WO1998015570A1 - Dna encoding galanin galr3 receptors and uses thereof - Google Patents
Dna encoding galanin galr3 receptors and uses thereof Download PDFInfo
- Publication number
- WO1998015570A1 WO1998015570A1 PCT/US1997/018222 US9718222W WO9815570A1 WO 1998015570 A1 WO1998015570 A1 WO 1998015570A1 US 9718222 W US9718222 W US 9718222W WO 9815570 A1 WO9815570 A1 WO 9815570A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- galr3 receptor
- receptor
- galr3
- cell
- compound
- Prior art date
Links
- 101800002068 Galanin Proteins 0.000 title claims description 67
- SLZIZIJTGAYEKK-CIJSCKBQSA-N molport-023-220-247 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CNC=N1 SLZIZIJTGAYEKK-CIJSCKBQSA-N 0.000 title claims description 66
- 102000019432 Galanin Human genes 0.000 title claims description 65
- 150000001875 compounds Chemical class 0.000 claims abstract description 571
- 238000000034 method Methods 0.000 claims abstract description 358
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 173
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 157
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 157
- 230000027455 binding Effects 0.000 claims abstract description 115
- 238000009739 binding Methods 0.000 claims abstract description 114
- 102000011392 Galanin receptor Human genes 0.000 claims abstract description 78
- 108050001605 Galanin receptor Proteins 0.000 claims abstract description 77
- 239000013598 vector Substances 0.000 claims abstract description 54
- 241001465754 Metazoa Species 0.000 claims abstract description 36
- 230000009261 transgenic effect Effects 0.000 claims abstract description 33
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims abstract description 32
- 239000002853 nucleic acid probe Substances 0.000 claims abstract description 32
- 230000000295 complement effect Effects 0.000 claims abstract description 23
- 239000000074 antisense oligonucleotide Substances 0.000 claims abstract description 9
- 238000012230 antisense oligonucleotides Methods 0.000 claims abstract description 9
- 101001072777 Homo sapiens Galanin receptor type 3 Proteins 0.000 claims description 893
- 102100036588 Galanin receptor type 3 Human genes 0.000 claims description 825
- 210000004027 cell Anatomy 0.000 claims description 431
- 230000008569 process Effects 0.000 claims description 174
- 108020004414 DNA Proteins 0.000 claims description 123
- 239000013612 plasmid Substances 0.000 claims description 120
- 230000004913 activation Effects 0.000 claims description 110
- 150000001413 amino acids Chemical group 0.000 claims description 98
- 108020003175 receptors Proteins 0.000 claims description 96
- 102000005962 receptors Human genes 0.000 claims description 94
- 230000014509 gene expression Effects 0.000 claims description 82
- 230000000694 effects Effects 0.000 claims description 81
- 239000005557 antagonist Substances 0.000 claims description 65
- 239000012528 membrane Substances 0.000 claims description 62
- 108020004999 messenger RNA Proteins 0.000 claims description 62
- 239000000523 sample Substances 0.000 claims description 62
- 102000048410 human GALR3 Human genes 0.000 claims description 60
- XOFLBQFBSOEHOG-UUOKFMHZSA-N γS-GTP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=S)[C@@H](O)[C@H]1O XOFLBQFBSOEHOG-UUOKFMHZSA-N 0.000 claims description 58
- 230000004044 response Effects 0.000 claims description 54
- 210000004962 mammalian cell Anatomy 0.000 claims description 51
- 239000008194 pharmaceutical composition Substances 0.000 claims description 48
- 230000005856 abnormality Effects 0.000 claims description 47
- 239000000556 agonist Substances 0.000 claims description 47
- 125000003729 nucleotide group Chemical group 0.000 claims description 47
- 101100447617 Rattus norvegicus Galr3 gene Proteins 0.000 claims description 44
- 239000002773 nucleotide Substances 0.000 claims description 44
- 229940044551 receptor antagonist Drugs 0.000 claims description 44
- 239000002464 receptor antagonist Substances 0.000 claims description 44
- 239000012634 fragment Substances 0.000 claims description 43
- 229940044601 receptor agonist Drugs 0.000 claims description 42
- 239000000018 receptor agonist Substances 0.000 claims description 42
- 210000000287 oocyte Anatomy 0.000 claims description 39
- 230000007423 decrease Effects 0.000 claims description 38
- 239000003937 drug carrier Substances 0.000 claims description 35
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 32
- 241000124008 Mammalia Species 0.000 claims description 32
- 230000003834 intracellular effect Effects 0.000 claims description 31
- 230000002441 reversible effect Effects 0.000 claims description 30
- 238000012216 screening Methods 0.000 claims description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 25
- 208000035475 disorder Diseases 0.000 claims description 25
- 230000037406 food intake Effects 0.000 claims description 25
- 235000012631 food intake Nutrition 0.000 claims description 25
- 230000001105 regulatory effect Effects 0.000 claims description 23
- 241000238631 Hexapoda Species 0.000 claims description 22
- 230000002829 reductive effect Effects 0.000 claims description 22
- 230000008859 change Effects 0.000 claims description 21
- 101001072780 Homo sapiens Galanin receptor type 2 Proteins 0.000 claims description 20
- 108091034117 Oligonucleotide Proteins 0.000 claims description 19
- 102100036584 Galanin receptor type 2 Human genes 0.000 claims description 18
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 18
- 210000003292 kidney cell Anatomy 0.000 claims description 18
- 230000036515 potency Effects 0.000 claims description 18
- 102100033063 G protein-activated inward rectifier potassium channel 1 Human genes 0.000 claims description 17
- 101000944266 Homo sapiens G protein-activated inward rectifier potassium channel 1 Proteins 0.000 claims description 17
- 102100021237 G protein-activated inward rectifier potassium channel 4 Human genes 0.000 claims description 16
- 101000614712 Homo sapiens G protein-activated inward rectifier potassium channel 4 Proteins 0.000 claims description 16
- 230000001965 increasing effect Effects 0.000 claims description 16
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 15
- 235000013305 food Nutrition 0.000 claims description 15
- 241000269370 Xenopus <genus> Species 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
- 230000004634 feeding behavior Effects 0.000 claims description 13
- 230000005764 inhibitory process Effects 0.000 claims description 12
- 230000009870 specific binding Effects 0.000 claims description 12
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 11
- 230000003247 decreasing effect Effects 0.000 claims description 11
- 239000011591 potassium Substances 0.000 claims description 11
- 229910052700 potassium Inorganic materials 0.000 claims description 11
- 208000002193 Pain Diseases 0.000 claims description 10
- 206010012601 diabetes mellitus Diseases 0.000 claims description 10
- 210000002569 neuron Anatomy 0.000 claims description 10
- 230000036407 pain Effects 0.000 claims description 10
- 229940123935 Galanin receptor agonist Drugs 0.000 claims description 9
- 238000013519 translation Methods 0.000 claims description 9
- 229940121970 Galanin receptor antagonist Drugs 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 238000012217 deletion Methods 0.000 claims description 8
- 230000037430 deletion Effects 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 102100029549 Neuropeptide Y receptor type 5 Human genes 0.000 claims description 7
- 101710198055 Neuropeptide Y receptor type 5 Proteins 0.000 claims description 7
- 210000000170 cell membrane Anatomy 0.000 claims description 7
- 230000001939 inductive effect Effects 0.000 claims description 7
- 230000001537 neural effect Effects 0.000 claims description 7
- 108700028369 Alleles Proteins 0.000 claims description 6
- 101000860415 Homo sapiens Galanin peptides Proteins 0.000 claims description 6
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 6
- 230000000692 anti-sense effect Effects 0.000 claims description 6
- 230000009137 competitive binding Effects 0.000 claims description 6
- 238000003745 diagnosis Methods 0.000 claims description 6
- 102000050963 human GAL Human genes 0.000 claims description 6
- 239000003446 ligand Substances 0.000 claims description 6
- 239000003550 marker Substances 0.000 claims description 6
- 230000020341 sensory perception of pain Effects 0.000 claims description 6
- 102000004257 Potassium Channel Human genes 0.000 claims description 5
- 230000003542 behavioural effect Effects 0.000 claims description 5
- 238000002744 homologous recombination Methods 0.000 claims description 5
- 230000006801 homologous recombination Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 210000003574 melanophore Anatomy 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 108020001213 potassium channel Proteins 0.000 claims description 5
- 230000001766 physiological effect Effects 0.000 claims description 4
- 241000701447 unidentified baculovirus Species 0.000 claims description 4
- 210000005253 yeast cell Anatomy 0.000 claims description 4
- 108020004491 Antisense DNA Proteins 0.000 claims description 3
- 108020005544 Antisense RNA Proteins 0.000 claims description 3
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 3
- 241000282465 Canis Species 0.000 claims description 3
- 108090000994 Catalytic RNA Proteins 0.000 claims description 3
- 102000053642 Catalytic RNA Human genes 0.000 claims description 3
- 239000003816 antisense DNA Substances 0.000 claims description 3
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 108091092562 ribozyme Proteins 0.000 claims description 3
- 238000004513 sizing Methods 0.000 claims description 3
- 206010037211 Psychomotor hyperactivity Diseases 0.000 claims description 2
- 241000283984 Rodentia Species 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 238000013268 sustained release Methods 0.000 claims description 2
- 239000012730 sustained-release form Substances 0.000 claims description 2
- 108020000948 Antisense Oligonucleotides Proteins 0.000 abstract 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 88
- 238000003752 polymerase chain reaction Methods 0.000 description 58
- 235000001014 amino acid Nutrition 0.000 description 49
- 229940024606 amino acid Drugs 0.000 description 48
- 108090000623 proteins and genes Proteins 0.000 description 37
- 241000700159 Rattus Species 0.000 description 30
- 108091026890 Coding region Proteins 0.000 description 24
- 239000000047 product Substances 0.000 description 23
- 210000001519 tissue Anatomy 0.000 description 22
- 238000009396 hybridization Methods 0.000 description 19
- 108090000765 processed proteins & peptides Proteins 0.000 description 19
- 210000004556 brain Anatomy 0.000 description 18
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 15
- 108020004705 Codon Proteins 0.000 description 13
- 238000003556 assay Methods 0.000 description 13
- 210000003016 hypothalamus Anatomy 0.000 description 13
- 238000000159 protein binding assay Methods 0.000 description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 12
- 239000002609 medium Substances 0.000 description 12
- SLZIZIJTGAYEKK-UHFFFAOYSA-N porcine galanin Chemical compound C=1NC=NC=1CC(C(=O)NC(CCCNC(N)=N)C(=O)NC(CO)C(=O)NC(CC=1C=CC=CC=1)C(=O)NC(CC=1NC=NC=1)C(=O)NC(CC(O)=O)C(=O)NC(CCCCN)C(=O)NC(CC=1C=CC(O)=CC=1)C(=O)NCC(=O)NC(CC(C)C)C(=O)NC(C)C(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CC(O)=O)NC(=O)C(C(C)CC)NC(=O)C(C)NC(=O)C(NC(=O)C1N(CCC1)C(=O)CNC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)C(C)NC(=O)C(CO)NC(=O)C(CC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)CN)C(C)O)CC1=CN=CN1 SLZIZIJTGAYEKK-UHFFFAOYSA-N 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 238000007792 addition Methods 0.000 description 11
- 238000013459 approach Methods 0.000 description 11
- 239000000499 gel Substances 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 10
- 102000001972 Galanin receptor 3 Human genes 0.000 description 10
- 102000043136 MAP kinase family Human genes 0.000 description 10
- 108091054455 MAP kinase family Proteins 0.000 description 10
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 10
- 101100447614 Rattus norvegicus Galr2 gene Proteins 0.000 description 10
- 102000030621 adenylate cyclase Human genes 0.000 description 10
- 108060000200 adenylate cyclase Proteins 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 210000003169 central nervous system Anatomy 0.000 description 10
- 238000005259 measurement Methods 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- 238000012163 sequencing technique Methods 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 208000024827 Alzheimer disease Diseases 0.000 description 9
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 9
- 108050009372 Galanin receptor 3 Proteins 0.000 description 9
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 9
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 9
- 229940114079 arachidonic acid Drugs 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- 244000309466 calf Species 0.000 description 9
- 229940095074 cyclic amp Drugs 0.000 description 9
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- -1 peptidyl compound Chemical class 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical class CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 229930182555 Penicillin Natural products 0.000 description 8
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 8
- 235000021342 arachidonic acid Nutrition 0.000 description 8
- 239000011575 calcium Substances 0.000 description 8
- 229910052791 calcium Inorganic materials 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 229960000367 inositol Drugs 0.000 description 8
- 229940049954 penicillin Drugs 0.000 description 8
- 230000000144 pharmacologic effect Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 8
- 210000000278 spinal cord Anatomy 0.000 description 8
- 229960005322 streptomycin Drugs 0.000 description 8
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 7
- 108091006027 G proteins Proteins 0.000 description 7
- 102000030782 GTP binding Human genes 0.000 description 7
- 108091000058 GTP-Binding Proteins 0.000 description 7
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 239000013589 supplement Substances 0.000 description 7
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 6
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 6
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- 235000013601 eggs Nutrition 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 238000002825 functional assay Methods 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 230000001817 pituitary effect Effects 0.000 description 6
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 101150082479 GAL gene Proteins 0.000 description 5
- 239000007995 HEPES buffer Substances 0.000 description 5
- 229930182816 L-glutamine Natural products 0.000 description 5
- 238000000636 Northern blotting Methods 0.000 description 5
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 5
- 102000014384 Type C Phospholipases Human genes 0.000 description 5
- 108010079194 Type C Phospholipases Proteins 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 230000036592 analgesia Effects 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 230000002267 hypothalamic effect Effects 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 239000002751 oligonucleotide probe Substances 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 229940124597 therapeutic agent Drugs 0.000 description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 108010001478 Bacitracin Proteins 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108020004635 Complementary DNA Proteins 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 108020004566 Transfer RNA Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 229960003071 bacitracin Drugs 0.000 description 4
- 229930184125 bacitracin Natural products 0.000 description 4
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 230000002496 gastric effect Effects 0.000 description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 4
- 210000001320 hippocampus Anatomy 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 238000005567 liquid scintillation counting Methods 0.000 description 4
- 239000003226 mitogen Substances 0.000 description 4
- OSGCBUDLRBUEGW-JZCUZNMGSA-N molport-023-276-806 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(N)=O)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CC=C(O)C=C1 OSGCBUDLRBUEGW-JZCUZNMGSA-N 0.000 description 4
- 210000001672 ovary Anatomy 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 210000002460 smooth muscle Anatomy 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 241000972773 Aulopiformes Species 0.000 description 3
- 108091006146 Channels Proteins 0.000 description 3
- 241000699802 Cricetulus griseus Species 0.000 description 3
- 229920002307 Dextran Polymers 0.000 description 3
- 208000030814 Eating disease Diseases 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 208000019454 Feeding and Eating disease Diseases 0.000 description 3
- 101100447612 Homo sapiens GALR2 gene Proteins 0.000 description 3
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 3
- 239000006137 Luria-Bertani broth Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- ZPHBZEQOLSRPAK-UHFFFAOYSA-N Phosphoramidon Natural products C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O ZPHBZEQOLSRPAK-UHFFFAOYSA-N 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 3
- 101500025322 Rattus norvegicus Galanin Proteins 0.000 description 3
- 238000012300 Sequence Analysis Methods 0.000 description 3
- 238000002105 Southern blotting Methods 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 210000000133 brain stem Anatomy 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000019771 cognition Effects 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 3
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 3
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 229960002086 dextran Drugs 0.000 description 3
- 150000001982 diacylglycerols Chemical class 0.000 description 3
- 235000014632 disordered eating Nutrition 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 3
- 108010052968 leupeptin Proteins 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 210000004498 neuroglial cell Anatomy 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 210000002963 paraventricular hypothalamic nucleus Anatomy 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000010412 perfusion Effects 0.000 description 3
- BWSDNRQVTFZQQD-AYVHNPTNSA-N phosphoramidon Chemical compound O([P@@](O)(=O)N[C@H](CC(C)C)C(=O)N[C@H](CC=1[C]2C=CC=CC2=NC=1)C(O)=O)[C@H]1O[C@@H](C)[C@H](O)[C@@H](O)[C@@H]1O BWSDNRQVTFZQQD-AYVHNPTNSA-N 0.000 description 3
- 108010072906 phosphoramidon Proteins 0.000 description 3
- 108091005981 phosphorylated proteins Proteins 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 235000019515 salmon Nutrition 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 208000019901 Anxiety disease Diseases 0.000 description 2
- 108010039627 Aprotinin Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 208000032841 Bulimia Diseases 0.000 description 2
- 206010006550 Bulimia nervosa Diseases 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 241001559589 Cullen Species 0.000 description 2
- 108010017826 DNA Polymerase I Proteins 0.000 description 2
- 102000004594 DNA Polymerase I Human genes 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 108090000862 Ion Channels Proteins 0.000 description 2
- 102000004310 Ion Channels Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 208000019695 Migraine disease Diseases 0.000 description 2
- 102000047918 Myelin Basic Human genes 0.000 description 2
- 101710107068 Myelin basic protein Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 208000008589 Obesity Diseases 0.000 description 2
- 108010081690 Pertussis Toxin Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 108020004518 RNA Probes Proteins 0.000 description 2
- 239000003391 RNA probe Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 210000001943 adrenal medulla Anatomy 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 2
- 210000004727 amygdala Anatomy 0.000 description 2
- 208000022531 anorexia Diseases 0.000 description 2
- 230000036506 anxiety Effects 0.000 description 2
- 229960004405 aprotinin Drugs 0.000 description 2
- 239000012131 assay buffer Substances 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 238000000376 autoradiography Methods 0.000 description 2
- 210000003050 axon Anatomy 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 208000015114 central nervous system disease Diseases 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 206010061428 decreased appetite Diseases 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 238000001400 expression cloning Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 230000003054 hormonal effect Effects 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 2
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 235000006109 methionine Nutrition 0.000 description 2
- 206010027599 migraine Diseases 0.000 description 2
- 238000001823 molecular biology technique Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 2
- 230000004973 motor coordination Effects 0.000 description 2
- 230000000955 neuroendocrine Effects 0.000 description 2
- 208000015706 neuroendocrine disease Diseases 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 235000020824 obesity Nutrition 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000027425 release of sequestered calcium ion into cytosol Effects 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000000284 resting effect Effects 0.000 description 2
- 238000003572 second messenger assay Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 208000019116 sleep disease Diseases 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- ZNJHFNUEQDVFCJ-UHFFFAOYSA-M sodium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical compound [OH-].[Na+].OCCN1CCN(CCS(O)(=O)=O)CC1 ZNJHFNUEQDVFCJ-UHFFFAOYSA-M 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000003594 spinal ganglia Anatomy 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000002889 sympathetic effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical group CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- KDELTXNPUXUBMU-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid boric acid Chemical compound OB(O)O.OB(O)O.OB(O)O.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KDELTXNPUXUBMU-UHFFFAOYSA-N 0.000 description 1
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 108060003345 Adrenergic Receptor Proteins 0.000 description 1
- 102000017910 Adrenergic receptor Human genes 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical compound OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 206010014418 Electrolyte imbalance Diseases 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 102100022466 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 description 1
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical group [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 108010041667 G Protein-Coupled Inwardly-Rectifying Potassium Channels Proteins 0.000 description 1
- 102000000542 G Protein-Coupled Inwardly-Rectifying Potassium Channels Human genes 0.000 description 1
- 101710169265 Galanin peptides Proteins 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 101150112014 Gapdh gene Proteins 0.000 description 1
- 208000017228 Gastrointestinal motility disease Diseases 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010078321 Guanylate Cyclase Proteins 0.000 description 1
- 102000014469 Guanylate cyclase Human genes 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 206010022714 Intestinal ulcer Diseases 0.000 description 1
- 102100024319 Intestinal-type alkaline phosphatase Human genes 0.000 description 1
- 101710184243 Intestinal-type alkaline phosphatase Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000001291 MAP Kinase Kinase Kinase Human genes 0.000 description 1
- 108060006687 MAP kinase kinase kinase Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 108050002826 Neuropeptide Y Receptor Proteins 0.000 description 1
- 102000012301 Neuropeptide Y receptor Human genes 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000000114 Pain Threshold Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 229940083963 Peptide antagonist Drugs 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 102000003923 Protein Kinase C Human genes 0.000 description 1
- 108090000315 Protein Kinase C Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108010019653 Pwo polymerase Proteins 0.000 description 1
- 238000010357 RNA editing Methods 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 101100121084 Rattus norvegicus Gal gene Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 208000020764 Sensation disease Diseases 0.000 description 1
- 108091081021 Sense strand Proteins 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- RZCIEJXAILMSQK-JXOAFFINSA-N TTP Chemical compound O=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 RZCIEJXAILMSQK-JXOAFFINSA-N 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 208000030886 Traumatic Brain injury Diseases 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 241000269368 Xenopus laevis Species 0.000 description 1
- ZHAFUINZIZIXFC-UHFFFAOYSA-N [9-(dimethylamino)-10-methylbenzo[a]phenoxazin-5-ylidene]azanium;chloride Chemical compound [Cl-].O1C2=CC(=[NH2+])C3=CC=CC=C3C2=NC2=C1C=C(N(C)C)C(C)=C2 ZHAFUINZIZIXFC-UHFFFAOYSA-N 0.000 description 1
- QPMSXSBEVQLBIL-CZRHPSIPSA-N ac1mix0p Chemical compound C1=CC=C2N(C[C@H](C)CN(C)C)C3=CC(OC)=CC=C3SC2=C1.O([C@H]1[C@]2(OC)C=CC34C[C@@H]2[C@](C)(O)CCC)C2=C5[C@]41CCN(C)[C@@H]3CC5=CC=C2O QPMSXSBEVQLBIL-CZRHPSIPSA-N 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 230000003502 anti-nociceptive effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 238000013320 baculovirus expression vector system Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 238000000339 bright-field microscopy Methods 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 230000003491 cAMP production Effects 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 108091006116 chimeric peptides Proteins 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012411 cloning technique Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 230000003931 cognitive performance Effects 0.000 description 1
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 229940009976 deoxycholate Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000003291 dopaminomimetic effect Effects 0.000 description 1
- 210000005110 dorsal hippocampus Anatomy 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 230000035611 feeding Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000003269 fluorescent indicator Substances 0.000 description 1
- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 108700039708 galantide Proteins 0.000 description 1
- TZOJVPDIYKRJSM-GKPUQKAJSA-N galantide Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CC=CC=C1 TZOJVPDIYKRJSM-GKPUQKAJSA-N 0.000 description 1
- 208000021302 gastroesophageal reflux disease Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 230000000971 hippocampal effect Effects 0.000 description 1
- 231100000508 hormonal effect Toxicity 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000008765 hyperinnervation Effects 0.000 description 1
- 230000004179 hypothalamic–pituitary–adrenal axis Effects 0.000 description 1
- YZXBAPSDXZZRGB-UHFFFAOYSA-N icosa-5,8,11,14-tetraenoic acid Chemical compound CCCCCC=CCC=CCC=CCC=CCCCC(O)=O YZXBAPSDXZZRGB-UHFFFAOYSA-N 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 206010022498 insulinoma Diseases 0.000 description 1
- 210000001153 interneuron Anatomy 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 150000002742 methionines Chemical class 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 230000008747 mitogenic response Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229960005181 morphine Drugs 0.000 description 1
- 201000003152 motion sickness Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000037040 pain threshold Effects 0.000 description 1
- 208000021255 pancreatic insulinoma Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 150000003906 phosphoinositides Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000003566 phosphorylation assay Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920006316 polyvinylpyrrolidine Polymers 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 230000009696 proliferative response Effects 0.000 description 1
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 description 1
- 210000004129 prosencephalon Anatomy 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000002287 radioligand Substances 0.000 description 1
- 238000003653 radioligand binding assay Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000003345 scintillation counting Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000001044 sensory neuron Anatomy 0.000 description 1
- 230000031893 sensory processing Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000012868 site-directed mutagenesis technique Methods 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 230000003238 somatosensory effect Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000013222 sprague-dawley male rat Methods 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 230000009529 traumatic brain injury Effects 0.000 description 1
- ZMCBYSBVJIMENC-UHFFFAOYSA-N tricaine Chemical compound CCOC(=O)C1=CC=CC(N)=C1 ZMCBYSBVJIMENC-UHFFFAOYSA-N 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 210000005111 ventral hippocampus Anatomy 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- 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.
- DRG dorsal root ganglia
- Galanin released from sympathetic nerve terminals in the pancreas is a potent regulator of insulin release in several species (Ahren and Lindskog, 1992; Boyle et al .
- 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 .
- Galanin receptors elsewhere in the CNS may also serve as therapeutic targets.
- galanin 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
- 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 2 ] - and [D-Phe 2 ] -galanin analogues (Lagny-Pourmir et al . , 1989).
- the chimeric galanin analogue M35 displaces 125 I-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).
- 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 proteolytic processing may release bioactive fragments of galanin to act at distinct receptors.
- the rat pituitary exhibits high-affinity binding for 125 I- Bolton and Hunter (N-terminus) -labeled galanin (1-29) but not for [ 125 I] Tyr 26 -porcine galanin (Wynick et al . , 1993), suggesting that the pituitary galanin receptor is a C- terminus-preferring subtype.
- a galanin receptor cDNA was recently isolated by expression cloning from a human Bowes melanoma cell line
- 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 GALRl.
- the cloned human GALRl receptor (“hGALRl") binds native human, porcine and rat galanin with ⁇ 1 nM affinity (K vs. 125 I -galanin) and porcine galanin 1-16 at a slightly lower affinity ( ⁇ 5nM) . Porcine galanin 3-29 does not bind to the receptor.
- the GALRl 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 ⁇ .M.
- rGALRl rat homologue of GALRl
- the pharmacologic data reported to date do not suggest substantial differences between the pharmacologic properties of the rat and human GALRl receptors.
- Localization studies reveal GALRl 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.
- GALRl appears to be distinct from the pituitary and hippocampal receptor subtypes described above .
- GALR2 novel galanin receptor
- GLR3 galanin receptor subtype
- 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 rat 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.
- treatment of such disorders may be effected by the administration of GALR3 receptor-selective compounds.
- 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.
- 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.
- This invention further provides a vector comprising the GALR3 receptor.
- 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.
- 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.
- 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 Figure 1 (Seq. ID No. 1) or (b) the reverse complement to the nucleic acid sequence shown in Figure 1 (Seq. ID No. 1) .
- the GALR3 receptor is the rat GALR3 receptor having substantially the same amino acid sequence as the amino acid sequence shown in Figure 2. In another embodiment, the GALR3 receptor is the rat GALR3 receptor having the amino acid sequence shown in Figure 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 Figure 3 (Seq. ID No. 3) or (b) the reverse complement to the nucleic acid sequence shown in Figure 3 (Seq. ID No. 3) .
- 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.
- 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.
- 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.
- This invention provides a pharmaceutical composition
- 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.
- the oligonucleotide is coupled to a substance which inactivates mRNA.
- the substance which inactivates mRNA is a ribozyme.
- This invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of a GALR3 receptor and a pharmaceutically acceptable carrier.
- This invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an amount of an agonist effective to increase activity of a GALR3 receptor and a pharmaceutically acceptable carrier.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 .
- a known GALR3 receptor agonist such as galanin
- This invention provides a compound determined by the above-described processes.
- the compound is not previously known.
- the compound is not known to bind a GALR3 receptor.
- 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.
- 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 .
- 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.
- 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 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.
- 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.
- the abnormality is obesity.
- the abnormality is bulimia.
- 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.
- the abnormal condition is anorexia.
- 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
- 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.
- the compound is a GALR3 receptor antagonist and the amount is effective to decrease the consumption of food by the subject.
- the compound is administered in combination with food.
- the compound is a GALR3 receptor agonist and the amount is effective to increase the consumption of food by the subject.
- the compound is administered in combination with food.
- the subject is a vertebrate, a mammal, a human or a canine.
- 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 ⁇ S, and with only GTP ⁇ S, under conditions permitting the activation of the GALR3 receptor, and detecting GTP ⁇ S binding to the membrane fraction, an increase in GTP ⁇ S binding in the presence of the compound indicating that the chemical compound activates the GALR3 receptor.
- 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 ⁇ S and a second chemical compound known to activate the GALR3 receptor, with GTP ⁇ S and only the second compound, and with GTP ⁇ S alone, under conditions permitting the activation of the GALR3 receptor, detecting GTP ⁇ S binding to each membrane fraction, and comparing the increase in GTP ⁇ S binding in the presence of the compound and the second compound relative to the binding of GTP ⁇ S alone, to the increase in GTP ⁇ S binding in the presence of the second chemical compound relative to the binding of GTP ⁇ S alone, a smaller increase in GTP ⁇ S binding in the presence of the compound and the second compound indicating that the compound is a GALR3 receptor antagonist.
- 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.
- 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 .
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 .
- 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 .
- 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.
- 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.
- 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.
- 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.
- 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.
- 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:
- 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 ⁇ S and a second chemical compound known to activate the GALR3 receptor, with GTP ⁇ S and only the second compound, and with GTP ⁇ S alone, under conditions permitting the activation of the GALR3 receptor, detecting GTP ⁇ S binding to each membrane fraction, and comparing the increase in GTP ⁇ S binding in the presence of the compound and the second compound relative to the binding of GTP ⁇ S alone, to the increase in GTP ⁇ S binding in the presence of the second chemical compound relative to the binding of GTP ⁇ S alone, a smaller increase in GTP ⁇ S binding in the presence of the compound and the second compound indicating that the compound is a GALR3 receptor antagonist .
- Ficrure 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.
- Ficrure 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 Figure 1.
- Ficrure 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.
- Ficrure 4 Deduced amino acid sequence of the human galanin GALR3 receptor (Seq. I.D. No. 4) encoded by the human nucleotide sequence shown in Figure 3.
- the nucleotide sequence shown in Figure 3 is translated from nucleotide 1 to the stop codon. Two possible starting methionines are underlined.
- 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 GALRl receptor (bottom row) (Seq. ID No. 5) .
- Transmembrane domains (TM 1-7) are indicated by brackets above the sequence .
- Figures 6A-6B Figure 6A: Long continuous trace (3 segments) demonstrates galanin responsivity and sensitivity to Ba ++ block in an oocyte expressing hGalR3 and GIRK1 and GIRK4. Switching from ND96 to l/2hK solution causes the appearance of a large resting (inward) K + current that increases further upon transient addition of 3 ⁇ M galanin. Subsequent addition of 300 ⁇ M Ba ++ largely blocks both the resting and galanin- stimulated K + currents. After removal of Ba ++ galanin responsivity is partially restored.
- Figure 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 .
- Figure 7 Pertussis toxin sensitivity of GalR3 and GalRl stimulation of GIRK currents. Normalized mean currents elicited by 0.1 ⁇ M (GalRl) and 1 ⁇ 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 ⁇ la receptors, the response amplitude was measured as the peak of the Cl" current stimulated by 1 ⁇ 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) .
- Figures 8A-8G Concentration-response relations for 6 peptides at GalR3 receptors expressed in oocytes .
- Figure 8A M32; Figure 8B: porcine galanin;
- Figure 8C C7 ;
- Figure 8D Gal -7-29;
- Figure 8E Gal 1-16;
- Figure 8F M40;
- Figure 8G human galanin.
- 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.
- Figures 9A-9B Figure 9A: Current-voltage relation for responses generated by galanin in oocytes expressing hGalR3, GIRK1 and GIRK . Voltage ramps from -100 to +20 mV were applied at a rate of 50 mV/s. Ramps were generated in l/2hK, l/2hK + 1 ⁇ M galanin, and l/2hK + galanin + 300 ⁇ M Ba ++ .
- FIG. 9B the galanin-sensitive current (I gal ) was derived by subtracting the background current (l/2hK) from the galanin current (+gal) ; the total inward rectifier current (I tot ) was similarly obtained by subtracting the current in the presence of Ba ++ from the galanin current. Both I gal and I tot display steep inward rectification and reverse at approximately -24 mV.
- 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.
- 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.
- 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.
- 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.
- the galanin receptor is a vertebrate or a mammalian GALR3 receptor.
- the galanin receptor is a rat GALR3 receptor.
- the galanin receptor is a human GALR3 receptor.
- 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 GALRl receptor.
- the GALR3 receptor is a rat GALR3 receptor. In another embodiment, the GALR3 receptor is a human GALR3 receptor.
- 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) .
- 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 substantially the same amino acid sequence as the amino acid sequence shown in Figure 2
- the GALR3 receptor is the rat GALR3 receptor having the amino acid sequence shown in Figure 2 (Seq. ID NO. 2) .
- the nucleic acid comprises at least an intron.
- the nucleic acid comprises alternately spliced nucleic acid transcribed from the nucleic acid contained in plasmid K1086.
- the alternately spliced nucleic acid is mRNA transcribed from DNA encoding a galanin receptor.
- 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 Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
- 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.
- 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 .
- 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 .
- nucleic acid is DNA.
- the DNA is cDNA.
- the DNA is genomic DNA.
- nucleic acid molecule is RNA. Methods for production and manipulation of nucleic acid molecules are well known in the art .
- This invention provides a vector encoding the nucleic acid of human GALR3 receptor.
- 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.
- 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.
- 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.
- the nucleic acid comprises a nucleotide sequence which is degenerate with respect to the nucleotide sequence of plasmid pEXJ- rGalR3T (ATCC Accession No. 97826) .
- the nucleic acid comprises a nucleotide sequence which is degenerate with respect to the nucleotide sequence of plasmid pEXJ-hGalR3 (ATCC Accession No. 97827) .
- 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.
- 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 .
- 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.
- 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.
- 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.
- 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.
- 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.
- the deleted amino acids are at the carboxy terminus of the third intracellular domain.
- modified receptors may be produced using methods well-known in the art such as site-directed mutagenesis or recombinant techniques using restriction enzymes.
- 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.
- the modified GALR3 receptor differs by having a deletion in the third intracellular domain.
- the modified GALR3 receptor differs by having an amino acid replacement or addition to the third intracellular domain.
- 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.
- 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.
- 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 Figure 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 Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427. This invention provides a vector comprising the above- described nucleic acid molecule.
- Suitable vectors comprise, but are not limited to, a plasmid or a virus. These 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.
- 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.
- 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 .
- 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.
- the vector is a baculovirus.
- 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.
- 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.
- 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.
- the vector is a plasmid.
- 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 .
- 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 .
- 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.
- This plasmid (K1086) was deposited on October 8, 1996, with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 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.
- ATCC American Type Culture Collection
- 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 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 .
- This plasmid was deposited December 17, 1996, with the ATCC, 12301 Parklawn Drive, Rockville, Maryland, 20852,
- 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.
- a vector or plasmid with untranslated sequences of varying lengths may express differing amounts of the receptor depending upon the host cell used.
- the vector or plasmid comprises the coding sequence of the GALR3 receptor and the regulatory elements necessary for expression in the host cell .
- 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.
- the cell is a Xenopus oocyte or melanophore cell.
- the cell is a neuronal cell such as the glial cell line C6.
- the mammalian cell is non-neuronal in origin.
- the mammalian cell is a COS-7 cell.
- the mammalian cell is a Chinese hamster ovary (CHO) cell.
- the cell is a mouse Yl cell.
- 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.
- 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 February 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.
- the mammalian cell is the LM(tk-) cell designated L-hGALR3-228 , which comprises the plasmid pEXJ-hGalR3.
- L-hGALR3-228 which comprises the plasmid pEXJ-hGalR3.
- This cell line was deposited with the ATCC on June 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.
- This invention also provides an insect cell comprising the above-described vector.
- the insect cell is an Sf9 cell.
- the insect cell is an Sf21 cell.
- This invention provides a membrane preparation isolated from any of the above-described cells.
- 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 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.
- 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 Figure 1 (Seq. ID NO. 1) or (b) the reverse complement to the nucleic acid sequence shown in Figure 1 (Seq. ID No. 1) .
- 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 Figure 3 (Seq. ID No. 3) or (b) the reverse complement to the nucleic acid sequence shown in Figure 3 (Seq. ID NO. 3) .
- 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.
- 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.
- the nucleic acid encoding a GALR3 receptor comprises an intron.
- 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.
- the nucleic acid probe is DNA. In another embodiment the nucleic acid probe is RNA.
- 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 .
- 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.
- 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 .
- 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 provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA molecule encoding a GALR3 receptor .
- This invention provides an antisense oligonucleotide comprising chemical analogues of nucleotides.
- 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.
- the rat GALR3 has an amino acid sequence substantially the same as an amino acid sequence encoded by plasmid K1086.
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same sequence as the sequence shown in Figure 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.
- 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.
- This invention provides a pharmaceutical composition
- 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.
- the oligonucleotide is coupled to a substance which inactivates mRNA.
- the substance which inactivates mRNA is a ribozyme.
- 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.
- the pharmaceutically acceptable carrier is capable of binding to a receptor which is specific for a selected cell type.
- This invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of a GALR3 receptor and a pharmaceutically acceptable carrier.
- This invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an amount of an agonist effective to increase activity of a GALR3 receptor and a pharmaceutically acceptable carrier.
- 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.
- 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.
- 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.
- This invention provides the above-described transgenic nonhuman mammal , wherein the DNA encoding a GALR3 receptor additionally comprises an inducible promoter.
- This invention provides the transgenic nonhuman mammal, wherein the DNA encoding a GALR3 receptor additionally comprises tissue specific regulatory elements.
- the transgenic nonhuman mammal is a mouse.
- 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 .
- One means available for producing a transgenic animal 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.
- microinjection needle which may be made from capillary tubing using a pipet puller
- 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.
- pseudopregnant mouse a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant
- 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.
- 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.
- 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.
- 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.
- 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 Figure 2 (Seq. ID NO. 2) . In another embodiment, the GALR3 receptor has the amino acid sequence shown in Figure 2 (Seq. ID NO. 2) .
- 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 Figure 1 (Seq. ID NO. 1) ) .
- the GALR3 receptor is a human GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827) .
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as the sequence shown in Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
- the above process further comprises determining whether the compound selectively binds to the GALR3 receptor relative to another galanin receptor.
- 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.
- the other galanin receptor is a GALRl receptor.
- the other galanin receptor is a GALR2 receptor.
- This invention provides a process for determining whether a chemical compound is a GALR3 receptor agonist which comprises contacting cells 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 .
- 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.
- 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 Figure 2 (Seq. ID No. 2) . In another embodiment, the GALR3 receptor has the amino acid sequence shown in Figure 2 (Seq. ID No. 2) . In another embodiment, the GALR3 receptor is a human GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827) .
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as the sequence shown in Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
- the cells are transfected with plasmid pEXJ-RGalR3T (ATCC Accession No. 97826) .
- This invention provides a process for determining whether a chemical compound is a GALR3 receptor antagonist which comprises contacting cells 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 .
- a known GALR3 receptor agonist such as galanin
- 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.
- a known GALR3 receptor agonist such as galanin
- 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. 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 Figure 2 (Seq. ID No. 2) . In another embodiment, the GALR3 receptor has the amino acid sequence shown in Figure 2 (Seq. ID No. 2) . In another embodiment, the GALR3 receptor is a human GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827).
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as the sequence shown in Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO . 4) from amino acid 60 through amino acid 427.
- the cell is a non-mammalian cell such as an insect cell or a Xenopus cell.
- the cell is a mammalian cell.
- the cell is non-neuronal in origin.
- the non-neuronal cell is a COS-7 cell, 293 human embryonic kidney cell, NIH-3T3 cell, a CHO cell, or LM(tk-) cell.
- the cell is a mouse Yl cell.
- This invention provides a compound determined by the above-described methods.
- the compound is not previously known to bind to a GALR3 receptor.
- 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.
- the cells are transfected with and expressing GIRK1 and GIRK4.
- the GALR3 receptor is a mammalian GALR3 receptor.
- 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.
- the GALR3 receptor agonist is not previously known.
- 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.
- the GALR3 receptor antagonist is not previously known.
- 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.
- 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.
- the agonist or antagonist is not previously known to bind to a GALR3 receptor.
- 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.
- 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.
- 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.
- 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 .
- the second messenger response comprises potassium channel activation and the change in second messenger is an increase in the level of inward potassium current.
- the second messenger response comprises adenylate cyclase activity and the change in second messenger response is a decrease in adenylate cyclase activity.
- adenylate cyclase activity is determined by measurement of cyclic AMP levels.
- the second messenger response comprises arachidonic acid release and the change in second messenger response is an increase in arachidonic acid levels.
- the second messenger response comprises intracellular calcium levels and the change in second messenger response is an increase in intracellular calcium levels.
- the second messenger response comprises inositol phospholipid hydrolysis and the change in second messenger response is an increase in inositol phospholipid hydrolysis.
- 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.
- 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.
- the second messenger response comprises potassium channel activation and the change in second messenger response is a smaller increase in the level of inward 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.
- 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.
- adenylate cyclase activity is determined by measurement of cyclic AMP levels.
- the second messenger response comprises arachidonic acid release
- 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 .
- the second messenger response comprises intracellular calcium levels
- 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.
- the second messenger response comprises inositol phospholipid hydrolysis
- 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.
- 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 Figure 2 (Seq. ID No. 2) . In another embodiment, the GALR3 receptor has the amino acid sequence shown in Figure 2 (Seq. ID No. 2) .
- the GALR3 receptor has the same or substantially the same amino acid sequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827) .
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as the sequence shown in Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
- the cells are transfected with plasmid pEXJ-RGalR3T (ATCC Accession No. 97826) .
- 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 Yl cell, NIH-3T3 cell or LM(tk-) cell.
- This invention further provides a compound determined by any of the above processes.
- the compound is not previously known to bind to a GALR3 receptor.
- 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.
- the GALR3 receptor agonist is not previously known.
- 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.
- the GALR3 receptor antagonist is not previously known.
- 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.
- 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.
- 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 Figure 2 (Seq. ID NO. 2) . In another embodiment, the GALR3 receptor has the amino acid sequence shown in Figure 2 (Seq. ID No. 2) . In another embodiment, the GALR3 receptor is a human GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827) .
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as the sequence shown in Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
- 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.
- 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.
- the cells are transfected with and expressing GIRK1 and GIRK4.
- the GALR3 receptor is a mammalian GALR3 receptor.
- the GALR3 receptor is a rat GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as the amino acid sequence encoded by plasmid K1086.
- the GALR3 receptor has substantially the same amino acid sequence as the amino acid sequence shown in Figure 2 (Seq. ID No . 2) .
- the GALR3 receptor has the amino acid sequence shown in Figure 2 (Seq. ID No . 2) .
- the GALR3 receptor is a human GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827) .
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as the sequence shown in Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
- 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.
- 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;
- the cells are transfected with and expressing GIRK1 and GIRK4.
- the GALR3 receptor is a mammalian GALR3 receptor.
- the GALR3 receptor is a rat GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as the amino acid sequence encoded by plasmid K1086.
- the GALR3 receptor has substantially the same amino acid sequence as the amino acid sequence shown in Figure 2 (Seq. ID No. 2) .
- the GALR3 receptor has the amino acid sequence shown in Figure 2 (Seq. ID No. 2) .
- the GALR3 receptor is a human GALR3 receptor.
- the GALR3 receptor has the same or substantially the same amino acid sequence as that encoded by plasmid pEXJ-hGalR3 (ATCC Accession No. 97827) .
- the human GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as the sequence shown in Figure 4 (Seq. I.D. No. 4) from amino acid 60 through amino acid 427.
- the GALR3 receptor has a sequence, which sequence comprises the sequence shown in Figure 4 (Seq. ID NO. 4) from amino acid 60 through amino acid 427.
- the cells are transfected with and expressing GIRK1 and GIRK4.
- receptor activation is determined by measurement of potassium channel activation.
- receptor activation is determined by measurement of an increase in inward potassium current.
- inhibition of receptor activation is determined by a smaller increase in inward potassium current in the presence of the compound and a galanin receptor agonist than in the presence of only the galanin receptor agonist .
- the galanin receptor agonist is galanin.
- This invention provides a pharmaceutical composition
- 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.
- This invention provides a pharmaceutical composition
- 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.
- 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.
- 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.
- such other galanin receptor is a GALRl receptor.
- such other galanin receptor is a GALR2 receptor.
- 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.
- 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;
- such other galanin receptor is a GALRl receptor.
- such other galanin receptor is a GALR2 receptor.
- the activation of the GALR3 receptor is determined by a second messenger assay.
- the second messenger assay measures adenylate cyclase activity.
- 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 ⁇ S (gamma thiol GTP) to membranes, which precedes and is therefore independent of second messenger coupling.
- 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 ⁇ S, and with only GTP ⁇ S, under conditions permitting the activation of the GALR3 receptor, and detecting GTP ⁇ S binding to the membrane fraction, an increase in GTP ⁇ S binding in the presence of the compound indicating that the chemical compound activates the GALR3 receptor.
- 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 ⁇ S and a second chemical compound known to activate the GALR3 receptor, with GTP ⁇ S and only the second compound, and with GTP ⁇ S alone, under conditions permitting the activation of the GALR3 receptor, detecting GTP ⁇ S binding to each membrane fraction, and comparing the increase in GTP ⁇ S binding in the presence of the compound and the second compound relative to the binding of GTP ⁇ S alone, to the increase in GTP ⁇ S binding in the presence of the second chemical compound relative to the binding of GTP ⁇ S alone, a smaller increase in GTP ⁇ S binding in the presence of the compound and the second compound indicating that the compound is a GALR3 receptor antagonist.
- the second chemical compound is a labeled compound. In another embodiment, the second chemical compound is a radiolabeled compound.
- 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 Figure 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) .
- the GALR3 receptor has a sequence, which sequence comprises substantially the same amino acid sequence as that shown in Figure 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 Figure 4 (Seq. ID No . 4) from amino acid 60 through amino acid 427.
- the cell is an insect cell.
- the cell is a mammalian cell.
- the mammalian cell is nonneuronal in origin.
- the nonneuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell.
- the nonneuronal cell is the 293 human embryonic kidney cell designated 293-rGALR3-105 (ATCC Accession No. CRL-12287) .
- the nonneuronal cell is the LM(tk-) cell designated L-hGALR3-228 (ATCC Accession No. CRL-12373) .
- GTP ⁇ 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.
- 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.
- This invention further provides a method of measuring GALR3 receptor activation in an oocyte expression system such as a Xenopus oocyte or melanophore.
- receptor activation is determined by measurement of ion channel activity, e.g., using the voltage clamp technique (St ⁇ hmer, 1992) .
- receptor activation is determined by the measurement of inward 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.
- 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.
- the cell is a non-mammalian cell such as an insect cell or Xenopus cell.
- the cell is a mammalian cell.
- the mammalian cell is non ⁇ neuronal in origin.
- the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell, a mouse Yl cell, a CHO cell, or an NIH-3T3 cell.
- This invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound identified by the above-described methods and a pharmaceutically acceptable carrier.
- the cell is non-neuronal in origin.
- the non-neuronal cell is a COS-7 cell, 293 human embryonic kidney cell, CHO cell, NIH-3T3 cell or LM(tk-) cell.
- the compound is not previously known to bind to a GALR3 receptor.
- 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.
- the cell is a non- mammalian cell, for example, a Xenopus oocyte or melanophore.
- the cell is a neuronal cell, for example, a glial cell line such as C6.
- the cell is non-neuronal in origin.
- 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.
- This invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a drug candidate identified by the above- described methods and a pharmaceutically acceptable carrier.
- 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 .
- 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.
- the animal is a non- human mammal.
- the animal is a rodent .
- 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.
- 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.
- the compound is an agonist.
- the compound is an antagonist .
- 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.
- the abnormality is obesity.
- the abnormality is bulimia.
- 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.
- the abnormal condition is anorexia.
- 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 GALRl 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.
- 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.
- 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.
- 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.
- 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 .
- 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.
- 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.
- 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.
- This invention provides an agonist identified by the above-described methods.
- This invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an agonist identified by the above-described methods and a pharmaceutically acceptable carrier.
- 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.
- 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 labelled with a detectable marker; (e) detecting labelled bands which have hybridized to DNA encoding a human GALR3 receptor 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
- a disorder associated with the activity of a specific human GALR3 receptor allele is diagnosed.
- 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.
- 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.
- 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;
- 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.
- the compound is a GALR3 receptor antagonist and the amount is effective to decrease the consumption of food by the subject.
- the compound is administered in combination with food.
- the compound is a GALR3 receptor agonist and the amount is effective to increase the consumption of food by the subject.
- the compound is administered in combination with food.
- the subject is a vertebrate, a mammal, a human or a canine.
- the compound binds selectively to a GALR3 receptor. In another embodiment, the compound binds to the GALR3 receptor with an affinity greater than tenfold higher than the affinity with which the compound binds to a GALRl 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 GALRl 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.
- 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.
- 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.
- 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.
- the galanin receptor agonist is a GALR3 receptor agonist and the amount of the compound is effective to produce analgesia in the subject.
- 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 .
- 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.
- 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.
- 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.
- the GALR3 antagonist and the Y5 antagonist are administered in combination.
- the GALR3 antagonist and the Y5 antagonist are administered once.
- the GALR3 antagonist and the Y5 antagonist are administered separately.
- the GALR3 antagonist and the Y5 antagonist are administered once.
- the galanin receptor antagonist is administered for about 1 week to 2 weeks.
- the Y5 receptor antagonist is administered for about 1 week to 2 weeks .
- 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.
- TM transmembrane
- TM 1, 2, 3, 4, 5, 6 and 7 overlapping transmembrane oligonucleotide probes derived from the rat GALR2 receptor cDNA.
- Overlapping oligomers were labeled with [ 32 P] dATP and [ 3 ] dCTP by synthesis with the large fragment of DNA polymerase, and comprised the following sequences:
- (+) strand 5 ' -GTTGCGCATCCTTTCACACCTAGTTTCCTATGCCAACTCCTGTGT-3 ' (SEQ ID NO: 18) ;
- Hybridization of phage lifts was performed at reduced stringency conditions: 40°C in a solution containing 37.5% formamide, 5x SSC (IX SSC is 0.15M sodium chloride, 0.015M sodium citrate), lx Denhardt's solution (0.02% polyvmylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin) , and 25 ⁇ g/ ⁇ L sonicated salmon sperm DNA.
- the filters were washed at 45°C in 0. lx SSC containing 0.1% sodium dodecyl sulfate and exposed at -70°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 ⁇ Zap II using helper phage Re704, as described by the manufacturer's protocol (Rapid Excision Kit, Stratagene, LaJolla, CA. ) . 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, WI) .
- GCG Genetics Computer Group, Madison, WI
- rHY35a 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 rGALRl 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:
- rHY35a i.e., rat GALR3: (forward primer) :
- 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 + RNA was purified with a FastTrack kit (Invitrogen Corp., San Diego, CA) and converted to single-stranded cDNA by random priming using Superscript reverse transcriptase (BRL, Gaithersburg, MD) .
- 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 . Construction and PCR screening of a RIN14B cell line plasmid library
- Total RNA was prepared from RIN14B cells by a modification of the guanidine thiocyanate method (Chirgwin et al . , 1979) .
- Poly A + RNA was purified with a FastTrack kit (Invitrogen Corp., San Diego, CA) .
- Double stranded (ds) cDNA was synthesized from 4 ⁇ g of poly A + 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.
- Glycerol stocks (2 ⁇ 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
- 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) .
- PCR was conducted on glycerol stocks (2 ⁇ 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.
- 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, CA) 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, WI) . 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.
- RNA was prepared from rat hypothalami by a modification of the guanidine thiocyanate method (Chirgwin, 1979) .
- Poly A + RNA was purified using a FastTrack kit (Invitrogen Corp., San Diego, CA) .
- Double stranded (ds) cDNA was synthesized from 6 ⁇ g of poly A + RNA according to Gubler and Hoffman (1983) with minor modifications.
- the resulting cDNA was ligated to BstXl/EcoRI adaptors (Invitrogen Corp.) and the excess adaptors removed by exclusion column chromatography.
- 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, CA) . Aliquots of each bacterial pool were stored at -85°C in 20% glycerol.
- 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 °C in a humidified 5% C0 2 atmosphere for 24 hours before transfection.
- DMEM Dulbecco's modified Eagle medium
- DMEM-C 2 mM L-glutamine
- 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) .
- 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)
- vector and gene-specific primers preceding section
- 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) . Colonies were transferred to nitrocellulose membranes
- COS-7 cells were plated in slide chambers (Lab- Tek) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% calf serum, lOOU/mL of penicillin, 100 ⁇ g/mL streptomycin, 2mM L-glutamine (DMEM-c) and grown at 37°C in a humidified 5% C0 2 atmosphere for 24 hours before transfection.
- DMEM Dulbecco's modified Eagle medium
- DMEM-c 2mM L-glutamine
- cells were rinsed with phosphate-buffered saline (PBS) then incubated with 2 nM 125 I -porcine galanin (NEN; specific activity -2200 Ci/mmol) in 20mM HEPES-NaOH, pH 7.4, containing 1.26 mM CaCl 2 , 0.81 mM MgS0 4 , 0.44 mM KH 2 P0 4 , 5.4 mM KC1, 10 mM NaCI, 0.1% BSA, and 0.1% bacitracin for one hour at room temperature.
- PBS phosphate-buffered saline
- Lambda phage clones hybridizing with the probe were plaque purified and DNA was prepared for Southern blot analysis (Southern, 1975; Sambrook et al . , 1989).
- phage clone contained a 2.7 kb Kpnl/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, WI) . DNA sequence analysis indicated greatest homology to the rat and human GALRl 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).
- 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:
- human fetal brain cDNA lambda ZAPII library (Stratagene) ; human testis cDNA lambda ZAPII library (Stratagene) ; human hypothalamus cDNA plasmid library (proprietary) --3 superpools identified; and human hippocampus cDNA plasmid library (proprietary) - -3 superpools identified.
- 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) .
- 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.
- restriction enzymes may be used 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.
- 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 .
- human cDNA expressing human GALR3 e.g., human brain
- 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 phage clone containing MET through the intron in the second intracellular loop i.e. TM 3/4 loop
- plc21a 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 6/7) or to TM 4, derived from the rat GALR3 sequence:
- plcl4a Another clone, plcl4a, 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 1.4kb Kpnl fragment which similarly hybridized to the above probes. Since the phage clone, plcl4a, also hybridized with a TM2/3 loop probe under high stringency, derived from sequence data of human GALR3 5' fragment (plc21a, see above),
- this 3' fragment (e.g. plcl4a) presumably corresponds to the 3' end of human GALR3 and is molecularly linked to the 5' fragment (e.g. plc21a 2.7kb Kpnl/EcoRI clone); however, an intron of unknown size separates the coding region, which is defined on the 5' (2.7kb Kpnl/EcoRI plc21a fragment) and 3' (1.4kb Kpnlplcl4a fragment) genomic pieces. Nucleotide sequence analysis was conducted on the 1.4 kb Kpnl plcl4a fragment, as described above, and indicated greatest homology to the rat and human GALRl and GALR2 genes .
- 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 Kpnl/EcoRI plc21a subclone) and the exon containing the 3/4 loop through the predicted STOP codon (e.g. housed on the 1.4 kb Kpnl plcl4a subclone). The sequence of this junction demonstrated the presence of a Kpnl site, which was utilized in the construction of the full-length gene.
- 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 Kpnl 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:
- the addition of the Kpnl site enabled the attachment of the 3' Kpnl fragment but preserved the sequence which was identified from human brain and liver cDNAs .
- the forward and reverse primers were used to amplify the 2.7kb Kpnl/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 Kpnl and purified further by phenol extraction. This BamHI/Kpnl PCR product was subcloned into BamHI/Kpnl -digested expression vector, pEXJ, and sequenced. The sequence of the PCR product was identical to that determined for the original genomic fragment .
- Northern Blots Rat multiple tissue northern blots (rat MTN blot, Clontech, Palo Alto, CA) , containing 2 ⁇ g poly A + RNA, or northern blots containing 5 ⁇ g poly + 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 [ ⁇ - 32 P] dCTP and [01- 32 P]dATP (3000Ci/mmol, NEN) were used. Northern blots were reprobed with a randomly-primed /3-actin probe to assess quantities of mRNA present in each lane.
- Human brain multiple tissue northern blots (MTN brain blots II and III, Clontech, Palo Alto, CA) and human peripheral MTN blot (Clontech, Palo Alto, CA) carrying mRNA (2 ⁇ 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 5 ' GATGGCTGATGCCCAGAACATTTCACTGGACAGCCCAGGGAGTGT 3 ' (SEQ ID NO. 51) and
- RNA samples 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 + -selected using FastTrack kit (Invitrogen), according to manufacturer's specifications.
- 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,
- 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.
- 0.5 ⁇ g of viral DNA (BaculoGold) and 3 ⁇ g of GALR3 construct may be co- transfected into 2 x 10 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°C.
- 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.
- 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 ⁇ g/mL streptomycin) at 37°C, 5% C0 2 . Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4 days.
- supplements Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 ⁇ g/mL streptomycin
- 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 ⁇ g/mL streptomycin) at 37°C, 5% C0 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 ⁇ g/mL streptomycin) at 37°C, 5% C0 2 . Stock plates of LM(tk-) cells are trypsinized and split 1:10 every 3-4 days.
- supplements Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 ⁇ g/mL streptomycin
- 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 6 cells/mL in suspension media (10% bovine calf serum, 10% 10X Medium 199 (Gibco) , 9 mM NaHC0 3 , 25 mM glucose, 2 mM L-glutamine, 100 units/mL penicillin/100 ⁇ g/mL streptomycin, and 0.05% methyl cellulose) .
- suspension media (10% bovine calf serum, 10% 10X Medium 199 (Gibco) , 9 mM NaHC0 3 , 25 mM glucose, 2 mM L-glutamine, 100 units/mL penicillin/100 ⁇ g/mL streptomycin, and
- Cell suspensions are maintained in a shaking incubator at 37°C, 5% C0 2 for 24 hours.
- Membranes harvested from cells grown in this manner may be stored as large, uniform batches in liquid nitrogen.
- 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°C, 5% C0 2 for 24 hours.
- Cells prepared in this manner generally yield a robust and reliable response in cAMP radio- immunoassays as further described hereinbelow.
- 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 ⁇ g/mL streptomycin) at 37°C, 5% C02. 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 ug/ml streptomycin) at 37°C, 5% C02. Stock plates of CHO cells were trypsinized and split 1:8 every 3-4 days.
- 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°C, no C0 2 .
- High Five insect cells are grown on 150 mm tissue culture dishes in Ex- Cell 400TM medium supplemented with L-Glutamine, also at 27°C, no C0 2 .
- All receptor subtypes studied may be transiently transfected into COS-7 cells by the DEAE-dextran method, using 1 ⁇ g of DNA /10 6 cells (Cullen, 1987) .
- 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.
- 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. GALRl receptors were expressed in cells using methods well-known in the art.
- binding buffer 50mM Tris-HCl, 5mM MgS0 4 , ImM EDTA at pH 7.5 supplemented with 0.1% BSA, 2 ⁇ g/ml aprotinin,
- 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 125 I- labeled peptide, non- labeled peptides and binding buffer to a final volume of 250 ⁇ l.
- membrane preparations may be incubated in the presence of increasing concentrations (e.g., 0.1 nM to 4 nM) of
- the binding affinities of the different galanin analogs may be determined in equilibrium competition binding assays, using 0.1-0.5 nM [ 125 I] 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 °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 lOOnM 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.
- 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 ⁇ M porcine galanin.
- cAMP cyclic AMP
- CHO cells stably transfected with the rat GALR3 receptor are seeded into 96 well plates and grown for 3 days in
- 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 ⁇ L of Fura-2/AM (10 ⁇ 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 40X 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.
- 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 ⁇ l of medium containing 1% serum and 0.5 ⁇ Ci [ 3 H]myo-inositol, and the plates are incubated overnight in a C0 2 incubator (5% C0 2 at 37°C) .
- arachidonic acid release may be measured if [ 3 H] arachidonic acid is substituted for the [H]myo- inositol. Immediately before the assay, the medium is removed and replaced by 200 ⁇ L of PBS containing 10 mM
- [ 3 H] inositol-phosphates accumulation from inositol phospholipid metabolism may be started by adding 10 ⁇ L of a solution containing the agonist. To the first well 10 ⁇ 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 C0 2 incubator for 1 hr. The reaction may be terminated by adding 15 ⁇ l of 50% v/v trichloroacetic acid (TCA) , followed by a 40 min. incubation at 4°C.
- TCA 50% v/v trichloroacetic acid
- 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 ⁇ 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 ⁇ L of water, followed by 2 x 200 ⁇ L of 5mM sodium tetraborate/60 mM ammonium formate.
- the filter plates are prepared adding 200 ⁇ 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 ⁇ L of water, followed by 2 x 200 ⁇ L of 5mM sodium tetraborate/60
- IPs are eluted into empty 96-well plates with 200 ⁇ l of 1.2 M ammonium formate/0.1 formic acid. The content of the wells is added to 3 L of scintillation cocktail, and the radioactivity is determined by liquid scintillation counting.
- GTP ⁇ 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.
- the binding and functional assays described herein may also be performed using GALRl and GALR2 receptors.
- the GALRl 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.
- 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 March 28, 1996) and L-rGALR2I-4 (ATCC Accession No. CRL-12223, deposited October 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 January 15, 1997) also stably expresses the rat GALR2 receptor.
- the human GALR2 receptor may be expressed using plasmid B029 (ATCC Accession No. 97735, deposited September 25, 1996) or plasmid B039 (ATCC Accession No. 97851, deposited January 15, 1997).
- Plasmid B039 comprises an intronless construct encoding the human GALR2 receptor.
- Xenopus laevis Female Xenopus laevis (Xenopus- 1, Ann Arbor, MI) 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) .
- Oocytes are defolliculated using 2 mg/ml collagenase (Worthington Biochemical Corp., Freehold, NJ) in a solution containing 87.5 mM NaCI, 2 mM KCl, 2 mM MgCl 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) .
- 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 + tail. After injection of mRNA, oocytes are incubated at 16° on a rotating platform for 3-8 days. Dual electrode voltage clamp ( "GeneClamp” , Axon Instruments Inc., Foster City, CA) is performed using 3 M KCl-filled glass microelectrodes having resistances of 1-3 Mohms .
- oocytes are voltage clamped at a holding potential of -80 mV.
- oocytes are bathed in continuously flowing (2-5 ml/min) medium containing 96 mM NaCI, 2 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , and 5 mM HEPES, pH 7.5 ("ND96"), or, in the case of oocytes expressing GIRK1 and GIRK4 , elevated K + containing 96 mM KCl, 2 mM NaCI, 2 mM CaCl 2 , 2 mM MgCl 2 , and 5 mM HEPES, pH 7.5 ( "hK" ) .
- Drugs are applied by switching from a series of gravity fed perfusion lines.
- 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 ⁇ 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.
- Oocytes expressing GalR3 plus the two GIRK subunits are tested for galanin responsivity using 1 ⁇ M galanin and measuring K + currents in elevated K + solution (hK) .
- Activation of inwardly rectifying currents that are sensitive to 300 ⁇ M Ba ++ signifies GALR3 coupling to a G ⁇ or G D pathway in the oocytes .
- 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 + channels 1 and 4 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 5' -CGCGGATCCATTATGTCTGCACTCCGAAGGAAATTTG-3' (SEQ ID NO. 54) and
- the upstream primer contained a BamHI site and the downstream primer contained an EcoRI site to facilitate cloning of the PCR product into pcDNAl-Amp (Invitrogen) .
- the transcription template for hGalR3 was obtained similarly by PCR using the cloned cDNA in combination with primers 5 ' -CCAAGCTTCTAATACGACTCACTATAGGGCCACCATGGCTGATGCCCAGA-3 '
- This plasmid was modified by adding the recognition sequence for the restriction enzyme Srfl 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.
- M54 a suitable clone
- the transcription template was produced by linearization of the plasmid DNA with Srfl.
- the plasmid M54 was deposited on September 30, 1997, with the American Type Culture
- GIRK1 and GIRK4 mRNA in combination with 25 ng of GalR3 mRNA.
- oocytes received injections of mRNAs encoding the human oflA adrenergic receptor, rGalRl or rGalR2 galanin receptors (Forray et al . , 1994; Parker et al . , 1995) with or without GIRKs 1 and 4.
- oocytes were incubated at 17° for 3-8 days.
- Dual electrode voltage clamp ( “GeneClamp” , Axon Instruments Inc., Foster City, CA) 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 + containing 48 mM KCl, 49 mM NaCI, 2 mM CaCl 2 , 2 mM MgCl 2 , and 5 mM HEPES, pH 7.5 (l/2hK) .
- Drugs were applied either by local perfusion from a 10 ⁇ l glass capillary tube fixed at a distance of 0.5 mm from the oocyte, or for calculation of steady-state EC 50 s, 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.
- MAP kinase mitogen activated 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 . Gi coupled receptors are also known to activate ras and subsequently produce an activation of MAP kinase.
- Receptors that activate phospholipase C (Gq and Gil) produce diacylglycerol (DAG) as a consequence of phosphatidyl inositol hydrolysis. DAG activates protein kinase C which in turn phosphorylates MAP kinase.
- DAG diacylglycerol
- 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.
- antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase.
- 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.
- 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°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 3 P0 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 32 P in a liquid scintillation counter.
- 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 carrried out for 10 min at 30°C.
- the extract can then by aspirated through the filter, which retains the phosphorylated myelin basic protein.
- the filter is washed and counted for 32 P by liquid scintillation counting.
- Receptor activation of a G protein coupled receptor may lead to a mitogenic or proliferative response which can be monitored via 3 H-thymidine uptake.
- 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.
- 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.
- the cells are incubated with 3 H-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 3 H by liquid scintillation counting.
- 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 3 H by liquid scintillation counting.
- 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/ ⁇ L, and stored at -20°C. Probe sequences may include DNA or RNA which is complementary to the mRNA which encodes the GALR3 receptor.
- Probes are 3' -end labeled with 35 S-dATP (1200 Ci/mmol, New England Nuclear, Boston, MA) to a specific activity of about 10 9 dpm/ ⁇ g using terminal deoxynucleotidyl transferase (Pharmacia) .
- the radiolabeled probes are purified on Biospin 6 chromatography columns (Bio-Rad; Richmond, CA) , and diluted in hybridization buffer to a concentration of 1.5 x 10 4 cpm/ ⁇ L.
- Solution hybridization/ribonuclease protection assay For solution hybridization 2-15 ⁇ 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 ⁇ g transfer RNA (tRNA) or no tissue blanks. Samples are placed in 1.5-ml microfuge tubes and vacuum dried. Hybridization buffer (40 ⁇ l of 400 mM NaCI, 20 mM Tris, pH 6.4 , 2 mM EDTA, in 80% formamide) containing 0.25-1.0 X 10 counts of each probe is added to each tube. Samples are heated at 90°C for 15 min, after which the temperature is lowered to 45°C for hybridization .
- Hybridization buffer 40 ⁇ l of 400 mM NaCI, 20 mM Tris, pH 6.4 , 2 mM EDTA, in 80% formamide
- RNA/probe mixtures are digested with RNAse A (Sigma) and RNAse Tl (Bethesda Research Labs, Gaithersburg, Maryland) .
- a mixture of 2.0 ⁇ g RNAse A and 1000 units of RNAse Tl in a buffer containing 330 mM NaCI, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 ⁇ l) is added to each sample and incubated for 90 min at room temperature.
- 20 ⁇ l of 10% SDS and 50 ⁇ g proteinase K are added to each tube and incubated at 37°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°C.
- tRNA is added to each tube (30 mg) as a carrier to facilitate 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 f ilm .
- RNA transcripts were obtained using a full length cDNA construct in pBluescript .
- 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
- riboprobes 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 ⁇ L linearized template (I ⁇ g/ ⁇ L) , 1.5 ⁇ L of ATP, GTP, UTP (10 mM each), 3 ⁇ L dithiothreitol (0.1 M) , 30 units RNAsin RNAse inhibitor, 0.5-1.0 ⁇ L (15-20 units/ ⁇ L) RNA polymerase, 7.0 ⁇ L transcription buffer (Promega Corp.), and 12.5 ⁇ L ⁇ 32 P-CTP (specific activity 3 , OOOCi/mmol) .
- RNAse-free DNAse Promega Corp.
- 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.
- RNA samples were placed in 1.5-ml microfuge tubes and vacuum dried.
- Hybridization buffer 40 ⁇ l of 400 mM NaCI, 20 mM Tris, pH 6.4 , 2 M EDTA, in 80% formamide
- Hybridization buffer 40 ⁇ l of 400 mM NaCI, 20 mM Tris, pH 6.4 , 2 M EDTA, in 80% formamide
- Hybridization buffer 40 ⁇ l of 400 mM NaCI, 20 mM Tris, pH 6.4 , 2 M EDTA, in 80% formamide
- RNA/probe mixtures were digested with RNAse A (Sigma) and RNAse Tl (Life kits).
- RNAse A A mixture of 2.0 ⁇ g RNAse A and 1000 units of RNAse Tl in a buffer containing 330 mM NaCI, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 ⁇ L) was added to each sample and incubated for 90 min at room temperature. After digestion with RNAses, 20 ⁇ L of 10% SDS and 50 ⁇ g proteinase K were added to each tube and incubated at 37°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°C.
- Pellet Paint Novagen was added to each tube (2.0 ⁇ 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°C.
- the effects of galanin, galanin derivatives, and related peptides and compounds may be evaluated by intracerebroventricular (i.e. 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.
- 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) .
- 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.
- Cell culture media and supplements are from Specialty Media (Lavallette, NJ) .
- Cell culture plates (150 mm and 96 -well microtiter) are from Corning (Corning, NY) .
- Sf9, Sf21, and High Five insect cells, as well as the baculovirus transfer plasmid, pBlueBacIIITM, are purchased from Invitrogen (San Diego, CA) .
- TMN-FH insect medium complemented with 10% fetal calf serum, and the baculovirus DNA, BaculoGoldTM, is obtained from Pharmingen (San Diego, CA. ) .
- Ex-Cell 400TM medium with L-Glutamine is purchased from JRH Scientific.
- Polypropylene 96-well microtiter plates are from Co- star (Cambridge, MA) . All radioligands are from New England Nuclear (Boston, MA) .
- Galanin and related peptide analogs were either from Bachem California (Torrance, CA) , Peninsula (Belmont, CA) ; or were synthesized by custom order from Chiron Mimotopes Peptide Systems (San Diego, CA) .
- Bio-Rad Reagent was from Bio-Rad (Hercules, CA) .
- Bovine serum albumin (ultra-fat free, A-7511) was from Sigma (St. Louis. MO) . All other materials were reagent grade .
- 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 l.Okb, 0.2kb, and 2.3kb EcoRI fragments), which hybridized with the second transmembrane domain oligonucleotide probe of rat GALR2.
- This gene fragment exhibited 52% and 66% nucleotide identity and 37% and 60% amino acid identity to the rat GALRl 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, rGALRl 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 GALRl and GALR2 suggested that this cDNA represented a partial gene fragment of a novel galanin-like receptor gene, referred to herein as GALR3.
- 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 GALRl 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.
- sequence 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.
- 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) .
- 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.
- Plasmid K1086 confirmed that it contained a full- length coding region in the correct orientation for expression.
- 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.
- 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. Table 1. Northern blot analyses of GALR3 mRNA in brain and various peripheral rat tissues.
- RNase protection assay to detect mRNA coding for rat GA R3 mRNA was isolated and assayed as described from: heart, striated muscle, liver, kidney, lung, stomach, spleen, pancreas, pituitary, adrenal medulla, adrenal cortex, trige inal 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.
- 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 Musculoskeletal disorders, muscle 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 + cerebral + Sensory integration, cognition cortex hypothalamus ++ Appetite/obesity, Neuroendocrine regulation hippocampus - Cognitio /memory spinal cord ++ Analgesia, sensory modulation and transmission cerebellum + Motor coordination medulla + Analgesia, motor coordination substantia Modulation of dopaminergic nigra function. Modulation
- 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 Figure 1) .
- pEXJ- RGalR3T a "trimmed" plasmid
- Rat GALR3 Peptide binding profiles for the rat GALRl, GALR2 and GALR3 receptor subtypes were derived from membranes prepared from transiently transfected COS- 7 cells.
- Rat GALR3 also displays low affinity for the D-Trp 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.
- 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 Kpnl/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%
- 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 18kb, may contain the 3' end of this gene, assuming a smaller size for the intron which serparates the 5' and 3' exons .
- 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 Kpnl site appended to the reverse PCR primer, and the 3' exon, contained on a 1.4 kb Kpnl 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 GALRl and GALR2 receptors, respectively. Furthermore, within the transmembrane domains, the human GALR3 receptor displays 46% and 74% amino acid identities with the human GALRl and GALR2 receptors, respectively. This relationship suggests that human GALR3 represents a novel receptor subtype within the galanin gene family.
- Membranes from the stably transfected cell line 293- rGalR3-105 bound porcine 125 I -galanin with a K d of 0.74 nM and an apparent B max of 450 fmol/mg membrane protein.
- Both the transiently and stably expressed rat GALR3 receptors were analyzed in competitive displacement assays using porcine 125 I -galanin (Table 5) .
- GALR3 appears to bind the N-terminally extended peptide galanin -7 to + 29 with affinity comparable to that for porcine galanin.
- the cDNA for the human GALR3 receptor was used to prepare both transiently and stably transfected cells.
- GPCRs in Xenopus oocytes have 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 Gi/G 0 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).
- oocytes injected with mRNAs for hGALR3 and GIRKs 1 and 4 responded with inward currents to local perfusion of porcine galanin (Fig. 6A) .
- Oocytes injected with mRNA encoding the rat GalR3 receptor also exhibited current responses to the 1 ⁇ M local application of M32 or porcine galanin.
- the pharmacology of the rat GalR3 receptor was not further evaluated in oocytes.
- evidence that galanin-induced currents were mediated by GIRK channels included: 1) dependency on elevated external K + , 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 + (-23 mV) , 4) sensitivity to block by 300 ⁇ M Ba ++ (Fig. 6A) , and 5) lack of galanin-sensitivity in oocytes injected with only hGALR3 mRNA (data not shown) .
- a series of galanin and galanin-related peptides were tested at the human GALR3 receptor for agonist and antagonist activities.
- 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 ⁇ M.
- D-Trp2-galanin and galanin 3-29 were inactive.
- EC 50 s were constructed from cumulative concentration-response measurements performed on a series of oocytes (Figs. 6B, 8).
- Table 8 Comparison of rank orders of EC 50 s for stimulation of GIRKs, apparent binding affinities (K A ) and EC 50 s for stimulation of feeding behavior in vivo .
- Peptide ligands were evaluated in binding and functional assays (Tables 9-11) .
- GMAP 16-41 > 1000 > 1000 > 1000 amide Peptide Ki (nM) from porcine 1251-galanin binding assay
- nucleic acids have been isolated encoding a novel galanin receptor, termed GALR3 , that is distinct from the previously cloned GALRl and GALR2 receptors.
- 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.
- 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 Figure 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
- the present invention includes both the receptor beginning at the downstream MET and the receptor beginning at the upstream MET.
- GALRl receptors The localization of GALRl 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.
- galanin is co-released with norepinephrine from sympathetic nerve terminals
- 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).
- 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.
- galanin receptors mediating effects on cognition, analgesia, neuroendocrine regulation, control of insulin release and control of feeding behavior.
- novel GALR3 receptor those functions mediated by galanin receptors in the rat hypothalamus.
- Peptide displacement assays indicate that the rat GALR3 receptor has a unique pharmacological profile.
- the low affinity for M40 invites further speculation as to the physiological role of the rat GALR3 receptor.
- 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).
- 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.
- the ⁇ -opioid receptor Isolation of a cDNA by expression cloning and . Proc. Natl. Acad. Sci. USA 89:12048-12052.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- General Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Neurology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU48125/97A AU741041B2 (en) | 1996-10-09 | 1997-10-09 | DNA encoding galanin GALR3 receptors and uses thereof |
DE0956291T DE956291T1 (en) | 1996-10-09 | 1997-10-09 | GALANIN GALR3 RECEPTOR-ENCODING DNA AND APPLICATIONS |
EP19970910850 EP0956291A4 (en) | 1996-10-09 | 1997-10-09 | Dna encoding galanin galr3 receptors and uses thereof |
JP51773898A JP2002514055A (en) | 1996-10-09 | 1997-10-09 | DNA encoding galanin GALR3 receptor and use thereof |
US09/058,333 US6368812B1 (en) | 1996-10-09 | 1998-04-09 | Process for determining the agonist or antagonist of galanin receptor (GALR3) |
US09/199,737 US6287788B1 (en) | 1996-10-09 | 1998-11-25 | DNA encoding galanin GALR3 receptors and uses thereof |
US10/006,343 US20030119096A1 (en) | 1997-01-24 | 2001-12-03 | Method of treating an abnormality using a GALR3 receptor antagonist |
US10/007,132 US7022489B2 (en) | 1996-10-09 | 2001-12-03 | Method of using cells expressing galanin receptor 3 (GALR3) |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US72813996A | 1996-10-09 | 1996-10-09 | |
US08/728,139 | 1996-10-09 | ||
US76796496A | 1996-12-17 | 1996-12-17 | |
US08/767,964 | 1996-12-17 | ||
US78726197A | 1997-01-24 | 1997-01-24 | |
US08/787,261 | 1997-01-24 | ||
US08/900,230 | 1997-07-23 | ||
US08/900,230 US6329197B2 (en) | 1996-10-09 | 1997-07-23 | DNA encoding galanin GALR3 receptors and uses thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/900,230 Continuation-In-Part US6329197B2 (en) | 1996-10-09 | 1997-07-23 | DNA encoding galanin GALR3 receptors and uses thereof |
Related Child Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/058,333 A-371-Of-International US6368812B1 (en) | 1996-10-09 | 1998-04-09 | Process for determining the agonist or antagonist of galanin receptor (GALR3) |
US09/058,333 Continuation-In-Part US6368812B1 (en) | 1996-10-09 | 1998-04-09 | Process for determining the agonist or antagonist of galanin receptor (GALR3) |
US09/199,737 Continuation US6287788B1 (en) | 1996-10-09 | 1998-11-25 | DNA encoding galanin GALR3 receptors and uses thereof |
US09/771,287 Continuation US20030082641A1 (en) | 1996-10-09 | 2001-01-26 | A method of treating depression using a galr3 receptor antagonist |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998015570A1 true WO1998015570A1 (en) | 1998-04-16 |
Family
ID=27505578
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/018222 WO1998015570A1 (en) | 1996-10-09 | 1997-10-09 | Dna encoding galanin galr3 receptors and uses thereof |
Country Status (8)
Country | Link |
---|---|
US (5) | US6329197B2 (en) |
EP (1) | EP0956291A4 (en) |
JP (1) | JP2002514055A (en) |
AU (1) | AU741041B2 (en) |
CA (1) | CA2248222A1 (en) |
DE (1) | DE956291T1 (en) |
ES (1) | ES2151465T1 (en) |
WO (1) | WO1998015570A1 (en) |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999031130A1 (en) * | 1997-12-17 | 1999-06-24 | Merck & Co., Inc. | Galanin receptor galr3 and nucleotides encoding same |
EP1141020A2 (en) * | 1998-12-31 | 2001-10-10 | Synaptic Pharmaceutical Corporation | Dna encoding a human melanin concentrating hormone receptor (mch1) and uses thereof |
WO2001087930A2 (en) * | 2000-05-18 | 2001-11-22 | Bayer Aktiengesellschaft | Human galanin receptor-like g protein coupled receptor |
US6395877B1 (en) | 1998-06-30 | 2002-05-28 | Millennium Pharmaceuticals, Inc. | 14273 receptor, a novel G-protein coupled receptor |
US6448005B1 (en) | 1998-06-30 | 2002-09-10 | Millennium Pharmaceuticals, Inc. | 14723 Receptor, a novel G-protein coupled receptor |
WO2004014376A1 (en) * | 2002-08-07 | 2004-02-19 | Synaptic Pharmaceutical Corporation | Gal3 receptor antagonists for the treatment of affective disorders |
US6733990B1 (en) | 1999-08-03 | 2004-05-11 | Millennium Pharmaceuticals, Inc. | Nucleic acid encoding 15571, a GPCR-like molecule of the secretin-like family |
US7057028B2 (en) | 1998-06-30 | 2006-06-06 | Millennium Pharmaceuticals, Inc. | 14273 Receptor, a novel G-protein coupled receptor |
US7220775B2 (en) | 2002-08-07 | 2007-05-22 | H. Lundbeck A/S | Compound useful for the treatment of neuropathic pain |
US7465750B2 (en) | 2001-01-31 | 2008-12-16 | H. Lundbeck A/S | Use of GAL3 antagonist for treatment of depression and/or anxiety and compounds useful in such methods |
US7534579B2 (en) | 1998-06-30 | 2009-05-19 | Millennium Pharmaceuticals, Inc. | 14273 receptor, a novel G-protein coupled receptor |
EP2140862A2 (en) | 2001-01-31 | 2010-01-06 | H.Lundbeck A/S | Use of GAL 3 receptor antagonists for the treatment of depression and /or anxiety and compounds useful in such methods |
US7868034B2 (en) | 2001-01-31 | 2011-01-11 | H. Lundbeck A/S | Use of GALR3 receptor antagonists for the treatment of depression and/or anxiety and compounds useful in such methods |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6329197B2 (en) * | 1996-10-09 | 2001-12-11 | Synaptic Pharmaceutical Corporation | DNA encoding galanin GALR3 receptors and uses thereof |
US20030119096A1 (en) * | 1997-01-24 | 2003-06-26 | Bard Jonathan A. | Method of treating an abnormality using a GALR3 receptor antagonist |
US20020137890A1 (en) * | 1997-03-31 | 2002-09-26 | Genentech, Inc. | Secreted and transmembrane polypeptides and nucleic acids encoding the same |
US6693182B1 (en) * | 1997-08-22 | 2004-02-17 | Schering Corporation | Mammalian galanin receptors |
US20030092042A1 (en) * | 2001-08-27 | 2003-05-15 | David Mu | Amplified oncogenes and their involvement in cancer |
US7642281B2 (en) * | 2002-08-07 | 2010-01-05 | Helicon Therapeutics, Inc. | Indolone compounds useful to treat cognitive impairment |
US7582673B2 (en) * | 2004-10-21 | 2009-09-01 | High Point Pharmaceuticals, Llc | Bissulfonamide compounds as agonists of GalR1, compositions, and methods of use |
WO2009144581A1 (en) | 2008-05-27 | 2009-12-03 | Dako Denmark A/S | Hybridization compositions and methods |
US9388456B2 (en) * | 2009-02-26 | 2016-07-12 | Dako Denmark A/S | Compositions and methods for performing a stringent wash step in hybridization applications |
US20100284977A1 (en) * | 2009-04-28 | 2010-11-11 | University Of South Carolina | Expression of Anti-Nociceptive Compounds from Endogenously Regulated Promoters |
JP5884736B2 (en) * | 2010-12-03 | 2016-03-15 | 株式会社ツムラ | Daikenchuto bioassay method and quality control method using the same |
WO2013046033A1 (en) | 2011-09-30 | 2013-04-04 | Dako Denmark A/S | Hybridization compositions and methods using formamide |
EP3252173A1 (en) | 2011-10-21 | 2017-12-06 | Dako Denmark A/S | Hybridization compositions and methods |
WO2013109713A1 (en) * | 2012-01-18 | 2013-07-25 | The General Hospital Corporation | Targeting rnas to microvesicles |
KR101514440B1 (en) * | 2013-12-13 | 2015-04-22 | 고려대학교 산학협력단 | Use of spexin as ligand of galanin receptor type 3 |
CN113512592A (en) * | 2021-04-23 | 2021-10-19 | 中国科学院深圳先进技术研究院 | Primer, probe and kit for detecting galanin secretory cells |
WO2024116101A1 (en) * | 2022-11-29 | 2024-06-06 | Neuracle Science Co., Ltd. | Use of galr2 agonists to treat gastrointestinal and/or endocrine disorders |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995022608A1 (en) * | 1994-02-17 | 1995-08-24 | Rhone-Poulenc Rorer S.A. | Galanin receptor, nucleic acids, transformed cells and uses thereof |
US5462856A (en) * | 1990-07-19 | 1995-10-31 | Bunsen Rush Laboratories, Inc. | Methods for identifying chemicals that act as agonists or antagonists for receptors and other proteins involved in signal transduction via pathways that utilize G-proteins |
Family Cites Families (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5401629A (en) | 1990-08-07 | 1995-03-28 | The Salk Institute Biotechnology/Industrial Associates, Inc. | Assay methods and compositions useful for measuring the transduction of an intracellular signal |
AU8951191A (en) | 1990-10-29 | 1992-05-26 | Dekalb Plant Genetics | Isolation of biological materials using magnetic particles |
WO1992012997A1 (en) | 1991-01-16 | 1992-08-06 | The General Hospital Corporation | Human galanin |
WO1992015015A1 (en) | 1991-02-25 | 1992-09-03 | Zymogenetic, Inc. | Methods for detecting galanin antagonists |
JP3135262B2 (en) | 1991-03-06 | 2001-02-13 | ガーヴァン インスティチュート オブ メディカル リサーチ | Human galanin, cDNA clone encoding human galanin, and method for producing human galanin |
US5576296A (en) | 1991-05-15 | 1996-11-19 | Aktiebolaget Astra | Galanin antagonist |
SE9101472D0 (en) | 1991-05-15 | 1991-05-15 | Trion Forskning & Utveckling | GALANIN ANTAGONIST |
US5436155A (en) | 1991-12-31 | 1995-07-25 | Arch Development Corporation | Isolated DNA encoding a somatostatin receptor |
US5290808A (en) | 1992-11-06 | 1994-03-01 | Carter-Wallace, Inc. | Method to control the intake of food |
US6399325B1 (en) | 1994-10-13 | 2002-06-04 | Takeda Chemical Industries, Ltd. | DNA encoding a galanin receptor |
US6562862B1 (en) | 1994-10-20 | 2003-05-13 | Eli Lilly And Company | Methods of inhibiting physiological conditions associated with an excess of neuropeptide Y |
US6410686B1 (en) | 1996-06-05 | 2002-06-25 | Bayer Corporation | Galanin receptor 2 protein |
SE9602822D0 (en) | 1996-07-19 | 1996-07-19 | Astra Pharma Inc | New receptor |
PT1342410E (en) | 1996-07-24 | 2010-05-11 | Neurotargets Ltd | Use of a galanin agonist in the preparation of a medicament for improving memory and other cognitive functions |
US6329197B2 (en) | 1996-10-09 | 2001-12-11 | Synaptic Pharmaceutical Corporation | DNA encoding galanin GALR3 receptors and uses thereof |
JP2002508169A (en) | 1997-12-17 | 2002-03-19 | メルク エンド カムパニー インコーポレーテッド | Galanin receptor GALR3 and nucleotides encoding the same |
-
1997
- 1997-07-23 US US08/900,230 patent/US6329197B2/en not_active Expired - Fee Related
- 1997-10-09 ES ES97910850T patent/ES2151465T1/en active Pending
- 1997-10-09 EP EP19970910850 patent/EP0956291A4/en not_active Withdrawn
- 1997-10-09 JP JP51773898A patent/JP2002514055A/en not_active Withdrawn
- 1997-10-09 WO PCT/US1997/018222 patent/WO1998015570A1/en not_active Application Discontinuation
- 1997-10-09 CA CA002248222A patent/CA2248222A1/en not_active Abandoned
- 1997-10-09 DE DE0956291T patent/DE956291T1/en active Pending
- 1997-10-09 AU AU48125/97A patent/AU741041B2/en not_active Ceased
-
1998
- 1998-04-09 US US09/058,333 patent/US6368812B1/en not_active Expired - Fee Related
- 1998-11-25 US US09/199,737 patent/US6287788B1/en not_active Expired - Fee Related
-
2001
- 2001-01-26 US US09/771,287 patent/US20030082641A1/en not_active Abandoned
- 2001-12-03 US US10/007,132 patent/US7022489B2/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5462856A (en) * | 1990-07-19 | 1995-10-31 | Bunsen Rush Laboratories, Inc. | Methods for identifying chemicals that act as agonists or antagonists for receptors and other proteins involved in signal transduction via pathways that utilize G-proteins |
WO1995022608A1 (en) * | 1994-02-17 | 1995-08-24 | Rhone-Poulenc Rorer S.A. | Galanin receptor, nucleic acids, transformed cells and uses thereof |
Non-Patent Citations (3)
Title |
---|
METHODS IN ENZYMOLOGY, 1995, Vol. 250, BOUVIER M. et al., "Dynamic Palmitoylation of G-Protein-Coupled Receptors in Eukaryotic Cells", pages 300-314. * |
MOLECULAR BRAIN RESEARCH, 28 December 1995, Vol. 34, No. 2, PARKER et al., "Cloning and Characterization of the Rat GA1R1 Galanin Receptor from Rin 14B Insulinoma Cells", pages 179-189. * |
See also references of EP0956291A4 * |
Cited By (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999031130A1 (en) * | 1997-12-17 | 1999-06-24 | Merck & Co., Inc. | Galanin receptor galr3 and nucleotides encoding same |
US6448005B1 (en) | 1998-06-30 | 2002-09-10 | Millennium Pharmaceuticals, Inc. | 14723 Receptor, a novel G-protein coupled receptor |
US7534579B2 (en) | 1998-06-30 | 2009-05-19 | Millennium Pharmaceuticals, Inc. | 14273 receptor, a novel G-protein coupled receptor |
US6395877B1 (en) | 1998-06-30 | 2002-05-28 | Millennium Pharmaceuticals, Inc. | 14273 receptor, a novel G-protein coupled receptor |
US7057028B2 (en) | 1998-06-30 | 2006-06-06 | Millennium Pharmaceuticals, Inc. | 14273 Receptor, a novel G-protein coupled receptor |
US7393655B2 (en) | 1998-12-31 | 2008-07-01 | H. Lundbeck A/S | Methods of identifying melanin concentrating hormone receptor antagonists |
EP1141020A4 (en) * | 1998-12-31 | 2002-09-04 | Synaptic Pharma Corp | Dna encoding a human melanin concentrating hormone receptor (mch1) and uses thereof |
EP1141020A2 (en) * | 1998-12-31 | 2001-10-10 | Synaptic Pharmaceutical Corporation | Dna encoding a human melanin concentrating hormone receptor (mch1) and uses thereof |
US6723552B2 (en) | 1998-12-31 | 2004-04-20 | Synaptic Pharmaceutical Corporation | DNA encoding a human melanin concentrating hormone receptor (MCH1) and uses thereof |
US6733990B1 (en) | 1999-08-03 | 2004-05-11 | Millennium Pharmaceuticals, Inc. | Nucleic acid encoding 15571, a GPCR-like molecule of the secretin-like family |
WO2001087930A2 (en) * | 2000-05-18 | 2001-11-22 | Bayer Aktiengesellschaft | Human galanin receptor-like g protein coupled receptor |
WO2001087930A3 (en) * | 2000-05-18 | 2002-08-29 | Bayer Ag | Human galanin receptor-like g protein coupled receptor |
US7465750B2 (en) | 2001-01-31 | 2008-12-16 | H. Lundbeck A/S | Use of GAL3 antagonist for treatment of depression and/or anxiety and compounds useful in such methods |
EP2140862A2 (en) | 2001-01-31 | 2010-01-06 | H.Lundbeck A/S | Use of GAL 3 receptor antagonists for the treatment of depression and /or anxiety and compounds useful in such methods |
EP2140864A2 (en) | 2001-01-31 | 2010-01-06 | H.Lundbeck A/S | Use of GAL3 receptor antagonists for the treatment of depression and / or anxiety and compounds useful in such methods |
EP2140862A3 (en) * | 2001-01-31 | 2010-02-17 | H.Lundbeck A/S | Use of GAL 3 receptor antagonists for the treatment of depression and /or anxiety and compounds useful in such methods |
US7868034B2 (en) | 2001-01-31 | 2011-01-11 | H. Lundbeck A/S | Use of GALR3 receptor antagonists for the treatment of depression and/or anxiety and compounds useful in such methods |
US7220775B2 (en) | 2002-08-07 | 2007-05-22 | H. Lundbeck A/S | Compound useful for the treatment of neuropathic pain |
WO2004014376A1 (en) * | 2002-08-07 | 2004-02-19 | Synaptic Pharmaceutical Corporation | Gal3 receptor antagonists for the treatment of affective disorders |
Also Published As
Publication number | Publication date |
---|---|
EP0956291A4 (en) | 2002-10-24 |
US6287788B1 (en) | 2001-09-11 |
ES2151465T1 (en) | 2001-01-01 |
US20030082641A1 (en) | 2003-05-01 |
JP2002514055A (en) | 2002-05-14 |
AU4812597A (en) | 1998-05-05 |
US20010009766A1 (en) | 2001-07-26 |
AU741041B2 (en) | 2001-11-22 |
US20030027254A1 (en) | 2003-02-06 |
US6329197B2 (en) | 2001-12-11 |
US7022489B2 (en) | 2006-04-04 |
DE956291T1 (en) | 2001-05-23 |
US6368812B1 (en) | 2002-04-09 |
EP0956291A1 (en) | 1999-11-17 |
CA2248222A1 (en) | 1998-04-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7022489B2 (en) | Method of using cells expressing galanin receptor 3 (GALR3) | |
US7371535B2 (en) | Processes for determining compounds that interact with snorf33 receptor | |
WO2000012692A1 (en) | Dna encoding a gababr2 polypeptide and uses thereof | |
US6790656B1 (en) | DNA encoding galanin GALR2 receptors | |
WO1997026853A9 (en) | Dna encoding galanin galr2 receptors and uses thereof | |
US6262246B1 (en) | DNA encoding mammalian neuropeptides FF (NPFF) receptors and uses thereof | |
US5972624A (en) | Method of identifying ligands which bind recombinant galanin receptor (GALR2) | |
AU782848B2 (en) | DNA encoding SNORF62 and SNORF72 receptors | |
WO1999020751A1 (en) | Dna encoding a gababr2 polypeptide and uses thereof | |
US20030119096A1 (en) | Method of treating an abnormality using a GALR3 receptor antagonist | |
US20020156265A1 (en) | DNA encoding a GABABR2 polypeptide and uses thereof | |
US20030139589A1 (en) | G protein coupled receptor A4 | |
EP1044265A1 (en) | Dna encoding a gaba br2 polypeptide and uses thereof | |
CA2284857A1 (en) | G protein coupled receptor a4 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 09058333 Country of ref document: US |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2248222 Country of ref document: CA Ref country code: CA Ref document number: 2248222 Kind code of ref document: A Format of ref document f/p: F |
|
CFP | Corrected version of a pamphlet front page |
Free format text: ADD INID NUMBER (63) "RELATED BY CONTINUATION (CON) OR CONTINUATION-IN-PART (CIP) TO EARLIER APPLICATION" WHICH WAS INADVERTENTLY OMITTED FROM THE FRONT PAGE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09199737 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1997910850 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1997910850 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1997910850 Country of ref document: EP |