining
whether the amount of food intake is reduced in the presence of the
chemical compound relative to the amount of food intake in the
absence of the compound, so as to thereby determine whether the
compound is a GALR3 antagonist.
[0232] This invention further provides a method of screening a
plurality of chemical compounds to identify a chemical compound
which is a GALR3 antagonist which comprises: (a) administering to
an animal a GALR3 agonist and measuring the amount of food intake
in the animal; (b) administering to a second animal the GALR3
agonist and at least one chemical compound of the plurality of
compounds, and measuring the amount of food intake in the animal;
(c) determining whether the amount of food intake is reduced in the
presence of at least one chemical compound of the plurality of
chemical compounds relative to the amount of food intake in the
absence of at least one of the compounds, and if so; (d) separately
determining whether each chemical compound is a GALR3 antagonist
according to the method described above, so as to thereby determine
if the chemical compound is a GALR3 antagonist. In another
embodiment the animal is a non-human mammal. In a further
embodiment, the animal is a rodent.
[0233] This invention provides a method of detecting expression of
a GALR3 receptor by detecting the presence of mRNA coding for the
GALR3 receptor which comprises obtaining total mRNA from a cell or
tissue sample and contacting the mRNA so obtained with the
above-described nucleic acid probe under hybridizing conditions,
detecting the presence of mRNA hybridized to the probe, and thereby
detecting the expression of the GALR3 receptor by the cell or in
the tissue.
[0234] This invention provides a method of treating an abnormality
in a subject, wherein the abnormality is alleviated by
administering to the subject an amount of a GALR3 selective
compound, effective to treat the abnormality. Abnormalities which
may be treated include cognitive disorder, pain, sensory disorder
(olfactory, visual), motor coordination abnormality, motion
sickness, neuroendocrine disorders, sleep disorders, migraine,
Parkinson's disease, hypertension, heart failure,
convulsion/epilepsy, traumatic brain injury, diabetes, glaucoma,
electrolyte imbalances, respiratory disorders (asthma, emphysema),
depression, reproductive disorders, gastric and intestinal ulcers,
gastroesophageal reflux disorder, gastric hypersecretion,
gastrointestinal motility disorders (diarrhea), inflammation,
immune disorders, and anxiety. In one embodiment the compound is an
agonist. In another embodiment the compound is an antagonist.
[0235] This invention provides a method of treating an abnormality
in a subject, wherein the abnormality is alleviated by the
inhibition of a GALR3 receptor which comprises administering to a
subject an effective amount of the above-described pharmaceutical
composition effective to decrease the activity of the GALR3
receptor in the subject, thereby treating the abnormality in the
subject. In an embodiment, the abnormality is obesity.
[0236] In another embodiment, the abnormality is bulimia.
[0237] This invention provides a method of treating an abnormality
in a subject wherein the abnormality is alleviated by the
activation of a GALR3 receptor which comprises administering to a
subject an effective amount of the above-described pharmaceutical
composition effective to activate the GALR3 receptor in the
subject.
[0238] In an embodiment, the abnormal condition is anorexia. In
another embodiment, the compound binds selectively to a GALR3
receptor. In yet another embodiment, the compound binds to the
GALR3 receptor with an affinity greater than ten-fold higher than
the affinity with which the compound binds to a GALR1 receptor. In
a still further embodiment, the compound binds to the GALR3
receptor with an affinity greater than ten-fold higher than the
affinity with which the compound binds to a GALR2 receptor.
[0239] This invention provides a method of detecting the presence
of a GALR3 receptor on the surface of a cell which comprises
contacting the cell with the above-described antibody under
conditions permitting binding of the antibody to the receptor,
detecting the presence of the antibody bound to the cell, and
thereby detecting the presence of a GALR3 receptor on the surface
of the cell.
[0240] This invention provides a method of determining the
physiological effects of varying levels of activity of GALR3
receptors which comprises producing a transgenic nonhuman mammal
whose levels of GALR3 receptor activity are varied by use of an
inducible promoter which regulates GALR3 receptor expression.
[0241] This invention provides a method of determining the
physiological effects of varying levels of activity of GALR3
receptors which comprises producing a panel of transgenic nonhuman
mammals each expressing a different amount of GALR3 receptor.
[0242] This invention provides a method for identifying an
antagonist capable of alleviating an abnormality wherein the
abnormality is alleviated by decreasing the activity of a GALR3
receptor comprising administering a compound to the above-described
transgenic nonhuman mammal and determining whether the compound
alleviates the physical and behavioral abnormalities displayed by
the transgenic nonhuman mammal as a result of overactivity of a
GALR3 receptor, the alleviation of the abnormality identifying the
compound as an antagonist.
[0243] This invention provides an antagonist identified by the
above-described methods. This invention provides a pharmaceutical
composition comprising an antagonist identified by the
above-described methods and a pharmaceutically acceptable
carrier.
[0244] This invention provides a method of treating an abnormality
in a subject wherein the abnormality is alleviated by decreasing
the activity of a GALR3 receptor which comprises administering to a
subject an effective amount of the above-described pharmaceutical
composition, thereby treating the abnormality.
[0245] This invention provides a method for identifying an agonist
capable of alleviating an abnormality in a subject wherein the
abnormality is alleviated by increasing the activity of a GALR3
receptor comprising administering a compound to a transgenic
nonhuman mammal and determining whether the compound alleviates the
physical and behavioral abnormalities displayed by the transgenic
nonhuman mammal, the alleviation of the abnormality identifying the
compound as an agonist.
[0246] This invention provides an agonist identified by the
above-described methods.
[0247] This invention provides a pharmaceutical composition
comprising an agonist identified by the above-described methods and
a pharmaceutically acceptable carrier.
[0248] This invention provides a method for treating an abnormality
in a subject wherein the abnormality is alleviated by increasing
the activity of a GALR3 receptor which comprises administering to a
subject an effective amount of the above-described pharmaceutical
composition, thereby treating the abnormality.
[0249] This invention provides a method for diagnosing a
predisposition to a disorder associated with the activity of a
specific human GALR3 receptor allele which comprises: (a) obtaining
DNA of subjects suffering from the disorder; (b) performing a
restriction digest of the DNA with a panel of restriction enzymes;
(c) electrophoretically separating the resulting DNA fragments on a
sizing gel; (d) contacting the resulting gel with a nucleic acid
probe capable of specifically hybridizing with a unique sequence
included within the sequence of a nucleic acid molecule encoding a
human GALR3 receptor and labeled with a detectable marker; (e)
detecting labeled bands which have hybridized to DNA encoding a
human GALR3 receptor labeled with a detectable marker to create a
unique band pattern specific to the DNA of subjects suffering from
the disorder; (f) preparing DNA obtained for diagnosis by steps
a-e; and (g) comparing the unique band pattern specific to the DNA
of subjects suffering from the disorder from step e and the DNA
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.
[0250] In an embodiment, a disorder associated with the activity of
a specific human GALR3 receptor allele is diagnosed. In another
embodiment, the above-described method may be used to identify a
population of patients having a specific GALR3 receptor allele, in
which population the disorder may be alleviated by administering to
the subjects a GALR3-selective compound.
[0251] This invention provides a method of preparing the purified
GALR3 receptor which comprises: (a) inducing cells to express GALR3
receptor; (b) recovering the receptor from the induced cells; and
(c) purifying the receptor so recovered.
[0252] This invention provides a method of preparing a purified
GALR3 receptor which comprises: (a) inserting nucleic acid encoding
the GALR3 receptor in a suitable vector; (b) introducing the
resulting vector in a suitable host cell; (c) placing the resulting
cell in suitable condition permitting the production of the
isolated GALR3 receptor; (d) recovering the receptor produced by
the resulting cell; and (e) purifying the receptor so
recovered.
[0253] This invention provides a method of modifying feeding
behavior of a subject which comprises administering to the subject
an amount of a compound which is a galanin receptor agonist or
antagonist effective to increase or decrease the consumption of
food by the subject so as to thereby modify feeding behavior of the
subject. In one embodiment, the compound is a GALR3 receptor
antagonist and the amount is effective to decrease the consumption
of food by the subject. In another embodiment the compound is
administered in combination with food.
[0254] In yet another embodiment the compound is a GALR3 receptor
agonist and the amount is effective to increase the consumption of
food by the subject. In a still further embodiment, the compound is
administered in combination with food. In other embodiments the
subject is a vertebrate, a mammal, a human or a canine.
[0255] In one embodiment, the compound binds selectively to a GALR3
receptor. In another embodiment, the compound binds to the GALR3
receptor with an affinity greater than ten-fold higher than the
affinity with which the compound binds to a GALR1 receptor. In
another embodiment, the compound binds to the GALR3 receptor with
an affinity greater than ten-fold higher than the affinity with
which the compound binds to a GALR2 receptor. In yet another
embodiment, the compound binds to the GALR3 receptor with an
affinity greater than one hundred-fold higher than the affinity
with which the compound binds to a GALR1 receptor. In another
embodiment, the compound binds to the GALR3 receptor with an
affinity greater than one hundred-fold higher than the affinity
with which the compound binds to a GALR2 receptor.
[0256] This invention provides a method of treating Alzheimer's
disease in a subject which comprises administering to the subject
an amount of a compound which is a galanin receptor antagonist
effective to treat the subject's Alzheimer's disease. In one
embodiment, the galanin receptor antagonist is a GALR3 receptor
antagonist and the amount of the compound is effective to treat the
subject's Alzheimer's disease.
[0257] This invention provides a method of producing analgesia in a
subject which comprises administering to the subject an amount of a
compound which is a galanin receptor agonist effective to produce
analgesia in the subject. In another embodiment, the galanin
receptor agonist is a GALR3 receptor agonist and the amount of the
compound is effective to produce analgesia in the subject.
[0258] This invention provides a method of decreasing nociception
in a subject which comprises administering to the subject an amount
of a compound which is a GALR3 receptor agonist effective to
decrease nociception in the subject.
[0259] This invention provides a method of treating pain in a
subject which comprises administering to the subject an amount of a
compound which is a GALR3 receptor agonist effective to treat pain
in the subject.
[0260] This invention provides a method of treating diabetes in a
subject which comprises administering to the subject an amount of a
compound which is a GALR3 receptor antagonist effective to treat
diabetes in the subject.
[0261] This invention provides a method of enhancing cognition in a
subject which comprises administering to the subject an amount of a
compound which is a GALR3 receptor antagonist effective to enhance
cognition in the subject.
[0262] This invention provides a method of decreasing feeding
behavior of a subject which comprises administering a compound
which is a GALR3 receptor antagonist and a compound which is a Y5
receptor antagonist, the amount of such antagonists being effective
to decrease the feeding behavior of the subject. In an embodiment,
the GALR3 antagonist and the Y5 antagonist are administered in
combination. In another embodiment, the GALR3 antagonist and the Y5
antagonist are administered once. In another embodiment, the GALR3
antagonist and the Y5 antagonist are administered separately. In
still another embodiment, the GALR3 antagonist and the Y5
antagonist are administered once. In another embodiment, the
galanin receptor antagonist is administered for about 1 week to 2
weeks. In another embodiment, the Y5 receptor antagonist is
administered for about 1 week to 2 weeks.
[0263] In yet another embodiment, the GALR3 antagonist and the Y5
antagonist are administered alternately. In another embodiment, the
GALR3 antagonist and the Y5 antagonist are administered repeatedly.
In a still further embodiment, the galanin receptor antagonist is
administered for about 1 week to 2 weeks. In another embodiment,
the Y5 receptor antagonist is administered for about 1 week to 2
weeks. This invention also provides a method as described above,
wherein the compound is administered in a pharmaceutical
composition comprising a sustained release formulation.
[0264] This invention will be better understood from the
Experimental Details which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims which follow thereafter.
[0265] Experimental Details
[0266] Materials and Methods
[0267] Cloning and Sequencing a Novel Rat Galanin Receptor
Fragment
[0268] A rat hypothalamus cDNA library in lambda ZAP II
(.apprxeq.2.5.times.10.sup.6 total recombinants; Stratagene,
LaJolla, Calif.) was screened using overlapping transmembrane (TM)
oligonucleotide probes (TM 1, 2, 3, 4, 5, 6 and 7) derived from the
rat GALR2 receptor cDNA. Overlapping oligomers were labeled with
[.sup.32P] DATP and [.sup.32P] dCTP by synthesis with the large
fragment of DNA polymerase, and comprised the following
sequences:
2 TM1: (+) strand: 5'TTGTACCCCTATTTTTCGCGCTCATCTTCC-
TCGTGGGCACCGTGG-3'; (SEQ ID NO:6) (-) strand:
5'-AGCACCGCCAGCACCAGCGCGTTGCCCACGGTGCCCACGAGGAAG-3'; (SEQ ID NO:7)
TM2: (+) strand: 5'-TCAGCACCACCAACCTGTTCATCCTCAACC-
TGGGCGTGGCCGACCTGTGT-3'; (SEQ ID NO:8) (-) strand:
5'-GGCCTGGAAAGGCACGCAGCACAGGATGAAACACAGGTCGGCCACGCCCA-3'; (SEQ ID
NO:9) TM3: (+) strand: 5'-CTGCAAGGCTGTTCATTTCCTCATC-
TTTCTCACTATGCACGCCAG-3'; (SEQ ID NO:10) (-) strand:
5'-GGAGACGGCGGCCAGCGTGAAGCTGCTGGCGTGCATAGTGAGAAA-3'; (SEQ ID NO:11)
TM4: (+) strand 5'-AACGCGCTGGCCGCCATCGGGCTCATCTGG-
GGGCTAGCACTGCTC-3'; (SEQ ID NO:12) (-) strand
5'-AGTAGCTCAGGTAGGGCCCGGAGAAGAGCAGTGCTAGCCCCCAGA-3'; (SEQ ID NO:13)
TM5: (+) strand: 5'-AGCCATGGACCTCTGCACCTTCGTCTTTA-
GCTACCTGCTGCCAGT-3'; (SEQ ID NO:14) (-) strand:
5'-CGCATAGGTCAGACTGAGGACTAGCACTGGCAGCAGGTAGCTAAA-3'; (SEQ ID NO:15)
TM6: (+) strand: 5'-GATCATCATCGTGGCGGTGCTTTTCTGCC-
TCTGTTGGATGCCCCA-3'; (SEQ ID NO:16) (-) strand:
5'-CCACACGCAGAGGATAAGCGCGTGGTGGGGCATCCAACAGAGGCA-3'; (SEQ ID NO:17)
TM7: (+) strand: 5'-GTTGCGCATCCTTTCACACCTAGTTTCCT-
ATGCCAACTCCTGTGT-3'; (SEQ ID NO:18) (-) strand:
5'-AGACCAGAGCGTAAACGATGGGGTTGACACAGGAGTTGGCATAGGA-3'. (SEQ ID
NO:19)
[0269] Hybridization of phage lifts was performed at reduced
stringency conditions: 40.degree. C. in a solution containing 37.5%
formamide, 5.times.SSC (1.times.SSC is 0.15M sodium chloride,
0.015M sodium citrate), 1.times. Denhardt's solution (0.02%
polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin),
and 25 .mu.g/.mu.L sonicated salmon sperm DNA. The filters were
washed at 45.degree. C. in 0.1.times. SSC containing 0.1% sodium
dodecyl sulfate and exposed at -70.degree. C. to Kodak BioMax film
in the presence of an intensifying screen. Lambda phage clones
hybridizing with the probes were plaque purified and pBluescript
recombinant DNAs were excision-rescued from .lambda. Zap II using
helper phage Re704, as described by the manufacturer's protocol
(Rapid Excision Kit, Stratagene, LaJolla, Calif.). Insert size was
confirmed by restriction enzyme digest analysis. The cDNA insert
was sequenced on both strands by cycle sequencing with AmpliTaq DNA
Polymerase, FS (Perkin Elmer) and products run on an automated
fluorescent sequencer, the ABI Prism 377 Sequencer (ABI).
Nucleotide and peptide sequence analyses were performed using the
Wisconsin Package (GCG, Genetics Computer Group, Madison, Wis.).
Sequence analyses indicated that one clone, named rHY35a, contained
an open reading frame from the starting MET codon to the middle of
a predicted seventh transmembrane domain. Because the high degree
of identity of rHY35a to rGALR1 and rGALR2 indicated that it might
represent a fragment of a novel galanin receptor (referred to
herein as "GALR3"), PCR primers directed to the amino terminus
(forward primer) and first extracellular loop (reverse primer) of
each of the corresponding receptor cDNA were synthesized having the
following sequences:
3 rGALR1: (forward primer): 5'-CCTCAGTGAAGGGAATGGGAG- CGA-3'; (SEQ
ID NO:20) (reverse primer): 5'-GTAGTGTATAAACTTGCAGATGAAGGC-3'; (SEQ
ID NO:21) rGALR2: (forward primer): 5'-ATGAATGGCTCCGGCAGCCAGGG-3';
(SEQ ID NO:22) (reverse primer): 5'-TTGCAGAGCAGCGAGCCGAACAC-3'; and
(SEQ ID NO:23) rHY35a (i.e., rat GALR3): (forward primer)
5'-GGCTGACATCCAGAACATTTCGCT-3'; (SEQ ID NO:24) (reverse primer):
5'-CAGATGTACCGTCTTGCACACGAA-3'. (SEQ ID NO:25)
[0270] Polymerase Chain Reaction (PCR) of cDNA
[0271] Total RNA was prepared from RIN14B cells (ATCC No. CCL 89)
by a modification of the guanidine thiocyanate method (Chirgwin et
al., 1979). Poly A.sup.+ RNA was purified with a FastTrack kit
(Invitrogen Corp., San Diego, Calif.) and converted to
single-stranded cDNA by random priming using Superscript reverse
transcriptase (BRL, Gaithersburg, Md.). An aliquot of the first
strand cDNA was diluted (1:50) in a 50 .mu.L PCR reaction mixture
containing a combination of Taq and Pwo DNA polymerases in the
buffer supplied by the manufacturer (for the Expand Long Template
PCR System, Boehringer Mannheim), and 300 nM each of the amino
terminus and first extracellular loop rGALR3 (rHY35a) primers
described above. The PCR amplification reaction was performed under
the following conditions: 30 sec. at 94.degree. C. and 1 min. 30
sec. at 68.degree. C. for 40 cycles, with a pre- and
post-incubation of 5 min. at 95.degree. C. and 2 min. 30 sec. at
68.degree. C., respectively. In order to control for the
amplification of DNA (potentially carried over during the RNA
extraction), control PCR reactions were run in parallel using
RIN14B RNA prepared as above but without reverse transcriptase, and
thus not converted to cDNA. The PCR products were separated on a
1.0% agarose gel and stained with ethidium bromide.
[0272] Construction and PCR Screening of a RIN14B Cell Line Plasmid
Library
[0273] Total RNA was prepared from RIN14B cells by a modification
of the guanidine thiocyanate method (Chirgwin et al., 1979). Poly
A.sup.+ RNA was purified with a FastTrack kit (Invitrogen Corp.,
San Diego, Calif.). Double stranded (ds) cDNA was synthesized from
4 .mu.g of poly A.sup.+ RNA according to Gubler and Hoffman (1983)
with minor modifications. The resulting cDNA was ligated to
BstXI/EcoRI adaptors (Invitrogen Corp.) and the excess adaptors
removed by exclusion column chromatography. High molecular weight
fractions of size-selected ds-cDNA were ligated in pEXJ.BS (an
Okayama and Berg expression vector) and electroporated in E.coli MC
1061 (Gene Pulser, Biorad). A total of 0.9.times.10.sup.6
independent clones with an insert mean size of 3.4 kb were
generated. The library was plated on agar plates (Ampicillin
selection) in 216 pools of 4,000 independent clones. After 18 hours
amplification, the bacteria from each pool were scraped,
resuspended in 4 mL of LB media, and 1.5 mL processed for plasmid
purification (Qiaprep, Qiagen, Inc., Chatsworth, Calif.). Aliquots
of each bacterial pool were stored at -85.degree. C. in 20%
glycerol.
[0274] Glycerol stocks (2 .mu.L) of the 216 primary pools for the
RIN14B plasmid library (designated "F") were screened for rGALR3 by
PCR using a forward primer from the third transmembrane domain of
rGALR3 (5'-CATCTGCTCATCTACCTCACCATG-3' (SEQ ID NO: 26)) and a
reverse primer from third intracellular loop of rGALR3
(5'-CATAGGAAACATAGCGTGCGTCCG-3' (SEQ ID NO: 27)). PCR was performed
with the Expand Long Template PCR System, as described in the
preceding section. Two positive pools, F105 and F212, were
subjected to further PCR analyses, using a forward primer to the
amino terminus of rat GALR3 (described above) with a reverse primer
from the third intracellular loop (described above), as well as
vector-anchored PCR (see below). These PCR analyses indicated that,
although these clones were full-length, they were in the incorrect
orientation in the expression vector (pEXJ.BS). Although these
pools were not further subdivided, the sequence missing from clone
rHY35a (i.e., from the middle of TM7 through the stop codon) was
determined from the F105 clone, using vector-anchored PCR, as
described below.
[0275] Vector-Anchored PCR
[0276] To determine the orientation and size of the F105 cDNA
insert (including the coding region, 5' untranslated (UT) and 3' UT
regions) PCR was conducted on glycerol stocks (2 .mu.L) using
combinations of vector-derived primers and gene-specific primers.
The vector-derived forward primer sequence was
5'-AAGCTTCTAGAGATCCCTCGACCTC-3' (SEQ ID NO: 28); the reverse primer
sequence was 5'-AGGCGCAGAACTGGTAGGTATGGAA-3' (SEQ ID NO: 29). The
rGALR3-specific forward primer (in the sixth transmembrane domain)
was 5'-GCTCATCCTCTGCTTCTGGTACG-3' (SEQ ID NO: 30); the reverse
primer (in the first extracellular loop) was
5'-CAGATGTACCGTCTTGCACACGAA-3' (SEQ ID NO: 31). PCR was performed
with the Expand Long Template PCR System, as described above. The
PCR products were separated on a 1.0% agarose gel and stained with
ethidium bromide.
[0277] A 1.2 kb vector-anchored PCR product generated from pool
F105 using the sixth TM forward primer from rGALR3 and the
vector-derived reverse primer was isolated from a 1% TAE gel using
a GENECLEAN III kit (BIO 101, Vista, Calif.) and sequenced using
AmpliTaq DNA Polymerase, FS (Perkin Elmer). Sequencing reactions
were run on an ABI PRISM 377 DNA Sequencer and analyzed using the
Wisconsin Package (GCG, Genetics Computer Group, Madison, Wis.).
The sequence information from this vector-anchored PCR product
corresponding to the predicted 3' end of the novel receptor gene
indicated an overlap with rHY35a within the first half of TM7.
Downstream of this overlap was new sequence, consistent with the
second half of TM7 and the carboxy terminus, including an in-frame
stop codon. Based on this newly acquired sequence, a reverse
primer, within the 3'UT, was synthesized (also containing a BamHI
site at the 5' end, as indicated by the underline):
5'-CGAGGATCCCAACTTTGCCTCTGCTTTTTGGTGG-3' (SEQ ID NO: 32).
[0278] Construction and PCR Screening of a Rat Hypothalamus Plasmid
Library
[0279] Total RNA was prepared from rat hypothalami by a
modification of the guanidine thiocyanate method (Chirgwin, 1979).
Poly A.sup.+ RNA was purified using a FastTrack kit (Invitrogen
Corp., San Diego, Calif.). Double stranded (ds) cDNA was
synthesized from 6 .mu.g of poly A.sup.+ RNA according to Gubler
and Hoffman (1983) with minor modifications. The resulting cDNA was
ligated to BstXI/EcoRI adaptors (Invitrogen Corp.) and the excess
adaptors removed by exclusion column chromatography. High molecular
weight fractions of size-selected ds-cDNA were ligated in pEXJ.T7
(an Okayama and Berg expression vector modified from pcEXV (Miller
& Germain, 1986) to contain BstXI and other additional
restriction sites and a T7 promoter (Stratagene)) and
electroporated in E.coli MC 1061 (Gene Pulser, Biorad). A total of
1.2.times.10.sup.6 independent clones with a mean insert size of
3.2 kb were generated. The library (designated "K") was plated on
agar plates (Ampicillin selection) in 373 primary pools of
.about.3,200 independent clones. After 18 hours amplification, the
bacteria from each pool were scraped, resuspended in 4 mL of LB
media and 0.75 mL processed for plasmid purification (QIAwell-96
ultra, Qiagen, Inc., Chatsworth, Calif.). Aliquots of each
bacterial pool were stored at -85.degree. C. in 20% glycerol.
[0280] To screen the library for galanin binding, COS-7 cells were
plated in slide chambers (Lab-Tek) in Dulbecco's modified Eagle
medium (DMEM) supplemented with 10% calf serum, 100 U/mL of
penicillin, 100 ug/mL streptomycin, 2 mM L-glutamine (DMEM-C) and
grown at 37.degree. C. in a humidified 5% CO.sub.2atmosphere for 24
hours before transfection. Cells were transfected with miniprep DNA
prepared from the primary pools (.about.3,200 cfu/pool) of the rat
hypothalamus cDNA library ("K" library ) using a modification of
the DEAE-dextran method (Warden & Thorne, 1968). Pools
containing GALR1 and GALR2 were identified by PCR prior to
screening. The galanin binding assay was carried out after 48
hours. Cells were rinsed twice with phosphate-buffered saline (PBS)
then incubated with 2 nM .sup.125I-porcine galanin (NEN; specific
activity .about.2200 Ci/mmol) in 20 mM HEPES-NaOH, pH 7.4,
containing 1.26 mM CaCl.sub.2, 0.81 mM MgSO.sub.4, 0.44 mM
KH.sub.2PO.sub.4, 5.4 mM KCl, 10 mM NaCl, 0.1% BSA, and 0.1%
bacitracin for one hour at room temperature. After rinsing and
fixation in 2.5% glutaraldehyde, slides were rinsed in PBS,
air-dried, and dipped in photoemulsion (Kodak, NTB-2). After a 4
day exposure slides were developed in Kodak D19 developer, fixed,
and coverslipped (Aqua-Mount, Lerner Laboratories), then inspected
for positive cells by brightfield microscopy (Leitz Laborlux,
25.times. magnification).
[0281] PCR Screening of the Rat Hypothalamus cDNA Library
[0282] Glycerol stocks of the primary pools were combined into 40
superpools of 10 primary pools and screened for rGALR3 by PCR using
the same primers as described for the screening of the RIN14B
plasmid library (see above). Primary pools from positive superpools
(#3 and #17) were inspected for galanin binding using the
photoemulsion binding assay described above and screened by PCR.
The slide corresponding to pool K163 exhibited positive galanin
binding. Pool K163 was then subjected to PCR with internal rGALR3
primers (TM3 forward primer and third intracellular loop reverse
primer; described above), full-length primers (forward primer to
the amino terminus, at the starting MET, and reverse primer to the
3' UT (containing a Bam HI site as above)) and with the vector and
gene-specific primers (preceding section). These PCR analyses
indicated that the primary pool K163 contained a full-length coding
region for rGALR3 in the correct orientation in the expression
vector, pEXJ.T7. Pool K163 was further analyzed by PCR and shown to
contain GALR3 but not GALR1 nor GALR2, indicating that a novel
galanin receptor cDNA was present in the pool and responsible for
the galanin binding. The PCR primers used to confirm the absence of
GALR1 and GALR2 in the pool are described below:
4 rGALR1: Forward primer, KS-1311: 5'-CCTCAGTGAAGGGAATGGGAGCGA;
(SEQ ID NO:33) Reverse primer, KS-1447:
5'-CTTGCTTGTACGCCTTCCGGAAGT; (SEQ ID NO:34) Human GALR1: Forward
primer, KS-1177: 5'-TGGGCAACAGCCTAGTGATCACCG-3'; (SEQ ID NO:35)
Reverse primer, KS-1178: 5'-CTGCTCCCAGCAGAAGGTCTGGTT-3'; (SEQ ID
NO:36) rGALR2: Forward primer, KS-1543:
5'-ATGAATGGCTCCGGCAGCCAGGG-3'; (SEQ ID NO:37) Reverse primer,
KS-1499: 5'-TTGGAGACCAGAGCGTAAACGATGG-3'. (SEQ ID NO:38)
[0283] The primary pool K163 was further subdivided and screened by
PCR. One positive subpool, 163-30, was subdivided into 15 pools of
150 clones and 15 pools of 500 clones and plated on agar plates
(ampicillin selection).
[0284] Colonies were transferred to nitrocellulose membranes
(Schleicher and Schuell, Keene, N.H.), denatured in 0.4 N NaOH, 1.5
M NaCl, renatured in 1M Tris, 1.5 M NaCl, and UV cross-linked.
Filters were hybridized overnight at 40.degree. C. in a buffer
containing 50% formamide, 5.times.SSC, 7 mM TRIS, 1.times.
Denhardt's solution and 25 .mu.g/mL salmon sperm DNA (Sigma
Chemical Co.) and 10.sup.6 cpm/ml of overlapping 45-mer
oligonucleotide probes, filled-in using [.alpha.-.sup.32P]dCTP and
[.alpha.-.sup.32P]dATP (800 Ci/mmol, NEN) and Klenow fragment of
DNA polymerase (Boehringer Mannheim). The following probe sequence
is directed to the amino terminus of rGALR3:
5 from the sense strand: 5'-AGATGGCTGACATCCAGAACATTTCGCTGGA- CA
(SEQ ID NO:39) GCCCAGGGAGCG-3'; from the antisense strand:
5'-ATCACAGGCACTGCCACAGCCCCTACGCTCCCT (SEQ ID NO:40)
GGGCTGTCCAGCG-3'.
[0285] Filters were washed 2.times.15 minutes at room temperature
in 2.times.SSC, 0.1% SDS, 2.times.15 minutes at 50.degree. C. in
0.1.times. SSC, 0.1% SDS, and exposed to BioMax MS X-ray film
(Kodak) with corresponding Kodak intensifying screens for 6 hours.
One positive colony, 163-30-17, was amplified overnight separately
in 100 mL LB media and in 100 mL TB media and processed for plasmid
purification using a standard alkaline lysis miniprep procedure
followed by a PEG precipitation. Clone K163-30-17 was sequenced on
both strands using AmpliTaq DNA Polymerase, FS (Perkin Elmer).
Sequencing reactions were run on an ABI PRISM 377 DNA Sequencer and
analyzed using the Wisconsin Package (GCG, Genetics Computer Group,
Madison, Wis.). Clone K163-30-17 was given the designation K1086
and deposited with the ATCC (Accession No. 97747).
[0286] Expression in COS-7 Cells for Whole Cell-Slide Binding
[0287] To test the ability of K163-30-17 to confer galanin binding,
COS-7 cells were plated in slide chambers (Lab-Tek) in Dulbecco's
modified Eagle medium (DMEM) supplemented with 10% calf serum, 100
U/mL of penicillin, 100 .mu.g/mL streptomycin, 2 mM L-glutamine
(DMEM-c) and grown at 37.degree. C. in a humidified 5% CO.sub.2
atmosphere for 24 hours before transfection. Cells were transfected
with 1 .mu.g of miniprep DNA from K163-30-17 or vector control
using a modification of the DEAE-dextran method (Warden and Thorne,
1968). 48 hours after transfection, cells were rinsed with
phosphate-buffered saline (PBS) then incubated with 2 nM
.sup.125I-porcine galanin (NEN; specific activity .about.2200
Ci/mmol) in 20 mM HEPES-NaOH, pH 7.4, containing 1.26 mM
CaCl.sub.2, 0.81 mM MgSO.sub.4, 0.44 mM KH.sub.2PO.sub.4, 5.4 mM
KCl, 10 mM NaCl, 0.1% BSA, and 0.1% bacitracin for one hour at room
temperature. After rinsing and fixation in 2.5% glutaraldehyde,
binding of .sup.125I-galanin to cells on the slide was detected by
autoradiography using BioMax MS film (Kodak) and an intensifying
screen (Kodak). The signal from K163-30-17 transfected cells was
compared with the signal from control vector transfected cells.
[0288] Cloning and Sequencing a Novel Human Galanin Receptor
Fragment
[0289] A human placenta genomic library in .lambda. dash II
(.apprxeq.1.5.times.10.sup.6 total recombinants; Stratagene,
LaJolla, Calif.) was screened using the same set of overlapping
oligonucleotide probes to TM regions 1-7 of rat GALR2 and under the
same hybridization and wash conditions as described for screening
the rat hypothalamus cDNA library (supra). Lambda phage clones
hybridizing with the probe were plaque purified and DNA was
prepared for Southern blot analysis (Southern, 1975; Sambrook et
al., 1989).
[0290] One phage clone, plc21a, contained a 2.7 kb KpnI/EcoRI
fragment which hybridized with the rat GALR2 TM2 oligonucleotide
probe and was subsequently subcloned into a pUC vector. Nucleotide
sequence analysis was accomplished by sequencing both strands using
cycle sequencing with AmpliTaq DNA Polymerase, FS (Perkin Elmer)
and products run on the automated fluorescent sequencer, the ABI
Prism 377 Sequencer (ABI), and sequence analyses were performed
using the Wisconsin Package (GCG, Genetics Computer Group, Madison,
Wis.). DNA sequence analysis indicated greatest homology to the rat
and human GALR1 and GALR2 genes. This clone was a partial
intron-containing gene fragment, encoding the starting MET through
to an intron in the second intracellular loop (i.e., TM 3/4
loop).
[0291] Isolation of the Full-Length Human GALR3 Receptor Gene
[0292] Sequence analyses of the cloned human genomic fragment
indicated the presence of a open reading frame from the starting
MET codon down to a predicted intron in the second intracellular
loop, with a nucleotide identity of 88% (93% amino acid identity)
with the rat GALR3 receptor described above (thus establishing this
human genomic clone to be the human homologue of rat GALR3).
Although this human genomic fragment was not full-length and
contained an intron downstream of TM3, it is anticipated that a
molecular biologist skilled in the art may isolate the full-length,
intronless version of the human GALR3 receptor gene using standard
molecular biology techniques and approaches such as those briefly
described below:
[0293] Approach #1: Using PCR to screen commercial human cDNA phage
libraries and in-house human cDNA plasmid libraries with primers to
the human GALR3 sequence (forward primer in amino terminus,
5'-ATGGCTGATGCCCAGAACATTTCAC-3' (SEQ ID NO: 41), and reverse primer
in first extracellular loop, 5'-AGCCAGGCATCCAGCGTGTAGAT-3' (SEQ ID
NO: 42), we have identified two commercial libraries and two
proprietary plasmid libraries that contain at least part of the
human GALR3 gene, as follows:
[0294] human fetal brain cDNA lambda ZAPII library
(Stratagene);
[0295] human testis cDNA lambda ZAPII library (Stratagene);
[0296] human hypothalamus cDNA plasmid library (proprietary)--3
superpools identified; and
[0297] human hippocampus cDNA plasmid library (proprietary)--3
superpools identified.
[0298] One may determine whether these libraries contain
full-length human GALR3 by: (1) obtaining a purified clone from the
lambda libraries by plaque-purification and then conducting
hybridization screening using probes derived from rat GALR3 under
reduced stringency, using standard protocols and/or (2) using PCR
to determine which pool of the human plasmid library superpools
contain the gene and then conducting vector-anchor PCR (as
described in this patent) to determine if these cDNAs are
full-length. One problem which may arise with vector-anchored PCR
is a false-positive result, in which the PCR product size is
consistent with a full-length clone but the product actually
contains an intron in the second intracellular loop. In this case,
sequencing of this product would identify whether this product
contains the intron or is intronless and full-length (also see
Approach #2 below).
[0299] Approach #2: We have also determined that the phage clone
containing MET thru the intron in the second intracellular loop
(i.e., TM3/4 loop), plc21a (see above), also contains at least part
of the 3' end of the gene, by using hybridization at reduced
stringency with a probe to the third extracellular loop (TM
{fraction (6/7)}) derived from the rat GALR3 sequence:
[0300] 5'-ACGGTCGCTTCGCCTTCAGCCCGGCCACCTACGCCTGTCGCCTGG-3' (SEQ ID
NO: 43).
[0301] Standard molecular biology techniques may be used to
subclone either the entire intron-containing full-length human
GALR3 (with confirmation that it contains an in-frame stop codon)
or subclone the part of the gene from the intron in the second
intracellular loop through the stop codon. This approach would
permit one to utilize sequence around the termination codon to
design a primer which can be used with the primer around the
starting MET, to generate the full-length intronless human GALR3
gene, using human cDNA as the target template. Alternatively, one
may use restriction enzymes to remove the intron and some adjacent
coding region from the intron-containing human GALR3 gene, and then
replace the removed coding region by inserting a restriction
enzyme-digested PCR fragment amplified from a tissue shown to
express the intronless form of the receptor.
[0302] Approach #3: As yet another alternative method, one could
utilize 3' RACE to generate a PCR product from human cDNA
expressing human GALR3 (e.g., human brain), using a forward primer
derived from known sequence between the starting MET thru the
second intracellular loop (from the fragment already isolated).
Such a PCR product could then be sequenced to confirm that it
contains the rest of the coding region (without an intron), and
then attached to the 5' end of the molecule, using an overlapping
restriction site, or alternatively, its sequence could be used to
design a reverse primer in the predicted 3' UT region to generate
the full-length, intronless human GALR3 receptor gene with use of
the primer at the starting MET codon and using human cDNA as target
template.
[0303] To this end, we have also determined that the phage clone
containing MET through the intron in the second intracellular loop
(i.e. TM 3/4 loop), plc21a (see above), also contains at least part
of the 3' end of the gene, by using hybridization at reduced
stringency with probes either to the third extracellular loop (TM
{fraction (6/7)}) or to TM 4, derived from the rat GALR3
sequence:
[0304] 5'-ACGGTCGCTTCGCCTTCAGCCCGGCCACCTACGCCTGTCGCCTGG-3' (SEQ ID
NO: 44)
[0305] 5'-GCGCAACGCGCGCGCCGCCGTGGGGCTCGTGTGGCTGCTGGCGGC-3' (SEQ ID
NO: 45).
[0306] Another clone, plc14a, which was essentially the same as
plc21a (i.e. possessed the identical restriction map and
hybridizing bands as plc21a), was further utilized by subcloning a
1.4 kb KpnI fragment which similarly hybridized to the above
probes. Since the phage clone, plc14a, also hybridized with a TM2/3
loop probe under high stringency, derived from sequence data of
human GALR3 5' fragment (plc21a, see above),
[0307] 5-ATCTACACGCTGGATGCCTGGCTCTTTGGGGCCCTCGTCTGCAAG-3' (SEQ ID
NO: 46),
[0308] this 3' fragment (e.g. plc14a) presumably corresponds to the
3' end of human GALR3 and is molecularly linked to the 5' fragment
(e.g. plc21a 2.7 kb KpnI/EcoRI clone); however, an intron of
unknown size separates the coding region, which is defined on the
5' (2.7 kb KpnI/EcoRI plc21a fragment) and 3' (1.4 kb KpnIplc14a
fragment) genomic pieces. Nucleotide sequence analysis was
conducted on the 1.4 kb KpnI plc14a fragment, as described above,
and indicated greatest homology to the rat and human GALR1 and
GALR2 genes.
[0309] To obtain sequence information from the region defined by
the intersection of these to exons as well as to prove that the 5'
and 3' fragments, putatively representing the entire full-length
coding region of human GALR3, are molecularly linked, we used a
forward oligonucleotide primer located on the 5' fragment (within
2/3 loop)
[0310] 5'-ATCTACACGCTGGATGCCCTGGCT-3' (SEQ ID NO: 47) and a reverse
oligonucleotide primer located on the 3' fragment (within the
predicted 4/5 loop),
[0311] 5'-CGTAGCGCACGGTGCCGTAGTA-3' (SEQ ID NO: 48),
[0312] to amplify human brain and liver cDNA (corresponding to 5 ng
of poly.sup.+ RNA). The predicted=250 nts. PCR products were
sequenced and demonstrated that: (1) the sequences were identical
between brain and liver cDNA, (2) the 5' and 3' genomic fragments
are linked and represent the 5' and 3' fragments of the human GALR3
gene, and (3) the sequence obtained defined the junction of the
exon containing the starting MET through the 3/4 loop (e.g., housed
on the 2.7 kb KpnI/EcoRI plc21a subclone) and the exon containing
the 3/4 loop through the predicted STOP codon (e.g. housed on the
1.4 kb KpnI plc14a subclone). The sequence of this junction
demonstrated the presence of a KpnI site, which was utilized in the
construction of the full-length gene.
[0313] The construction of the full-length human GALR3 gene first
involved the generation of the 5' end of the gene using PCR to
synthetically create a KpnI site at the 3' end of the PCR product.
To this end, we designed a forward oligonucleotide primer located
at the starting MET of the 5' fragment and added a consensus Kozak
sequence as well as a BamHI site to be used for subcloning:
[0314] 5'-GATGGATCCGCCACCATGGCTGATGCCCAGAACATTTCAC-3' (SEQ ID NO:
49),
[0315] and a reverse oligonucleotide primer, within the 3/4 loop,
containing a KpnI site that generated the joint between the 5' and
3' KpnI fragment:
[0316] 5'-GCAGGTACCTGTCCACGGAGACAGCAGC-3' (SEQ ID NO: 50).
[0317] The addition of the KpnI site enabled the attachment of the
3' KpnI fragment but preserved the sequence which was identified
from human brain and liver cDNAs.
[0318] The forward and reverse primers were used to amplify the 2.7
kb KpnI/EcoRI5' genomic-containing plasmid (plc21a) using PCR, as
described in a previous section but utilizing Expand High Fidelity
PCR System (Boehringer Manniheim). The PCR product was isolated
from a low melting gel, purified by phenol extraction, digested
with BamHI and KpnI and purified further by phenol extraction. This
BamHI/KpnI PCR product was subcloned into BamHI/KpnI-digested
expression vector, PEXJ, and sequenced. The sequence of the PCR
product was identical to that determined for the original genomic
fragment. The subclone was then digested with KpnI, treated with
calf intestinal alkaline phosphatase, and ligated with the 1.4 KpnI
3' genomic fragment. Correct orientation was determined by both
restriction mapping and sequencing. Therefore, the full-length
human GALR3 construct contained=1.7 kb genomic insert, containing
1107 bp of coding region and =600 bp of 3' non-coding region.
[0319] Northern Blots
[0320] Rat multiple tissue northern blots (rat MTN blot, Clontech,
Palo Alto, Calif.), containing 2 .mu.g poly A.sup.+ RNA, or
northern blots containing 5 .mu.g poly.sup.+ RNA, either purchased
from Clontech or purified from various rat peripheral tissues and
brain regions, respectively, were similarly hybridized at high
stringency with a probe directed to the amino-terminus of rGalR3
(SEQ ID NO 39 and 40), according to the manufacturer's
specifications. Probe was labeled as previously described (supra),
using Klenow fragment of DNA polymerase, except
[.alpha.-.sup.32P]dCTP and [.alpha.-.sup.32P]dATP (3000 Ci/mmol,
NEN) were used. Northern blots were reprobed with a randomly-primed
.beta.-actin probe to assess quantities of mRNA present in each
lane.
[0321] Human brain multiple tissue northern blots (MTN brain blots
II and III, Clontech, Palo Alto, Calif.) and human peripheral MTN
blot (Clontech, Palo Alto, Calif.) carrying mRNA (2 .mu.g) purified
from various human brain areas and peripheral tissues,
respectively, were hybridized at high stringency with overlapping
probes directed to the amino-terminus of hGALR3
[0322] 5' GATGGCTGATGCCCAGAACATTTCACTGGACAGCCCAGGGAGTGT 3' (SEQ ID
NO. 51) and
[0323] 5' GACCACAGGCACTGCCACGGCCCCCACACTCCCTGGGCTGTCCAG 3' (SEQ ID
NO. 52), according to the manufacturer's specifications.
[0324] RT-PCR Analyses of GALR3 mRNA
[0325] Tissues were homogenized and total RNA extracted using the
guanidine isothiocyanate/CsCl cushion method. RNA was then treated
with DNase to remove any contaminating genomic DNA and poly
A.sup.+-selected using FastTrack kit (Invitrogen), according to
manufacturer's specifications. cDNA was prepared from mRNA with
random hexanucleotide primers using reverse transcriptase
Superscript II (BRL, Gaithersburg, Md.). First strand cDNA
(corresponding to .apprxeq.5 ng of poly A.sup.+ RNA) was amplified
in a 50 .mu.L PCR reaction mixture with 300 nM of forward (directed
to the amino-terminus: SEQ ID NO. 24) and reverse (directed to the
third intracellular loop: SEQ ID NO. 27) primers, using the thermal
cycling program and conditions described above.
[0326] The PCR products were run on a 1.5% agarose gel and
transferred to charged nylon membranes (Zetaprobe GT, BioRad), and
analyzed as Southern blots. GALR3 primers were screened for the
absence of cross-reactivity with the other galanin receptors.
Filters were hybridized with a radiolabeled probe directed to the
first intracellular loop,
5'-TGCAGCCTGGCCCAAGTGCCTGGCAGGAGCCAAGCAGTACCACAG-3' (Seq. I.D. No.
53), and washed under high stringency. Labeled PCR products were
visualized on X-ray film. Similar PCR and Southern blot analyses
were conducted with primers and probes directed to the housekeeping
gene, glyceraldehyde phosphate dehydrogenase (G3PDH; Clontech, Palo
Alto, Calif.), to normalize the amount of cDNA used from the
different tissues.
[0327] RT-PCR was performed on human pituitary cDNA (two sources:
Clontech cDNA and cDNA prepared from poly A+RNA purchased from ABS)
using the following conditions: 94.degree. C. for 30 sec and
68.degree. C. for 2 min, for 40 cycles, with a preincubation at
94.degree. C. for 2 min and a postincubation at 68.degree. C. for 5
minutes. Primers specific for human GALR1 were used (KS1177; SEQ ID
NO. 35 and KS1178; SEQ ID NO. 36). Primers specific for human GALR2
were used (BB183; SEQ ID NO. 60 and BB184; SEQ ID NO. 61). Primers
specific for human GALR3 were used (BB444; SEQ ID NO. 62 and BB445;
SEQ ID NO. 63). Primers specific for human prolactin were used
(BB446; SEQ ID NO. 64 and BB447; SEQ ID NO. 65).
6 Primers used: BB183: 5'-TCAGCGGCACCATGAACGTCTCGGGC- T-3' (SEQ ID
NO.60) BB184: 5'-GGCCACATCAACCGTCAGGAT- GCT-3' (SEQ ID NO.61)
BB444: 5'-ATGGCTGATGCCCAGAACATTTCAC-3' (SEQ ID NO.62) BB445:
5'-TAGCGCACGGTGCCGTAGTAGCTGAGGT-3' (SEQ ID NO.63) BB446:
5'-ATGAAAGGGTCCCTCCTGCTGCTGCT-3' (SEQ ID NO.64) BB447:
5'-TATCAGCTCCATGCCCTCTAGAAGCC-3' (SEQ ID NO.65)
[0328] Production of Recombinant Baculovirus
[0329] The coding region of GALR3 may be subcloned into pBlueBacIII
into existing restriction sites, or sites engineered into sequences
5' and 3' to the coding region of GALR3, for example, a 5' EcoRI
site and a 3' EcoRI site. To generate baculovirus, 0.5 .mu.g of
viral DNA (BaculoGold) and 3 .mu.g of GALR3 construct may be
co-transfected into 2.times.10.sup.6 Spodoptera frugiperda insect
Sf9 cells by the calcium phosphate co-precipitation method, as
outlined in by Pharmingen (in "Baculovirus Expression Vector
System: Procedures and Methods Manual"). The cells then are
incubated for 5 days at 27.degree. C.
[0330] The supernatant of the co-transfection plate may be
collected by centrifugation and the recombinant virus plaque
purified. The procedure to infect cells with virus, to prepare
stocks of virus and to titer the virus stocks are as described in
Pharmingen's manual.
[0331] Cell Culture
[0332] COS-7 cells are grown on 150 mm plates in DMEM with
supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf
serum, 4 mM glutamine, 100 units/mL penicillin/100 .mu.g/mL
streptomycin) at 37.degree. C., 5% CO.sub.2. Stock plates of COS-7
cells are trypsinized and split 1:6 every 3-4 days. Human embryonic
kidney 293 cells are grown on 150 mm plates in D-MEM with
supplements (minimal essential medium) with Hanks' salts and
supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf
serum, 4 mM glutamine, 100 units/mL penicillin/100 .mu.g/mL
streptomycin) at 37.degree. C., 5% CO.sub.2. Stock plates of 293
cells are trypsinized and split 1:6 every 3-4 days. Mouse
fibroblast LM(tk-) cells are grown on 150 mm plates in D-MEM with
supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf
serum, 4 mM glutamine, 100 units/mL penicillin/100 .mu.g/mL
streptomycin) at 37.degree. C., 5% CO.sub.2. Stock plates of
LM(tk-) cells are trypsinized and split 1:10 every 3-4 days.
[0333] LM(tk-) cells stably transfected with the GALR3 receptor may
be routinely converted from an adherent monolayer to a viable
suspension. Adherent cells are harvested with trypsin at the point
of confluence, resuspended in a minimal volume of complete DMEM for
a cell count, and further diluted to a concentration of 10.sup.6
cells/mL in suspension media (10% bovine calf serum, 10% 10.times.
Medium 199 (Gibco), 9 mM NaHCO.sub.3, 25 mM glucose, 2 mM
L-glutamine, 100 units/mL penicillin/100 .mu.g/mL streptomycin, and
0.05% methyl cellulose). Cell suspensions are maintained in a
shaking incubator at 37.degree. C., 5% CO.sub.2 for 24 hours.
Membranes harvested from cells grown in this manner may be stored
as large, uniform batches in liquid nitrogen. Alternatively, cells
may be returned to adherent cell culture in complete DMEM by
distribution into 96-well microtiter plates coated with
poly-D-lysine (0.01 mg/mL) followed by incubation at 37.degree. C.,
5% CO.sub.2 for 24 hours. Cells prepared in this manner generally
yield a robust and reliable response in cAMP radio-immunoassays as
further described hereinbelow.
[0334] Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm
plates in Dulbecco's Modified Eagle Medium (DMEM) with supplements
(10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100
.mu.g/mL streptomycin) at 37.degree. C., 5% CO2. Stock plates of
NIH-3T3 cells are trypsinized and split 1:15 every 3-4 days.
Chinese hamster ovary (CHO) cells were grown on 150 mm plates in
HAM's F-12 medium with supplements (10% bovine calf serum, 4 mM
L-glutamine and 100 units/mL penicillin/100 .mu.g/ml streptomycin)
at 37.degree. C., 5% CO2. Stock plates of CHO cells were
trypsinized and split 1:8 every 3-4 days.
[0335] Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue
culture dishes in TMN-FH media supplemented with 10% fetal calf
serum, at 27.degree. C., no CO2. High Five insect cells are grown
on 150 mm tissue culture dishes in Ex-Cell 400.TM. medium
supplemented with L-Glutamine, also at 27.degree. C., no
CO.sub.2.
[0336] Transfection
[0337] All receptor subtypes studied may be transiently transfected
into COS-7 cells by the DEAE-dextran method, using 1 .mu.g of
DNA/10.sup.6 cells (Warden, D., et al., 1968). In addition,
Schneider 2 Drosophila cells may be cotransfected with vectors
containing the receptor gene, under control of a promoter which is
active in insect cells, and a selectable resistance gene, eg., the
G418 resistant neomycin gene, for expression of the galanin
receptor.
[0338] Stable Transfection
[0339] The GALR3 receptor may be co-transfected with a G-418
resistant gene into the human embryonic kidney 293 cell line by a
calcium phosphate transfection method (Cullen, 1987). Stably
transfected cells are selected with G-418. GALR3 receptors may be
similarly transfected into mouse fibroblast LM(tk-) cells, Chinese
hamster ovary (CHO) cells and NIH-3T3 cells, or other suitable host
cells. GALR1 receptors were expressed in cells using methods
well-known in the art.
[0340] Radioligand Binding Assays
[0341] Transfected cells from culture flasks are scraped into of 20
mM Tris-HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. The cell
lysates are centrifuged at 1000 rpm for 5 min. at 4.degree. C., and
the supernatant centrifuged at 30,000.times.g for 20 min. at
4.degree. C. The pellet is suspended in binding buffer (50 mM
Tris-HCl, 5 mM MgSO.sub.4, 1 mM EDTA at pH 7.5 supplemented with
0.1% BSA, 2 .mu.g/ml aprotinin, 0.5 mg/ml leupeptin, and 10
.mu.g/ml phosphoramidon). Optimal membrane suspension dilutions,
defined as the protein concentration required to bind less than 10%
of the added radioligand, are added to 96-well polpropylene
microtiter plates containing .sup.125I-labeled peptide, non-labeled
peptides and binding buffer to a final volume of 250 .mu.l. In
equilibrium saturation binding assays membrane preparations may be
incubated in the presence of increasing concentrations (e.g., 0.1
nM to 4 nM) of [.sup.125I] porcine galanin (specific activity about
2200 Ci/mmol). The binding affinities of the different galanin
analogs may be determined in equilibrium competition binding
assays, using 0.1-0.5 nM [.sup.125I] porcine galanin in the
presence of e.g., twelve different concentrations of the displacing
ligands. Binding reaction mixtures are incubated for 1 hr at
30.degree. C., and the reaction stopped by filtration through GF/B
filters treated with 0.5% polyethyleneimine, using a cell
harvester. Radioactivity may be measured by scintillation counting
and the data analyzed by a computerized non-linear regression
program. Non-specific binding may be defined as the amount of
radioactivity remaining after incubation of membrane protein in the
presence of 100 nM of unlabeled porcine galanin. Protein
concentration may be measured by the Bradford method using Bio-Rad
Reagent, with bovine serum albumin as a standard. Such competitive
binding assays are well-known in the art, and may also include the
use of non-hydrolyzable analogues of GTP, which may reduce the
binding of agonists to the GALR3 receptors of the present
invention.
[0342] The binding assays used to generate the data shown in Table
4 were conducted as described above, with certain modifications.
Assays were conducted at room temperature for 120 minutes, and
leupeptin, aprotonin and phosphoramidon were omitted from the rat
GALR3 assay, while bacitracin was added to 0.1%. In addition,
nonspecific binding was defined in the presence of 1 .mu.M porcine
galanin.
[0343] Functional Assays
[0344] Cyclic AMP (cAMP) Formation
[0345] The receptor-mediated inhibition of cyclic AMP (cAMP)
formation may be assayed in LM(tk-) cells expressing the galanin
receptors. Cells are plated in 96-well plates and incubated in
Dulbecco's phosphate buffered saline (PBS) supplemented with 10 mM
HEPES, 5 mM theophylline, 2 .mu.g/ml aprotinin, 0.5 mg/ml
leupeptin, and 10 .mu.g/ml phosphoramidon for 20 min at 37.degree.
C., in 5% CO.sub.2. Galanin or the test compounds are added and
incubated for an additional 10 min at 37.degree. C. The medium is
then aspirated and the reaction stopped by the addition of 100 mM
HCl The plates are stored at 4.degree. C. for 15 min, and the cAMP
content in the stopping solution measured by radioimmunoassay.
Radioactivity may be quantified using a gamma counter equipped with
data reduction software.
[0346] Arachidonic Acid Release
[0347] CHO cells stably transfected with the rat GALR3 receptor are
seeded into 96 well plates and grown for 3 days in HAM's F-12 with
supplements. .sup.3H-arachidonic acid (specific activity -0.75
uCi/ml) is delivered as a 100 uL aliquot to each well and samples
were incubated at 37.degree. C., 5% CO.sub.2 for 18 hours. The
labeled cells are washed three times with 200 uL HAM's F-12. The
wells are then filled with medium (200 uL) and the assay is
initiated with the addition of peptides or buffer (22 uL). Cells
are incubated for 30 min at 37.degree. C., 5% CO2. Supernatants are
transferred to a microtiter plate and evaporated to dryness at
75.degree. C. in a vacuum oven. Samples are then dissolved and
resuspended in 25 uL distilled water. Scintillant (300 uL) is added
to each well and samples are counted for .sup.3H in a Trilux plate
reader. Data are analyzed using nonlinear regression and
statistical techniques available in the GraphPAD Prism package (San
Diego, Calif.).
[0348] Intracellular Calcium Mobilization
[0349] The intracellular free calcium concentration may be measured
by microspectroflourometry using the fluorescent indicator dye
Fura-2/AM (Bush et al. 1991). Stably transfected cells are seeded
onto a 35 mm culture dish containing a glass coverslip insert.
Cells are washed with HBS and loaded with 100 .mu.L of Fura-2/AM
(10 .mu.M) for 20 to 40 min. After washing with HBS to remove the
Fura-2/AM solution, cells are equilibrated in HBS for 10 to 20 min.
Cells are then visualized under the 40.times. objective of a Leitz
Fluovert FS microscope and fluorescence emission is determined at
510 nM with excitation wavelengths alternating between 340 nM and
380 nM. Raw fluorescence data are converted to calcium
concentrations using standard calcium concentration curves and
software analysis techniques.
[0350] Phosphoinositide Metabolism
[0351] LM(tk-) cells stably expressing the rat GALR3 receptor cDNA
are plated in 96-well plates and grown to confluence. The day
before the assay the growth medium is changed to 100 .mu.l of
medium containing 1% serum and 0.5 .mu.Ci [.sup.3H]myo-inositol,
and the plates are incubated overnight in a CO.sub.2 incubator (5%
CO.sub.2 at 37.degree. C.). Alternatively, arachidonic acid release
may be measured if [.sup.3H] aracliidonic acid is substituted for
the .sup.3[ H] myo-insoditol. Immediately before the assay, the
medium is removed and replaced by 200 .mu.L of PBS containing 10 mM
LiCl, and the cells are equilibrated with the new medium for 20
min. During this interval cells are also equilibrated with the
antagonist, added as a 10 .mu.L aliquot of a 20-fold concentrated
solution in PBS. The [.sup.3H]inositol-phosphates accumulation from
inositol phospholipid metabolism may be started by adding 10 .mu.L
of a solution containing the agonist. To the first well 10 .mu.L
may be added to measure basal accumulation, and 11 different
concentrations of agonist are assayed in the following 11 wells of
each plate row. All assays are performed in duplicate by repeating
the same additions in two consecutive plate rows. The plates are
incubated in a CO.sub.2 incubator for 1 hr. The reaction may be
terminated by adding 15 .mu.l of 50% v/v trichloroacetic acid (TCA)
followed by a 40 min. incubation at 4.degree. C. After neutralizing
TCA with 40 .mu.l of 1M Tris, the content of the wells may be
transferred to a Multiscreen HV filter plate (Millipore) containing
Dowex AG1-X8 (200-400 mesh, formate form). The filter plates are
prepared adding 200 .mu.L of Dowex AG1-X8 suspension (50% v/v,
water: resin) to each well. The filter plates are placed on a
vacuum manifold to wash or elute the resin bed. Each well is washed
2 times with 200 .mu.L of water, followed by 2.times.200 .mu.L of 5
mM sodium tetraborate/60 mM ammonium formate. The [.sup.3H]IPs are
eluted into empty 96-well plates with 200 .mu.l of 1.2 M ammonium
formate/0.1 formic acid. The content of the wells is added to 3 mL
of scintillation cocktail, and the radioactivity is determined by
liquid scintillation counting.
[0352] GTPVS Functional Assay
[0353] Membranes from cells transfected with the GALR3 receptors
are suspended in assay buffer (50 mM Tris, 100 mM NaCl, 5 mM
MgCl.sub.2, pH 7.4) supplemented with 0.1% BSA, 0.1% bacitracin and
10 .mu.M GDP. Membranes are incubated on ice for 20 minutes,
transferred to a 96-well Millipore microtiter GF/C filter plate and
mixed with GTP.gamma..sup.35S (e.g., 250,000 cpm/sample, specific
activity .about.1000 Ci/mmol) plus or minus GTP.gamma.S (final
concentration=100 .mu.M). Final membrane protein
concentration.apprxeq.90 .mu.g/mL. Samples are incubated in the
presence or absence of porcine galanin (final concentration=1
.mu.M) for 30 min. at room temperature, then filtered on a
Millipore vacuum manifold and washed three times with cold assay
buffer. Samples collected in the filter plate are treated with
scintillant and counted for .sup.35S in a Trilux (Wallac) liquid
scintillation counter. It is expected that optimal results are
obtained when the GALR3 receptor membrane preparation is derived
from an appropriately engineered heterologous expression system,
i.e., an expression system resulting in high levels of expression
of the GALR3 receptor and/or expressing G-proteins having high
turnover rates (for the exchange of GDP for GTP) GTP.mu.s assays
are well-known in the art, and it is expected that variations on
the method described above, such as are described by e.g., Tian et
al. (1994) or Lazareno and Birdsall (1993), may be used by one of
ordinary skill in the art.
[0354] The binding and functional assays described herein may also
be performed using GALR1 and GALR2 receptors. The GALR1 receptors
are well-known in the art and may be prepared and transfected into
cells (transiently and stably) using standard methods. Applicants
have isolated and cloned the rat and human GALR2 receptors, and
have deposited several plasmids expressing GALR2 receptors, as well
as cell lines stably expressing the rat GALR2 receptor. Plasmids
expressing GALR2 receptors may be transiently or stably transfected
into cell using methods well-known in the art, examples of which
are provided herein. The rat GALR2 receptor may be expressed using
plasmid K985 (ATCC Accession No. 97426, deposited Jan. 24, 1996),
or using plasmid K1045 (ATCC Accession No. 97778, deposited Oct.
30, 1996). Plasmid K1045 comprises an intronless construct encoding
the rat GALR2 receptor. Cell lines stably expressing the rat GALR2
receptor have also been prepared, for example, the LM(tk-) cell
lines L-rGALR2-8 (ATCC Accession No. CRL-12074, deposited Mar. 28,
1996) and L-rGALR2I-4 (ATCC Accession No. CRL-12223, deposited Oct.
30, 1996). L-rGALR2I-4 comprises an intronless construct expressing
the rat GALR2 receptor. The CHO cell line C-rGalR2-79 (ATCC
Accession No. CRL-12262, deposited Jan. 15, 1997) also stably
expresses the rat GALR2 receptor. The human GALR2 receptor may be
expressed using plasmid BO29 (ATCC Accession No. 97735, deposited
Sep. 25, 1996) or plasmid BO39 (ATCC Accession No. 97851, deposited
Jan. 15, 1997). Plasmid BO39 comprises an intronless construct
encoding the human GALR2 receptor.
[0355] The plasmids and cell lines described above were deposited
with the American Type Culture Collection (ATCC), 12301 Parklawn
Drive, Rockville, Md. 20852, U.S.A. under the provisions of the
Budapest Treaty for the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure.
[0356] It is to be understood that the cell lines described herein
are merely illustrative of the methods used to evaluate the binding
and function of the galanin receptors of the present invention, and
that other suitable cells may be used in the assays described
herein.
[0357] Methods for Recording Currents in Xenopus oocytes
[0358] Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) are
anesthetized in 0.2% tricain (3-aminobenzoic acid ethyl ester,
Sigma Chemical Corp.) and a portion of ovary is removed using
aseptic technique (Quick and Lester, 1994).
[0359] Oocytes are defolliculated using 2 mg/ml collagenase
(Worthington Biochemical Corp., Freehold, N.J.) in a solution
containing 87.5 mM NaCl, 2 mM KCl, 2 mM MgCl.sub.2 and 5 mM HEPES,
pH 7.5. Oocytes are injected (Nanoject, Drummond Scientific,
Broomall, Pa.) with 50 nL of rat GalR3 mRNA. Other oocytes are
injected with a mixture of GalR3 mRNA and mRNA encoding the genes
for G-protein-activated inward rectifiers (GIRK1 and GIRK4). Genes
encoding GIRK1 and GIRK4 are obtained using conventional PCR-based
cloning techniques based on published sequences (Kubo et al., 1993;
Dascal et al., 1993; Krapivinsky et al., 1995). RNAs are prepared
from separate DNA plasmids containing the complete coding regions
of GalR3, GIRK1 and GIRK4. Plasmids are linearized and transcribed
using the T7 polymerase ("Message Machine", Ambion) Alternatively,
mRNA may be translated from a template generated by PCR,
incorporating a T7 promoter and a poly A.sup.+ tail. After
injection of mRNA, oocytes are incubated at 160 on a rotating
platform for 3-8 days. Dual electrode voltage clamp ("GeneClamp",
Axon Instruments Inc., Foster City, Calif.) is performed using 3 M
KCl-filled glass microelectrodes having resistances of 1-3 Mohms.
Unless otherwise specified, oocytes are voltage clamped at a
holding potential of -80 mV. During recordings, oocytes are bathed
in continuously flowing (2-5 ml/min) medium containing 96 mM NaCl,
2 mM KCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, and 5 mM HEPES, pH 7.5
("ND96"), or, in the case of oocytes expressing GIRK1 and GIRK4,
elevated K.sup.+containing 96 mM KCl, 2 mM NaCl, 2 mM CaCl.sub.2, 2
mM MgCl.sub.2, and 5 mM HEPES, pH 7.5 ("hK"). Drugs are applied by
switching from a series of gravity fed perfusion lines.
[0360] Heterologous expression of GPCRs in Xenopus oocytes has been
widely used to determine the identity of signaling pathways
activated by agonist stimulation (Gundersen et al., 1983; Takahashi
et al., 1987). Activation of the phospholipase C (PLC) pathway is
assayed by applying 1 .mu.M galanin in ND96 solution to oocytes
previously injected with mRNA for the GalR3 receptor and observing
inward currents at a holding potential of -80 mV. The appearance of
currents that reverse at -25 mV and display other properties of the
Ca.sup.++-activated Cl- channel is indicative of GalR3
receptor-activation of PLC and release of IP3 and intracellular
Ca.sup.++. Subsequently, measurement of inwardly rectifying K.sup.+
channel (GIRK) activity is monitored in oocytes that have been
co-injected with mRNAs encoding GALR3, GIRK1 and GIRK4. These two
GIRK gene products co-assemble to form a G-protein activated
potassium channel known to be activated (i.e., stimulated) by a
number of GPCRs that couple to G.alpha..sub.i or G.alpha..sub.o
(Kubo et al., 1993; Dascal et al., 1993). Oocytes expressing GalR3
plus the two GIRK subunits are tested for galanin responsivity
using 1 .mu.M galanin and measuring K.sup.+ currents in elevated
K.sup.+ solution (hK). Activation of inwardly rectifying currents
that are sensitive to 300 .mu.M Ba.sup.++ signifies GALR3 coupling
to a G.alpha..sub.i or G.alpha..sub.o pathway in the oocytes.
[0361] Oocytes were isolated as described above, except that 3
mg/mL collagenase was used to defolliculate the oocytes. Genes
encoding G-protein inwardly rectifying K.sup.+ channels 1 and 4
(GIRK1 and GIRK4) were obtained by PCR using the published
sequences (Kubo et al., 1993; Dascal et al., 1993; Krapivinsky et
al., 1995b) to derive appropriate 5' and 3' primers. Human heart
cDNA was used as template together with the primers
[0362] 5'-CGCGGATCCATTATGTCTGCACTCCGAAGGAAATTTG-3' (SEQ ID NO. 54)
and
[0363] 5'-CGCGAATTCTTATGTGAAGCGATCAGAGTTCATTTTTC -3' (SEQ ID NO.
55) for GIRK1 and
[0364] 5'-GCGGGATCCGCTATGGCTGGTGATTCTAGGAATG-3' (SEQ ID NO. 56)
and
[0365] 5'-CCGGAATTCCCCTCACACCGAGCCCCTGG-3' (SEQ ID NO. 57) for
GIRK4. In each primer pair, the upstream primer contained a BamHI
site and the downstream primer contained an EcoRI site to
facilitate cloning of the PCR product into pcDNA1-Amp (Invitrogen).
The transcription template for hGalR3 was obtained similarly by PCR
using the cloned cDNA in combination with primers
[0366] 5'-CCAAGCTTCTAATACGACTCACTATAGGGCCACCATGGCTGATGCCCAGA-3'
(SEQ ID NO. 58) and
[0367] 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAGGG
TTTATTCCGGTCCTCG-3' (SEQ ID NO. 59). Alternatively, the complete
coding region of hGalR3 is subcloned into the high-efficiency
transcription vector pBS KS.sup.+AMV-pA50 (Nowak et al., 1995).
This plasmid was modified by adding the recognition sequence for
the restriction enzyme SrfI downstream of the poly A sequence in
the plasmid. The new plasmid was designated M52. Subcloning
involved the isolation of a 1.1 kb NcoI/EcoRI restriction fragment
encoding the entire hGALR3 gene followed by its ligation into
NcoI/EcoRI digested M52. After identification of a suitable clone
(M54), the transcription template was produced by linearization of
the plasmid DNA with SrfI. The plasmid M54 was deposited on Sep.
30, 1997, with the American Type Culture Collection (ATCC), 12301
Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions
of the Budapest Treaty for the International Recognition of the
Deposit of Microorganisms for the Purposes of Patent Procedure and
was accorded ATCC Accession No. 209312. The sequence comprising the
coding region of rat GALR3 was subcloned into pBS KS.sup.+AMV-pA50
(Nowak, et al., 1995) to produce M67. The transcription template
was produced by linearization of the plasmid DNA with SrfI. The
plasmid M67 was deposited on Mar. 27, 1998, with the American Type
Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.
20852, U.S.A. under the provisions of the Budapest Treaty for the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure and was accorded ATCC Accession No.
xxxxxx. mRNAs were transcribed using the T7 polymerase ("Message
Machine", Ambion). Each oocyte received 2 ng each of GIRK1 and
GIRK4 mRNA in combination with 25 ng of GalR3 mRNA. In other
experiments oocytes received injections of mRNAs encoding the human
.alpha.1A adrenergic receptor, rGalR1 or rGalR2 galanin receptors
(Forray et al., 1994; Parker et al., 1995) with or without GIRKs 1
and 4. After injection of mRNAs, oocytes were incubated at
17.degree. C. for 3-8 days.
[0368] Dual electrode voltage clamp ("GeneClamp", Axon Instruments
Inc., Foster City, Calif.) was performed as described above, with
the following modifications: during recordings, oocytes were bathed
in continuously flowing (1-3 mL/min) ND96 medium or, in the case of
oocytes expressing GIRKs 1 and 4, elevated K.sup.+ containing 48 mM
KCl, 49 mM NaCl, 2 mM CaCl.sub.2, 2 mM MgCl.sub.2, and 5 mM HEPES,
pH 7.5 (1/2 hK). Drugs were applied either by local perfusion from
a 10 .mu.l glass capillary tube fixed at a distance of 0.5 mm from
the oocyte, or for calculation of steady-state EC.sub.50s, by
switching from a series of gravity fed perfusion lines. Experiments
were carried out at room temperature. All values are expressed as
mean .+-. standard error of the mean.
[0369] MAP Kinase
[0370] MAP kinase (mitogen activated kinase) may be monitored to
evaluate receptor activation. MAP kinase is activated by multiple
pathways in the cell. A primary mode of activation involves the
ras/raf/MEK/MAP kinase pathway. Growth factor (tyrosine kinase)
receptors feed into this pathway via SHC/Grb-2/SOS/ras. Gai coupled
receptors are also known to activate ras and subsequently produce
an activation of MAP kinase. Receptors that activate phospholipase
C (through G.alpha.q and G.alpha.11) produce diacylglycerol (DAG)
as a consequence of phosphatidyl inositol hydrolysis. DAG activates
protein kinase C which in turn phosphorylates MAP kinase.
[0371] MAP kinase activation can be detected by several approaches.
One approach is based on an evaluation of the phosphorylation
state, either unphosphorylated (inactive) or phosphorylated
(active). The phosphorylated protein has a slower mobility in
SDS-PAGE and can therefore be compared with the unstimulated
protein using Western blotting. Alternatively, antibodies specific
for the phosphorylated protein are available (New England Biolabs)
which can be used to detect an increase in the phosphorylated
kinase. In either method, cells are stimulated with the mitogen and
then extracted with Laemmli buffer. The soluble fraction is applied
to an SDS-PAGE gel and proteins are transferred electrophoretically
to nitrocellulose or Immobilon. Immunoreactive bands are detected
by standard Western blotting technique. Visible or chemiluminescent
signals are recorded on film and may be quantified by
densitometry.
[0372] Another approach is based on evaluation of the MAP kinase
activity via a phosphorylation assay. Briefly, cells are stimulated
with the mitogen and a soluble extract is prepared. The extract is
incubated at 30.degree. C. for 10 min with gamma-32-ATP, an ATP
regenerating system, and a specific substrate for MAP kinase such
as phosphorylated heat and acid stable protein regulated by
insulin, or PHAS-I. The reaction is terminated by the addition of
H.sub.3PO.sub.4 and samples are transferred to ice. An aliquot is
spotted onto Whatman P81 chromatography paper, which retains the
phosphorylated protein. The chromatrography paper is washed and
counted for .sup.32P in a liquid scintillation counter.
Alternatively, the cell extract is incubated with gamma-32-ATP, an
ATP regenerating system, and biotinylated myelin basic protein
bound by streptavidin to a filter support. The myelin basic protein
is a substrate for activated MAP kinase. The phosphorylation
reaction is carried out for 10 min at 30.degree. C. The extract can
then by aspirated through the filter, which retains the
phosphorylated myelin basic protein. The filter is washed and
counted for .sup.32P by liquid scintillation counting.
[0373] Cell Proliferation Assay
[0374] Receptor activation of a G protein coupled receptor may lead
to a mitogenic or proliferative response which can be monitored via
.sup.3H-thymidine uptake. When cultured cells are incubated with
.sup.3H-thymidine, the thymidine translocates into the nuclei where
it is phosphorylated to thymidine triphosphate. The nucleotide
triphosphate is then incorporated into the cellular DNA at a rate
that is proportional to the rate of cell growth. Typically, cells
are grown in culture for 1-3 days. Cells are forced into quiescence
by the removal of serum for 24 hrs. A mitogenic agent is then added
to the media. 24 hrs later, the cells are incubated with
.sup.3H-thymidine at specific activities ranging from 1 to 10
uCi/ml for 2-6 hrs Harvesting procedures may involve trypsinization
and trapping of cells by filtration over GF/C filters with or
without a prior incubation in TCA to extract soluble thymidine. The
filters are processed with scintillant and counted for .sup.3H by
liquid scintillation counting. Alternatively, adherant cells are
fixed in MeOH or TCA, washed in water, and solubilized in 0.05%
deoxycholate/0.1 N NaOH. The soluble extract is transferred to
scintillation vials and counted for .sup.3H by liquid scintillation
counting.
[0375] Receptor/G Protein Co-Transfection Studies
[0376] A strategy for determining whether GALR3 can couple
preferentially to selected G proteins involves co-transfection of
GALR3 receptor cDNA into a host cell together with the cDNA for a G
protein alpha sub-unit. Examples of G alpha sub-units include
members of the G.alpha.i/G.alpha.o class (including G.alpha.t2 and
G.alpha.z), the G.alpha.q class, the G.alpha.s class, and
G.alpha.12/13 class. A typical procedure involves transient
transfection into a host cell such as COS-7. Other host cells may
be used. A key consideration is whether the cell has a downstream
effector (a particular adenylate cyclase, phospholipase C, or
channel isoform, for example) to support a functional response
through the G protein under investigation. G protein beta gamma
sub-units native to the cell are presumed to complete the G protein
heterotrimer; otherwise specific beta and gamma sub-units may be
co-transfected as well. Additionally, any individual or combination
of alpha, beta, or gamma subunits may be co-transfected to optimize
the functional signal mediated by the receptor.
[0377] The receptor/G alpha co-transfected cells are evaluated in a
binding assay in which case the radioligand binding may be enhanced
by the presence of the optimal G protein coupling or in a
functional assay designed to test the receptor/ G protein
hypothesis. In one example, GALR3 may be hypothesized to inhibit
cAMP accumulation through coupling with G alpha sub-units of the
G.alpha.i/C.alpha.o class. Host cells co-transfected with GALR3 and
appropriate G alpha sub-unit cDNA are stimulated with forskolin +/-
GALR3 agonist, as described above in cAMP methods. Intracellular
cAMP is extracted for analysis by radioimmunoassay. Other assays
may be substituted for cAMP inhibition, including
GTP.gamma..sup.35S binding assays and inositol phosphate hydrolysis
assays. Host cells transfected with GALR3 minus Galpha or with
GaLpha minus GALR3 would be tested simultaneously as negative
controls. GALR3 receptor expression in transfected cells may be
confirmed in .sup.125I-galanin binding studies using membranes from
transfected cells. G alpha expression in transfected cells may be
confirmed by Western blot analysis of membranes from transfected
cells, using antibodies specific for the G protein of interest.
[0378] The efficiency of the transient transfection procedure is a
critical factor for signal to noise in an inhibitory assay, much
more so than in a stimulatory assay. If a positive signal present
in all cells (such as forskolin -stimulated cAMP accumulation) is
inhibited only in the fraction of cells successfully transfected
with receptor and G alpha, the signal to noise ratio will be poor.
One method for improving the signal to noise ratio is to create a
stably transfected cell line in which 100% of the cells express
both the receptor and the G alpha subunit. Another method involves
transient co-transfection with a third cDNA for a G protein-coupled
receptor which positively regulates the signal which is to be
inhibited. If the co-transfected cells simultaneously express the
stimulatory receptor, the inhibitory receptor, and a requisite G
protein for the inhibitory receptor, then a positive signal may be
elevated selectively in transfected cells using a receptor-specific
agonist. An example involves co-transfection of COS-7 with 5HT4,
GALR3, and a G alpha sub-unit. Transfected cells are stimulated
with a 5HT4 agonist +/- galanin. Cyclic AMP is expected to be
elevated only in the cells also expressing GALR3 and the G alpha
subunit of interest, and a galanin-dependent inhibition may be
measured with an improved signal to noise ratio.
[0379] Tissue Preparation for Neuroanatomical Studies
[0380] Male Sprague-Dawley rats (Charles River, Wilmington, Mass.)
are decapitated and the brains rapidly removed and frozen in
isopentane. Coronal sections may be cut at 11 .mu.m on a cryostat
and thaw-mounted onto poly-L-lysine coated slides and stored at
-80.degree. C. until use. Prior to hybridization, tissues are fixed
in 4% paraformaldehyde, treated with 5 mM dithiothreitol,
acetylated in 0.1 M triethanolamine containing 0.25% acetic
anhydride, delipidated with chloroform, and dehydrated in graded
ethanols.
[0381] Probes
[0382] Oligonucleotide probes employed to characterize the
distribution of the rat GALR3 receptor mRNA may be synthesized, for
example, on a Millipore Expedite 8909 Nucleic Acid Synthesis
System. The probes are then lyophilized, reconstituted in sterile
water, and purified on a 12% polyacrylamide denaturing gel. The
purified probes are again reconstituted to a concentration of 100
ng/.mu.L, and stored at -20.degree. C. Probe sequences may include
DNA or RNA which is complementary to the mRNA which encodes the
GALR3 receptor.
[0383] In Situ Hybridization
[0384] Probes are 3'-end labeled with .sup.35S-dATP (1200 Ci/mmol,
New England Nuclear, Boston, Mass.) to a specific activity of about
10.sup.9 dpm/.mu.g using terminal deoxynucleotidyl transferase
(Pharmacia). The radiolabeled probes are purified on Biospin 6
chromatography columns (Bio-Rad; Richmond, Calif.), and diluted in
hybridization buffer to a concentration of 1.5.times.10.sup.4
cpm/.mu.L. The hybridization buffer consists of 50% formamide,
4.times.sodium citrate buffer (1.times. SSC=0.15 M NaCl and 0.015 M
sodium citrate), 1.times. Denhardt's solution (0.2%
polyvinylpyrrolidine, 0.2% Ficoll, 0.2% bovine serum albumin), 50
mM dithiothreitol, 0.5 mg/ml salmon sperm DNA, 0.5 mg/ml yeast
tRNA, and 10% dextran sulfate. About one hundred .mu.L of the
diluted radiolabeled probe is applied to each section, which is
then covered with a Parafilm coverslip. Hybridization is carried
out overnight in humid chambers at 40 to 55.degree. C. The
following day the sections are washed in two changes of 2.times.
SSC for one hour at room temperature, in 2.times.SSC for 30 min at
50-60.degree. C., and finally in 0.1.times. SSC for 30 min at room
temperature. Tissues are dehydrated in graded ethanols and apposed
to Kodak XAR-5 film for 3 days to 3 weeks at -20.degree. C., then
dipped in Kodak NTB3 autoradiography emulsion diluted 1:1 with 0.2%
glycerol water. After exposure at 4.degree. C. for 2 to 8 weeks,
the slides are developed in Kodak D-19 developer, fixed, and
counterstained with cresyl violet.
[0385] Solution Hybridization/Ribonuclease Protection Assay
[0386] For solution hybridization 2-15 .mu.g of total RNA isolated
from tissues may be used. Sense RNA synthesized using the
full-length coding sequence of the rGalR2 is used to characterize
specific hybridization. Negative controls may consist of 30 .mu.g
transfer RNA (tRNA) or no tissue blanks. Samples are placed in
1.5-ml microfuge tubes and vacuum dried. Hybridization buffer (40
.mu.l of 400 mM NaCl, 20 mM Tris, pH 6.4, 2 mM EDTA, in 80%
formamide) containing 0.25-1.0.times.10.sup.6 counts of each probe
is added to each tube. Samples are heated at 90.degree. C. for 15
min, after which the temperature is lowered to 45.degree. C. for
hybridization.
[0387] After hybridization for 14-18 hr, the RNA/probe mixtures are
digested with RNAse A (Sigma) and RNAse T1 (Bethesda Research Labs,
Gaithersburg, Md.). A mixture of 2.0 .mu.g RNAse A and 1000 units
of RNAse T1 in a buffer containing 330 mM NaCl, 10 mM Tris (pH 8.0)
and 5 mM EDTA (400 .mu.l) is added to each sample and incubated for
90 min at room temperature. After digestion with RNAses, 20 .mu.l
of 10% SDS and 50 .mu.g proteinase K are added to each tube and
incubated at 37.degree. C. for 15 min. Samples are then extracted
with phenol/chloroform:isoamyl alcohol and precipitated in 2
volumes of ethanol for 1 hr at -70.degree. C. tRNA is added to each
tube (30 mg) as a carrier to facilitate precipitation. Following
precipitation, samples are centrifuged, washed with cold 70%
ethanol, and vacuum dried. Samples are dissolved in formamide
loading buffer and size-fractionated on a urea/acrylamide
sequencing gel (7.6 M urea, 6% acrylamide in Tris-borate-EDTA).
Gels are dried and apposed to Kodak XAR-5 x-ray film.
[0388] Development of probes: Using full length cDNA encoding the
rat Gal R3 receptor as a template, PCR was used to amplify a 445
base pair fragment corresponding to nucleotides 1061-1506 of the
coding sequence. Primers used in PCR contained both sp6 and T7 RNA
polymerase promoter sequences, and the PCR generated fragments were
subcloned into a plasmid vector (pUC-18). This construct was
linearized with Bam HI or Hind III. sp6 and T7 RNA polymerases were
used to synthesize the sense and antisense strands of RNA
respectively. Full length RNA transcripts were obtained using a
full length cDNA construct in pBluescript.
[0389] A probe coding for rat glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) gene, a constitutively expressed protein, is
used concurrently. GAPDH is expressed at a relatively constant
level in most tissue and its detection is used to compare
expression levels of the rat GalR3 gene in different tissue.
[0390] Extraction of RNA: Tissue harvested from rat peripheral
tissue as well as regions of the CNS was frozen using liquid
N.sub.2 and stored at -70.degree. C. until needed Tissue was
homogenized in buffer containing detergent, protein and RNase
degrader. The homogenate was incubated with Oligo(dT) cellulose
powder, and washed extensively. mRNA was eluted from the Oligo(dT)
cellulose with 10 mM Tris, and precipitated after the addition of
NaCl. Yield and relative purity were assessed by measuring
absorbance A.sub.260/A.sub.280.
[0391] Synthesis of probes: rGALR3 and GAPDH CDNA sequences
preceded by phage polymerase promoter sequences were used to
synthesize radiolabeled riboprobes. Conditions for the synthesis of
riboprobes were: 0.5-1.0 .mu.L linearized template (1 .mu.g/.mu.L),
1.5 .mu.L of ATP, GTP, UTP (10 mM each), 3 .mu.L dithiothreitol
(0.1 M), 30 units RNAsin RNAse inhibitor, 0.5-1.0 .mu.L (15-20
units/.mu.L) RNA polymerase, 7.0 .mu.L transcription buffer
(Promega Corp.), and 12.5 .mu.L .alpha..sup.32P-CTP (specific
activity 3,000 Ci/mmol). 0.1 mM CTP (0.02-1.0 .mu.L) were added to
the reactions, and the volume were adjusted to 35 .mu.L with
DEPC-treated water. Labeling reactions were incubated at 37.degree.
C. for 90 min, after which 3 units of RQ1 RNAse-free DNAse (Promega
Corp.) were added to digest the template. The riboprobes were
separated from unincorporated nucleotide by a spun G-50 column
(Select D G-50(RF); 5 Prime-3 Prime, Inc.). TCA precipitation and
liquid scintillation spectrometry were used to measure the amount
of label incorporated into the probe. A fraction of all riboprobes
synthesized were size-fractionated on 0.4 mm thick 5% acrylamide
sequencing gels and autoradiographed to confirm that the probes
synthesized were full-length and not degraded.
[0392] Solution Hybridization/Ribonuclease Protection Assay:
[0393] For solution hybridization 2.0 .mu.g of total RNA isolated
from tissues were used. Sense RNA synthesized using the full-length
coding sequence of the rGalR3 was used to characterize specific
hybridization. Negative controls consisted of 30 .mu.g transfer RNA
(tRNA) or no tissue blanks. All mRNA samples were placed in 1.5-ml
microfuge tubes and vacuum dried. Hybridization buffer (40 .mu.l of
400 mM NaCl, 20 mM Tris, pH 6.4, 2 mM EDTA, in 80% formamide)
containing 0.25-1.0.times.10.sup.6 counts of each probe were added
to each tube. Samples were heated at 90.degree. C. for 15 min,
after which the temperature was lowered to 42.degree. C. for
hybridization.
[0394] After hybridization for 14-18 hr, the RNA/probe mixtures
were digested with RNAse A (Sigma) and RNAse T1 (Life
Technologies). A mixture of 2.0 .mu.g RNAse A and 1000 units of
RNAse T1 in a buffer containing 330 mM NaCl, 10 mM Tris (pH 8.0)
and 5 mM EDTA (400 .mu.L) was added to each sample and incubated
for 90 min at room temperature. After digestion with RNAses, 20
.mu.L of 10% SDS and 50 .mu.g proteinase K were added to each tube
and incubated at 37.degree. C. for 15 min. Samples were then
extracted with phenol/chloroform:isoamyl alcohol and precipitated
in 2 volumes of ethanol for 1 hr at -70.degree. C. Pellet Paint
(Novagen) was added to each tube (2.0 .mu.g) as a carrier to
facilitate precipitation. Following precipitation, samples were
centrifuged, washed with cold 70% ethanol, and vacuum dried.
Samples were dissolved in formamide loading buffer and
size-fractionated on a urea/acrylamide sequencing gel (7.6 M urea,
6% acrylamide in Tris-borate-EDTA). Gels were dried and apposed to
Kodak XAR-5 x-ray or BioMax film and exposed at -70.degree. C.
[0395] An additional set of solution hybridization/ribonuclease
protection assays (RPA) were used to detect rGALR3 receptor
transcripts in mRNA isolated from rat tissues. Poly A+ RNA was
isolated using either the Trizol reagent (Life Technologies,
Gaithersburg, Md.) followed by oligo dT chromatography, or the Fast
Track RNA isolation kit (Invitrogen, Carslbad, Calif.). A 445 bp
fragment of the rat GALR3 cDNA (nucleotides 1061-1506) flanked by
RNA polymerase promoter sequences was used to synthesize a
radiolabeled GALR3 cRNA probe using standard methods and reagents
(Promega). The quality of each probe was confirmed by
polyacrylamide gel electrophoresis. For solution hybridization, two
.mu.g poly A+ RNA from each tissue were incubated in 40 .mu.l
hybridization buffer (20 mM Tris, pH 6.4 containing 400 mM NaCl and
2 mM EDTA in 80% formamide) with radiolabeled cRNA probe
(0.25-1.25.times.10.sup.6 cpm) at 90.degree. C. for 15 min. prior
to overnight hybridization at 45 or 55.degree. C. Negative controls
consisted of 30 .mu.g transfer RNA (tRNA) or blanks with no poly A+
RNA. Hybridization mixtures were digested for 90 min. at room
temperature with RNAses A (Sigma) and T1 (Life Technologies), then
treated with 10% SDS and 50 .mu.g proteinase K, extracted with
phenol/chloroform, and precipitated in ethanol. Samples were
separated by urea/acrylamide gel electrophoresis (7.6 M urea, 6%
acrylamide in Tris-borate-EDTA); gels were dried and apposed to a
phosphorimager screen (Molecular Dynamics) or Kodak BioMax film at
-70.degree. C.
[0396] In Vivo Methods
[0397] The effects of galanin, galanin derivatives, and related
peptides and compounds may be evaluated by intracerebroventricular
(i.c.v.) injection of the peptide or compound followed by
measurement of food intake in the animal. Measurement of food
intake was performed for 3 hours after injection, but other
protocols may also be used. Saline was injected as a control, but
it is understood that other vehicles may be required as controls
for some peptides and compounds. In order to determine whether a
compound is a GALR3 antagonist, food intake in rats may be
stimulated by administration of (for example) a galanin receptor
agonist through an intracerebroventricular (i.c.v.) cannula. A
preferred anatomic location for injection is the hypothalamus, in
particular, the paraventricular nucleus. Methods of cannulation and
food intake measurements are well-known in the art, as are i.c.v.
modes of administration (Kyrkouli et al., 1990, Ogren et al.,
1992). To determine whether a compound reduces agonist-stimulated
food intake, the compound may be administered either simultaneously
with the peptide, or separately, either through cannula, or by
subcutaneous, intramuscular, or intraperitoneal injection, or more
preferably, orally.
[0398] Materials
[0399] Cell culture media and supplements are from Specialty Media
(Lavallette, N.J.). Cell culture plates (150 mm and 96-well
microtiter) are from Corning (Corning, N.Y.). Sf9, Sf21, and High
Five insect cells, as well as the baculovirus transfer plasmid,
pBlueBacIII.TM., are purchased from Invitrogen (San Diego, Calif.).
TMN-FH insect medium complemented with 10% fetal calf serum, and
the baculovirus DNA, BaculoGold.TM., is obtained from Pharmingen
(San Diego, Calif.). Ex-Cell 400.TM. medium with L-Glutamine is
purchased from JRH Scientific. Polypropylene 96-well microtiter
plates are from Co-star (Cambridge, Mass.). All radioligands are
from New England Nuclear (Boston, Mass.).
[0400] Galanin and related peptide analogs were either from Bachem
California (Torrance, Calif.), Peninsula (Belmont, Calif.); or were
synthesized by custom order from Chiron Mimotopes Peptide Systems
(San Diego, Calif.).
[0401] Bio-Rad Reagent was from Bio-Rad (Hercules, Calif.). Bovine
serum albumin (ultra-fat free, A-7511) was from Sigma (St. Louis.
Mo.). All other materials were reagent grade.
[0402] Experimental Results
[0403] Isolation of a Partial GALR3 cDNA from Rat Hypothalamus
[0404] In order to clone additional members of the galanin receptor
family, a homology cloning strategy based on the potential presence
of multiple galanin receptors in hypothalamus was designed.
Although recent evidence indicated that GALR1 and GALR2 receptor
mRNAs were present in rat hypothalamus (Gustafson et al., 1996;
Parker et al., 1995), not all aspects of the cloned GALR1 and GALR2
pharmacological profiles match that observed for galanin-mediated
feeding (Crawley et al., 1993). These results suggested that the
regulation of galanin-induced feeding may not be explained by the
presence of only GALR1 or GALR2 (or both) in the rat
hypothalamus.
[0405] In order to attempt to isolate additional galanin receptors,
a rat hypothalamus cDNA phage library was screened, under reduced
stringency conditions, with oligonucleotide probes directed to the
transmembrane regions of the rat GALR2 neuropeptide receptor gene.
Five positively-hybridizing clones were isolated, plaque-purified
and characterized by Southern blot analysis and sequencing. One
clone, rHY35a, contained a 3.5 kb insert (consisting of a 1.0 kb,
0.2 kb, and 2.3 kb EcoRI fragments), which hybridized with the
second transmembrane domain oligonucleotide probe of rat GALR2. DNA
sequence analysis indicated greatest homology to the published rat
GALR1 gene (Burgevin, et al., 1995) and the novel rat GALR2
receptor gene we have recently identified. This clone was a partial
intronless gene fragment, containing an open reading frame and
encoding a predicted starting MET through the middle of the
predicted seventh transmembrane domain, with .apprxeq.150
nucleotides of 5' UT. Hydropathy analysis of the predicted
translated protein is consistent with a putative topography of at
least six transmembrane domains (the predicted sequence ended in
the middle of TM7), indicative of the G protein-coupled receptor
family. This gene fragment exhibited 52% and 66% nucleotide
identity and 37% and 60% amino acid identity to the rat GALR1 and
rat GALR2 receptors, respectively. Furthermore, PCR primers
directed to the amino terminus (forward primer) and first
extracellular loop (reverse primer) of each of the corresponding
receptor genes, rGALR1 and rGALR2, were unable to amplify this
clone, whereas primers directed to this clone resulted in the
correct size PCR product. The putative six (or seven) transmembrane
topography and the high degree of identity to rat GALR1 and GALR2
suggested that this cDNA represented a partial gene fragment of a
novel galanin-like receptor gene, referred to herein as GALR3.
[0406] In order to obtain the full-length gene, PCR on cDNA derived
from the RIN14B cell line, using internal primers directed to TM3
and third intracellular loop of rat GALR3 was first conducted. It
was hypothesized that since previous data indicated that this cell
line expressed both GALR1 and GALR2, it may also contain further
subtypes. PCR analyses revealed the presence of at least a portion
of GALR3 in cDNA from RIN14B cells; the absence of reverse
transcriptase did not result in PCR amplification, indicating the
ability to amplify RIN14B cDNA was due to authentic GALR3 mRNA and
not any contaminating genomic DNA in the RNA source.
[0407] To isolate a cDNA molecule from RIN14B which expresses
GALR3, a RIN14B plasmid library was screened by PCR (using internal
primers) and two pools, F105 and F212, were identified which
contained a PCR product of the correct size. To determine if the
insert was in the correct orientation for expression and to
determine the size of the cDNA insert (including the coding region,
5'UT and 3'UT), vector-anchored PCR was conducted on each pool. The
PCR analyses suggested that both pools contained full-length GALR3
but in the incorrect orientation and thus would be predicted not to
express the GALR3 receptor. Examination of slides of COS-7 cells
which had been transfected with DNA from each of these pools and
subsequently bound with radioligand confirmed the absence of
binding of radiolabeled galanin, presumably due to its incorrect
orientation.
[0408] Although the full-length clone of rat GALR3 in the correct
orientation from the RIN14B plasmid library was not obtained, it
was reasoned that the sequence of the missing 3' end (i.e., from
the middle of TM7 through the stop codon) could be obtained by
sequencing the vector-anchored PCR product corresponding to the 3'
end of the molecule. An .apprxeq.1.2 kb PCR product from a
vector-anchored amplification of bacterial glycerol stock of the
F105 pool was obtained, using a vector-derived reverse primer and a
rGALR3-specific forward primer from TM6. This PCR product was
sequenced with the gene-specific primer to reveal an overlap within
TM7 with the sequence known from rHY35a. In addition, further
sequence was obtained representing an open reading frame
corresponding to the missing second half of TM7 and the carboxy
terminus. The sequence obtained showed an overall 47% nucleotide
identity to rGalR2, and a 62% nucleotide identity to rGalR2 from
the third extracellular domain to the 5' end of the COOH terminus,
confirming the existence of an open reading frame from a starting
MET through a stop codon, with the presence of seven putative
transmembrane domains. Furthermore, this sequence permitted us to
design an oligonucleotide primer in the 3' UT which could serve as
a diagnostic tool for determination of full-length characterization
of additional pools of DNA (see below).
[0409] Since the most convenient method to obtain the full-length
rGALR3 clone in the correct orientation in an expression vector is
to locate a full-length clone in preexisting libraries, and it was
known that this gene was expressed in rat hypothalamus, we screened
a rat hypothalamus plasmid library ("K") by PCR. Two superpools
from the K library (#3 and #17) were identified as containing
rGALR3. A primary pool, K163 (from superpool #17), was identified
to be positive and full-length using internal and full-length PCR
primers, and vector-anchor primers were used to determine the
orientation. These data were consistent with primary pool K163
(made up of 3200 primary clones), containing full-length rGALR3 in
the correct orientation in the expression vector, pEXJ.T7.
Furthermore, this pool failed to amplify with GALR1- and
GALR2-specific primers and yet exhibited galanin binding when DNA
from this pool was used to transfect COS cells and tested for
radiolabeled galanin binding. These data suggested that a pool from
a rat hypothalamus plasmid cDNA library which contains the novel
sequence initially identified from rat hypothalamus as a
galanin-like receptor had been identified, which, in addition,
exhibits galanin binding, thereby identifying the pool as
containing a novel galanin receptor, referred to herein as GALR3,
or more specifically, rGALR3.
[0410] The pool K163 was then sib selected through one round by PCR
and a second round by colony hybridization, using a probe directed
to the amino terminus of the sequence from rHY35a, resulting in the
isolation of a single clone (i.e., a bacterial colony containing
rat GALR3), called K163-30-17, representing the full-length rat
GALR3 in the correct orientation. The rGALR3 recombinant bacterial
colony was grown up in broth with ampicillin and DNA extracted.
Restriction enzyme digestion suggested a 2.1 kb insert, consistent
with the clone comprising the full-length coding region.
[0411] Furthermore, sequence analysis on K163-30-17 DNA (plasmid
K1086) confirmed that it contained a full-length coding region in
the correct orientation for expression.
[0412] Northern Blot Analyses of GALR3 mRNA
[0413] To define the size and distribution of the mRNA encoding
GALR3, Northern blot analyses of poly A.sup.+ RNA from various rat
tissues and brain regions was carried out. A radiolabeled 70-mer
oligonucleotide probe directed to the amino terminus of the rat
GALR3 coding region was used as a hybridization probe under high
stringency. This probe failed to cross-hybridize with either the
GALR1 or GALR2 genes under similar hybrization conditions,
demonstrating its specificity for GALR3 receptor. A single
transcript of .apprxeq.3.3 kb is detected after a 5 day exposure of
the autoradiogram at -80.degree. C. using Kodak Biomax MS film with
a Biomax MS intensifying screen. GALR3 mRNA was not detected by
Northern analysis in the brain nor in various regions of the brain
(see Table 1). Among various rat tissues, the GALR3 transcript had
a restricted distribution; GALR3 mRNA was predominantly observed in
kidney with a faint signal detected in liver (see Table 1). This
distribution was the same upon a longer exposure of the
autoradiogram (14 days). Northern blots were reprobed with G3PDH
probe to assess whether similar amounts of mRNA were present in
each lane.
[0414] Northern blot analyses of poly A+ RNA from various human
brain regions and peripheral tissues were carried out with a
radiolabeled 70-mer oligonucleotide probe directed to the amino
terminus of the human GALR3 coding region under high stringency. As
demonstrated for the corresponding rat probe, this human probe
failed to cross-hybridize with either the human GALR1 or GALR2
genes under similar hybridization conditions, demonstrating its
specificity for human GALR3 receptor. No transcript was observed
even after 14 day exposure of the autoradiogram in any of the human
brain regions or peripheral tissues, by Northern blot analyses. The
regions of the brain and periphery included in this analysis, as
contained in the MTN blots from Clontech, included: amygdala,
caudate nucleus, corpus callosum, hippocampus, total brain,
substantia nigra, subthalamic brain, thalamus nucleus, cerebellum,
cerebral cortex, medulla, spinal cord, occipital pole, frontal
lobe, temporal lobe, putamen, heart, total brain, placenta, lung,
liver, skeletal muscle, kidney, and pancreas.
[0415] Reverse-Transcription PCR of GALR3 mRNA
[0416] Amplification of cDNA derived from mRNA of various rat
peripheral and brain regions demonstrated the presence of GALR3
mRNA in various regions of the brain, including hypothalamus (see
Table 2), as well as several peripheral tissues tested, such as
pancreas and liver. It was anticipated that we would identify GALR3
mRNA in hypothalamus since the gene was cloned from this region of
the brain (supra). Therapeutic indications implied from
localization of GALR3 mRNA for several of these regions are also
indicated in Table 2.
[0417] RT-PCR was performed on human pituitary cDNA from two
sources (Clontech cDNA and cDNA from poly A+RNA purchased from ABS)
using primers from human GALR1, human GALR2, human GALR3, and human
prolactin. The results from the two sources of human pituitary cDNA
were similar. GALR1 and prolactin were amplified from human
pituitary, while GALR2 and GALR3 were not. Since GALR2 (Fathi et
al., 1997) and GALR3 (FIG. 10) transcripts have been detected in
rat pituitary, these findings suggest that the localization of
these receptors in humans is distinct from that in rats.
7TABLE 1 Northern blot analyses of GALR3 mRNA in brain and various
peripheral rat tissues. Intensity Therapeutic Tissue of Signal
Indications Heart (-) Brain (-) Spleen (-) Lung (-) Liver +
Diabetes Skeletal Muscle (-) Kidney ++ Hypertension, electrolyte
balance, diuretic, anti -diuretic Testis (-) Spinal cord (-)
Periaqueductal Grey (-) Cerebellum (-) Cortex (-) Brain Stem (-)
Hypothalamus (-) Amygdala (-) RIN14B cell line (-)
[0418]
8TABLE 2 RT-PCR analyses of GALR3 mRNA in brain and various
peripheral rat tissues. Intensity Tissue of Signal Therapeutic
Indications Heart (-) Brain + Obesity/feeding, analgesia, cognition
enhancement, Alzheimer's disease, depression, anxiety, sleep
disorders, Parkinson's disease, traumatic brain injury,
convulsion/epilepsy Spleen + Immune functions, hematopoiesis Lung +
Respiratory disorders, asthma, emphysema, lung cancer diagnostics
Liver + Diabetes Skeletal Muscle (-) Diabetes Smooth Muscle +
regulation of gastrointestinal motility Kidney + Hypertension,
electrolyte balance, diuretic, anti- diuretic Pancreas +++
Appetite/obesity, diabetes, gastrointestinal disorders,
neuroendocrine regulation Retina (-) vision disorders Testis +
Reproductive function Ventral ++ movement disorders, spinal
regulation of cord parasympathetic nervous system function Dorsal
spinal ++ cord Periaqueductal (-) Grey Cerebellum + Motor disorders
Cortex (-) Brain Stem + Autonomic disorders Lower midbrain +
analgesia, sensory transmission, regulation of cardiovascular and
respiratory systems Hypothalamus ++ Neuroendocrine regulation,
appetite/obesity Amygdala (-) RIN14B cell + Neuroendocrine line
regulation, including diabetes
[0419] RNase Protection Assay to Detect mRNA Coding for Rat
GALR3
[0420] mRNA was isolated and assayed as described from: heart,
striated muscle, liver, kidney, lung, stomach, spleen, pancreas,
pituitary, adrenal medulla, adrenal cortex, trigeminal ganglion and
CNS regions. CNS regions included: whole brain, spinal cord,
medulla, hypothalamus, cerebral cortex, cerebellum, hippocampus,
caudate-putamen, and substantia nigra. Levels of rat GALR3 mRNA
were extremely low in all areas assayed. The highest levels of rat
GALR3 mRNA were detected in the hypothalamus. Lower amounts were
found in: kidney, liver, stomach, pancreas, spleen, pituitary,
adrenal medulla, adrenal cortex, whole brain, spinal cord, medulla,
cerebellum and caudate/putamen. At the present time, mRNA coding
for the rat GALR3 has not been detected in RNA extracted from other
regions (Table 3).
[0421] To further assess the distribution of GALR3 mRNA in the rat,
further solution hybridization/RNAse protection assays (RPA) on
poly A.sup.+ RNA isolated from a variety of tissues and brain
regions were carried out (FIG. 11; Table 3). GALR3 transcripts were
broadly distributed but present at low abundance within the rat
central nervous system and many peripheral tissues. Within the CNS,
the highest levels of GALR3 mRNA were found in the rat
hypothalamus, with lower levels in the olfactory bulb, cerebral
cortex, medulla oblongata, caudate putamen, cerebellum, and spinal
cord. GALR3 mRNA was not detected in hippocampus or substantia
nigra. In peripheral tissues, highest levels of GALR3 mRNA were
found in the pituitary gland; areas containing low levels of GALR3
included liver, kidney, stomach, testicle (not shown in FIG. 11),
and the adrenal cortex. Additionally GALR3 mRNA was found in lung,
adrenal medulla, spleen, and pancreas (not shown in FIG. 11). GALR3
transcripts were not detected in RNA extracted from heart, uterus,
vas deferens, choroid plexus or dorsal root ganglion. Other areas
not expressing GALR3 mRNA in our assay include striated muscle,
urinary bladder, trigeminal ganglion, duodenum, and superior
cervical ganglion (not all shown in FIG. 11). This localization
pattern suggests that GALR3 may contribute more to galanin-mediated
physiology in the rat hypothalamus and pituitary than in other
areas. However, the up-regulation of galanin peptide expression in
a variety of pathophysiological states (Chan-Palay, 1990; Schreiber
et al., 1994; Xu et al., 1996; Xu et al., 1997; Sten Shi et al.,
1997) leaves open the possibility that GALR3 receptor expression
could be similarly plastic. The broad distribution of rat GALR3
transcripts also indicates that multiple galanin receptors are
likely to be present in many tissues.
9TABLE 3 Distribution of mRNA coding for rat GALR3 receptors.
Region rGalR3 Potential applications liver + Diabetes kidney +
Hypertension, Electrolyte balance lung + Respiratory disorders,
asthma heart - Cardiovascular indications stomach +
Gastrointestinal disorders duodenum - Gastrointestinal disorders
spleen + Immune function pancreas + Diabetes, endocrine disorders
testicle + Reproductive function striated - muscoloskeletal
disorders; muscle glucose metabolism (e.g., diabetes) pituitary +
Endocrine/neuroendocrine regulation adrenal + Regulation of
epinephrine medulla release adrenal + Regulation of steroid
hormones cortex trigeminal - Analgesia, sensory ganglion
transmission, migraine whole brain + Degenerative diseases of the
central nervous system (e.g., Parkinson's disease, Huntington's
disease, and Alzheimer's disease) anxiety, manic depression,
schizophrenia, epilepsy, stroke and see below for specific regions
of whole brain cerebral + Sensory integration, cognition cortex
hypothalamus ++ Appetite/obesity, Neuroendocrine regulation
hippocampus - Cognition/memory/Alzheimer's disease spinal cord ++
Analgesia, sensory modulation and transmission cerebellum + Motor
coordination medulla + Analgesia; sensory modulation and
transmission; regulation of cardiovascular and respiratory systems
substantia - Modulation of dopaminergic nigra function. Modulation
of motor coordination. Parkinson's disease. Dorsal root -
Analgesia, sensory ganglion transmission superior - Regulation of
sympathetic cervical nervous system function ganglion urinary -
control of micturition bladder uterus - reproductive function vas
deferens - reproductive function chloroid - Regulation of
intracerebral plexus fluid volume and composition caudate- +
Modulation of dopaminergic putamen function; Parkinson's disease;
Huntington's disease
[0422] Pharmacological Characterization of GALR3
[0423] The pharmacology of GALR3 was studied in COS-7 cells
transiently transfected with the GALR3 cDNA, K163-30-17 (or
"K1086"). COS-7 cells transfected with the single clone K1086
exhibit specific binding of .sup.125I-galanin in comparison with
COS-7 cells transfected with control vector. In preliminary
radioligand binding experiments, porcine .sup.125I-galanin bound to
membranes from COS-7 cells transfected with K1086, with a specific
binding of 90 fmol/mg, when the membranes (0.17 mg/mL) were
incubated with 2.1 nM porcine .sup.125I-galanin for 60 min at room
temperature. (Specific binding was decreased by as much as 70% when
the incubation temperature was raised to 30.degree. C., suggesting
receptor instability and/or protease activity in the membrane
preparation.) In this experiment, the binding buffer used was that
described for the whole cell slide binding assay. No specific
binding was detected to membranes from mock-transfected COS-7 cells
when tested under the same conditions.
[0424] In another experiment, COS-7 cells were transiently
transfected with a "trimmed" plasmid (designated pEXJ-RGalR3T),
which comprises the entire coding region of rat GALR3, but in which
the 5' initiating ATG is joined directly to the vector, and which
comprises only 100 nucleotides from the 3' untranslated region,
after the stop codon (i.e., up to and including nucleotide 1275 in
FIG. 1). A full saturation binding analysis using .sup.125I-galanin
was performed using the COS-7 cells transfected with plasmid
pEXJ-RGalR3T, and yielded a K.sub.d (dissociation constant) of 0.34
nM and an apparent B.sub.max as high as 570 fmol/mg. The use of the
"trimmed" plasmid provides for greater expression and therefore
greater convenience and accuracy in binding assays.
[0425] Peptide displacement assays yielded a distinct rank order of
binding affinity (Table 4). Porcine galanin bound with relatively
high affinity (K.sub.i=5 nM), C-terminal truncation to porcine
galanin 1-16 was disruptive (Ki=86 nM), and galanin 3-29 as well as
D-Trp.sup.2-galanin analogs were without demonstrable binding. Two
chimeric peptides displayed high affinity for GALR3 (M32 and M35)
whereas galantide was slightly less active and the putative
"antagonists" C7 and M40 were relatively weak ligands.
[0426] Peptide binding profiles for the rat GALR1, GALR2 and GALR3
receptor subtypes were derived from membranes prepared from
transiently transfected COS-7 cells. Rat GALR3 is distinguished
from the other receptor subtypes by having 40-fold lower affinity
for M40 vs. galanin, whereas the rat GALR1 and GALR2 receptor
subtypes display <=8-fold lower affinity for M40 vs. galanin.
Rat GALR3 also displays low affinity for the D-Trp.sup.2-galanin
analogs, which appear to be primarily useful for distinguishing the
rat GALR2 receptor. It is concluded that the rat GALR3 displays a
distinctive pharmacological profile which can be used to evaluate
receptor expression in native cells and tissues.
10TABLE 4 Peptide binding profile of rat GALR1, GALR2 and GALR3
receptors transiently expressed in COS-7 cell membranes and labeled
with porcine .sup.125I-galanin. Values are reported as K.sub.i
(nM). GALR1 (K.sub.i, GALR2 (K.sub.i, GALR3 (K.sub.i, Peptide nM)
nM) nM) porcine 0.46 0.45 5.1 galanin M32 0.62 12 2.1 M35 0.33 0.57
6.7 galantide 9.5 2.0 18 C7 16 19 68 M40 3.6 0.72 210 porcine 2.2
7.2 86 galanin 1-16 D-Trp.sup.2-galanin 3700 52 >1000 1-29
D-Trp.sup.2-galanin 40 000 23 >1000 1-16 porcine >100 000
>100 000 >1000 galanin 3-29
[0427] Isolation of the Human GALR3 Gene
[0428] A human placenta genomic library in .lambda. dash II
(.apprxeq.1.5.times.10.sup.6 total recombinants) was screened using
the same set of overlapping oligonucleotide probes to TM regions
1-7 of rat GALR2 and under the same hybridization and wash
conditions as described for screening the rat hypothalamus cDNA
library. Lambda phage clones hybridizing with the probe were plaque
purified and DNA was prepared for Southern blot analysis. One phage
clone, plc21a, contained a 2.7 kb KpnI/EcoRI fragment which
hybridized with the rat GALR2 TM2 oligonucleotide probe and was
subsequently subcloned into a pUC vector for sequence analysis. The
cloned human genomic fragment contains an a open reading frame from
the starting MET codon to a predicted intron in the second
intracellular loop, with a nucleotide identity of 88% (93% aa
identity) with the rat GALR3 receptor described above (thus
establishing this human genomic clone to be the human homologue of
rat GALR3). Although this human genomic fragment was not
full-length and contained an intron downstream of TM3, it is
anticipated that the full-length, intronless version of the human
GALR3 receptor gene may be isolated using standard molecular
biology techniques, as described in Materials and Methods.
[0429] Since the human genomic fragment was not full-length and
contained an intron downstream of TM3, it was hypothesized that the
original phage clone, which contains an average insert size of
about 18 kb, may contain the 3' end of this gene, assuming a
smaller size for the intron which serparates the 5' and 3' exons.
The presence of the exon, representing the 3' end of the human
GALR3, on the original phage clone, was demonstrated by positive
hybridization signals of the phage clone, plc21a, with probes
directed to the third extracellular loop or TM4 of the rat GALR3
gene.
[0430] The full-length human GALR3 gene was constructed by ligating
a PCR-derived product of the 5' exon, representing the starting MET
through the 3/4 loop with a synthetically-created KpnI site
appended to the reverse PCR primer, and the 3' exon, contained on a
1.4 kb KpnI genomic fragment. The full-length human GALR3 gene
contains 1107 bp within its coding region, encoding for a predicted
protein of 368 aa. The rat homologue contains two additional aa and
encodes for a predicted protein of 370 aa. The human and rat GALR3
homologues exhibit 86% nucleotide and 92% amino acid identities,
consistent with designating these genes as species homologues of
the same gene within the GPCR family. The amino acid identity
increases to 96% when restricting the comparison to within the
transmembrane domains. The human GALR3 gene exhibits 52% and 67%
nucleotide identities and 36% and 58% amino acid identities to the
human GALR1 and GALR2 receptors, respectively. Furthermore, within
the transmembrane domains, the human GALR3 receptor displays 46%
and 74% amino acid identities with the human GALR1 and GALR2
receptors, respectively. This relationship suggests that human
GALR3 represents a novel receptor subtype within the galanin gene
family.
[0431] Pharmacological Characterization of Human GALR3
[0432] The pharmacology of human GALR3 was studied in COS-7 cells
transiently transfected with pEXJ-hGalR3. In preliminary
radioligand binding experiments using membranes prepared from COS-7
cells transfected with pEXJ-hGalR3, specific binding of galanin was
observed with binding of 6 fmol/mg when the membranes (0.31 mg/mL)
were incubated with 0.32 nM porcine .sup.125I-galanin for 2 hrs. at
room temperature. No mock transfection was performed in this assay
because no galanin binding to COS-7 cells was observed previously
in binding experiments using similar conditions (supra).
[0433] In a subsequent experiment, when membranes from transiently
transfected cells (membrane protein=0.15 mg/ml) were incubated with
porcine .sup.125I-galanin (0.32 nM), specific binding was measured
as 110 fmol/mg. Therefore, it is concluded that the human GALR3
receptor cDNA leads to expression of functional GALR3 receptors,
thereby providing an important tool with which to evaluate ligand
selectivity for human GALR1, GALR2 and GALR3 receptor subtypes.
[0434] In further experiments, cell lines stably expressing the rat
and human GALR3 receptors were prepared. Membranes from the stably
transfected cell line 293-rGalR3-105 bound porcine
.sup.125I-galanin with a K.sub.d of 0.74 nM and an apparent
B.sub.max of 450 fmol/mg membrane protein. Data generated from
experiments in which porcine .sup.125I-galanin concentrations
ranged from 0.5 pM to 3.0 nM were best fit to a 1-site model. It
should be noted that the use of an iodinated agonist such as
porcine .sup.125I-galanin may underestimate actual receptor
expression levels, however, given the potential for agonists to
discriminate receptor conformation and the practical radioligand
concentration limit of approximately 3 nM. Both the transiently and
stably expressed rat GALR3 receptors were analyzed in competitive
displacement assays using porcine .sup.125I-galanin (Table 5). Like
GALR2, GALR3 appears to bind the N-terminally extended peptide
galanin -7 to +29 with affinity comparable to that for porcine
galanin. These data provide a pharmacological fingerprint which
should be useful for characterizing GALR3-dependent processes in
vivo.
[0435] Next, the cDNA for the human GALR3 receptor was used to
prepare both transiently and stably transfected cells. Membranes
from COS-7 cells transiently transfected with human GALR3 cDNA
bound porcine .sup.125I-galanin with a K.sub.d of 1.25 nM and an
apparent B.sub.max of 750 fmol/mg membrane protein. Membranes
LM(tk-) cells stably transfected with human GALR3 receptor cDNA
(L-hGalR3-228) bound porcine .sup.125I-galanin with a K.sub.d of
2.57 nM and an apparent B.sub.max of 1700 fmol/mg membrane protein.
Data generated from experiments in which porcine .sup.125I-galanin
concentrations ranged from 0.5 pM to 3.0 nM were best fit to a
1-site model. It should be noted that the use of an iodinated
agonist such as porcine .sup.125I-galanin may underestimate actual
receptor expression levels, however, given the potential for
agonists to discriminate receptor conformation and the practical
radioligand concentration limit of approximately 3 nM. Specific
binding measured in the presence of 0.3 nM porcine
.sup.125I-galanin was reduced by 40% in the presence of
nonhydrolyzable guanine nucleotides such as GTP.gamma.s or Gpp(NH)p
at concentrations up to 100 .mu.M. These data suggest that the
human GALR3 receptor interacts with one or more G proteins in the
LMTK-cell, and furthermore, that receptor stimulation by galanin
might lead to a functional response in the LMTK-cell at the level
of the G-protein or further downstream in the signal transduction
pathway. Preliminary analyses in peptide displacement assays using
porcine .sup.125I-galanin as the radioligand indicate that the
human GALR3 receptor, sharing 92% amino acid identity with the rat
GALR3 receptor, binds galanin and related analogs with affinities
resembling those for the rat receptor. A similar pharmacological
profile for both the human and rat GALR3 receptor homologs suggests
that the rat may be used to model the therapeutic value of
GALR3-directed ligands. A noteworthy feature of the pharmacology is
that the GALR3 receptor, whether human or rat, binds human galanin
with lower affinity compared to rat and porcine galanin. Human
galanin is also somewhat less potent than porcine galanin in both
in vitro functional and in vivo feeding assays. This relationship
differentiates the GALR3 receptor from the GALR1 and GALR2
subtypes, and may be useful in further investigations.
11 TABLE 5 Ki (nM) Rat Human GalR3 293-rGalR3- GalR3 L-hGalR3-
Peptide COS7 105 COS7 228 M32 1.9 1.0 6.0 M35 3.7 3.2 19 7.8 rat
4.3 5.7 galanin porcine 5.1 5.8 5.3 14 galanin human 10.5 53 19 69
galanin galantide 9.0 7.6 23 40 C-7 23 9.6 8.1 M40 103 85 130
porcine 52 138 300 320 galanin 1- 16 D-Trp2- >1000 >1000
galanin galanin 3.3 21 29 -7 to + 29
[0436] Signal Transduction Pathway of hGalR3: Stimulation of
K.sup.+ Currents
[0437] Heterologous expression of GPCRs in Xenopus oocytes has been
widely used to determine the identity of signaling pathways
activated by agonist stimulation (Gundersen et al., 1983; Takahashi
et al., 1987; Dascal et al., 1993). A large family of GPCRs that
naturally couple to heterotrimeric G-proteins of the
G.alpha..sub.i/G.alpha..sub.o class activate GIRK channels (North,
1989) in native neurons (Kofuji et al., 1995) and in the Xenopus
expression system (Dascal et al., 1993; Kubo et al., 1993;
Krapivinsky et al., 1995). Under voltage clamp conditions, oocytes
injected with mRNAs for hGALR3 and GIRKs 1 and 4 responded with
inward currents to local perfusion of porcine galanin (FIG. 6A).
Average currents were 51.3.+-.9.4 nA (n=16) in the presence of 1
.mu.M porcine galanin, whereas oocytes injected with mRNAs for
GIRKs 1 and 4 alone produced little or no inward current
(2.5.+-.1.2 nA, n=8) in response to 1 .mu.M galanin. Oocytes
injected with mRNA encoding the rat GalR3 receptor also exhibited
current responses to the 1 .mu.M local application of M32 or
porcine galanin. The pharmacology of the rat GalR3 receptor was not
further evaluated in oocytes. In oocytes expressing human GalR3,
evidence that galanin-induced currents were mediated by GIRK
channels included: 1) dependency on elevated external K.sup.+, 2)
strong inward rectification of the current-voltage (I/V) relation,
3) reversal potential (-26.+-.-2 mV) close to the predicted
equilibrium potential for K.sup.+ (-23 mV), 4) sensitivity to block
by 300 .mu.M Ba.sup.++ (FIG. 6A), and 5) lack of
galanin-sensitivity in oocytes injected with only hGALR3 mRNA (data
not shown). Currents having these same properties, but larger in
amplitude, were also evoked by galanin in oocytes expressing GALR1
receptors in combination with GIRKs 1 and 4 (Table 6). Thus, GALR1
and GALR3 receptors appear to have a related signal transduction
pathway.
[0438] Other GPCRs, when expressed in Xenopus oocytes, activate a
Ca.sup.++-dependent Cl.sup.- conductance that results from the
activation of phospholipase C and the subsequent release of
Ca.sup.++ from intracellular stores. This pathway was not activated
in oocytes expressing hGALR3 since Cl.sup.- currents were never
observed following application of galanin (n =20). (Cl.sup.-
currents were also not observed in oocytes expressing the GALR1
receptor.) In contrast, in oocytes expressing mRNAs encoding GALR2
or .alpha..sub.1a receptors, 1 .mu.M galanin or epinephrine,
respectively, stimulates transient Cl.sup.- currents (data not
shown). To provide further evidence that hGALR3 couples to the
G.alpha.o/G.alpha.i/G.alpha.t family of G-proteins, batches of
oocytes, previously injected with hGALR3 and GIRK mRNAs, were
injected with pertussis toxin (2 ng/oocyte) and tested for receptor
coupling to K.sup.+ currents. In oocytes treated with the toxin,
galanin currents were completely abolished (FIG. 7); oocytes
injected with buffer alone displayed normal galanin-induced
currents. A similar sensitivity to pertussis toxin was observed for
oocytes expressing GALR1 receptors. Agonist responses in oocytes
expressing GALR2 or .alpha..sub.1a adrenergic receptors were
unaffected by pertussis toxin (FIG. 7, Table 6). Taken together,
these results support the conclusion that GALR1 and GALR3 receptors
couple to a G.alpha.o/G.alpha.i/G.alpha.t pathway, and that GALR2
(like the .alpha..sub.1a adrenergic receptor) couples to a
.sub.qG.alpha.-type pathway (Table 7). Although these data reveal a
functional similarity between GALR1 and GALR3 in oocytes despite a
low level of primary sequence identity, exactly which G proteins
are involved in the oocyte or would be involved in mammalian cells
remain to be determined for each receptor subtype.
12TABLE 6 Effects of pertussis toxin treatment on currents
generated by stimulation of galanin or alpha adrenergic receptors
expressed in oocytes. Current is presented in nA (nanoamperes)
Receptor rat human rat GALR1 GALR2 GALR3 Alpha 1a Control 1775 .+-.
278 229 .+-. 60 24 .+-. 5 5483 .+-. 1154 PTX 17 .+-. 3 238 .+-. 51
0 .+-. 0 6350 .+-. 1318
[0439]
13TABLE 7 Comparison of intracellular signaling pathways for three
galanin receptors expressed in oocytes. Signaling pathway Activates
Activates Receptor CL.sup.- current GIRKs PTX sensitive rGALR1 no
yes yes rGALR2 yes no no hGALR3 no yes yes
[0440] Pharmacology of hGALR3 in oocytes
[0441] A series of galanin and galanin-related peptides were tested
at the human GALR3 receptor for agonist and antagonist activities.
Of these peptides, porcine galanin, human galanin, M32, C7, M35,
M15 (spantide), galanin -7-29, galanin 1-16, and M40 evoked agonist
activity at a fixed dose of 1 .mu.M. D-Trp2-galanin and galanin
3-29 were inactive. EC.sub.50s were constructed from cumulative
concentration-response measurements performed on a series of
oocytes (FIGS. 6B, 8). EC.sub.50s (in rank order) for M32, porcine
galanin, C7, galanin -7 to 29, galanin 1-16, and M40 were 45, 222,
343, 1906, 2030, and 2265 nM, respectively (Table 8). This rank
order of potency was similar to that observed for K.sub.i values in
binding assays using the human GalR3 receptor in LM(tk-) cell.
[0442] We have observed that the peptide galanin -7-29, which binds
selectively to GALR3 over GALR1 and to GALR2 over GALR1, induces
feeding in rats when injected i.c.v. Another peptide, shown in
binding and functional studies to selectively bind to the GALR2
receptor over both GALR1 and GALR3, did not stimulate feeding when
injected i.c.v. Taken together, these results suggest a role for
GALR3 in mediating galanin-induced feeding.
14TABLE 8 Comparison of rank orders of EC.sub.50s for stimulation
of GIRKs and apparent binding affinities (K.sub.i). rat GALR3 human
Oocyte Cos-7 GALR3LM (tk-) EC.sub.50 K.sub.i Ki Peptide (nM) (nM)
(nM) M32 45 1.9 6.0 p-Galanin 222 5.1 C7 343 23.0 8.1 gal -7 to 29
1,906 3.3 28.8 gal 1-16 2,030 51.9 319 M40 2,265 103.0 281
[0443] With respect to the rank orders of EC50s shown in Table 8,
it is to be understood that there is no difference in rank order
between any of M32, p-Galanin, and C7 or any of gal -7 to 29, gal
1-16, and M40. Therefore the rank order of potency of the peptides
at GALR3 are: M32.congruent.p-Galanin .congruent.C7>gal-7 to
29.congruent.gal 1-16.congruent.M40. With respect to the rank
orders of Ki values shown in Table 8, it is to be understood that
there is no difference in rank order between any of M32, C7, and
gal -7 to 29 or any of gal 1-16 and M40.
[0444] Pharmacology of rGALR3 in oocytes
[0445] The functional activity of rat GALR3 receptors in Xenopus
oocytes co-expressing GIRK potassium channels by
electrophysiological recordings was assessed. Under voltage clamp
conditions, oocytes injected with mRNAs for rat GALR3 and GIRKs 1
and 4 responded with inward currents to local perfusion of porcine
galanin (FIG. 12). Average currents were 34.+-.6 nA (n=6) in the
presence of 1 .mu.M porcine galanin (FIG. 12), whereas oocytes
injected with mRNAs for GIRKs 1 and 4 alone produced little or no
inward current (2.5.+-.1.2 nA, n=8) in response to 1 .mu.M
galanin.
[0446] Further Pharmacologic Characterization Galaninergic
Peptides
[0447] Peptide ligands were evaluated in binding and functional
assays (Tables 9-11).
15TABLE 9 Binding Data for Rat Galanin Receptors Ki (nM) from
porcine 125I-galanin binding assay Rat GalR1 Rat GALR2 Rat GALR3
Peptide CHO LMTK #4 293 #105 (3-iodo-L- Tyr9)-(3-iodo- L-Tyr26) -
galanin M32 0.70 0.69 1.32 C7 1.44 0.56 11.75 Rat galanin 1- 0.31
1.43 2.73 29 porcine galanin 0.32 1.02 2.81 M35 0.37 4.27 3.24 (-7)
to (+29) 36.31 3.16 21.38 galanin, porcine galantide 0.67 2.14
11.48 (-) 9 to (+) 29 51.29 3.47 4.17 galanin, porcine Human
galanin 0.62 2.54 53.09 Tyr9-iodo-M35 M40 7.76 3.76 85.11 Porcine
galanin 1-12 Porcine galanin 1-15 porcine galanin 2.45 2.75 138.04
1-16 D-Trp2-(d-iodo- >1000 1.51 181.97 L-Tyr9)-(3- iodo-L-Tyr26)
- galanin porcine galanin >1000 >1000 >1000 3-29 porcine
D-Trp2- 407 .+-. 94 28 .+-. 9 >1000 galanin
[0448]
16TABLE 10 Binding Data for Human Receptors Ki (nM) from porcine
125I-galanin binding assay Hum GALR1 Hum GALR2 Hum GALR3 Peptide
LM(tk-) CHO LMTK- #8 (3-iodo-L- 0.21 0.40 1.43 Tyr9)-(3-iodo-
L-Tyr26)- galanin M32 0.26 1.45 6.03 C7 0.26 0.63 8.13 Rat galanin
1- 0.29 1.62 8.81 29 porcine galanin 0.23 0.97 8.97 M35 0.11 1.95
14.62 (-7) to (+29) 6.84 4.95 28.84 galanin, porcine galantide 0.25
1.08 40.18 (-) 9 to (+) 29 7.85 5.43 50.12 galanin, porcine Human
galanin 0.44 2.34 69.41 Tyr9-iodo-M35 0.83 1.45 87.10 M40 2.38 4.04
280.54 Porcine galanin 61.66 5.17 306.67 1-12 Porcine galanin 3.98
6.13 309.03 1-15 porcine galanin 1.89 5.37 319.15 1-16
D-Trp2-(d-iodo- 169.82 21.38 933.25 L-Tyr9)-(3- iodo-L-Tyr26)-
galanin Porcine galanin >1000 >1000 >1000 3-29 Porcine
galanin >1000 >1000 >1000 1-9 human galanin >1000
>1000 >1000 3-30 porcine galanin 7.94 28.18 >1000 1-13
GMAP 44-59 >1000 >1000 >1000 amide GMAP 25-41 >1000
>1000 >1000 amide GMAP 16-41 >1000 >1000 >1000 amide
GMAP 1-41 amide >1000 >1000 >1000 porcine D-Trp2- >1000
galanin
[0449] With respect to the rank orders of Ki values shown in Table
10, it is to be understood that there is no difference in rank
order between any of porcine galanin, M32, M35, (-7) to (+29)
porcine galanin, galantide, and human galanin or any of M40 and
porcine galain 1-16 or any of porcine D-Trp2-galanin and porcine
galanin 3-29.
17TABLE 11 Functional Data at Galanin Receptors EC.sub.50 (nM) Rat
GALR1 Rat GALR2 Human LM (tk-) CHO #79 GALR3 Peptide cAMP AA GIRK p
gal 1-16 0.34 2.63 2000 p galanin 0.06 1.25 238 human galanin 0.21
0.74 7340 C7 0.52 2.41 343 M40 0.82 2.69 5030 M32 0.34 2.51 45 rat
gal 0.06 0.71
[0450] Of particular note, human galanin is an order of magnitude
less potent than porcine galanin as determined by either receptor
binding or functional activation of GIRKs. The low potency of
galanin and related peptides overall in oocytes did not seem to be
related to a low efficiency of receptor coupling inherent to
oocytes since galanin exhibited an EC50 of 2 nM in oocytes
expressing GALR1. This value corresponds closely to that previously
reported for galanin at GALR1 in a cyclic AMP assay (Habert-Ortoli,
E., et al., 1994).
[0451] Human GalR3/G Protein Interactions in Mammalian Systems
[0452] Binding in LMTK-:
[0453] The ability of GALR3 to modulate GIRK activity in the oocyte
was blocked by pre-treatment with pertussis toxin. Pertussis toxin
ADP-ribosylates G proteins of the Gi/Go class (except for
G.alpha.z) and thereby prevents them from coupling to receptors,
leading to a loss of receptor function. Our aim was to determine
whether pertussis toxin reduced GALR3-G protein coupling in
hGALR3-LMTK-#228. A 7 TM receptor such as hGALR3 receptor may adopt
a combination of "high affinity" and "low affinity" conformations
in a membrane preparation. A 7 TM receptor coupled to a G protein
may have a "high affinity" conformation but will adopt a "low
affinity" conformation if the coupling is disrupted either by
pertussis toxin, or by the binding of GTP analogs to the G protein.
A 7 TM receptor not coupled to a G protein is not expected to be
sensitive to pertussis toxin or guanine nucleotides.
[0454] Human GALR3-LMTK-#228 cells were cultured for 16 hrs in the
absence or presence of pertussis toxin (100 ng/ml) and membranes
were subsequently prepared. Porcine .sup.125I-galanin binding
assays were conducted using 0.3 nM radioligand and 40 ug membrane
protein/sample (total volume=250 ul). Under these conditions,
specific binding measured for control membranes (no pertussis
toxin) was 14280 cpm=145 fmol/mg membrane protein. Specific binding
was decreased approximately 53% by GTP.gamma.S (IC.sub.50=4.7 nM)
and 56% by GDP (IC.sub.50=160 nM) but not by GMP (FIG. 13). A
similar effect of guanine nucleotides has been reported for native
galanin receptors in rat brain (Chen, Y., et al., 1992). Because
the binding of porcine .sup.125I-galanin was reduced by GTP
analogs, we conclude that hGALR3 is coupled to a G protein in the
LMTK-cell.
[0455] Membranes from pertussis toxin-treated cells, studied under
the same conditions in the same porcine .sup.125I-galanin binding
assay, specifically bound only 3940 cpm=40 fmol/mg membrane
protein. The reduction in specific binding is consistent with an
"uncoupling" effect of pertussis toxin, or a separation of hGALR3
from G protein. Porcine .sup.125I-galanin binding to the fraction
of receptors detected after toxin treatment was unaffected by
GTP.gamma.S or GDP, consistent with the absence of hGALR3/G protein
coupling. From these data, we conclude that hGALR3 binds to a G
protein in LMTK-cells of the G.alpha..sub.i/G.alpha..- sub.o class
(FIG. 13). Exactly which of the several known homologs in the
G.alpha..sub.i/G.alpha..sub.o class may be involved
(G.alpha..sub.i1, G.alpha..sub.i2, G.alpha..sub.i3,
G.alpha..sub.oA, G.alpha..sub.oB,G.alpha..sub.t1, G.alpha..sub.t2,
G.alpha..sub.z, G.alpha..sub.gust or some as yet undescribed
G.alpha. subunit) remains to be determined.
[0456] Experimental Discussion
[0457] Using a combination of homology and expression cloning
strategies, nucleic acids have been isolated encoding a novel
galanin receptor, termed GALR3, that is distinct from the
previously cloned GALRl and GALR2 receptors.
[0458] The rat GALR3 gene, whose sequence is derived from cDNA,
does not have any other MET upstream of the proposed starting MET,
in any of the three possible reading frames.
[0459] The human GALR3 gene contains two in-frame METs: the first
(as one reads 5' to 3') will be referred to herein as the "upstream
MET" and the second (i.e., closer to TM1) will be referred to
herein as the "downstream MET." Both the upstream and downstream
METs are shown in FIG. 4 (Seq. ID No. 4). Based on data currently
available, it is believed that the downstream MET is likely to be
the correct initiating methionine. It is theoretically possible
that the upstream MET might be the initiating MET. It is to be
understood that the present invention includes both the receptor
beginning at the downstream MET and the receptor beginning at the
upstream MET.
[0460] Both rat and human GALR3 receptor sequences contain a single
consensus site for N-linked glycosylation at position six in the
N-terminus and several predicted intracellular sites for
phosphorylation by protein kinases; one putative phosphorylation
site common to rat and human GALR3 in the third intracellular loop
is absent in GALR1 and GALR2.
[0461] The existence of multiple galanin receptor subtypes suggests
the potential for the design and discovery of novel subtype
selective compounds. In this regard, the expression of the cDNA
encoding the GALR3 receptor in cultured cell lines and other cells
provides a unique tool for the discovery of therapeutic agents
targeted at galanin receptors.
[0462] The localization of GALR1 receptors to multiple brain
regions (Gustafson, et al., 1996; Parker, et al., 1995) and the
identification of GALR3 in a hypothalamic cDNA library, suggests
multiple therapeutic indications for the use of galanin
receptor-selective drugs. These include feeding, cognition,
analgesia and/or sensory processing, and anxiety and
depression.
[0463] The observation that galanin is co-released with
norepinephrine from sympathetic nerve terminals suggests that
galanin could act via galanin receptors in the periphery to
modulate nearly every physiological process controlled by
sympathetic innervation. Additional therapeutic indications not
directly related to localization include diabetes, hypertension,
cardiovascular disorders, regulation of growth hormone release,
regulation of fertility, gastric ulcers, gastrointestinal
motility/transit/absorption/secretion, glaucoma, inflammation,
immune disorders, respiratory disorders (e.g., asthma,
emphysema).
[0464] The localization, functional coupling, and pharmacology of
GALR3 suggest a number of physiological roles for this receptor in
the regulation of feeding, inhibition of neurotransmitter release
(i.e, acetylcholine, serotonin, and norepinephrine), regulation of
pituitary endocrine release, inhibition of glucose-stimulated
insulin release, and regulation of spinal cord excitability (Kask
et al., 1997). The involvement of GALR3 mRNA in spinal cord
function is particularly intriguing. GALR3 shows high binding
affinity (relative to galanin) for the alternately processed
galanin -7 to +29. This peptide and galanin -9 to +29 are found in
adrenal gland (Bersani et al., 1991) and modulate spinal
excitability, albeit with weaker potency than full length galanin
(Bedecs et al., 1994). Furthermore, a galanin-dependent inward
current appears in DRG only after axotomy, when galanin mRNA is
upregulated but GALR1 and GALR2 mRNA are decreased (Xu et al.,1997;
Sten Shi et al., 1997).
[0465] The physiological and anatomical distribution of
galanin-containing neurons suggests potential roles of galanin
receptors mediating effects on cognition, analgesia, neuroendocrine
regulation, control of insulin release and control of feeding
behavior. Of particular relevance to the role of the novel GALR3
receptor, are those functions mediated by galanin receptors in the
rat hypothalamus.
[0466] Studies in rats indicate that the injection of galanin in
the hypothalamus increases food intake (Kyrouli et al, 1990, and
Schick et al, 1993) and that this stimulatory effect of galanin is
blocked by prior administration of M40 and C7 (Liebowitz and Kim,
1992; and Corwin, 1993). The expression of the mRNA encoding the
GALR1 receptor in the rat hypothalamus (Parker et al., 1995;
Gustafson et al., 1996), and the fact that the novel GALR3 receptor
was identified in a cDNA library prepared from rat hypothalamus
argues in favor of the involvement of one or more galanin receptor
subtypes in the regulation of feeding behavior. However, the
original evidence against the involvement of GALR1 in the
stimulation of feeding behavior stems from the fact that M40 and C7
are known to be agonists, and not antagonists, in cell lines
expressing human and rat GALR1 receptors (Heuillet et al. 1994;
Hale et al. 1993; and Bartfai et al. 1993).
[0467] Peptide displacement assays indicate that the rat GALR3
receptor has a unique pharmacological profile. The low affinity for
M40, in particular, invites further speculation as to the
physiological role of the rat GALR3 receptor. It is noted that M40
was reported to be inactive, for example, when tested for
antagonism of galaninergic inhibition of glucose-stimulated insulin
release in rat pancreas, (Bartfai, 1993). In another example,
intrathecal M40 was a weak antagonist of the galanin-facilitated
flexor reflex in rat (Xu, 1995). It was observed in feeding assays
that M40 was less potent but as effective as galanin in stimulating
food intake when injected i.c.v. into rat brain. The data are
consistent with a role for the GALR3 receptor in a range of
physiologic and pathophysiologic functions including diabetes,
pain, obesity and eating disorders, and furthermore suggest that
the rat GALR3 receptor may represent a target for the design of
therapeutic compounds. The cloning of the rat GALR3 receptor
further enables the design and development of in vitro functional
assays to determine the agonist or antagonist properties of
peptides and drug development candidates.
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Sequence CWU 1
1
65 1 1280 DNA Rattus norvegicus 1 agctccagcc taggcgttct acctggaaga
atgcaggggc ccagtaccta ggactgagga 60 agatggctga catccagaac
atttcgctgg acagcccagg gagcgtaggg gctgtggcag 120 tgcctgtgat
ctttgccctc atcttcctgt tgggcatggt gggcaatggg ctggtgttgg 180
ctgtgctact gcagcctggc ccaagtgcct ggcaggagcc aagcagtacc acagatctct
240 tcatcctcaa cttggccgtg gccgaccttt gcttcatcct gtgctgcgtg
cccttccagg 300 cagccatcta cacactggat gcctggctct ttggggcttt
cgtgtgcaag acggtacatc 360 tgctcatcta cctcaccatg tatgccagca
gcttcaccct ggcggccgtc tccctggaca 420 ggtacctggc tgtgcggcac
ccactgcgct ccagagccct gcgcaccccg cgcaacgcgc 480 gcgccgccgt
ggggctcgtg tggctgctgg cggctctctt ttccgcgccc tacctaagct 540
attacggcac ggtgcgctac ggcgcgctcg agctctgcgt gcccgcttgg gaggacgcgc
600 ggcggcgcgc gctggacgtg gccaccttcg ccgcgggcta cctgctgccg
gtggccgtgg 660 tgagcctggc ctacggacgc acgctatgtt tcctatgggc
cgccgtgggt cccgcgggcg 720 cggcggcagc agaggcgcgc agacgggcga
ccggccgggc gggacgcgcc atgctggcag 780 tggccgcgct ctacgcgctt
tgctggggcc cgcaccacgc gctcatcctc tgcttctggt 840 acggccgctt
cgccttcagc ccggccacct acgcctgtcg cctggcctcg cactgcctcg 900
cctacgccaa ctcctgcctt aacccgctcg tctactcgct cgcctcgcgc cacttccgcg
960 cgcgcttccg ccgcctgtgg ccctgcggcc gtcgccgcca ccgccaccac
caccgcgctc 1020 atcgagccct ccgtcgtgtc cagccggcgt cttcgggccc
cgccggttat cccggcgacg 1080 ccaggcctcg tggttggagt atggagccca
gaggggatgc tctgcgtggt ggtggagaga 1140 ctagactaac cctgtccccc
aggggacctc aataaccctg cccgcttgga ctctgacgtc 1200 tgtcagaatg
ccaccaagga acatctaggg aacggcagtc tcgccaggct ccaccaaaaa 1260
gcagaagcaa agttgcaggg 1280 2 370 PRT Rattus norvegicus 2 Met Ala
Asp Ile Gln Asn Ile Ser Leu Asp Ser Pro Gly Ser Val Gly 1 5 10 15
Ala Val Ala Val Pro Val Ile Phe Ala Leu Ile Phe Leu Leu Gly Met 20
25 30 Val Gly Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro
Ser 35 40 45 Ala Trp Gln Glu Pro Ser Ser Thr Thr Asp Leu Phe Ile
Leu Asn Leu 50 55 60 Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys
Val Pro Phe Gln Ala 65 70 75 80 Ala Ile Tyr Thr Leu Asp Ala Trp Leu
Phe Gly Ala Phe Val Cys Lys 85 90 95 Thr Cys His Leu Leu Ile Tyr
Leu Thr Met Tyr Ala Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val Ser
Leu Asp Arg Tyr Leu Ala Val Arg His Pro Leu 115 120 125 Arg Ser Arg
Ala Leu Arg Thr Pro Arg Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu
Val Trp Leu Leu Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150
155 160 Tyr Gly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala
Trp 165 170 175 Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe
Ala Ala Gly 180 185 190 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala
Tyr Gly Arg Thr Leu 195 200 205 Cys Phe Leu Trp Ala Ala Val Gly Pro
Ala Gly Ala Ala Ala Ala Glu 210 215 220 Ala Arg Arg Arg Ala Thr Gly
Arg Ala Gly Arg Ala Met Leu Ala Val 225 230 235 240 Ala Ala Leu Tyr
Ala Leu Cys Trp Gly Pro His His Ala Leu Ile Leu 245 250 255 Cys Phe
Trp Tyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys 260 265 270
Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro 275
280 285 Leu Val Tyr Ser Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg
Arg 290 295 300 Leu Trp Pro Cys Gly Arg Arg Arg His Arg His His His
Arg Ala His 305 310 315 320 Arg Ala Leu Arg Arg Val Gln Pro Ala Ser
Ser Gly Pro Ala Gly Tyr 325 330 335 Pro Gly Asp Ala Arg Pro Arg Gly
Trp Ser Met Glu Pro Arg Gly Asp 340 345 350 Ala Leu Arg Gly Gly Gly
Glu Thr Arg Leu Thr Leu Ser Pro Arg Gly 355 360 365 Pro Gln 370 3
1417 DNA Homo sapiens 3 cactcagcga tgactttggc tctgctctcc cctcctccat
ctcccacgag cttccagccc 60 agaacacctg gccagaccca ggtcggggga
gttagatccc ggggtcaagc aaccagaact 120 gggggctctt gcctgaggat
tccagcttct cttcccaggt gcccgtctga tggggagatg 180 gctgatgccc
agaacatttc actggacagc ccagggagtg tgggggccgt ggcagtgcct 240
gtggtctttg ccctaatctt cctgctgggc acagtgggca atgggctggt gctggcagtg
300 ctcctgcagc ctggcccgag tgcctggcag gagcctggca gcaccacgga
cctgttcatc 360 ctcaacctgg cggtggctga cctctgcttc atcctgtgct
gcgtgccctt ccaggccacc 420 atctacacgc tggatgcctg gctctttggg
gccctcgtct gcaaggccgt gcacctgctc 480 atctacctca ccatgtacgc
cagcagcttt acgctggctg ctgtctccgt ggacaggtac 540 ctggccgtgc
ggcacccgct gcgctcgcgc gccctgcgca cgccgcgtaa cgcccgcgcc 600
gcagtggggc tggtgtggct gctggcggcg ctcttctcgg cgccctacct cagctactac
660 ggcaccgtgc gctacggcgc gctggagctc tgcgtgcccg cctgggagga
cgcgcgccgc 720 cgcgccctgg acgtggccac cttcgctgcc ggctacctgc
tgcccgtggc tgtggtgagc 780 ctggcctacg ggcgcacgct gcgcttcctg
tgggccgccg tgggtcccgc gggcgcggcg 840 gcggccgagg cgcggcggag
ggcgacgggc cgcgcggggc gcgccatgct ggcggtggcc 900 gcgctctacg
cgctctgctg gggtccgcac cacgcgctca tcctgtgctt ctggtacggc 960
cgcttcgcct tcagcccggc cacctacgcc tgccgcctgg cctcacactg cctggcctac
1020 gccaactcct gcctcaaccc gctcgtctac gcgctcgcct cgcgccactt
ccgcgcgcgc 1080 ttccgccgcc tgtggccgtg cggccgccga cgccgccacc
gtgcccgccg cgccttgcgt 1140 cgcgtccgcc ccgcgtcctc gggcccaccc
ggctgccccg gagacgcccg gcctagcggg 1200 aggctgctgg ctggtggcgg
ccagggcccg gagcccaggg agggacccgt ccacggcgga 1260 gaggctgccc
gaggaccgga ataaaccctg ccgcctggac tccgcctgtg tccgtctgtc 1320
tcactcccgt tctccgaagg cgggacgcca ccgggccagg gatggggcaa tgccacgagc
1380 tctctgaggg gcgttgagtg gagcgacttg tccccgc 1417 4 427 PRT Homo
sapiens 4 His Ser Ala Met Thr Leu Ala Leu Leu Ser Pro Pro Pro Ser
Pro Thr 1 5 10 15 Ser Phe Gln Pro Arg Thr Pro Gly Gln Thr Gln Val
Gly Gly Val Arg 20 25 30 Ser Arg Gly Gln Ala Thr Arg Thr Gly Gly
Ser Cys Leu Arg Ile Pro 35 40 45 Ala Ser Leu Pro Arg Cys Pro Ser
Asp Gly Glu Met Ala Asp Ala Gln 50 55 60 Asn Ile Ser Leu Asp Ser
Pro Gly Ser Val Gly Ala Val Ala Val Pro 65 70 75 80 Val Val Phe Ala
Leu Ile Phe Leu Leu Gly Thr Val Gly Asn Gly Leu 85 90 95 Val Leu
Ala Val Leu Leu Gln Pro Gly Pro Ser Ala Trp Gln Glu Pro 100 105 110
Gly Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu Ala Val Ala Asp Leu 115
120 125 Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala Thr Ile Tyr Thr
Leu 130 135 140 Asp Ala Trp Leu Phe Gly Ala Leu Val Cys Lys Ala Val
His Leu Leu 145 150 155 160 Ile Tyr Leu Thr Met Tyr Ala Ser Ser Phe
Thr Leu Ala Ala Val Ser 165 170 175 Val Asp Arg Tyr Leu Ala Val Arg
His Pro Leu Arg Ser Arg Ala Leu 180 185 190 Arg Thr Pro Arg Asn Ala
Arg Ala Ala Val Gly Leu Val Trp Leu Leu 195 200 205 Ala Ala Leu Phe
Ser Ala Pro Tyr Leu Ser Tyr Tyr Gly Thr Val Arg 210 215 220 Tyr Gly
Ala Leu Glu Leu Cys Val Pro Ala Trp Glu Asp Ala Arg Arg 225 230 235
240 Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly Tyr Leu Leu Pro Val
245 250 255 Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu Arg Phe Leu
Trp Ala 260 265 270 Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Glu Ala
Arg Arg Arg Ala 275 280 285 Thr Gly Arg Ala Gly Arg Ala Met Leu Ala
Val Ala Ala Leu Tyr Ala 290 295 300 Leu Cys Trp Gly Pro His His Ala
Leu Ile Leu Cys Phe Trp Tyr Gly 305 310 315 320 Arg Phe Ala Phe Ser
Pro Ala Thr Tyr Ala Cys Arg Leu Ala Ser His 325 330 335 Cys Leu Ala
Tyr Ala Asn Ser Cys Leu Asn Pro Leu Val Tyr Ala Leu 340 345 350 Ala
Ser Arg His Phe Arg Ala Arg Phe Arg Arg Leu Trp Pro Cys Gly 355 360
365 Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu Arg Arg Val Arg Pro
370 375 380 Ala Ser Ser Gly Pro Pro Gly Cys Pro Gly Asp Ala Arg Pro
Ser Gly 385 390 395 400 Arg Arg Leu Ala Gly Gly Gly Gln Gly Pro Glu
Pro Arg Glu Gly Pro 405 410 415 Val His Gly Gly Glu Ala Ala Arg Gly
Pro Glu 420 425 5 346 PRT Rattus norvegicus 5 Met Glu Leu Ala Pro
Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu Pro Pro
Ala Glu Pro Arg Pro Leu Phe Gly Ile Gly Val Glu Asn 20 25 30 Phe
Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu 35 40
45 Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Lys
50 55 60 Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile
Ala Asp 65 70 75 80 Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala
Thr Val Tyr Ala 85 90 95 Leu Pro Thr Trp Val Leu Gly Ala Phe Ile
Cys Lys Phe Ile His Tyr 100 105 110 Phe Phe Thr Val Ser Met Leu Val
Ser Ile Phe Thr Leu Ala Ala Met 115 120 125 Ser Val Asp Arg Tyr Val
Ala Ile Val His Ser Arg Arg Ser Ser Ser 130 135 140 Leu Arg Val Ser
Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp Ala 145 150 155 160 Leu
Ser Ile Ala Met Ala Ser Pro Val Ala Tyr Tyr Gln Arg Leu Phe 165 170
175 His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu His Trp Pro Asn Gln
180 185 190 Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly
Tyr Leu 195 200 205 Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys
Val Leu Asn His 210 215 220 Leu His Lys Lys Leu Lys Asn Met Ser Lys
Lys Ser Glu Ala Ser Lys 225 230 235 240 Lys Lys Thr Ala Gln Thr Val
Leu Val Val Val Val Val Phe Gly Ile 245 250 255 Ser Trp Leu Pro His
His Val Ile His Leu Trp Ala Glu Phe Gly Ala 260 265 270 Phe Pro Leu
Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His Cys 275 280 285 Leu
Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe Leu 290 295
300 Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys Arg Val
305 310 315 320 Cys Asn Glu Ser Pro His Gly Asp Ala Lys Glu Lys Asn
Arg Ile Asp 325 330 335 Thr Pro Pro Ser Thr Asn Cys Thr His Val 340
345 6 45 DNA Artificial Sequence Description of Artificial Sequence
oligonucleotide/probe 6 ttgtacccct atttttcgcg ctcatcttcc tcgtgggcac
cgtgg 45 7 45 DNA Artificial Sequence Description of Artificial
Sequence oligonucleotide/probe 7 agcaccgcca gcaccagcgc gttgcccacg
gtgcccacga ggaag 45 8 50 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide/probe 8 tcagcaccac caacctgttc
atcctcaacc tgggcgtggc cgacctgtgt 50 9 50 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide/probe 9
ggcctggaaa ggcacgcagc acaggatgaa acacaggtcg gccacgccca 50 10 45 DNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide/probe 10 ctgcaaggct gttcatttcc tcatctttct
cactatgcac gccag 45 11 45 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide/probe 11 ggagacggcg gccagcgtga
agctgctggc gtgcatagtg agaaa 45 12 45 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide/probe 12
aacgcgctgg ccgccatcgg gctcatctgg gggctagcac tgctc 45 13 45 DNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide/probe 13 agtagctcag gtagggcccg gagaagagca
gtgctagccc ccaga 45 14 45 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide/probe 14 agccatggac ctctgcacct
tcgtctttag ctacctgctg ccagt 45 15 45 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide/probe 15
cgcataggtc agactgagga ctagcactgg cagcaggtag ctaaa 45 16 45 DNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide/probe 16 gatcatcatc gtggcggtgc ttttctgcct
ctgttggatg cccca 45 17 45 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide/probe 17 ccacacgcag aggataagcg
cgtggtgggg catccaacag aggca 45 18 45 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide/probe 18
gttgcgcatc ctttcacacc tagtttccta tgccaactcc tgtgt 45 19 46 DNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide/probe 19 agaccagagc gtaaacgatg gggttgacac
aggagttggc atagga 46 20 24 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 20 cctcagtgaa gggaatggga gcga 24 21
27 DNA Artificial Sequence Description of Artificial Sequence PCR
primer 21 gtagtgtata aacttgcaga tgaaggc 27 22 23 DNA Artificial
Sequence Description of Artificial Sequence PCR primer 22
atgaatggct ccggcagcca ggg 23 23 23 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 23 ttgcagagca
gcgagccgaa cac 23 24 24 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 24 ggctgacatc cagaacattt cgct 24 25
24 DNA Artificial Sequence Description of Artificial Sequence PCR
primer 25 cagatgtacc gtcttgcaca cgaa 24 26 24 DNA Artificial
Sequence Description of Artificial Sequence PCR primer 26
catctgctca tctacctcac catg 24 27 24 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 27 cataggaaac
atagcgtgcg tccg 24 28 25 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 28 aagcttctag agatccctcg acctc 25 29
25 DNA Artificial Sequence Description of Artificial Sequence PCR
primer 29 aggcgcagaa ctggtaggta tggaa 25 30 23 DNA Artificial
Sequence Description of Artificial Sequence PCR primer 30
gctcatcctc tgcttctggt acg 23 31 24 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 31 cagatgtacc
gtcttgcaca cgaa 24 32 34 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 32 cgaggatccc aactttgcct ctgctttttg
gtgg 34 33 24 DNA Artificial Sequence Description of Artificial
Sequence PCR primer 33 cctcagtgaa gggaatggga gcga 24 34 24 DNA
Artificial Sequence Description of Artificial Sequence PCR primer
34 cttgcttgta cgccttccgg aagt 24 35 24 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 35 tgggcaacag
cctagtgatc accg 24 36 24 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 36 ctgctcccag cagaaggtct ggtt 24 37
23 DNA Artificial Sequence Description of Artificial Sequence PCR
primer 37 atgaatggct ccggcagcca ggg 23 38 25 DNA Artificial
Sequence Description of Artificial Sequence oligonucleotide/probe
38 ttggagacca gagcgtaaac gatgg 25 39 45 DNA Artificial Sequence
Description of Artificial Sequence oligonucleotide/probe 39
agatggctga catccagaac atttcgctgg acagcccagg gagcg 45 40 46 DNA
Artificial Sequence Description of Artificial Sequence primer 40
atcacaggca ctgccacagc ccctacgctc cctgggctgt ccagcg 46 41 25 DNA
Artificial Sequence Description of Artificial Sequence primer 41
atggctgatg cccagaacat ttcac 25 42 23 DNA Artificial Sequence
Description of Artificial Sequence primer 42 agccaggcat ccagcgtgta
gat 23 43 45 DNA Artificial Sequence Description of Artificial
Sequence probe 43 acggtcgctt cgccttcagc ccggccacct acgcctgtcg cctgg
45 44 45 DNA Artificial Sequence Description of Artificial Sequence
probe
44 acggtcgctt cgccttcagc ccggccacct acgcctgtcg cctgg 45 45 45 DNA
Artificial Sequence Description of Artificial Sequence probe 45
gcgcaacgcg cgcgccgccg tggggctcgt gtggctgctg gcggc 45 46 45 DNA
Artificial Sequence Description of Artificial Sequence
oligonucleotide/primer 46 atctacacgc tggatgcctg gctctttggg
gccctcgtct gcaag 45 47 24 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide/primer 47 atctacacgc tggatgccct
ggct 24 48 22 DNA Artificial Sequence Description of Artificial
Sequence oligonucleotide/primer 48 cgtagcgcac ggtgccgtag ta 22 49
40 DNA Artificial Sequence Description of Artificial Sequence
oligonucleotide/primer 49 gatggatccg ccaccatggc tgatgcccag
aacatttcac 40 50 28 DNA Artificial Sequence Description of
Artificial Sequence oligonucleotide/primer 50 gcaggtacct gtccacggag
acagcagc 28 51 45 DNA Artificial Sequence Description of Artificial
Sequence probe 51 gatggctgat gcccagaaca tttcactgga cagcccaggg agtgt
45 52 45 DNA Artificial Sequence Description of Artificial Sequence
probe 52 gaccacaggc actgccacgg cccccacact ccctgggctg tccag 45 53 45
DNA Artificial Sequence Description of Artificial Sequence probe 53
tgcagcctgg cccaagtgcc tggcaggagc caagcagtac cacag 45 54 37 DNA
Artificial Sequence Description of Artificial Sequence primer 54
cgcggatcca ttatgtctgc actccgaagg aaatttg 37 55 38 DNA Artificial
Sequence Description of Artificial Sequence primer 55 cgcgaattct
tatgtgaagc gatcagagtt catttttc 38 56 34 DNA Artificial Sequence
Description of Artificial Sequence primer 56 gcgggatccg ctatggctgg
tgattctagg aatg 34 57 29 DNA Artificial Sequence Description of
Artificial Sequence primer 57 ccggaattcc cctcacaccg agcccctgg 29 58
50 DNA Artificial Sequence Description of Artificial Sequence
primer 58 ccaagcttct aatacgactc actatagggc caccatggct gatgcccaga 50
59 57 DNA Artificial Sequence Description of Artificial Sequence
primer 59 tttttttttt tttttttttt tttttttttt tttttgcagg gtttattccg
gtcctcg 57 60 27 DNA Artificial Sequence Description of Artificial
Sequence primer 60 tcagcggcac catgaacgtc tcgggct 27 61 24 DNA
Artificial Sequence Description of Artificial Sequence primer 61
ggccacatca accgtcagga tgct 24 62 25 DNA Artificial Sequence
Description of Artificial Sequence primer 62 atggctgatg cccagaacat
ttcac 25 63 28 DNA Artificial Sequence Description of Artificial
Sequence primer 63 tagcgcacgg tgccgtagta gctgaggt 28 64 26 DNA
Artificial Sequence Description of Artificial Sequence primer 64
atgaaagggt ccctcctgct gctgct 26 65 26 DNA Artificial Sequence
Description of Artificial Sequence primer 65 tatcagctcc atgccctcta
gaagcc 26
* * * * *