CA2383177A1 - Human g-protein coupled receptor - Google Patents
Human g-protein coupled receptor Download PDFInfo
- Publication number
- CA2383177A1 CA2383177A1 CA002383177A CA2383177A CA2383177A1 CA 2383177 A1 CA2383177 A1 CA 2383177A1 CA 002383177 A CA002383177 A CA 002383177A CA 2383177 A CA2383177 A CA 2383177A CA 2383177 A1 CA2383177 A1 CA 2383177A1
- Authority
- CA
- Canada
- Prior art keywords
- igs3
- polypeptide
- seq
- nucleotide sequence
- receptor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 102000005962 receptors Human genes 0.000 title claims abstract description 53
- 108020003175 receptors Proteins 0.000 title claims abstract description 53
- 101000887490 Homo sapiens Guanine nucleotide-binding protein G(z) subunit alpha Proteins 0.000 title description 3
- 102000052301 human GNAZ Human genes 0.000 title description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 183
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 176
- 229920001184 polypeptide Polymers 0.000 claims abstract description 173
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 91
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 91
- 239000002157 polynucleotide Substances 0.000 claims abstract description 90
- 238000000034 method Methods 0.000 claims abstract description 78
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 70
- 241001465754 Metazoa Species 0.000 claims abstract description 48
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 34
- 150000001875 compounds Chemical class 0.000 claims abstract description 28
- 241000282414 Homo sapiens Species 0.000 claims abstract description 20
- 239000000556 agonist Substances 0.000 claims abstract description 20
- 239000005557 antagonist Substances 0.000 claims abstract description 17
- 238000011282 treatment Methods 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 72
- 125000003729 nucleotide group Chemical group 0.000 claims description 63
- 239000002773 nucleotide Substances 0.000 claims description 62
- 108020004414 DNA Proteins 0.000 claims description 56
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 44
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 40
- 230000014509 gene expression Effects 0.000 claims description 39
- 230000000694 effects Effects 0.000 claims description 38
- 201000010099 disease Diseases 0.000 claims description 28
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 21
- 239000003446 ligand Substances 0.000 claims description 14
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 11
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 10
- 230000035772 mutation Effects 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 8
- 150000007523 nucleic acids Chemical group 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 238000001727 in vivo Methods 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 5
- 108700028369 Alleles Proteins 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000004102 animal cell Anatomy 0.000 claims description 2
- 210000005253 yeast cell Anatomy 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims 5
- 230000002508 compound effect Effects 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
- 230000003292 diminished effect Effects 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 abstract description 39
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 abstract description 38
- 239000013598 vector Substances 0.000 abstract description 16
- 238000003556 assay Methods 0.000 abstract description 15
- 230000009261 transgenic effect Effects 0.000 abstract description 15
- 238000012216 screening Methods 0.000 abstract description 13
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 230000003213 activating effect Effects 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 description 36
- 235000018102 proteins Nutrition 0.000 description 29
- 239000000203 mixture Substances 0.000 description 27
- 239000013615 primer Substances 0.000 description 19
- 235000001014 amino acid Nutrition 0.000 description 17
- 230000027455 binding Effects 0.000 description 16
- 208000035475 disorder Diseases 0.000 description 16
- 108700019146 Transgenes Proteins 0.000 description 15
- 238000009472 formulation Methods 0.000 description 15
- 229940024606 amino acid Drugs 0.000 description 14
- 150000001413 amino acids Chemical class 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 14
- 108010064300 Receptor Activity-Modifying Proteins Proteins 0.000 description 13
- 102000015146 Receptor Activity-Modifying Proteins Human genes 0.000 description 13
- 108091006027 G proteins Proteins 0.000 description 12
- 102000030782 GTP binding Human genes 0.000 description 12
- 108091000058 GTP-Binding Proteins 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 244000005700 microbiome Species 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- -1 e.g. Proteins 0.000 description 9
- 230000002068 genetic effect Effects 0.000 description 9
- 238000009396 hybridization Methods 0.000 description 9
- 230000001404 mediated effect Effects 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 238000013459 approach Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 229960005486 vaccine Drugs 0.000 description 8
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 241000124008 Mammalia Species 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 6
- 230000000890 antigenic effect Effects 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000002759 chromosomal effect Effects 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 108010016616 cysteinylglycine Proteins 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000006467 substitution reaction Methods 0.000 description 6
- 230000035899 viability Effects 0.000 description 6
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 5
- 238000012300 Sequence Analysis Methods 0.000 description 5
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 5
- 230000000692 anti-sense effect Effects 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 230000026731 phosphorylation Effects 0.000 description 5
- 238000006366 phosphorylation reaction Methods 0.000 description 5
- 238000007423 screening assay Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 108090000994 Catalytic RNA Proteins 0.000 description 4
- 102000053642 Catalytic RNA Human genes 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 229930010555 Inosine Natural products 0.000 description 4
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 4
- 102000006382 Ribonucleases Human genes 0.000 description 4
- 108010083644 Ribonucleases Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 108091007498 Transmembrane domain 2 Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000001594 aberrant effect Effects 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 102000030621 adenylate cyclase Human genes 0.000 description 4
- 108060000200 adenylate cyclase Proteins 0.000 description 4
- 230000004075 alteration Effects 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- 238000005755 formation reaction Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000001415 gene therapy Methods 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 229960003786 inosine Drugs 0.000 description 4
- 230000010354 integration Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 230000000069 prophylactic effect Effects 0.000 description 4
- 230000001177 retroviral effect Effects 0.000 description 4
- 108091092562 ribozyme Proteins 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- SUMYEVXWCAYLLJ-GUBZILKMSA-N Ala-Leu-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O SUMYEVXWCAYLLJ-GUBZILKMSA-N 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- UUYBFNKHOCJCHT-VHSXEESVSA-N Gly-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN UUYBFNKHOCJCHT-VHSXEESVSA-N 0.000 description 3
- KUIDCYNIEJBZBU-AJNGGQMLSA-N Leu-Ile-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O KUIDCYNIEJBZBU-AJNGGQMLSA-N 0.000 description 3
- IFMPDNRWZZEZSL-SRVKXCTJSA-N Leu-Leu-Cys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(O)=O IFMPDNRWZZEZSL-SRVKXCTJSA-N 0.000 description 3
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 3
- BTWMICVCQLKKNR-DCAQKATOSA-N Val-Leu-Ser Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C([O-])=O BTWMICVCQLKKNR-DCAQKATOSA-N 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000010363 gene targeting Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000007857 nested PCR Methods 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- MLGCXEBRWGEOQX-UHFFFAOYSA-N tetradifon Chemical compound C1=CC(Cl)=CC=C1S(=O)(=O)C1=CC(Cl)=C(Cl)C=C1Cl MLGCXEBRWGEOQX-UHFFFAOYSA-N 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 230000009452 underexpressoin Effects 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 208000019901 Anxiety disease Diseases 0.000 description 2
- GMFAGHNRXPSSJS-SRVKXCTJSA-N Arg-Leu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O GMFAGHNRXPSSJS-SRVKXCTJSA-N 0.000 description 2
- NJSNXIOKBHPFMB-GMOBBJLQSA-N Asn-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)N)N NJSNXIOKBHPFMB-GMOBBJLQSA-N 0.000 description 2
- KTTCQQNRRLCIBC-GHCJXIJMSA-N Asp-Ile-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O KTTCQQNRRLCIBC-GHCJXIJMSA-N 0.000 description 2
- 208000036864 Attention deficit/hyperactivity disease Diseases 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 2
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 102000013446 GTP Phosphohydrolases Human genes 0.000 description 2
- 108091006109 GTPases Proteins 0.000 description 2
- OREPWMPAUWIIAM-ZPFDUUQYSA-N Gln-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N OREPWMPAUWIIAM-ZPFDUUQYSA-N 0.000 description 2
- WBBVTGIFQIZBHP-JBACZVJFSA-N Gln-Trp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)NC(=O)[C@H](CCC(=O)N)N WBBVTGIFQIZBHP-JBACZVJFSA-N 0.000 description 2
- YPHPEHMXOYTEQG-LAEOZQHASA-N Glu-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O YPHPEHMXOYTEQG-LAEOZQHASA-N 0.000 description 2
- YOBGUCWZPXJHTN-BQBZGAKWSA-N Gly-Ser-Arg Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YOBGUCWZPXJHTN-BQBZGAKWSA-N 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- NUKXXNFEUZGPRO-BJDJZHNGSA-N Ile-Leu-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N NUKXXNFEUZGPRO-BJDJZHNGSA-N 0.000 description 2
- XLXPYSDGMXTTNQ-UHFFFAOYSA-N Ile-Phe-Leu Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 XLXPYSDGMXTTNQ-UHFFFAOYSA-N 0.000 description 2
- DZMWFIRHFFVBHS-ZEWNOJEFSA-N Ile-Tyr-Phe Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)N DZMWFIRHFFVBHS-ZEWNOJEFSA-N 0.000 description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 2
- IASQBRJGRVXNJI-YUMQZZPRSA-N Leu-Cys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)NCC(O)=O IASQBRJGRVXNJI-YUMQZZPRSA-N 0.000 description 2
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 2
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 2
- FBNPMTNBFFAMMH-AVGNSLFASA-N Leu-Val-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-AVGNSLFASA-N 0.000 description 2
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 2
- AAKRWBIIGKPOKQ-ONGXEEELSA-N Leu-Val-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AAKRWBIIGKPOKQ-ONGXEEELSA-N 0.000 description 2
- 239000006142 Luria-Bertani Agar Substances 0.000 description 2
- QFGVDCBPDGLVTA-SZMVWBNQSA-N Lys-Gln-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCCN)C(O)=O)=CNC2=C1 QFGVDCBPDGLVTA-SZMVWBNQSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- DNDVVILEHVMWIS-LPEHRKFASA-N Met-Asp-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N DNDVVILEHVMWIS-LPEHRKFASA-N 0.000 description 2
- 108060003370 Neurotensin receptor Proteins 0.000 description 2
- 238000000636 Northern blotting Methods 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 208000021384 Obsessive-Compulsive disease Diseases 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- HGNGAMWHGGANAU-WHOFXGATSA-N Phe-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 HGNGAMWHGGANAU-WHOFXGATSA-N 0.000 description 2
- XALFIVXGQUEGKV-JSGCOSHPSA-N Phe-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 XALFIVXGQUEGKV-JSGCOSHPSA-N 0.000 description 2
- KCNSGAMPBPYUAI-CIUDSAMLSA-N Ser-Leu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O KCNSGAMPBPYUAI-CIUDSAMLSA-N 0.000 description 2
- JAWGSPUJAXYXJA-IHRRRGAJSA-N Ser-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=CC=C1 JAWGSPUJAXYXJA-IHRRRGAJSA-N 0.000 description 2
- HHJFMHQYEAAOBM-ZLUOBGJFSA-N Ser-Ser-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O HHJFMHQYEAAOBM-ZLUOBGJFSA-N 0.000 description 2
- ZESGVALRVJIVLZ-VFCFLDTKSA-N Thr-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O ZESGVALRVJIVLZ-VFCFLDTKSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 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 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 108010062796 arginyllysine Proteins 0.000 description 2
- 208000015802 attention deficit-hyperactivity disease Diseases 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- YKPUWZUDDOIDPM-SOFGYWHQSA-N capsaicin Chemical compound COC1=CC(CNC(=O)CCCC\C=C\C(C)C)=CC=C1O YKPUWZUDDOIDPM-SOFGYWHQSA-N 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 208000015114 central nervous system disease Diseases 0.000 description 2
- 206010008118 cerebral infarction Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000004186 co-expression Effects 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 108010074027 glycyl-seryl-phenylalanine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 108091006093 heterotrimeric G proteins Proteins 0.000 description 2
- 102000034345 heterotrimeric G proteins Human genes 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 208000002551 irritable bowel syndrome Diseases 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000004481 post-translational protein modification Effects 0.000 description 2
- 208000028173 post-traumatic stress disease Diseases 0.000 description 2
- 230000001323 posttranslational effect Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 108010080629 tryptophan-leucine Proteins 0.000 description 2
- 238000010798 ubiquitination Methods 0.000 description 2
- 230000034512 ubiquitination Effects 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- AZUYLZMQTIKGSC-UHFFFAOYSA-N 1-[6-[4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methylindazol-5-yl)pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl]prop-2-en-1-one Chemical compound ClC=1C(=C2C=NNC2=CC=1C)C=1C(=NN(C=1C)C1CC2(CN(C2)C(C=C)=O)C1)C=1C=C2C=NN(C2=CC=1)C AZUYLZMQTIKGSC-UHFFFAOYSA-N 0.000 description 1
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- LVRVABPNVHYXRT-BQWXUCBYSA-N 52906-92-0 Chemical compound C([C@H](N)C(=O)N[C@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(O)=O)C(C)C)C1=CC=CC=C1 LVRVABPNVHYXRT-BQWXUCBYSA-N 0.000 description 1
- 230000005730 ADP ribosylation Effects 0.000 description 1
- KQFRUSHJPKXBMB-BHDSKKPTSA-N Ala-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)C)C(O)=O)=CNC2=C1 KQFRUSHJPKXBMB-BHDSKKPTSA-N 0.000 description 1
- YCRAFFCYWOUEOF-DLOVCJGASA-N Ala-Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 YCRAFFCYWOUEOF-DLOVCJGASA-N 0.000 description 1
- LFFOJBOTZUWINF-ZANVPECISA-N Ala-Trp-Gly Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C)C(=O)NCC(O)=O)=CNC2=C1 LFFOJBOTZUWINF-ZANVPECISA-N 0.000 description 1
- YXXPVUOMPSZURS-ZLIFDBKOSA-N Ala-Trp-Leu Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](C)N)=CNC2=C1 YXXPVUOMPSZURS-ZLIFDBKOSA-N 0.000 description 1
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 101000772461 Arabidopsis thaliana Thioredoxin reductase 1, mitochondrial Proteins 0.000 description 1
- 101000772460 Arabidopsis thaliana Thioredoxin reductase 2 Proteins 0.000 description 1
- VWVPYNGMOCSSGK-GUBZILKMSA-N Arg-Arg-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O VWVPYNGMOCSSGK-GUBZILKMSA-N 0.000 description 1
- OVVUNXXROOFSIM-SDDRHHMPSA-N Arg-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O OVVUNXXROOFSIM-SDDRHHMPSA-N 0.000 description 1
- IGULQRCJLQQPSM-DCAQKATOSA-N Arg-Cys-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IGULQRCJLQQPSM-DCAQKATOSA-N 0.000 description 1
- JUWQNWXEGDYCIE-YUMQZZPRSA-N Arg-Gln-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O JUWQNWXEGDYCIE-YUMQZZPRSA-N 0.000 description 1
- NVCIXQYNWYTLDO-IHRRRGAJSA-N Arg-His-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCCN=C(N)N)N NVCIXQYNWYTLDO-IHRRRGAJSA-N 0.000 description 1
- PZBSKYJGKNNYNK-ULQDDVLXSA-N Arg-Leu-Tyr Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCN=C(N)N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O PZBSKYJGKNNYNK-ULQDDVLXSA-N 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- QQEWINYJRFBLNN-DLOVCJGASA-N Asn-Ala-Phe Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QQEWINYJRFBLNN-DLOVCJGASA-N 0.000 description 1
- CUQUEHYSSFETRD-ACZMJKKPSA-N Asn-Asp-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N CUQUEHYSSFETRD-ACZMJKKPSA-N 0.000 description 1
- ZPMNECSEJXXNBE-CIUDSAMLSA-N Asn-Cys-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O ZPMNECSEJXXNBE-CIUDSAMLSA-N 0.000 description 1
- DXVMJJNAOVECBA-WHFBIAKZSA-N Asn-Gly-Asn Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O DXVMJJNAOVECBA-WHFBIAKZSA-N 0.000 description 1
- JQSWHKKUZMTOIH-QWRGUYRKSA-N Asn-Gly-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N JQSWHKKUZMTOIH-QWRGUYRKSA-N 0.000 description 1
- FTNRWCPWDWRPAV-BZSNNMDCSA-N Asn-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CC(N)=O)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FTNRWCPWDWRPAV-BZSNNMDCSA-N 0.000 description 1
- ZJIFRAPZHAGLGR-MELADBBJSA-N Asn-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC(=O)N)N)C(=O)O ZJIFRAPZHAGLGR-MELADBBJSA-N 0.000 description 1
- NYQHSUGFEWDWPD-ACZMJKKPSA-N Asp-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N NYQHSUGFEWDWPD-ACZMJKKPSA-N 0.000 description 1
- WBDWQKRLTVCDSY-WHFBIAKZSA-N Asp-Gly-Asp Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O WBDWQKRLTVCDSY-WHFBIAKZSA-N 0.000 description 1
- CUQDCPXNZPDYFQ-ZLUOBGJFSA-N Asp-Ser-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O CUQDCPXNZPDYFQ-ZLUOBGJFSA-N 0.000 description 1
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 206010003805 Autism Diseases 0.000 description 1
- 208000020706 Autistic disease Diseases 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010004446 Benign prostatic hyperplasia Diseases 0.000 description 1
- 208000020925 Bipolar disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101800004538 Bradykinin Proteins 0.000 description 1
- 102400000967 Bradykinin Human genes 0.000 description 1
- 201000006474 Brain Ischemia Diseases 0.000 description 1
- 208000032841 Bulimia Diseases 0.000 description 1
- 206010006550 Bulimia nervosa Diseases 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 102100038518 Calcitonin Human genes 0.000 description 1
- 108090000932 Calcitonin Gene-Related Peptide Proteins 0.000 description 1
- 102000056906 Calcitonin Receptor-Like Human genes 0.000 description 1
- 101710118454 Calcitonin gene-related peptide type 1 receptor Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000006029 Cardiomegaly Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 206010008120 Cerebral ischaemia Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- KKZHXOOZHFABQQ-UWJYBYFXSA-N Cys-Ala-Tyr Chemical compound SC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 KKZHXOOZHFABQQ-UWJYBYFXSA-N 0.000 description 1
- UCMIKRLLIOVDRJ-XKBZYTNZSA-N Cys-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CS)N)O UCMIKRLLIOVDRJ-XKBZYTNZSA-N 0.000 description 1
- UDDITVWSXPEAIQ-IHRRRGAJSA-N Cys-Phe-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UDDITVWSXPEAIQ-IHRRRGAJSA-N 0.000 description 1
- SRZZZTMJARUVPI-JBDRJPRFSA-N Cys-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N SRZZZTMJARUVPI-JBDRJPRFSA-N 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 206010012218 Delirium Diseases 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 206010051153 Diabetic gastroparesis Diseases 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 102000015554 Dopamine receptor Human genes 0.000 description 1
- 108050004812 Dopamine receptor Proteins 0.000 description 1
- 208000012661 Dyskinesia Diseases 0.000 description 1
- 208000032928 Dyslipidaemia Diseases 0.000 description 1
- 208000014094 Dystonic disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000002045 Endothelin Human genes 0.000 description 1
- 108050009340 Endothelin Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 208000007530 Essential hypertension Diseases 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 102400001370 Galanin Human genes 0.000 description 1
- 101800002068 Galanin Proteins 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- JXFLPKSDLDEOQK-JHEQGTHGSA-N Gln-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O JXFLPKSDLDEOQK-JHEQGTHGSA-N 0.000 description 1
- RGAOLBZBLOJUTP-GRLWGSQLSA-N Gln-Ile-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)NC(=O)[C@H](CCC(=O)N)N RGAOLBZBLOJUTP-GRLWGSQLSA-N 0.000 description 1
- RONJIBWTGKVKFY-HTUGSXCWSA-N Gln-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O RONJIBWTGKVKFY-HTUGSXCWSA-N 0.000 description 1
- IYAUFWMUCGBFMQ-CIUDSAMLSA-N Glu-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)CN=C(N)N IYAUFWMUCGBFMQ-CIUDSAMLSA-N 0.000 description 1
- KRGZZKWSBGPLKL-IUCAKERBSA-N Glu-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)O)N KRGZZKWSBGPLKL-IUCAKERBSA-N 0.000 description 1
- RLFSBAPJTYKSLG-WHFBIAKZSA-N Gly-Ala-Asp Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O RLFSBAPJTYKSLG-WHFBIAKZSA-N 0.000 description 1
- LCNXZQROPKFGQK-WHFBIAKZSA-N Gly-Asp-Ser Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O LCNXZQROPKFGQK-WHFBIAKZSA-N 0.000 description 1
- SABZDFAAOJATBR-QWRGUYRKSA-N Gly-Cys-Phe Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SABZDFAAOJATBR-QWRGUYRKSA-N 0.000 description 1
- NNCSJUBVFBDDLC-YUMQZZPRSA-N Gly-Leu-Ser Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O NNCSJUBVFBDDLC-YUMQZZPRSA-N 0.000 description 1
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 1
- MHZXESQPPXOING-KBPBESRZSA-N Gly-Lys-Phe Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O MHZXESQPPXOING-KBPBESRZSA-N 0.000 description 1
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 1
- VDCRBJACQKOSMS-JSGCOSHPSA-N Gly-Phe-Val Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O VDCRBJACQKOSMS-JSGCOSHPSA-N 0.000 description 1
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 1
- RZEDHGORCKRINR-STQMWFEESA-N Gly-Trp-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)CN RZEDHGORCKRINR-STQMWFEESA-N 0.000 description 1
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical group C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- UQTKYYNHMVAOAA-HJPIBITLSA-N His-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N UQTKYYNHMVAOAA-HJPIBITLSA-N 0.000 description 1
- JMSONHOUHFDOJH-GUBZILKMSA-N His-Ser-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 JMSONHOUHFDOJH-GUBZILKMSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000591385 Homo sapiens Neurotensin receptor type 1 Proteins 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- RSDHVTMRXSABSV-GHCJXIJMSA-N Ile-Asn-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N RSDHVTMRXSABSV-GHCJXIJMSA-N 0.000 description 1
- LNJLOZYNZFGJMM-DEQVHRJGSA-N Ile-His-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N LNJLOZYNZFGJMM-DEQVHRJGSA-N 0.000 description 1
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- XLXPYSDGMXTTNQ-DKIMLUQUSA-N Ile-Phe-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(C)C)C(O)=O XLXPYSDGMXTTNQ-DKIMLUQUSA-N 0.000 description 1
- AGGIYSLVUKVOPT-HTFCKZLJSA-N Ile-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N AGGIYSLVUKVOPT-HTFCKZLJSA-N 0.000 description 1
- CNMOKANDJMLAIF-CIQUZCHMSA-N Ile-Thr-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O CNMOKANDJMLAIF-CIQUZCHMSA-N 0.000 description 1
- VBGCPJBKUXRYDA-DSYPUSFNSA-N Ile-Trp-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCCCN)C(=O)O)N VBGCPJBKUXRYDA-DSYPUSFNSA-N 0.000 description 1
- MITYXXNZSZLHGG-OBAATPRFSA-N Ile-Trp-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N MITYXXNZSZLHGG-OBAATPRFSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 206010021518 Impaired gastric emptying Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- 108010093008 Kinins Proteins 0.000 description 1
- 102000002397 Kinins Human genes 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 101000591392 Leishmania infantum Probable flavin mononucleotide-dependent alkene reductase Proteins 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- YORLGJINWYYIMX-KKUMJFAQSA-N Leu-Cys-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YORLGJINWYYIMX-KKUMJFAQSA-N 0.000 description 1
- VPKIQULSKFVCSM-SRVKXCTJSA-N Leu-Gln-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VPKIQULSKFVCSM-SRVKXCTJSA-N 0.000 description 1
- KEVYYIMVELOXCT-KBPBESRZSA-N Leu-Gly-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KEVYYIMVELOXCT-KBPBESRZSA-N 0.000 description 1
- QLDHBYRUNQZIJQ-DKIMLUQUSA-N Leu-Ile-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O QLDHBYRUNQZIJQ-DKIMLUQUSA-N 0.000 description 1
- OMHLATXVNQSALM-FQUUOJAGSA-N Leu-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(C)C)N OMHLATXVNQSALM-FQUUOJAGSA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- DNDWZFHLZVYOGF-KKUMJFAQSA-N Leu-Leu-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O DNDWZFHLZVYOGF-KKUMJFAQSA-N 0.000 description 1
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- BGZCJDGBBUUBHA-KKUMJFAQSA-N Leu-Lys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O BGZCJDGBBUUBHA-KKUMJFAQSA-N 0.000 description 1
- YESNGRDJQWDYLH-KKUMJFAQSA-N Leu-Phe-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N YESNGRDJQWDYLH-KKUMJFAQSA-N 0.000 description 1
- WXDRGWBQZIMJDE-ULQDDVLXSA-N Leu-Phe-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(O)=O WXDRGWBQZIMJDE-ULQDDVLXSA-N 0.000 description 1
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 1
- GOFJOGXGMPHOGL-DCAQKATOSA-N Leu-Ser-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(C)C GOFJOGXGMPHOGL-DCAQKATOSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- ZDJQVSIPFLMNOX-RHYQMDGZSA-N Leu-Thr-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N ZDJQVSIPFLMNOX-RHYQMDGZSA-N 0.000 description 1
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 1
- AIQWYVFNBNNOLU-RHYQMDGZSA-N Leu-Thr-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O AIQWYVFNBNNOLU-RHYQMDGZSA-N 0.000 description 1
- CNWDWAMPKVYJJB-NUTKFTJISA-N Leu-Trp-Ala Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)CC(C)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 CNWDWAMPKVYJJB-NUTKFTJISA-N 0.000 description 1
- FPFOYSCDUWTZBF-IHPCNDPISA-N Leu-Trp-Leu Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H]([NH3+])CC(C)C)C(=O)N[C@@H](CC(C)C)C([O-])=O)=CNC2=C1 FPFOYSCDUWTZBF-IHPCNDPISA-N 0.000 description 1
- ONHCDMBHPQIPAI-YTQUADARSA-N Leu-Trp-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N3CCC[C@@H]3C(=O)O)N ONHCDMBHPQIPAI-YTQUADARSA-N 0.000 description 1
- JGKHAFUAPZCCDU-BZSNNMDCSA-N Leu-Tyr-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=C(O)C=C1 JGKHAFUAPZCCDU-BZSNNMDCSA-N 0.000 description 1
- XOEDPXDZJHBQIX-ULQDDVLXSA-N Leu-Val-Phe Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XOEDPXDZJHBQIX-ULQDDVLXSA-N 0.000 description 1
- MSFITIBEMPWCBD-ULQDDVLXSA-N Leu-Val-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 MSFITIBEMPWCBD-ULQDDVLXSA-N 0.000 description 1
- NRQRKMYZONPCTM-CIUDSAMLSA-N Lys-Asp-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NRQRKMYZONPCTM-CIUDSAMLSA-N 0.000 description 1
- WGLAORUKDGRINI-WDCWCFNPSA-N Lys-Glu-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WGLAORUKDGRINI-WDCWCFNPSA-N 0.000 description 1
- MYZMQWHPDAYKIE-SRVKXCTJSA-N Lys-Leu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O MYZMQWHPDAYKIE-SRVKXCTJSA-N 0.000 description 1
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- DBXMFHGGHMXYHY-DCAQKATOSA-N Met-Leu-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O DBXMFHGGHMXYHY-DCAQKATOSA-N 0.000 description 1
- XPVCDCMPKCERFT-GUBZILKMSA-N Met-Ser-Arg Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O XPVCDCMPKCERFT-GUBZILKMSA-N 0.000 description 1
- 208000019695 Migraine disease Diseases 0.000 description 1
- 102400001357 Motilin Human genes 0.000 description 1
- 101800002372 Motilin Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 1
- WYBVBIHNJWOLCJ-UHFFFAOYSA-N N-L-arginyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCCN=C(N)N WYBVBIHNJWOLCJ-UHFFFAOYSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- 102000017921 NTSR1 Human genes 0.000 description 1
- 102000017938 NTSR2 Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102400001103 Neurotensin Human genes 0.000 description 1
- 101800001814 Neurotensin Proteins 0.000 description 1
- 102000017922 Neurotensin receptor Human genes 0.000 description 1
- 102400001111 Nociceptin Human genes 0.000 description 1
- 108090000622 Nociceptin Proteins 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 108050002069 Olfactory receptors Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000010175 Opsin Human genes 0.000 description 1
- 108050001704 Opsin Proteins 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241000282520 Papio Species 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 108091093037 Peptide nucleic acid Chemical group 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- PLNHHOXNVSYKOB-JYJNAYRXSA-N Phe-Arg-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CC=CC=C1)N PLNHHOXNVSYKOB-JYJNAYRXSA-N 0.000 description 1
- DJPXNKUDJKGQEE-BZSNNMDCSA-N Phe-Asp-Phe Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DJPXNKUDJKGQEE-BZSNNMDCSA-N 0.000 description 1
- ALHULIGNEXGFRM-QWRGUYRKSA-N Phe-Cys-Gly Chemical compound OC(=O)CNC(=O)[C@H](CS)NC(=O)[C@@H](N)CC1=CC=CC=C1 ALHULIGNEXGFRM-QWRGUYRKSA-N 0.000 description 1
- QEPZQAPZKIPVDV-KKUMJFAQSA-N Phe-Cys-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N QEPZQAPZKIPVDV-KKUMJFAQSA-N 0.000 description 1
- WFDAEEUZPZSMOG-SRVKXCTJSA-N Phe-Cys-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O WFDAEEUZPZSMOG-SRVKXCTJSA-N 0.000 description 1
- RJYBHZVWJPUSLB-QEWYBTABSA-N Phe-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N RJYBHZVWJPUSLB-QEWYBTABSA-N 0.000 description 1
- VZFPYFRVHMSSNA-JURCDPSOSA-N Phe-Ile-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=CC=C1 VZFPYFRVHMSSNA-JURCDPSOSA-N 0.000 description 1
- BYAIIACBWBOJCU-URLPEUOOSA-N Phe-Ile-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O BYAIIACBWBOJCU-URLPEUOOSA-N 0.000 description 1
- TXKWKTWYTIAZSV-KKUMJFAQSA-N Phe-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N TXKWKTWYTIAZSV-KKUMJFAQSA-N 0.000 description 1
- MSHZERMPZKCODG-ACRUOGEOSA-N Phe-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MSHZERMPZKCODG-ACRUOGEOSA-N 0.000 description 1
- OKQQWSNUSQURLI-JYJNAYRXSA-N Phe-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CC=CC=C1)N OKQQWSNUSQURLI-JYJNAYRXSA-N 0.000 description 1
- NJJBATPLUQHRBM-IHRRRGAJSA-N Phe-Pro-Ser Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CO)C(=O)O NJJBATPLUQHRBM-IHRRRGAJSA-N 0.000 description 1
- XDMMOISUAHXXFD-SRVKXCTJSA-N Phe-Ser-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O XDMMOISUAHXXFD-SRVKXCTJSA-N 0.000 description 1
- YFXXRYFWJFQAFW-JHYOHUSXSA-N Phe-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O YFXXRYFWJFQAFW-JHYOHUSXSA-N 0.000 description 1
- 206010034912 Phobia Diseases 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- QBFONMUYNSNKIX-AVGNSLFASA-N Pro-Arg-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O QBFONMUYNSNKIX-AVGNSLFASA-N 0.000 description 1
- PTLOFJZJADCNCD-DCAQKATOSA-N Pro-Glu-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H]1CCCN1 PTLOFJZJADCNCD-DCAQKATOSA-N 0.000 description 1
- VTFXTWDFPTWNJY-RHYQMDGZSA-N Pro-Leu-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VTFXTWDFPTWNJY-RHYQMDGZSA-N 0.000 description 1
- VDHGTOHMHHQSKG-JYJNAYRXSA-N Pro-Val-Phe Chemical compound CC(C)[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O VDHGTOHMHHQSKG-JYJNAYRXSA-N 0.000 description 1
- FIODMZKLZFLYQP-GUBZILKMSA-N Pro-Val-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O FIODMZKLZFLYQP-GUBZILKMSA-N 0.000 description 1
- 208000004403 Prostatic Hyperplasia Diseases 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102000003923 Protein Kinase C Human genes 0.000 description 1
- 108090000315 Protein Kinase C Proteins 0.000 description 1
- 208000010362 Protozoan Infections Diseases 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 208000003782 Raynaud disease Diseases 0.000 description 1
- 208000012322 Raynaud phenomenon Diseases 0.000 description 1
- 108091005682 Receptor kinases Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 102100040756 Rhodopsin Human genes 0.000 description 1
- 108090000820 Rhodopsin Proteins 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 241000555745 Sciuridae Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- HBZBPFLJNDXRAY-FXQIFTODSA-N Ser-Ala-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O HBZBPFLJNDXRAY-FXQIFTODSA-N 0.000 description 1
- QVOGDCQNGLBNCR-FXQIFTODSA-N Ser-Arg-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O QVOGDCQNGLBNCR-FXQIFTODSA-N 0.000 description 1
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 1
- CXBFHZLODKPIJY-AAEUAGOBSA-N Ser-Gly-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N CXBFHZLODKPIJY-AAEUAGOBSA-N 0.000 description 1
- LQESNKGTTNHZPZ-GHCJXIJMSA-N Ser-Ile-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O LQESNKGTTNHZPZ-GHCJXIJMSA-N 0.000 description 1
- RIAKPZVSNBBNRE-BJDJZHNGSA-N Ser-Ile-Leu Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O RIAKPZVSNBBNRE-BJDJZHNGSA-N 0.000 description 1
- RRVFEDGUXSYWOW-BZSNNMDCSA-N Ser-Phe-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RRVFEDGUXSYWOW-BZSNNMDCSA-N 0.000 description 1
- KKKVOZNCLALMPV-XKBZYTNZSA-N Ser-Thr-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O KKKVOZNCLALMPV-XKBZYTNZSA-N 0.000 description 1
- VLMIUSLQONKLDV-HEIBUPTGSA-N Ser-Thr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VLMIUSLQONKLDV-HEIBUPTGSA-N 0.000 description 1
- BDMWLJLPPUCLNV-XGEHTFHBSA-N Ser-Thr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O BDMWLJLPPUCLNV-XGEHTFHBSA-N 0.000 description 1
- YEDSOSIKVUMIJE-DCAQKATOSA-N Ser-Val-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O YEDSOSIKVUMIJE-DCAQKATOSA-N 0.000 description 1
- ODRUTDLAONAVDV-IHRRRGAJSA-N Ser-Val-Tyr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ODRUTDLAONAVDV-IHRRRGAJSA-N 0.000 description 1
- 208000036623 Severe mental retardation Diseases 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 208000005392 Spasm Diseases 0.000 description 1
- 241000256248 Spodoptera Species 0.000 description 1
- 241000295644 Staphylococcaceae Species 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000032851 Subarachnoid Hemorrhage Diseases 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- QWMPARMKIDVBLV-VZFHVOOUSA-N Thr-Cys-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O QWMPARMKIDVBLV-VZFHVOOUSA-N 0.000 description 1
- ONNSECRQFSTMCC-XKBZYTNZSA-N Thr-Glu-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O ONNSECRQFSTMCC-XKBZYTNZSA-N 0.000 description 1
- ADPHPKGWVDHWML-PPCPHDFISA-N Thr-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N ADPHPKGWVDHWML-PPCPHDFISA-N 0.000 description 1
- FLPZMPOZGYPBEN-PPCPHDFISA-N Thr-Leu-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FLPZMPOZGYPBEN-PPCPHDFISA-N 0.000 description 1
- XKWABWFMQXMUMT-HJGDQZAQSA-N Thr-Pro-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XKWABWFMQXMUMT-HJGDQZAQSA-N 0.000 description 1
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- RIOVOFZXVOWCCX-SBCJRHGPSA-N Trp-Ile-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=3C4=CC=CC=C4NC=3)[C@@H](C)CC)C(O)=O)=CNC2=C1 RIOVOFZXVOWCCX-SBCJRHGPSA-N 0.000 description 1
- VCGOTJGGBXEBFO-FDARSICLSA-N Trp-Pro-Ile Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VCGOTJGGBXEBFO-FDARSICLSA-N 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- SEFNTZYRPGBDCY-IHRRRGAJSA-N Tyr-Arg-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N)O SEFNTZYRPGBDCY-IHRRRGAJSA-N 0.000 description 1
- GULIUBBXCYPDJU-CQDKDKBSSA-N Tyr-Leu-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CC1=CC=C(O)C=C1 GULIUBBXCYPDJU-CQDKDKBSSA-N 0.000 description 1
- NSGZILIDHCIZAM-KKUMJFAQSA-N Tyr-Leu-Ser Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N NSGZILIDHCIZAM-KKUMJFAQSA-N 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010046555 Urinary retention Diseases 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- UDNYEPLJTRDMEJ-RCOVLWMOSA-N Val-Asn-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N UDNYEPLJTRDMEJ-RCOVLWMOSA-N 0.000 description 1
- XJFXZQKJQGYFMM-GUBZILKMSA-N Val-Cys-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)O)N XJFXZQKJQGYFMM-GUBZILKMSA-N 0.000 description 1
- NXRAUQGGHPCJIB-RCOVLWMOSA-N Val-Gly-Asn Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O NXRAUQGGHPCJIB-RCOVLWMOSA-N 0.000 description 1
- BMOFUVHDBROBSE-DCAQKATOSA-N Val-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N BMOFUVHDBROBSE-DCAQKATOSA-N 0.000 description 1
- WLHIIWDIDLQTKP-IHRRRGAJSA-N Val-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)C(C)C WLHIIWDIDLQTKP-IHRRRGAJSA-N 0.000 description 1
- RFKJNTRMXGCKFE-FHWLQOOXSA-N Val-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC(C)C)C(O)=O)=CNC2=C1 RFKJNTRMXGCKFE-FHWLQOOXSA-N 0.000 description 1
- QPPZEDOTPZOSEC-RCWTZXSCSA-N Val-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](C(C)C)N)O QPPZEDOTPZOSEC-RCWTZXSCSA-N 0.000 description 1
- HJSLDXZAZGFPDK-ULQDDVLXSA-N Val-Phe-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C(C)C)N HJSLDXZAZGFPDK-ULQDDVLXSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- ZNGPROMGGGFOAA-JYJNAYRXSA-N Val-Tyr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=C(O)C=C1 ZNGPROMGGGFOAA-JYJNAYRXSA-N 0.000 description 1
- ZHWZDZFWBXWPDW-GUBZILKMSA-N Val-Val-Cys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(O)=O ZHWZDZFWBXWPDW-GUBZILKMSA-N 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 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
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical class C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000001800 adrenalinergic effect Effects 0.000 description 1
- 239000000464 adrenergic agent Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- PPQRONHOSHZGFQ-LMVFSUKVSA-N aldehydo-D-ribose 5-phosphate Chemical group OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PPQRONHOSHZGFQ-LMVFSUKVSA-N 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 230000010516 arginylation Effects 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-L aspartate group Chemical group N[C@@H](CC(=O)[O-])C(=O)[O-] CKLJMWTZIZZHCS-REOHCLBHSA-L 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000001593 cAMP accumulation Effects 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 229960002504 capsaicin Drugs 0.000 description 1
- 235000017663 capsaicin Nutrition 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000019788 craving Nutrition 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 108010041898 cytomegalovirus receptor Proteins 0.000 description 1
- 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 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229960002086 dextran Drugs 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
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 208000010118 dystonia Diseases 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000009762 endothelial cell differentiation Effects 0.000 description 1
- ZUBDGKVDJUIMQQ-UBFCDGJISA-N endothelin-1 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)NC(=O)[C@H]1NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H](C(C)C)NC(=O)[C@H]2CSSC[C@@H](C(N[C@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N2)=O)NC(=O)[C@@H](CO)NC(=O)[C@H](N)CSSC1)C1=CNC=N1 ZUBDGKVDJUIMQQ-UBFCDGJISA-N 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 230000001667 episodic effect Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 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 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 230000022244 formylation Effects 0.000 description 1
- 238000006170 formylation reaction Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000006251 gamma-carboxylation Effects 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 108010019832 glycyl-asparaginyl-glycine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 150000003278 haem Chemical group 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 238000002169 hydrotherapy Methods 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000009399 inbreeding Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 208000001286 intracranial vasospasm Diseases 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000026045 iodination Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 1
- 108010049589 leucyl-leucyl-leucine Proteins 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 208000024714 major depressive disease Diseases 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000007246 mechanism 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
- 230000002503 metabolic effect Effects 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical group CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 206010027599 migraine Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 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 description 1
- 230000004899 motility Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000003551 muscarinic effect Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 230000007498 myristoylation Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 239000003176 neuroleptic agent Substances 0.000 description 1
- PCJGZPGTCUMMOT-ISULXFBGSA-N neurotensin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 PCJGZPGTCUMMOT-ISULXFBGSA-N 0.000 description 1
- PULGYDLMFSFVBL-SMFNREODSA-N nociceptin Chemical compound C([C@@H](C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O)[C@@H](C)O)NC(=O)CNC(=O)CNC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 PULGYDLMFSFVBL-SMFNREODSA-N 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000009400 out breeding Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000001314 paroxysmal effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 235000008729 phenylalanine Nutrition 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 208000019899 phobic disease Diseases 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000013823 prenylation Effects 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 108010053725 prolylvaline Proteins 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 208000002815 pulmonary hypertension Diseases 0.000 description 1
- 229940043131 pyroglutamate Drugs 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000011536 re-plating Methods 0.000 description 1
- 108091006082 receptor inhibitors Proteins 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 206010038464 renal hypertension Diseases 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 210000002265 sensory receptor cell Anatomy 0.000 description 1
- 235000004400 serine Nutrition 0.000 description 1
- 150000003355 serines Chemical class 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 208000019116 sleep disease Diseases 0.000 description 1
- 208000020685 sleep-wake disease Diseases 0.000 description 1
- YZHUMGUJCQRKBT-UHFFFAOYSA-M sodium chlorate Chemical compound [Na+].[O-]Cl(=O)=O YZHUMGUJCQRKBT-UHFFFAOYSA-M 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000019635 sulfation Effects 0.000 description 1
- 238000005670 sulfation reaction Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical group [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 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 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 235000002374 tyrosine Nutrition 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Diabetes (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Endocrinology (AREA)
- Neurology (AREA)
- Zoology (AREA)
- Oncology (AREA)
- Biomedical Technology (AREA)
- Communicable Diseases (AREA)
- Neurosurgery (AREA)
- Heart & Thoracic Surgery (AREA)
- Emergency Medicine (AREA)
- Cardiology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Pulmonology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Pain & Pain Management (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
Abstract
The present invention relates to the IGS3 G-protein coupled receptor family, and to polynucleotides encoding said IGS3 proteins. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides, to a vector containing said polynucleotides, a host cell containing such vector and non-human transgenic animals where the IGS3-gene is either overexpressed, misexpressed, underexpressed or suppressed (knock-out animals). The invention further relates to a method for screening compounds capable to act as an agonist or an antagonist of said G-protein coupled receptor family IGS3 and the use of IGS3 polypeptides and polynucleotides and agonists or antagonists to the IGS3 receptor family in the treatment of a broad range of disorders and diagnostic assays for such conditions.
Description
human G-protein coupled receptor Description The present invention relates to novel identified polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production. More particularly, the polynucleotides and polypeptides of the present invention relate to a G-protein coupled receptor (GPCR), hereinafter referred to as IGS3. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides, to a vector containing said polynucleotides, a host cell containing such vector and transgenic animals where the IGS3-gene is either overexpressed, misexpressed, underexpressed and/or suppressed (knock-out animals).
The invention further relates to a method for screening compounds capable to act as an agonist or an antagonist of said G-protein coupled receptor IGS3.
BACKGROUND OF THE INVENTION
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers; e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl. Acad. Sci., USA, 1987, 84:46-50; Kobilka, B.K., et al., Science, 1987, 238:650-656; Bunzow, J.R., et al., Nature, 1988, 336:783-787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenylate cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
For example, in one form of signal transduction, upon hormone binding to a GPCR the receptor interacts with the heterotrimeric G-protein and induces the dissociation of GDP from the guanine nucleotide-binding site. At normal cellular concentrations of guanine nucleotides, GTP
fills the site immediately. Binding of GTP to the a subunit of the G-protein causes the dissociation of the G-protein from the receptor and the dissociation of the G-protein into a and (3y subunits. The GTP-carrying form then binds to activated adenylate cyclase.
Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself (a subunit possesses an intrinsic GTPase activity), returns the G-protein to its basal, inactive form. The GTPase activity of the a subunit is, in essence, an internal clock that controls an on/off switch. The GDP bound form of the a subunit has high affinity for /3y and subsequent reassociation of aGDP with py returns the system to the CONFIRMATION COPY
The invention further relates to a method for screening compounds capable to act as an agonist or an antagonist of said G-protein coupled receptor IGS3.
BACKGROUND OF THE INVENTION
It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers; e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K., et al., Proc. Natl. Acad. Sci., USA, 1987, 84:46-50; Kobilka, B.K., et al., Science, 1987, 238:650-656; Bunzow, J.R., et al., Nature, 1988, 336:783-787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenylate cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8).
For example, in one form of signal transduction, upon hormone binding to a GPCR the receptor interacts with the heterotrimeric G-protein and induces the dissociation of GDP from the guanine nucleotide-binding site. At normal cellular concentrations of guanine nucleotides, GTP
fills the site immediately. Binding of GTP to the a subunit of the G-protein causes the dissociation of the G-protein from the receptor and the dissociation of the G-protein into a and (3y subunits. The GTP-carrying form then binds to activated adenylate cyclase.
Hydrolysis of GTP
to GDP, catalyzed by the G-protein itself (a subunit possesses an intrinsic GTPase activity), returns the G-protein to its basal, inactive form. The GTPase activity of the a subunit is, in essence, an internal clock that controls an on/off switch. The GDP bound form of the a subunit has high affinity for /3y and subsequent reassociation of aGDP with py returns the system to the CONFIRMATION COPY
basal state. Thus the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector (in this example adenylate cyclase), and as a clock that controls the duration of the signal.
The membrane bound superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane a-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
The G-protein coupled receptor family includes dopamine receptors which bind to neuroleptic drugs used for treating CNS disorders. Other examples of members of this family include, but are not limited to calcitonin, adrenergic, neuropeptideY, somastotatin, neurotensin, neurokinin, capsaicin, VIP, CGRP, CRF, CCK, bradykinin, galanin, motilin, nociceptin, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsin, endothelial differentiation gene-1, rhodopsin, odorant, and cytomegalovirus receptors.
Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structures. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TMS, TM6 and TM7. The cytoplasmic loop which connects TM5 and TM6 may be a major component of the G-protein binding domain.
Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the (3-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
Recently, it was discovered that certain GPCRs, like the calcitonin-receptor like receptor, might interact with small single pass membrane proteins called receptor activity modifying proteins (RAMP's). This interaction of the GPCR with a certain RAMP is determining which natural ligands have relevant affinity for the GPCR-RAMP combination and regulate the functional signaling activity of the complex (McLathie, L.M. et al., Nature (1998) 393:333-339).
The membrane bound superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane a-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors.
The G-protein coupled receptor family includes dopamine receptors which bind to neuroleptic drugs used for treating CNS disorders. Other examples of members of this family include, but are not limited to calcitonin, adrenergic, neuropeptideY, somastotatin, neurotensin, neurokinin, capsaicin, VIP, CGRP, CRF, CCK, bradykinin, galanin, motilin, nociceptin, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsin, endothelial differentiation gene-1, rhodopsin, odorant, and cytomegalovirus receptors.
Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structures. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TMS, TM6 and TM7. The cytoplasmic loop which connects TM5 and TM6 may be a major component of the G-protein binding domain.
Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the (3-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
Recently, it was discovered that certain GPCRs, like the calcitonin-receptor like receptor, might interact with small single pass membrane proteins called receptor activity modifying proteins (RAMP's). This interaction of the GPCR with a certain RAMP is determining which natural ligands have relevant affinity for the GPCR-RAMP combination and regulate the functional signaling activity of the complex (McLathie, L.M. et al., Nature (1998) 393:333-339).
For some receptors, the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said sockets being surrounded by hydrophobic residues of the G-protein coupled receptors. The hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form a polar ligand-binding site. TM3 has been implicated in several G-protein coupled receptors as having a ligand-binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding.
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331 ). Different G-protein a-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.
Receptors - primarily the GPCR class - have led to more than half of the currently known drugs (brews, Nature Biotechnology, 1996, 14: 1516). This indicates that these receptors have an established, proven history as therapeutic targets. The new IGS3 GPCR
described in this invention clearly satisfies a need in the art for identification and characterization of further receptors that can play a role in diagnosing, preventing, ameliorating or correcting dysfunctions, disorders, or diseases, hereafter generally referred to as "the Diseases". The Diseases include, but are not limited to, psychiatric and CNS disorders, including schizophrenia, episodic paroxysmal anxiety (EPA) disorders such as obsessive compulsive disorder (OCD), post traumatic stress disorder (PTSD), phobia and panic, major depressive disorder, bipolar disorder, Parkinson's disease, general anxiety disorder, autism, delirium, multiple sclerosis, Alzheimer disease/dementia and other neurodegenerative diseases, severe mental retardation, dyskinesias, Huntington's disease, Tourett's syndrome, tics, tremor, dystonia, spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleep disorder, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmias, myocardial infarction, cardiac hypertrophy, hypotension, hypertension -e.g. essential hypertension, renal hypertension, or pulmonary hypertension, thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, Raynaud's disease, kidney disease -e.g. renal failure;
dyslipidemias; obesity; emesis; gastrointestinal disorders, including irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD), motility disorders and conditions of delayed gastric emptying, such as post operative or diabetic gastroparesis, and diabetes, ulcers - e.g. gastric ulcer; diarrhoea; other diseases including osteoporosis; inflammations; infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; chemotherapy induced injury;
tumor invasion; immune disorders; urinary retention; asthma; allergies;
arthritis; benign prostatic hypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus; and gynaecological disorders.
SUMMARY OF THE INVENTION
In one aspect, the invention relates to IGS3 polypeptides, polynucleotides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such IGS3 polypeptides, polynucleotides and recombinant materials. Such uses include, but are not limited to, use as a therapeutic target and for treatment of one of the Diseases as mentioned above.
In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with IGS3 imbalance with the identified compounds. Yet another aspect of the invention -relates to diagnostic assays for detecting diseases associated with inappropriate IGS3 activity or levels. A
further aspect of the invention relates to animal-based systems which act as models for disorders arising from aberrant expression or activity of IGS3.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1. Schematic representation of the relative positions of the different DNA clones that were isolated to generate the consensus IGS3 cDNA sequence. HNT1370 represents the "founding" genomic clone. ~,-IGS3.1A,B etc. indicate separate (nearly) overlapping sequence contigs obtained from sequence analysis of DNA from lambda clone IGS3.1. PCR
primers that have been described in this document are indicated (1P#). CONSENSUS denotes the contig that was obtained after merging all obtained sequences. The part of the CONSENSUS
contig that was fully validated by sequence analysis of at least three independent clones is represented by IGS3DNA (SEQ ID NO: 1 ). The 330 amino acids long open reading frame present in IGS3DNA
is indicated with "**". The position of EST AF003828 is indicated with "_- .
Table 1: IGS3-DNA of SEO ID NO: 1 5'-TTAATCTCTTCAAGCCTCTGATTTCCTCTCCTGTAAAACAGGGGCGGTAATTACCACATA
CAGGGGCACCAGTGGAGGTTTTCTGAGCATGGATCCAACCACCCCGGCCTGGGGAACAGA
AAGTACAACAGTGAATGGAAATGACCAAGCCCTTCTTCTGCTTTGTGGCAAGGAGACCCT
GATCCCGGTCTTCCTGATCCTTTTCATTGCCCTGGTCGGGCTGGTAGGAAACGGGTTTGT
GCTCTGGCTCCTGGGCTTCCGCATGCGCAGGAACGCCTTCTCTGTCTACGTCCTCAGCCT
O GGCCGGGGCCGACTTCCTCTTCCTCTGCTTCCAGATTATAAATTGCCTGGTGTACCTCAG
TAACTTCTTCTGTTCCATCTCCATCAATTTCCCTAGCTTCTTCACCACTGTGATGACCTG
TGCCTACCTTGCAGGCCTGAGCATGCTGAGCACCGTCAGCACCGAGCGCTGCCTGTCCGT
CCTGTGGCCCATCTGGTATCGCTGCCGCCGCCCCAGACACCTGTCAGCGGTCGTGTGTGT
CCTGCTCTGGGCCCTGTCCCTACTGCTGAGCATCTTGGAAGGGAAGTTCTGTGGCTTCTT
ATTTAGTGATGGTGACTCTGGTTGGTGTCAGACATTTGATTTCATCACTGCAGCGTGGCT
GATTTTTTTATTCATGGTTCTCTGTGGGTCCAGTCTGGCCCTGCTGGTCAGGATCCTCTG
TGGCTCCAGGGGTCTGCCACTGACCAGGCTGTACCTGACCATCCTGCTCACAGTGCTGGT
GTTCCTCCTCTGCGGCCTGCCCTTTGGCATTCAGTGGTTCCTAATATTATGGATCTGGAA
GGATTCTGATGTCTTATTTTGTCATATTCATCCAGTTTCAGTTGTCCTGTCATCTCTTAA
GCAGCAGCCGATCCTCAAGCTGGCTCTCCAGAGGGCTCTGCAGGACATTGCTGAGGTGGA
TCACAGTGAAGGATGCTTCCGTCAGGGCACCCCGGAGATGTCGAGAAGCAGTCTGGTGTA
GAGATGGACAGCCTCTACTTCCATCAGATATATGTG-3' Table 2: IGS3-protein of SEQ ID NO: 2 MDPTTPAWGTESTTVNGNDQALLLLCGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMR
RNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLSML
STVSTERCLSVLWPIWYRCRRPRHLSAWCVLLWALSLLLSILEGKFCGFLFSDGDSGWC
QTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVFLLCGLPFG
IQWFLILWIWKDSDVLFCHIHPVSWLSSLNSSANPIIYFFVGSFRKQWRLQQPILKLAL
QRALQDIAEVDHSEGCFRQGTPEMSRSSLV
DESCRIPTION OF THE INVENTION
Structural and chemical similarity, in the context of sequences and motifs, exists among the IGS3 GPCR of the invention and other human GPCR's. Therefore, IGS3 is implied to play a role among other things in the Diseases mentioned above.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
Definitions The following definitions are provided to facilitate understanding of certain terms used frequently herein.
"IGS3" refers, among others, to a polypeptide comprising the amino acid sequence set forth in SEQ ID N0:2, or a Variant thereof.
"Receptor Activity" or "Biological Activity of the Receptor" refers to the metabolic or physiologic function of said IGS3 including similar activities or improved activities or these activities with decreased undesirable side effects. Also included are antigenic and immunogenic activities of said IGS3.
"IGS3-gene" refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID N0:1 or Variants thereof and/or their complements.
"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library.
"Isolated" means altered "by the hand of man" from the natural state and/or separated from the natural environment. Thus, if an "isolated" composition or substance that occurs in nature has been "isolated", it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides"
include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
In addition, "polynucleotide" may also include triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide"
embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.
"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins, and/or to combinations thereof. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
"Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well-described in basic texts and in more detailed monographs, as well as in voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol; cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth. Enzymol. (1990) 182:626-646 and Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann. NY Acad. Sci.
(1992) 663:48-62.
"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties such as essential biological, structural, regulatory or biochemical properties. A
typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination. A
substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A
variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Identity" is a measure of the identity of nucleotide sequences or amino acid sequences.
In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.:
(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed.;
Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994;
SEQUENCE ANALYSIS
IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE
ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ).
While there exist a number of methods to measure identity between two polynucleotide or 5 polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math. (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math. (1988) 48:1073. Methods to determine identity and 10 similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J. Molec. Biol. (1990) 215:403). The word "homology" may substitute for the words "identity".
As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five nucleotide differences per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to any 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to any 5%
of the total nucleotides in the reference sequence may be inserted into the reference sequence, or in a number of nucleotides of up to any 5% of the total nucleotides in the reference sequence there may be a combination of deletion, insertion and substitution. These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID N0:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to any 5%
of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to any 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
Polypeptides of the Invention In one aspect, the present invention relates to IGS3 polypeptides (including proteins). The IGS3 polypeptides include the polypeptide of SEQ ID N0:2 and the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS
102196, deposited on September 15, 1999 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands); as well as polypeptides comprising the amino acid sequence of SEO
ID N0:2 and the polypeptide having the amino acid sequence encoded by the DNA
insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), and polypeptides comprising an amino acid sequence having at least 80%
identity to that of SEQ ID N0:2 and/or to the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands) over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to said amino acid sequence. Furthermore, those with at least 97%, in particular at least 99%, are highly preferred.
Also included within IGS3 polypeptides are polypeptides having the amino acid sequence which has at least 80% identity to the polypeptide having the amino acid sequence of SEO ID NO: 2 or the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands) over its entire length, and still more preferably at least 90%
identity, and even still more preferably at least 95% identity to SEO ID NO: 2. Furthermore, those with at least 97%, in particular at least 99% are highly preferred. Preferably IGS3 polypeptides exhibit at least one biological activity of the receptor.
In an additional embodiment of the invention, the IGS3 polypeptides may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, sequences which aid in detection such as antigenic peptide tags (such as the haemagglutinin (HA) tag), or an additional sequence for stability during recombinant production.
Fragments of the IGS3 polypeptides are also included in the invention. A
fragment is a polypeptide having an amino acid sequence that is the same as part of, but not all of, the amino acid sequence of the aforementioned IGS3 polypeptides. As with IGS3 polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20;
21-40, 41-60, 61-80, 81-100; and 101 to the end of IGS3 polypeptide. In this context "about"
includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of IGS3 polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments.
Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
Thus, the polypeptides of the invention include polypeptides having an amino acid sequence that is at least 80% identical to either that of SEQ ID N0:2 and/or the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS
102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), or fragments thereof with at least 80% identity to the corresponding fragment. Preferably, all of these polypeptide fragments retain the biological activity of the receptor, including antigenic activity.
Variants of the defined sequence and fragments also form part of the present invention.
Preferred variants are those that vary from the referents by conservative amino acid substitutions -- i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
The IGS3 polypeptides of the invention can be prepared in any suitable manner.
Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for preparing such polypeptides are well known in the art.
Polynucleotides of the Invention A further aspect of the invention relates to IGS3 polynucleotides. IGS3 polynucleotides include isolated polynucleotides which encode the IGS3 polypeptides and fragments, and polynucleotides closely related thereto. More specifically, the IGS3 polynucleotide of the invention includes a polynucleotide comprising the nucleotide sequence contained in SEQ ID
N0:1, such as the one capable of encoding a IGS3 polypeptide of SEQ ID NO: 2, polynucleotides having the particular sequence of SEQ ID NO: 1 and polynucleotides which essentially correspond to the DNA insert contained in the deposit no. CBS
102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands).
IGS3 polynucleotides further include polynucleotides comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the IGS3 polypeptide of SEQ ID N0:2, polynucleotides comprising a nucleotide sequence that is at least 80% identical to that of SEQ ID N0:1 over its entire length and a polynucleotide which essentially corresponds to the DNA insert contained in the deposit no. CBS
102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands).
In this regard, polynucleotides with at least 90% identity are particularly preferred, and those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred. Also included under IGS3 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEO ID NO: 1 or to the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands) to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such IGS3 polynucleotides.
IGS3 of the invention is structurally related to other proteins of the G-protein coupled receptor family, as shown by the results of BLAST searches in the public databases. The amino acid sequence of Table 2 (SEO ID N0:2) has about 35 % identity (using BLAST, Altschul S.F. et al. Nucleic Acids Res. (1997) 25:3389-3402) over most of its length (amino acid residues 2-306 ) with the protein encoded by the human mas oncogene (Sequence 1 in patent application WO
8707472). The sequence is 37% identical (amino acid residues 35-315) with the G-protein coupled receptor published in patent application WO 9616087 (GENESEQ 96P-897222 ). The nucleotide sequence of Table 1 (SEQ ID NO: 1 ) has 52 % and 54 % identity over most of its length to the two receptors above (GENESEO 87N-70685 and 96N-T28807 respectively). Also there is 48% identity to the human Somatostatin-3 receptor in residues 104-1144 (WO 9313130;
93N-Q45657). Hydropathy analysis (Kyte J. et al., J. Mol. Biol. (1982) 157:
105-132; Klein P.et al., Biochim. Biophys. Acta (1985) 815: 468-476) of the IGS3 protein sequence expectedly showed the presence of 7 transmembrane domains. Thus, IGS3 polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.
Polynucleotides of the invention can be obtained from natural sources such as genomic DNA. In particular, degenerated PCR primers can be designed that encode conserved regions within a particular GPCR gene subfamily. PCR amplification reactions on genomic DNA or cDNA
using the degenerate primers will result in the amplification of several members (both known and novel) of the gene family under consideration (the degenerated primers must be located within the same exon, when a genomic template is used). (Libert et al., Science, 1989, 244: 569-572).
Polynucleotides of the invention can also be synthesized using well-known and commercially available techniques.
The nucleotide sequence encoding the IGS3 polypeptide of SEQ ID N0:2 may be identical to the polypeptide encoding sequence contained in SEQ ID N0:1 (nucleotide number 149 to 1138), or it may be a different nucleotide sequence, which as a result of the redundancy (degeneracy) of the genetic code might also show alterations compared to the polypeptide encoding sequence contained in SEO ID N0:1, but also encodes the polypeptide of SEQ ID
N0:2.
When the polynucleotides of the invention are used for the recombinant production of the IGS3 polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may 5 also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
Further preferred embodiments are polynucleotides encoding IGS3 variants comprising the amino acid sequence of the IGS3 polypeptide of SEO ID N0:2 in which several, 5-10, 1-5, 1-10 3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.
The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter IGS3-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
15 shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create amino acid substitutions, create new restriction sites, alter modification (e.g. glycosylation or phosphorylation) patterns, change codon preference, produce splice variants, and so forth.
The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the polynucleotides described above. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably at least 97%, in particular at least 99% identity between the sequences.
Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEO ID N0:1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding IGS3 and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the IGS3 gene. People skilled in the art are well aware of such hybridization techniques. Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent. The probes generally will comprise at least 5 nucleotides, and preferably at least 8 nucleotides, and more preferably at least 10 nucleotides, yet even more preferably at least 12 nucleotides, in particular at least 15 nucleotides. Most preferred, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
One embodiment, to obtain a polynucleotide encoding the IGS3 polypeptide, including homologs and orthologs from species other than human, comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the SEQ
ID NO: 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 °C in a solution comprising: 50% formamide, 5xSSC (150mM NaCI, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 xSSC at about 65°C.
The polynucleotides and polypeptides of the present invention may be used as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
Vectors, Host Cells, Expression The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention.
For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY
(1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;
animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells;
and plant cells.
A great variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression.
Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals, i.e. derived from a different species.
If the IGS3 polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. In case the affinity or functional activity of the IGS3 polypeptide is modified by receptor activity modifying proteins (RAMP), coexpression of the relevant RAMP most likely at the surface of the cell is preferred and often required. Also in this event harvesting of cells expressing the IGS3 polypeptide and the relevant RAMP prior to use in screening assays is required. If the IGS3 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered. Membranes expressing the IGS3 polypeptide can be recovered by methods that are well known to a person skilled in the art. In general, such methods include harvesting of the cells expressing the IGS3 polypeptide and homogenization of the cells by a method such as, but not limited to, pottering.
The membranes may be recovered by washing the suspension one or several times.
IGS3 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well-known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
Diagnostic Assays This invention also relates to the use of IGS3 polynucleotides for use as diagnostic reagents. Detection of a mutated form of the IGS3 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of IGS3.
Also in this event co-expression of relevant receptor activity modifying proteins can be required to obtain diagnostic assays of desired quality. Individuals carrying mutations in the IGS3 gene may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to labeled IGS3 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science (1985) 230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method. See Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401. In another embodiment, an array of oligonucleotide probes comprising the IGS3 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
The diagnostic assays offer a process for diagnosing or determining a susceptibility to among other things the Diseases as mentioned above, through detection of mutation in the IGS3 gene by the methods described.
In addition, among other things, the Diseases as mentioned above can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of the IGS3 polypeptide or IGS3 mRNA.
Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
Assay techniques that can be used to determine levels of a protein, such as an IGS3, in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA
assays.
In another aspect, the present invention relates to a diagnostic kit for among other things the Diseases or suspectability to one of the Diseases as mentioned above.
The kit may comprise:
(a) an IGS3 polynucleotide, preferably the nucleotide sequence of SEQ ID N0:1, or a fragment thereof; and/or (b) a nucleotide sequence complementary to that of (a); and/or (c) an IGS3 polypeptide, preferably the polypeptide of SEO ID N0:2, or a fragment thereof; and/or (d) an antibody to an IGS3 polypeptide, preferably to the polypeptide of SEQ
ID NO: 2;
and/or (e) a RAMP polypeptide required for the relevant biological or antigenic properties of an IGS3 polypeptide.
It will be appreciated that in any such kit, (a), (b), (c) (d) or (e) may comprise a substantial component.
Chromosome Assays The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location 5 on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in 10 Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected 15 individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
Antibodies 20 The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them if required together with relevant RAMP's, may also be used as immunogens to produce antibodies immunospecific for the IGS3 polypeptides. The term "immunospecific"
means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
Antibodies generated against the IGS3 polypeptides may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique, which provides antibodies produced by continuous cell line cultures, may be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al., MONOCLONAL
ANTIBODIES AND
CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
Antibodies against IGS3 polypeptides as such, or against IGS3 polypeptide-RAMP
complexes, may also be employed to treat among other things the Diseases as mentioned above.
Animals Another aspect of the invention relates to non-human animal-based systems which act as models for disorders arising from aberrant expression or activity of IGS3. Non-human animal-based model systems may also be used to further characterize the activity of the IGS3 gene.
Such systems may be utilized as part of screening strategies designed to identify compounds which are capable to treat IGS3 based disorders such as among other things the Diseases as mentioned above.
In this way the animal-based models may be used to identify pharmaceutical compounds, therapies and interventions which may be effective in treating disorders of aberrant expression or activity of IGS3. In addition such animal models may be used to determine the LDSO and the EDSO in animal subjects. These data may be used to determine the in vivo efficacy of potential IGS3 disorder treatments.
Animal-based model systems of IGS3 based disorders, based on aberrant IGS3 expression or activity, may include both non-recombinant animals as well as recombinantly engineered transgenic animals.
Animal models for IGS3 disorders may include, for example, genetic models.
Animal models exhibiting IGS3 based disorder-like symptoms may be engineered by utilizing, for example, IGS3 sequences such as those described, above, in conjunction with techniques for producing transgenic animals that are well known to persons skilled in the art. For example, IGS3 sequences may be introduced into, and overexpressed and/or misexpressed in, the genome of the animal of interest, or, if endogenous IGS3 sequences are present, they may either be overexpressed, misexpressed, or, alternatively, may be disrupted in order to underexpress or inactivate IGS3 gene expression.
In order to overexpress or misexpress a IGS3 gene sequence, the coding portion of the IGS3 gene sequence may be ligated to a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal type of interest. Such regulatory regions will be well known to those skilled in the art, and may be utilized in the absence of undue experimentation.
For underexpression of an endogenous IGS3 gene sequence, such a sequence may be isolated and engineered such that when reintroduced into the genome of the animal of interest, the endogenous IGS3 gene alleles will be inactivated, or "knocked-out".
Preferably, the engineered IGS3 gene sequence is introduced via gene targeting such that the endogenous IGS3 sequence is disrupted upon integration of the engineered IGS3 gene sequence into the animal's genome.
Animals of any species, including, but not limited to, mice, rats, rabbits, squirrels, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.~c ., baboons, monkeys, and chimpanzees may be used to generate animal models of IGS3 related disorders.
Any technique known in the art may be used to introduce a IGS3 transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat.
No. 4,873,191);
retrovirus mediated gene transfer into germ lines (van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152, 1985); gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321, 1989,); electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-1814, 1983); and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723, 1989); etc.
For a review of such techniques, see Cordon, Transgenic Animals, Intl. Rev. Cytol.115:171-229, 1989.
The present invention provides for transgenic animals that carry the IGS3 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals. (See, for example, techniques described by Jakobovits, Curr.
Biol. 4:761-763, 1994) The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al.
(Lasko, M..et al., Proc. Natl. Acad. Sci. USA 89:6232-6236, 1992).
The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
When it is desired that the IGS3 transgene be integrated into the chromosomal site of the endogenous IGS3 gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous IGS3 gene of interest (e.g., nucleotide sequences of the mouse IGS3 gene) are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of, the nucleotide sequence of the endogenous IGS3 gene or gene allele.
The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following, for example, the teaching of Gu et al. (Gu, H. et al.-, Science 265:103-106, 1994). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene and protein may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA
expression of the IGS3 transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of target gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the target gene transgene product of interest. The IGS3 transgenic animals that express IGS3 gene mRNA
or IGS3 transgene peptide (detected immunocytochemically, using antibodies directed against target gene product epitopes) at easily detectable levels may then be further evaluated to identify those animals which display characteristic IGS3 based disorder symptoms.
Once IGS3 transgenic founder animals are produced i.e., those animals which express IGS3 proteins in cells or tissues of interest, and which, preferably, exhibit symptoms of IGS3 based disorders), they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include but are not limited to:
outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound IGS3 transgenics that express the IGS3 transgene of interest at higher levels because of the effects of additive expression of each IGS3 transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the possible need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the IGS3 transgene and the development of IGS3-like symptoms. One such approach is to cross the IGS3 transgenic founder animals with a wild type strain to produce an F1 generation that exhibits IGS3 related disorder-like symptoms, such as those described above. The F1 generation may then be inbred in order to develop a homozygous line, if it is found that homozygous target gene transgenic animals are viable.
Vaccines Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises administering to (for example by inoculation) the mammal the IGS3 polypeptide, or a fragment thereof, if required together with a RAMP
polypeptide, adequate to produce antibody and/or T cell immune response to protect said animal from among other things one of the Diseases as mentioned above.
Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises delivering the IGS3 polypeptide via a vector directing expression of the IGS3 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
A further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to an IGS3 polypeptide wherein the composition comprises an IGS3 polypeptide or IGS3 gene. Such immunological/vaccine formulations (compositions) may be either therapeutic immunological/vaccine formulations or prophylactic immunological/vaccine formulations. The vaccine formulation may further comprise a suitable carrier.
Since the IGS3 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc.
injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Screening Assays The IGS3 polypeptide of the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists) the receptor polypeptide of the present invention. Thus, polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics.
IGS3 polypeptides are responsible for biological functions, including pathologies.
Accordingly, it is desirable to find compounds and drugs which stimulate IGS3 on the one hand and which can inhibit the function of IGS3 on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as among other things the 10 Diseases as mentioned above.
Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as among other things the Diseases as mentioned above.
15 In general, such screening procedures involve producing appropriate cells, which express the receptor polypeptide of the present invention on the surface thereof and, if essential co-expression of RAMP's at the surface thereof. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding, or stimulation or inhibition 20 of a functional response.
One screening technique includes the use of cells which express the receptor of this invention (for example, transfected CHO cells) in a system which measures extracellular pH, intracellular pH, or intracellular calcium changes caused by receptor activation. In this technique, 25 compounds may be contacted with cells expressing the receptor polypeptide of the present invention. A second messenger response, e.g., signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.
Another method involves screening for receptor inhibitors by determining modulation of a receptor-mediated signal, such as cAMP accumulation and/or adenylate cyclase activity. Such a method involves transfecting an eukaryotic cell with the receptor of this invention to express the receptor on the cell surface. The cell is then exposed to an agonist to the receptor of this invention in the presence of a potential antagonist. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the agonist-mediated signal will be modulated.
Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast-based technology as described in U.S. Patent 5,482,835.
The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor, using detection systems appropriate to the cells bearing the receptor at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing an IGS3 polypeptide to form a mixture, measuring the IGS3 activity in the mixture, and comparing the IGS3 activity of the mixture to a standard.
The IGS3 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of IGS3 mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of IGS3 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of IGS3 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well known in the art.
Examples of potential IGS3 antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the IGS3, e.g., a fragment of the ligand, or small molecules which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented.
Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for IGS3 polypeptides; or compounds which decrease, increase and/or otherwise enhance the production of polypeptides, which comprises:
(a) an IGS3 polypeptide, preferably that of SEO ID N0:2;
(b) a recombinant cell expressing an IGS3 polypeptide, preferably that of SEO
ID N0:2;
(c) a cell membrane expressing an IGS3 polypeptide, preferably that of SEQ ID
N0:2; or (d) antibody to an IGS3 polypeptide, preferably that of SEQ ID NO: 2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
Prophylactic and Therapeutic Methods This invention provides methods of treating abnormal conditions related to both an excess of and insufficient amounts of IGS3 activity.
If the activity of IGS3 is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the IGS3, or by inhibiting interaction with a RAMP
polypeptide or a second signal, and thereby alleviating the abnormal condition.
In another approach, soluble forms of IGS3 polypeptides still capable of binding the ligand in competition with endogenous IGS3 may be administered. Typical embodiments of such competitors comprise fragments of the IGS3 polypeptide.
In still another approach, expression of the gene encoding endogenous IGS3 can be inhibited using expression-blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered.
See, for example, O'Connor, J Neurochem (1991 ) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Florida USA (1988). Alternatively, oligonucleotides, which form triple helices with the gene, can be supplied. See, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al, Science (1991 ) 251:1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo. Synthetic antisense or triplex oligonucleotides may comprise modified bases or modified backbones. Examples of the latter include methylphosphonate, phosphorothioate or peptide nucleic acid backbones. Such backbones are incorporated in the antisense or triplex oligonucleotide in order to provide protection from degradation by nucleases and are well known in the art. Antisense and triplex molecules synthesized with these or other modified backbones also form part of the present invention.
In addition, expression of the IGS3 polypeptide may be prevented by using ribozymes specific to the IGS3 mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct.
Biol (1996) 6(4), 527-33.) Synthetic ribozymes can be designed to specifically cleave IGS3 mRNAs at selected positions thereby preventing translation of the IGS3 mRNAs into functional polypeptide.
Ribozymes may be synthesized with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribosymes may be synthesized with non-natural backbones to provide protection from ribonuclease degradation, for example, 2'-O-methyl RNA, and may contain modified bases.
For treating abnormal conditions related to an under-expression of IGS3 and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates IGS3, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of IGS3 by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA
encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, Strachan T. and Read A.P., BIOS Scientific Publishers Ltd (1996).
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
Formulation and Administration Peptides, such as the soluble form of IGS3 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Formulation should suit the mode of administration, and is well within the skill of the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible.
The dosage range required depends on the choice of peptide or compound, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages are in the range of 0.1-100 ~g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration.
For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
The following examples are only intended to further illustrate the invention in more detail, and therefore these examples are not deemed to restrict the scope of the invention in any way.
EXAMPLE 1. THE CLONING OF GENOMIC DNA ENCODING A NOVEL G PROTEIN-COUPLED RECEPTOR.
5 Example 1a. Homology PCR cloning of a genomic fragment encoding a novel G-protein coupled receptor (GPCR).
A PCR based homology cloning strategy was used to isolate partial genomic DNA
sequences encoding novel G-protein coupled receptors (GPCR). The following forward (F20) 10 and reverse (R42, R43) degenerate PCR primers were designed in conserved areas of the neurotensin receptor gene family (Vita N. et al. [1993] Febs Lett. 317: 139-142; Vita N. et al.
[1998] Eur. J. Pharmacol. 360: 265-272) at the boundary of intracellular loop n°1 (11) with transmembrane domain 2 (TM2) and at the boundary of transmembrane domain 3 with intracellular loop n°2 (TM3/12) respectively:
F20 (11/TM2):
5'-CTGCACTACCACGTGCTC(A or T)(G or C)(A,C,G or T)(C or T)T(A,C,G or T)GC -3' (SEQ ID NO: 3) R42 (TM3/12):
5'-GGGTGGCAGATGGCCA(A or G)(A or G)(C or T)A(A,C,G or T)C(G or T)(C or T)TC( C
or Inosine)(C,G or T) (SEQ ID NO: 4) R43 (TM3/12):
5'-GTGGCAGATGGCCAGGCAGCG(A or G)TC(A,C,G or T)(A or G)C(A or G)CT(A,G or T) -3' (SEQ ID NO: 5) In order to suppress amplification of known members of the neurotensin receptor family, the 3' ultimate nucleotide position of primers R42 and R43 was chosen in such a way that it was either not complementary to the corresponding position of the human NTR1 cDNA (R42) or to the corresponding position of both NTR1 and NTR2 cDNA (R43).
The primary PCR reaction was carried out in a 60p1 volume and contained 100 ng human genomic DNA (Clontech), 6 p1 GeneAmpT"~ 10 x PCR buffer II (100mM Tris-HCI pH 8.3;
500 mM KCI, Perkin Elmer), 3.6 p1 25 mM MgCl2, 0.36N1 dNTPs (25mM of each dNTP), 1.5 units AmpIiTaq Gold T"' polymerise (Perkin Elmer) and 30 pmoles of each of the degenerated forward (F20) and reverse primer (R42). Reaction tubes were heated at 95°C for 10 min and then subjected to 35 cycles of denaturation (95°C, 1 min), annealing (55°C, 2 min) and extension (72°C, 3min). Finally reaction tubes were heated for 10 min at 72°C.
For the semi-nested PCR reaction 1 p1 of a 1/50 dilution of the primary PCR
reaction was used as a template using the degenerate forward and reverse primers F20 and R43 respectively. The semi-nested PCR reaction was carried out under the same conditions as the primary PCR reaction.
Semi-nested PCR reaction products were size fractionated on an agarose gel and stained with ethidium bromide. A fragment of ~ 220 by was identified, purified from gel using the Qiaex-IIT"~ purification kit (Qiagen) and ligated into the pGEM-T plasmid according to the procedure recommended by the supplier (pGEM-T kit, Promega). The recombinant plasmids thus produced were used to transform competent E. coli SURET"~ 2 bacteria (Stratagene).
Transformed cells were plated on LB agar plates containing ampicillin (100 Ng/ml). Plasmid DNA
was purified from mini-cultures of individual colonies using a Qiagen-tip 20 miniprep kit (Qiagen).
DNA sequencing reactions were carried out on the purified plasmid DNA with the ABI PrismT"~
BigDyeT"~ Terminator Cycle Sequencing Ready Reaction kit (PE-ABI), using insert-flanking.
Sequencing reaction products were purified via EtOH/NaOAc precipitation and analysed on an ABI 377 automated sequences.
A computer-assisted homology search of the insert sequence of clone HNT1370 against public domain sequence databanks (Blastn; Altschul S.F. et al. [1997], Nucleic Acids Res.
25:3389-3402) revealed strong indications that it encoded (part of) a novel member of the GPCR
family. Although HNT1370 had been cloned from a ~ 220 by fragment the insert size was only ~
130 by as a result of a cloning artefact. We refer to this novel GPCR sequence as IGS3.
Table 3: Overview of oligo primers used.
SEQ ID NO: F20: 5'-CTGCACTACCACGTGCTC(A or T)(G or C)(A,C,G
3 or T)(C or T)T(A,C,G or T)GC -3' SEQ ID NO: R42: 5'-GGGTGGCAGATGGCCA(A or G)(A or G)(C or 4 T)A(A,C,G or T)C(G
or T)(C or T)TC( C or Inosine)(C,G or T) SEQ ID NO: R43: 5'-GTGGCAGATGGCCAGGCAGCG(A or G)TC(A,C,G
5 or T)(A or G)C(A
or G)CT(A,G or T) -3' SEQ ID NO: IP11969: 5'GGGGCCGACTTCCTCTTCCTCTGCTTCC-3' SEQ ID NO: IP12008: 5'-GCAAGGTAGGCACAGGTCATCACAGTGG-3' SEQ ID NO: IP12936: 5'-ATAAGCTTCTCCCTGGCCCTTAATAAATGAC-3' SEQ ID NO: IP12937: 5'-AGGAATTCAGACAGACAGGGGCAAAGTTG-3' Example 1b. Cloning of genomic DNA fragments containing the complete IGS3 coding sequence.
The complete coding sequence of IGS3 was obtained via hybridization screening of a human genomic library. A human genomic DNA library (Clontech #HL1067j), constructed in the lambda EMBL3 SP6/T7 phage vector was screened by hybridization using an IGS3 specific probe. This probe was derived from a 130 by PCR fragment amplified from the plasmid (which contained an identical insert as HNT1370) using IGS3 specific primers IP11969 (SEQ ID NO: 6) and IP12008 (SEQ ID NO: 7) (Fig.1 ). The 130 by fragment was purified from gel using the Qiaex-IIT"" purification kit (Qiagen) and radiolabelled via random primed incorporation of [a 32P]dCTP to a specific activity of > 109 cpm/ug using the Prime-It II
kitT"~ (Stratagene) according to the instructions provided by the supplier. Aproximately 550,000 plaques were screened with the 130 by probe according to the Lambda Library User Manual of Clontech (PT1010-1 ). Three positive clones (~,-IGS3.1, ~-IGS3.3 and 7~-IGS3.5) were plaque-purified and recombinant phage DNA was prepared from small-scale liquid cultures as described by Maniatis et al. (Sambrook, J. et al. Molecular Cloning: A Laboratory Manual Second Edition [1989], CSH
Laboratory Press).
Sequence analysis of the recombinant phage DNA using IGS3 specific primers showed that the inserts of all 3 lambda clones contained a long open reading frame encoding a novel putative (intron-less) GPCR of 330 amino acids (the postulated start of translation was preceded by an in-frame stop codon). The IGS3 coding sequence was subcloned into a plasmid vector after PCR amplification. PCR reactions were carried out on the isolated 7~-IGS3.1, 7~-IGS3.3 and 7~-IGS3.5 phage DNA (500 ng) with the IP12936 (SEO ID NO: 8) and IP12937 (SEQ
ID NO: 9) oligonucleotide primers using the ExpandT"" High Fidelity PCR system (Boehringer). PCR
reaction tubes were heated at 95°C for 2 min and then subjected to 35 cycles of denaturation (95°C, 30 sec), annealing (58°C, 30 sec) and extension (72°C, 1 min). There was a final elongation step at 72°C (10 min). A ~ 1,200 by PCR product was purified from gel and ligated into the pGEM-T vector. The recombinant DNA was then used to transform E.coli bacterial strain DHSaF'. This yielded bacterial clones HB4971, HB4972 (both subcloned from 7~-IGS3.1 ), HB4973 and HB4974 (both subcloned from ~-IGS3.3) and HB4975 and HB4976 (both subcloned from ~-IGS3.5). The inserts of all plasmid clones were completely sequenced. A meld of all sequence data yielded a consensus sequence, which confirmed the existence of a long open reading frame of 330 amino acids that encoded a putative novel GPCR
receptor (IGS3) (Fig.1 ). The consensus cDNA and protein sequence of IGS3 are presented here as IGS3DNA
(SEO ID NO: 1 ) and IGS3PROT (SEQ ID NO: 2) respectively. Homology searches of DNA
databanks with the IGS3DNA sequence showed one EST sequence (accession n° AF003828) which partially overlapped with IGS3DNA at the 3' end (Fig.1 ).
The bacterial strain harboring plasmid HNT4971 (containing the IGS3DNA
sequence) was recloned after replating on LB agar plates containing 100 Ng ampicillin/ml and deposited both in the Innogenetics N.V. strain list (ICCG4319) and at the "Centraalbureau voor Schimmelculturen (CBS)" in Baarn, The Netherlands (accession n°
102196). Plasmid DNA was prepared from the recloned isolate and the insert was resequenced and found to be identical to the IGS3DNA sequence.
PCT
Original (for SUBMISSION) - printed on 15.09.2000 04:00:52 PM
0-1 Form - PCT/R0/134 (EASY) Indications Relating to Deposited Microorganisms) or Other Biological Material (PCT Rule l3bis) 0-1-1 Prepared using PCT-EASY Version 2 . 90 (updated 15.12.1999) onal Aoolication No.
0-~Aoolicant's or a4ent's file reference I SPW99 . 07 1 The indications made below relate to the deposited microorganisms) or other biological material referred to in the description on:
1-1 page 33 1-2 line 3>
1-3 Identification of Deposit 1-3-1Name of depositary Centraalbureau voor Schimmelcultures institution 1-3-2Address of depositaryOOSterStraat 1, PostbuS 273 , NL-3740 AG
institution Baarn, Netherlands 1-3-3Date of deposit 15 September 1999 (15.09.1999) 1-3-4Accession Number CBS 102196 1~t AdditionallndicationsNONE
1-5 Designated States all designated States for which Indications are Made 1-6 Separate Furnishing rjONE
of Indications These indications will be submitted to the International Bureau later FOR RECEIVING OFFICE USE ONLY
0-4 This form was received with the international application:
(yes or no) 0-4-1 Authorized officer FOR INTERNATIONAL BUREAU USE ONLY
0-5 ~ This form was received by the international Bureau on' 0-5-1 Authorized officer WO 01/19983 3r~ PCT/EP00/09116 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Duphar International Research B.V. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
POStbUS 900 issued pursuant to Rule 7.1 by the identified at the bottom of this page The Netherlands name and address of depositor I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by Accession number given by the the 7EPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
E. coli DH5 alpha F' pGEM-ThIGS3 CBS 102196 II. SCIENTIFIC DESCRIPTION AND/OR
PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied by:
a scientific description a proposed taxonomic designation (mark with a cross where applicable) III. RECEIPT AND ACCEPTANCE
This International Depositary accepts the microorganism identified under I above, which received by it on 15-09-99 (date dd-mm-yy of the original deposit) IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Depositary Authority on notappliC2ble (date dd-mm-yy of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on notappliCable (date dd-mm-yy of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY
Name : Centraalbureau voor Schimmelculturessignature ( s ) of person ( s ) having the power to represent the International Depositary Authorit or_of authorized official(s):
Address: posterstraat 1 P.O. Box 273 3740 AG BAARN Mrs F.B. Snippe- laus Dr J. Stalper The Netherlands Date (cLd-mm-yy) : 17-09-99 o ~ ~
.
1 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
Form BP/4 (sole page) CBS/9107 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Duphar International Research B.V. VIABILITY STATEMENT
POStIJUS 900 issued pursuant to Rule 10.2 by the The Netherlands identified on the following page name and address of the party to whom the viability statement is issued I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Duphar International B.V. Accession number given by the Research INTERNATIONAL DEPOSITARY AUTHORITY:
Address: POStbUS 90O
1380 DA WEESP Date (dd-mm-yy) of the deposit or of the The Netherlands transfer: 1 III. VIABILITY STATEMENT
The viability of the microorganismidentified under II
above was tested on 17-09-99 2 . On that date (dd-mm-yy), the said microorganism was n3 viable ~
r--,3 no longer viable lIndicate the date of the original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most recent viability test.
Mark with a cross the applicable box.
Form BP/9 (first page) WO 01/19983 3~ PCT/EP00/09116 i IV. CONDITIONS UNDER WHICH THE VIABILITY HAS BEEN PERFORMED 4 I
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name : Centraalbureau voor Schimmelcultures signature ( s ) of person ( s ) having the power to represent the International Depositary Authorit~of authorized official(s):
Addres s : posterstraat 1 ,~ ..--P.O. Box 273 3740 AG BAARN Mrs F.B. Snippe-Claus Dr .~ ers -The Netherlands Date (dd-mm-yy) : 17-09-99 p ~, R ftrSe~.
4Fi11 in if the information has been requested and if the results of the test were negative.
Form BP/9 (second and last page) WO 01/19983 ~ ~6 PCT/EP00/09116 SEQUENCE LISTING
<110> SOLVAY PHARMACEUTICALS B.V.
<120> Novel Human G-Protein coupled Receptor <130> SPW 99.07 <140>
<141>
<160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 1176 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (149)..(1138) <400> 1 ttaatctctt caagcctctg atttcctctc ctgtaaaaca ggggcggtaa ttaccacata 60 acaggctggt catgaaaatc agtgaacatg cagcaggtgc tcaagtcttg tttttgtttc 120 caggggcacc agtggaggtt ttctgagc atg gat cca acc acc ccg gcc tgg 172 Met Asp Pro Thr Thr Pro Ala Trp gga aca gaa agt aca aca gtg aat gga aat gac caa gcc ctt ctt ctg 220 Gly Thr Glu Ser Thr Thr Val Asn Gly Asn Asp Gln Ala Leu Leu Leu ctt tgt ggc aag gag acc ctg atc ccg gtc ttc ctg atc ctt ttc att 268 Leu Cys Gly Lys Glu Thr Leu Ile Pro Val Phe Leu Ile Leu Phe.Ile gcc ctg gtc ggg ctg gta gga aac ggg ttt gtg ctc tgg ctc ctg ggc 316 Ala Leu Val Gly Leu Val Gly Asn Gly Phe Val Leu Trp Leu Leu Gly ttc cgc atg cgc agg aac gcc ttc tct gtc tac gtc ctc agc ctg gcc 364 Phe Arg Met Arg Arg Asn Ala Phe Ser Val Tyr Val Leu Ser Leu Ala ggg gcc gac ttc ctc ttc ctc tgc ttc cag att ata aat tgc ctg gtg 412 Gly Ala Asp Phe Leu Phe Leu Cys Phe Gln Ile Ile Asn Cys Leu Val WO 01/19983 2~6 PCT/EP00/09116 tac ctc agt aac ttc ttc tgt tcc atc tcc atc aat ttc cct agc ttc 460 Tyr Leu Ser Asn Phe Phe Cys Ser Ile Ser Ile Asn Phe Pro Ser Phe ttc acc act gtg atg acc tgt gcc tac ctt gca ggc ctg agc atg ctg 508 Phe Thr Thr Val Met Thr Cys Ala Tyr Leu Ala Gly Leu Ser Met Leu agc acc gtc agc acc gag cgc tgc ctg tcc gtc ctg tgg ccc atc tgg 556 0 Ser Thr Val Ser Thr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile Trp tat cgc tgc cgc cgc ccc aga cac ctg tca gcg gtc gtg tgt gtc ctg 604 Tyr Arg Cys Arg Arg Pro Arg His Leu Ser Ala Val Val Cys Val Leu ctc tgg gcc ctg tcc cta ctg ctg agc atc ttg gaa ggg aag ttc tgt 652 Leu Trp Ala Leu Ser Leu Leu Leu Ser Ile Leu Glu Gly Lys Phe Cys ggc ttc tta ttt agt gat ggt gac tct ggt tgg tgt cag aca ttt gat 700 Gly Phe Leu Phe Ser Asp Gly Asp Ser Gly Trp Cys Gln Thr Phe Asp ttc atc act gca gcg tgg ctg att ttt tta ttc atg gtt ctc tgt ggg 748 Phe Ile Thr Ala Ala Trp Leu Ile Phe Leu Phe Met Val Leu Cys Gly tcc agt ctg gcc ctg ctg gtc agg atc ctc tgt ggc tcc agg ggt ctg 796 Ser Ser Leu Ala Leu Leu Val Arg Ile Leu Cys Gly Ser Arg Gly Leu cca ctg acc agg ctg tac ctg acc atc ctg ctc aca gtg ctg gtg ttc 844 Pro Leu Thr Arg Leu Tyr Leu Thr Ile Leu Leu Thr Val Leu Val Phe ctc ctc tgc ggc ctg ccc ttt ggc att cag tgg ttc cta ata tta tgg 892 Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Trp Phe Leu Ile Leu Trp atc tgg aag gat tct gat gtc tta ttt tgt cat att cat cca gtt tca 940 Ile Trp Lys Asp Ser Asp Val Leu Phe Cys His Ile His Pro Val Ser gtt gtc ctg tca tct ctt aac agc agt gcc aac ccc atc att tac ttc 988 Val Val Leu Ser Ser Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr Phe ttc gtg ggc tct ttt agg aag cag tgg cgg ctg cag cag ccg atc ctc 1036 Phe Val Gly Ser Phe Arg Lys Gln Trp Arg Leu Gln Gln Pro Ile Leu 2g5 290 295 aag ctg get ctc cag agg get ctg cag gac att get gag gtg gat cac 1084 Lys Leu Ala Leu Gln Arg Ala Leu Gln Asp Ile Ala Glu Val Asp His WO 01/19983 3~6 PCT/EP00/09116 agt gaa gga tgc ttc cgt cag ggc acc ccg gag atg tcg aga agc agt 1132 Ser Glu Gly Cys Phe Arg Gln Gly Thr Pro Glu Met Ser Arg Ser Ser ctg gtg tagagatgga cagcctctac ttccatcaga tatatgtg 1176 Leu Val <210> 2 <211> 330 <212> PRT
<213> Homo Sapiens <400> 2 Met Asp Pro Thr Thr Pro Ala Trp Gly Thr Glu Ser Thr Thr Val Asn Gly Asn Asp Gln Ala Leu Leu Leu Leu Cys Gly Lys Glu Thr Leu Ile Pro ValPhe LeuIleLeu PheIleAla LeuValGlyLeu ValGlyAsn Gly PheVal LeuTrpLeu LeuGlyPhe ArgMetArgArg AsnAlaPhe Ser ValTyr ValLeuSer LeuAlaGly AlaAspPheLeu PheLeuCys Phe GlnIle IleAsnCys LeuValTyr LeuSerAsnPhe PheCysSer Ile SerIle AsnPhePro SerPhePhe ThrThrValMet ThrCysAla Tyr LeuAla GlyLeuSer MetLeuSer ThrValSerThr GluArgCys Leu SerVal LeuTrpPro IleTrpTyr ArgCysArgArg ProArgHis Leu SerAla ValValCys ValLeuLeu TrpAlaLeuSer LeuLeuLeu Ser IleLeu GluGlyLys PheCysGly PheLeuPheSer AspGlyAsp WO 01/19983 4~6 PCT/EP00/09116 Ser Gly Trp Cys Gln Thr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile Phe Leu Phe Met Val Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg Ile Leu Cys Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr Ile Leu Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Trp Phe Leu Ile Leu Trp Ile Trp Lys Asp Ser Asp Val Leu Phe Cys His Ile His Pro Val Ser Val Val Leu Ser Ser Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln Trp Arg Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg Ala Leu Gln Asp Ile Ala Glu Val Asp His Ser Glu Gly Cys Phe Arg Gln Gly Thr Pro Glu Met Ser Arg Ser Ser Leu Val <210> 3 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Degenerated primers <220>
<221> variation <222> (21) <223> A,C,G or T
<220>
<221> variation <222> (24) <223> A,C,G or T
<400> 3 ctgcactacc acgtgctcws nytngc 26 WO 01/19983 5~6 PCT/EP00/09116 <210> 4 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Degenerated primers <220>
<221> variation <222> (21) <223> A,C,G or T
<220>
<221> variation <222> (27) <223> C or Inosine <400> 4 gggtggcaga tggccarrya nckytcnb 28 <210> 5 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Degenerated primers <220>
<221> variation <222> (25) <223> A,C,G or T
<400> 5 gtggcagatg gccaggcagc grtcnrcrct d 31 <210> 6 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 6 ggggccgact tcctcttcct ctgcttcc 28 <210> 7 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400>
gcaaggtagg cacaggtcat cacagtgg 28 <210> 8 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 8 ataagcttct ccctggccct taataaatga c 31 <210> 9 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 9 aggaattcag acagacaggg gcaaagttg 29
G-protein coupled receptors can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331 ). Different G-protein a-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors has been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.
Receptors - primarily the GPCR class - have led to more than half of the currently known drugs (brews, Nature Biotechnology, 1996, 14: 1516). This indicates that these receptors have an established, proven history as therapeutic targets. The new IGS3 GPCR
described in this invention clearly satisfies a need in the art for identification and characterization of further receptors that can play a role in diagnosing, preventing, ameliorating or correcting dysfunctions, disorders, or diseases, hereafter generally referred to as "the Diseases". The Diseases include, but are not limited to, psychiatric and CNS disorders, including schizophrenia, episodic paroxysmal anxiety (EPA) disorders such as obsessive compulsive disorder (OCD), post traumatic stress disorder (PTSD), phobia and panic, major depressive disorder, bipolar disorder, Parkinson's disease, general anxiety disorder, autism, delirium, multiple sclerosis, Alzheimer disease/dementia and other neurodegenerative diseases, severe mental retardation, dyskinesias, Huntington's disease, Tourett's syndrome, tics, tremor, dystonia, spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleep disorder, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmias, myocardial infarction, cardiac hypertrophy, hypotension, hypertension -e.g. essential hypertension, renal hypertension, or pulmonary hypertension, thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, Raynaud's disease, kidney disease -e.g. renal failure;
dyslipidemias; obesity; emesis; gastrointestinal disorders, including irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), gastroesophagal reflux disease (GERD), motility disorders and conditions of delayed gastric emptying, such as post operative or diabetic gastroparesis, and diabetes, ulcers - e.g. gastric ulcer; diarrhoea; other diseases including osteoporosis; inflammations; infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; chemotherapy induced injury;
tumor invasion; immune disorders; urinary retention; asthma; allergies;
arthritis; benign prostatic hypertrophy; endotoxin shock; sepsis; complication of diabetes mellitus; and gynaecological disorders.
SUMMARY OF THE INVENTION
In one aspect, the invention relates to IGS3 polypeptides, polynucleotides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such IGS3 polypeptides, polynucleotides and recombinant materials. Such uses include, but are not limited to, use as a therapeutic target and for treatment of one of the Diseases as mentioned above.
In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with IGS3 imbalance with the identified compounds. Yet another aspect of the invention -relates to diagnostic assays for detecting diseases associated with inappropriate IGS3 activity or levels. A
further aspect of the invention relates to animal-based systems which act as models for disorders arising from aberrant expression or activity of IGS3.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1. Schematic representation of the relative positions of the different DNA clones that were isolated to generate the consensus IGS3 cDNA sequence. HNT1370 represents the "founding" genomic clone. ~,-IGS3.1A,B etc. indicate separate (nearly) overlapping sequence contigs obtained from sequence analysis of DNA from lambda clone IGS3.1. PCR
primers that have been described in this document are indicated (1P#). CONSENSUS denotes the contig that was obtained after merging all obtained sequences. The part of the CONSENSUS
contig that was fully validated by sequence analysis of at least three independent clones is represented by IGS3DNA (SEQ ID NO: 1 ). The 330 amino acids long open reading frame present in IGS3DNA
is indicated with "**". The position of EST AF003828 is indicated with "_- .
Table 1: IGS3-DNA of SEO ID NO: 1 5'-TTAATCTCTTCAAGCCTCTGATTTCCTCTCCTGTAAAACAGGGGCGGTAATTACCACATA
CAGGGGCACCAGTGGAGGTTTTCTGAGCATGGATCCAACCACCCCGGCCTGGGGAACAGA
AAGTACAACAGTGAATGGAAATGACCAAGCCCTTCTTCTGCTTTGTGGCAAGGAGACCCT
GATCCCGGTCTTCCTGATCCTTTTCATTGCCCTGGTCGGGCTGGTAGGAAACGGGTTTGT
GCTCTGGCTCCTGGGCTTCCGCATGCGCAGGAACGCCTTCTCTGTCTACGTCCTCAGCCT
O GGCCGGGGCCGACTTCCTCTTCCTCTGCTTCCAGATTATAAATTGCCTGGTGTACCTCAG
TAACTTCTTCTGTTCCATCTCCATCAATTTCCCTAGCTTCTTCACCACTGTGATGACCTG
TGCCTACCTTGCAGGCCTGAGCATGCTGAGCACCGTCAGCACCGAGCGCTGCCTGTCCGT
CCTGTGGCCCATCTGGTATCGCTGCCGCCGCCCCAGACACCTGTCAGCGGTCGTGTGTGT
CCTGCTCTGGGCCCTGTCCCTACTGCTGAGCATCTTGGAAGGGAAGTTCTGTGGCTTCTT
ATTTAGTGATGGTGACTCTGGTTGGTGTCAGACATTTGATTTCATCACTGCAGCGTGGCT
GATTTTTTTATTCATGGTTCTCTGTGGGTCCAGTCTGGCCCTGCTGGTCAGGATCCTCTG
TGGCTCCAGGGGTCTGCCACTGACCAGGCTGTACCTGACCATCCTGCTCACAGTGCTGGT
GTTCCTCCTCTGCGGCCTGCCCTTTGGCATTCAGTGGTTCCTAATATTATGGATCTGGAA
GGATTCTGATGTCTTATTTTGTCATATTCATCCAGTTTCAGTTGTCCTGTCATCTCTTAA
GCAGCAGCCGATCCTCAAGCTGGCTCTCCAGAGGGCTCTGCAGGACATTGCTGAGGTGGA
TCACAGTGAAGGATGCTTCCGTCAGGGCACCCCGGAGATGTCGAGAAGCAGTCTGGTGTA
GAGATGGACAGCCTCTACTTCCATCAGATATATGTG-3' Table 2: IGS3-protein of SEQ ID NO: 2 MDPTTPAWGTESTTVNGNDQALLLLCGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMR
RNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLSML
STVSTERCLSVLWPIWYRCRRPRHLSAWCVLLWALSLLLSILEGKFCGFLFSDGDSGWC
QTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVFLLCGLPFG
IQWFLILWIWKDSDVLFCHIHPVSWLSSLNSSANPIIYFFVGSFRKQWRLQQPILKLAL
QRALQDIAEVDHSEGCFRQGTPEMSRSSLV
DESCRIPTION OF THE INVENTION
Structural and chemical similarity, in the context of sequences and motifs, exists among the IGS3 GPCR of the invention and other human GPCR's. Therefore, IGS3 is implied to play a role among other things in the Diseases mentioned above.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
Definitions The following definitions are provided to facilitate understanding of certain terms used frequently herein.
"IGS3" refers, among others, to a polypeptide comprising the amino acid sequence set forth in SEQ ID N0:2, or a Variant thereof.
"Receptor Activity" or "Biological Activity of the Receptor" refers to the metabolic or physiologic function of said IGS3 including similar activities or improved activities or these activities with decreased undesirable side effects. Also included are antigenic and immunogenic activities of said IGS3.
"IGS3-gene" refers to a polynucleotide comprising the nucleotide sequence set forth in SEQ ID N0:1 or Variants thereof and/or their complements.
"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of a Fab or other immunoglobulin expression library.
"Isolated" means altered "by the hand of man" from the natural state and/or separated from the natural environment. Thus, if an "isolated" composition or substance that occurs in nature has been "isolated", it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
"Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides"
include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
In addition, "polynucleotide" may also include triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide"
embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.
"Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins, and/or to combinations thereof. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
"Polypeptides" include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well-described in basic texts and in more detailed monographs, as well as in voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol; cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth. Enzymol. (1990) 182:626-646 and Rattan et al., "Protein Synthesis: Posttranslational Modifications and Aging", Ann. NY Acad. Sci.
(1992) 663:48-62.
"Variant" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties such as essential biological, structural, regulatory or biochemical properties. A
typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination. A
substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A
variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Identity" is a measure of the identity of nucleotide sequences or amino acid sequences.
In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.:
(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed.;
Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994;
SEQUENCE ANALYSIS
IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE
ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ).
While there exist a number of methods to measure identity between two polynucleotide or 5 polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math. (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J. Applied Math. (1988) 48:1073. Methods to determine identity and 10 similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J. Molec. Biol. (1990) 215:403). The word "homology" may substitute for the words "identity".
As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five nucleotide differences per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to any 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to any 5%
of the total nucleotides in the reference sequence may be inserted into the reference sequence, or in a number of nucleotides of up to any 5% of the total nucleotides in the reference sequence there may be a combination of deletion, insertion and substitution. These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Similarly, by a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID N0:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to any 5%
of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to any 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
Polypeptides of the Invention In one aspect, the present invention relates to IGS3 polypeptides (including proteins). The IGS3 polypeptides include the polypeptide of SEQ ID N0:2 and the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS
102196, deposited on September 15, 1999 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands); as well as polypeptides comprising the amino acid sequence of SEO
ID N0:2 and the polypeptide having the amino acid sequence encoded by the DNA
insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), and polypeptides comprising an amino acid sequence having at least 80%
identity to that of SEQ ID N0:2 and/or to the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands) over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to said amino acid sequence. Furthermore, those with at least 97%, in particular at least 99%, are highly preferred.
Also included within IGS3 polypeptides are polypeptides having the amino acid sequence which has at least 80% identity to the polypeptide having the amino acid sequence of SEO ID NO: 2 or the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands) over its entire length, and still more preferably at least 90%
identity, and even still more preferably at least 95% identity to SEO ID NO: 2. Furthermore, those with at least 97%, in particular at least 99% are highly preferred. Preferably IGS3 polypeptides exhibit at least one biological activity of the receptor.
In an additional embodiment of the invention, the IGS3 polypeptides may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, sequences which aid in detection such as antigenic peptide tags (such as the haemagglutinin (HA) tag), or an additional sequence for stability during recombinant production.
Fragments of the IGS3 polypeptides are also included in the invention. A
fragment is a polypeptide having an amino acid sequence that is the same as part of, but not all of, the amino acid sequence of the aforementioned IGS3 polypeptides. As with IGS3 polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20;
21-40, 41-60, 61-80, 81-100; and 101 to the end of IGS3 polypeptide. In this context "about"
includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of IGS3 polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Other preferred fragments are biologically active fragments.
Biologically active fragments are those that mediate receptor activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
Thus, the polypeptides of the invention include polypeptides having an amino acid sequence that is at least 80% identical to either that of SEQ ID N0:2 and/or the polypeptide having the amino acid sequence encoded by the DNA insert contained in the deposit no. CBS
102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), or fragments thereof with at least 80% identity to the corresponding fragment. Preferably, all of these polypeptide fragments retain the biological activity of the receptor, including antigenic activity.
Variants of the defined sequence and fragments also form part of the present invention.
Preferred variants are those that vary from the referents by conservative amino acid substitutions -- i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
The IGS3 polypeptides of the invention can be prepared in any suitable manner.
Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for preparing such polypeptides are well known in the art.
Polynucleotides of the Invention A further aspect of the invention relates to IGS3 polynucleotides. IGS3 polynucleotides include isolated polynucleotides which encode the IGS3 polypeptides and fragments, and polynucleotides closely related thereto. More specifically, the IGS3 polynucleotide of the invention includes a polynucleotide comprising the nucleotide sequence contained in SEQ ID
N0:1, such as the one capable of encoding a IGS3 polypeptide of SEQ ID NO: 2, polynucleotides having the particular sequence of SEQ ID NO: 1 and polynucleotides which essentially correspond to the DNA insert contained in the deposit no. CBS
102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands).
IGS3 polynucleotides further include polynucleotides comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the IGS3 polypeptide of SEQ ID N0:2, polynucleotides comprising a nucleotide sequence that is at least 80% identical to that of SEQ ID N0:1 over its entire length and a polynucleotide which essentially corresponds to the DNA insert contained in the deposit no. CBS
102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands).
In this regard, polynucleotides with at least 90% identity are particularly preferred, and those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred. Also included under IGS3 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEO ID NO: 1 or to the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands) to hybridize under conditions useable for amplification or for use as a probe or marker. The invention also provides polynucleotides which are complementary to such IGS3 polynucleotides.
IGS3 of the invention is structurally related to other proteins of the G-protein coupled receptor family, as shown by the results of BLAST searches in the public databases. The amino acid sequence of Table 2 (SEO ID N0:2) has about 35 % identity (using BLAST, Altschul S.F. et al. Nucleic Acids Res. (1997) 25:3389-3402) over most of its length (amino acid residues 2-306 ) with the protein encoded by the human mas oncogene (Sequence 1 in patent application WO
8707472). The sequence is 37% identical (amino acid residues 35-315) with the G-protein coupled receptor published in patent application WO 9616087 (GENESEQ 96P-897222 ). The nucleotide sequence of Table 1 (SEQ ID NO: 1 ) has 52 % and 54 % identity over most of its length to the two receptors above (GENESEO 87N-70685 and 96N-T28807 respectively). Also there is 48% identity to the human Somatostatin-3 receptor in residues 104-1144 (WO 9313130;
93N-Q45657). Hydropathy analysis (Kyte J. et al., J. Mol. Biol. (1982) 157:
105-132; Klein P.et al., Biochim. Biophys. Acta (1985) 815: 468-476) of the IGS3 protein sequence expectedly showed the presence of 7 transmembrane domains. Thus, IGS3 polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.
Polynucleotides of the invention can be obtained from natural sources such as genomic DNA. In particular, degenerated PCR primers can be designed that encode conserved regions within a particular GPCR gene subfamily. PCR amplification reactions on genomic DNA or cDNA
using the degenerate primers will result in the amplification of several members (both known and novel) of the gene family under consideration (the degenerated primers must be located within the same exon, when a genomic template is used). (Libert et al., Science, 1989, 244: 569-572).
Polynucleotides of the invention can also be synthesized using well-known and commercially available techniques.
The nucleotide sequence encoding the IGS3 polypeptide of SEQ ID N0:2 may be identical to the polypeptide encoding sequence contained in SEQ ID N0:1 (nucleotide number 149 to 1138), or it may be a different nucleotide sequence, which as a result of the redundancy (degeneracy) of the genetic code might also show alterations compared to the polypeptide encoding sequence contained in SEO ID N0:1, but also encodes the polypeptide of SEQ ID
N0:2.
When the polynucleotides of the invention are used for the recombinant production of the IGS3 polypeptide, the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad. Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may 5 also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
Further preferred embodiments are polynucleotides encoding IGS3 variants comprising the amino acid sequence of the IGS3 polypeptide of SEO ID N0:2 in which several, 5-10, 1-5, 1-10 3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.
The polynucleotides of the invention can be engineered using methods generally known in the art in order to alter IGS3-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA
15 shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create amino acid substitutions, create new restriction sites, alter modification (e.g. glycosylation or phosphorylation) patterns, change codon preference, produce splice variants, and so forth.
The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the polynucleotides described above. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably at least 97%, in particular at least 99% identity between the sequences.
Polynucleotides of the invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEO ID N0:1 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding IGS3 and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similarity to the IGS3 gene. People skilled in the art are well aware of such hybridization techniques. Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent. The probes generally will comprise at least 5 nucleotides, and preferably at least 8 nucleotides, and more preferably at least 10 nucleotides, yet even more preferably at least 12 nucleotides, in particular at least 15 nucleotides. Most preferred, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
One embodiment, to obtain a polynucleotide encoding the IGS3 polypeptide, including homologs and orthologs from species other than human, comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the SEQ
ID NO: 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to those of skill in the art. Stringent hybridization conditions are as defined above or alternatively conditions under overnight incubation at 42 °C in a solution comprising: 50% formamide, 5xSSC (150mM NaCI, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 xSSC at about 65°C.
The polynucleotides and polypeptides of the present invention may be used as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
Vectors, Host Cells, Expression The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention.
For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY
(1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;
animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells;
and plant cells.
A great variety of expression systems can be used. Such systems include, among others, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression.
Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals, i.e. derived from a different species.
If the IGS3 polypeptide is to be expressed for use in screening assays, generally, it is preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. In case the affinity or functional activity of the IGS3 polypeptide is modified by receptor activity modifying proteins (RAMP), coexpression of the relevant RAMP most likely at the surface of the cell is preferred and often required. Also in this event harvesting of cells expressing the IGS3 polypeptide and the relevant RAMP prior to use in screening assays is required. If the IGS3 polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypeptide is recovered. Membranes expressing the IGS3 polypeptide can be recovered by methods that are well known to a person skilled in the art. In general, such methods include harvesting of the cells expressing the IGS3 polypeptide and homogenization of the cells by a method such as, but not limited to, pottering.
The membranes may be recovered by washing the suspension one or several times.
IGS3 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well-known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
Diagnostic Assays This invention also relates to the use of IGS3 polynucleotides for use as diagnostic reagents. Detection of a mutated form of the IGS3 gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of IGS3.
Also in this event co-expression of relevant receptor activity modifying proteins can be required to obtain diagnostic assays of desired quality. Individuals carrying mutations in the IGS3 gene may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to labeled IGS3 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science (1985) 230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method. See Cotton et al., Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401. In another embodiment, an array of oligonucleotide probes comprising the IGS3 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al., Science, Vol 274, pp 610-613 (1996)).
The diagnostic assays offer a process for diagnosing or determining a susceptibility to among other things the Diseases as mentioned above, through detection of mutation in the IGS3 gene by the methods described.
In addition, among other things, the Diseases as mentioned above can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of the IGS3 polypeptide or IGS3 mRNA.
Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
Assay techniques that can be used to determine levels of a protein, such as an IGS3, in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA
assays.
In another aspect, the present invention relates to a diagnostic kit for among other things the Diseases or suspectability to one of the Diseases as mentioned above.
The kit may comprise:
(a) an IGS3 polynucleotide, preferably the nucleotide sequence of SEQ ID N0:1, or a fragment thereof; and/or (b) a nucleotide sequence complementary to that of (a); and/or (c) an IGS3 polypeptide, preferably the polypeptide of SEO ID N0:2, or a fragment thereof; and/or (d) an antibody to an IGS3 polypeptide, preferably to the polypeptide of SEQ
ID NO: 2;
and/or (e) a RAMP polypeptide required for the relevant biological or antigenic properties of an IGS3 polypeptide.
It will be appreciated that in any such kit, (a), (b), (c) (d) or (e) may comprise a substantial component.
Chromosome Assays The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location 5 on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in 10 Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected 15 individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
Antibodies 20 The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them if required together with relevant RAMP's, may also be used as immunogens to produce antibodies immunospecific for the IGS3 polypeptides. The term "immunospecific"
means that the antibodies have substantiall greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
Antibodies generated against the IGS3 polypeptides may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique, which provides antibodies produced by continuous cell line cultures, may be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C., Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al., MONOCLONAL
ANTIBODIES AND
CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
Antibodies against IGS3 polypeptides as such, or against IGS3 polypeptide-RAMP
complexes, may also be employed to treat among other things the Diseases as mentioned above.
Animals Another aspect of the invention relates to non-human animal-based systems which act as models for disorders arising from aberrant expression or activity of IGS3. Non-human animal-based model systems may also be used to further characterize the activity of the IGS3 gene.
Such systems may be utilized as part of screening strategies designed to identify compounds which are capable to treat IGS3 based disorders such as among other things the Diseases as mentioned above.
In this way the animal-based models may be used to identify pharmaceutical compounds, therapies and interventions which may be effective in treating disorders of aberrant expression or activity of IGS3. In addition such animal models may be used to determine the LDSO and the EDSO in animal subjects. These data may be used to determine the in vivo efficacy of potential IGS3 disorder treatments.
Animal-based model systems of IGS3 based disorders, based on aberrant IGS3 expression or activity, may include both non-recombinant animals as well as recombinantly engineered transgenic animals.
Animal models for IGS3 disorders may include, for example, genetic models.
Animal models exhibiting IGS3 based disorder-like symptoms may be engineered by utilizing, for example, IGS3 sequences such as those described, above, in conjunction with techniques for producing transgenic animals that are well known to persons skilled in the art. For example, IGS3 sequences may be introduced into, and overexpressed and/or misexpressed in, the genome of the animal of interest, or, if endogenous IGS3 sequences are present, they may either be overexpressed, misexpressed, or, alternatively, may be disrupted in order to underexpress or inactivate IGS3 gene expression.
In order to overexpress or misexpress a IGS3 gene sequence, the coding portion of the IGS3 gene sequence may be ligated to a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in the animal type of interest. Such regulatory regions will be well known to those skilled in the art, and may be utilized in the absence of undue experimentation.
For underexpression of an endogenous IGS3 gene sequence, such a sequence may be isolated and engineered such that when reintroduced into the genome of the animal of interest, the endogenous IGS3 gene alleles will be inactivated, or "knocked-out".
Preferably, the engineered IGS3 gene sequence is introduced via gene targeting such that the endogenous IGS3 sequence is disrupted upon integration of the engineered IGS3 gene sequence into the animal's genome.
Animals of any species, including, but not limited to, mice, rats, rabbits, squirrels, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.~c ., baboons, monkeys, and chimpanzees may be used to generate animal models of IGS3 related disorders.
Any technique known in the art may be used to introduce a IGS3 transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat.
No. 4,873,191);
retrovirus mediated gene transfer into germ lines (van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152, 1985); gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321, 1989,); electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-1814, 1983); and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723, 1989); etc.
For a review of such techniques, see Cordon, Transgenic Animals, Intl. Rev. Cytol.115:171-229, 1989.
The present invention provides for transgenic animals that carry the IGS3 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals. (See, for example, techniques described by Jakobovits, Curr.
Biol. 4:761-763, 1994) The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al.
(Lasko, M..et al., Proc. Natl. Acad. Sci. USA 89:6232-6236, 1992).
The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
When it is desired that the IGS3 transgene be integrated into the chromosomal site of the endogenous IGS3 gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous IGS3 gene of interest (e.g., nucleotide sequences of the mouse IGS3 gene) are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of, the nucleotide sequence of the endogenous IGS3 gene or gene allele.
The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene of interest in only that cell type, by following, for example, the teaching of Gu et al. (Gu, H. et al.-, Science 265:103-106, 1994). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene and protein may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA
expression of the IGS3 transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of target gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the target gene transgene product of interest. The IGS3 transgenic animals that express IGS3 gene mRNA
or IGS3 transgene peptide (detected immunocytochemically, using antibodies directed against target gene product epitopes) at easily detectable levels may then be further evaluated to identify those animals which display characteristic IGS3 based disorder symptoms.
Once IGS3 transgenic founder animals are produced i.e., those animals which express IGS3 proteins in cells or tissues of interest, and which, preferably, exhibit symptoms of IGS3 based disorders), they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include but are not limited to:
outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound IGS3 transgenics that express the IGS3 transgene of interest at higher levels because of the effects of additive expression of each IGS3 transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the possible need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; breeding animals to different inbred genetic backgrounds so as to examine effects of modifying alleles on expression of the IGS3 transgene and the development of IGS3-like symptoms. One such approach is to cross the IGS3 transgenic founder animals with a wild type strain to produce an F1 generation that exhibits IGS3 related disorder-like symptoms, such as those described above. The F1 generation may then be inbred in order to develop a homozygous line, if it is found that homozygous target gene transgenic animals are viable.
Vaccines Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises administering to (for example by inoculation) the mammal the IGS3 polypeptide, or a fragment thereof, if required together with a RAMP
polypeptide, adequate to produce antibody and/or T cell immune response to protect said animal from among other things one of the Diseases as mentioned above.
Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises delivering the IGS3 polypeptide via a vector directing expression of the IGS3 polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
A further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to an IGS3 polypeptide wherein the composition comprises an IGS3 polypeptide or IGS3 gene. Such immunological/vaccine formulations (compositions) may be either therapeutic immunological/vaccine formulations or prophylactic immunological/vaccine formulations. The vaccine formulation may further comprise a suitable carrier.
Since the IGS3 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc.
injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Screening Assays The IGS3 polypeptide of the present invention may be employed in a screening process for compounds which bind the receptor and which activate (agonists) or inhibit activation of (antagonists) the receptor polypeptide of the present invention. Thus, polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics.
IGS3 polypeptides are responsible for biological functions, including pathologies.
Accordingly, it is desirable to find compounds and drugs which stimulate IGS3 on the one hand and which can inhibit the function of IGS3 on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as among other things the 10 Diseases as mentioned above.
Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as among other things the Diseases as mentioned above.
15 In general, such screening procedures involve producing appropriate cells, which express the receptor polypeptide of the present invention on the surface thereof and, if essential co-expression of RAMP's at the surface thereof. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells expressing the receptor (or cell membrane containing the expressed receptor) are then contacted with a test compound to observe binding, or stimulation or inhibition 20 of a functional response.
One screening technique includes the use of cells which express the receptor of this invention (for example, transfected CHO cells) in a system which measures extracellular pH, intracellular pH, or intracellular calcium changes caused by receptor activation. In this technique, 25 compounds may be contacted with cells expressing the receptor polypeptide of the present invention. A second messenger response, e.g., signal transduction, pH changes, or changes in calcium level, is then measured to determine whether the potential compound activates or inhibits the receptor.
Another method involves screening for receptor inhibitors by determining modulation of a receptor-mediated signal, such as cAMP accumulation and/or adenylate cyclase activity. Such a method involves transfecting an eukaryotic cell with the receptor of this invention to express the receptor on the cell surface. The cell is then exposed to an agonist to the receptor of this invention in the presence of a potential antagonist. If the potential antagonist binds the receptor, and thus inhibits receptor binding, the agonist-mediated signal will be modulated.
Another method for detecting agonists or antagonists for the receptor of the present invention is the yeast-based technology as described in U.S. Patent 5,482,835.
The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the receptor is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the receptor, using detection systems appropriate to the cells bearing the receptor at their surfaces. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
Further, the assays may simply comprise the steps of mixing a candidate compound with a solution containing an IGS3 polypeptide to form a mixture, measuring the IGS3 activity in the mixture, and comparing the IGS3 activity of the mixture to a standard.
The IGS3 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of IGS3 mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of IGS3 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of IGS3 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well known in the art.
Examples of potential IGS3 antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligand of the IGS3, e.g., a fragment of the ligand, or small molecules which bind to the receptor but do not elicit a response, so that the activity of the receptor is prevented.
Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for IGS3 polypeptides; or compounds which decrease, increase and/or otherwise enhance the production of polypeptides, which comprises:
(a) an IGS3 polypeptide, preferably that of SEO ID N0:2;
(b) a recombinant cell expressing an IGS3 polypeptide, preferably that of SEO
ID N0:2;
(c) a cell membrane expressing an IGS3 polypeptide, preferably that of SEQ ID
N0:2; or (d) antibody to an IGS3 polypeptide, preferably that of SEQ ID NO: 2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
Prophylactic and Therapeutic Methods This invention provides methods of treating abnormal conditions related to both an excess of and insufficient amounts of IGS3 activity.
If the activity of IGS3 is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acceptable carrier in an amount effective to inhibit activation by blocking binding of ligands to the IGS3, or by inhibiting interaction with a RAMP
polypeptide or a second signal, and thereby alleviating the abnormal condition.
In another approach, soluble forms of IGS3 polypeptides still capable of binding the ligand in competition with endogenous IGS3 may be administered. Typical embodiments of such competitors comprise fragments of the IGS3 polypeptide.
In still another approach, expression of the gene encoding endogenous IGS3 can be inhibited using expression-blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered.
See, for example, O'Connor, J Neurochem (1991 ) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Florida USA (1988). Alternatively, oligonucleotides, which form triple helices with the gene, can be supplied. See, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al, Science (1991 ) 251:1360. These oligomers can be administered per se or the relevant oligomers can be expressed in vivo. Synthetic antisense or triplex oligonucleotides may comprise modified bases or modified backbones. Examples of the latter include methylphosphonate, phosphorothioate or peptide nucleic acid backbones. Such backbones are incorporated in the antisense or triplex oligonucleotide in order to provide protection from degradation by nucleases and are well known in the art. Antisense and triplex molecules synthesized with these or other modified backbones also form part of the present invention.
In addition, expression of the IGS3 polypeptide may be prevented by using ribozymes specific to the IGS3 mRNA sequence. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al., Curr. Opin. Struct.
Biol (1996) 6(4), 527-33.) Synthetic ribozymes can be designed to specifically cleave IGS3 mRNAs at selected positions thereby preventing translation of the IGS3 mRNAs into functional polypeptide.
Ribozymes may be synthesized with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribosymes may be synthesized with non-natural backbones to provide protection from ribonuclease degradation, for example, 2'-O-methyl RNA, and may contain modified bases.
For treating abnormal conditions related to an under-expression of IGS3 and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates IGS3, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of IGS3 by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA
encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, Strachan T. and Read A.P., BIOS Scientific Publishers Ltd (1996).
Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
Formulation and Administration Peptides, such as the soluble form of IGS3 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. Formulation should suit the mode of administration, and is well within the skill of the art. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible.
The dosage range required depends on the choice of peptide or compound, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages are in the range of 0.1-100 ~g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration.
For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
The following examples are only intended to further illustrate the invention in more detail, and therefore these examples are not deemed to restrict the scope of the invention in any way.
EXAMPLE 1. THE CLONING OF GENOMIC DNA ENCODING A NOVEL G PROTEIN-COUPLED RECEPTOR.
5 Example 1a. Homology PCR cloning of a genomic fragment encoding a novel G-protein coupled receptor (GPCR).
A PCR based homology cloning strategy was used to isolate partial genomic DNA
sequences encoding novel G-protein coupled receptors (GPCR). The following forward (F20) 10 and reverse (R42, R43) degenerate PCR primers were designed in conserved areas of the neurotensin receptor gene family (Vita N. et al. [1993] Febs Lett. 317: 139-142; Vita N. et al.
[1998] Eur. J. Pharmacol. 360: 265-272) at the boundary of intracellular loop n°1 (11) with transmembrane domain 2 (TM2) and at the boundary of transmembrane domain 3 with intracellular loop n°2 (TM3/12) respectively:
F20 (11/TM2):
5'-CTGCACTACCACGTGCTC(A or T)(G or C)(A,C,G or T)(C or T)T(A,C,G or T)GC -3' (SEQ ID NO: 3) R42 (TM3/12):
5'-GGGTGGCAGATGGCCA(A or G)(A or G)(C or T)A(A,C,G or T)C(G or T)(C or T)TC( C
or Inosine)(C,G or T) (SEQ ID NO: 4) R43 (TM3/12):
5'-GTGGCAGATGGCCAGGCAGCG(A or G)TC(A,C,G or T)(A or G)C(A or G)CT(A,G or T) -3' (SEQ ID NO: 5) In order to suppress amplification of known members of the neurotensin receptor family, the 3' ultimate nucleotide position of primers R42 and R43 was chosen in such a way that it was either not complementary to the corresponding position of the human NTR1 cDNA (R42) or to the corresponding position of both NTR1 and NTR2 cDNA (R43).
The primary PCR reaction was carried out in a 60p1 volume and contained 100 ng human genomic DNA (Clontech), 6 p1 GeneAmpT"~ 10 x PCR buffer II (100mM Tris-HCI pH 8.3;
500 mM KCI, Perkin Elmer), 3.6 p1 25 mM MgCl2, 0.36N1 dNTPs (25mM of each dNTP), 1.5 units AmpIiTaq Gold T"' polymerise (Perkin Elmer) and 30 pmoles of each of the degenerated forward (F20) and reverse primer (R42). Reaction tubes were heated at 95°C for 10 min and then subjected to 35 cycles of denaturation (95°C, 1 min), annealing (55°C, 2 min) and extension (72°C, 3min). Finally reaction tubes were heated for 10 min at 72°C.
For the semi-nested PCR reaction 1 p1 of a 1/50 dilution of the primary PCR
reaction was used as a template using the degenerate forward and reverse primers F20 and R43 respectively. The semi-nested PCR reaction was carried out under the same conditions as the primary PCR reaction.
Semi-nested PCR reaction products were size fractionated on an agarose gel and stained with ethidium bromide. A fragment of ~ 220 by was identified, purified from gel using the Qiaex-IIT"~ purification kit (Qiagen) and ligated into the pGEM-T plasmid according to the procedure recommended by the supplier (pGEM-T kit, Promega). The recombinant plasmids thus produced were used to transform competent E. coli SURET"~ 2 bacteria (Stratagene).
Transformed cells were plated on LB agar plates containing ampicillin (100 Ng/ml). Plasmid DNA
was purified from mini-cultures of individual colonies using a Qiagen-tip 20 miniprep kit (Qiagen).
DNA sequencing reactions were carried out on the purified plasmid DNA with the ABI PrismT"~
BigDyeT"~ Terminator Cycle Sequencing Ready Reaction kit (PE-ABI), using insert-flanking.
Sequencing reaction products were purified via EtOH/NaOAc precipitation and analysed on an ABI 377 automated sequences.
A computer-assisted homology search of the insert sequence of clone HNT1370 against public domain sequence databanks (Blastn; Altschul S.F. et al. [1997], Nucleic Acids Res.
25:3389-3402) revealed strong indications that it encoded (part of) a novel member of the GPCR
family. Although HNT1370 had been cloned from a ~ 220 by fragment the insert size was only ~
130 by as a result of a cloning artefact. We refer to this novel GPCR sequence as IGS3.
Table 3: Overview of oligo primers used.
SEQ ID NO: F20: 5'-CTGCACTACCACGTGCTC(A or T)(G or C)(A,C,G
3 or T)(C or T)T(A,C,G or T)GC -3' SEQ ID NO: R42: 5'-GGGTGGCAGATGGCCA(A or G)(A or G)(C or 4 T)A(A,C,G or T)C(G
or T)(C or T)TC( C or Inosine)(C,G or T) SEQ ID NO: R43: 5'-GTGGCAGATGGCCAGGCAGCG(A or G)TC(A,C,G
5 or T)(A or G)C(A
or G)CT(A,G or T) -3' SEQ ID NO: IP11969: 5'GGGGCCGACTTCCTCTTCCTCTGCTTCC-3' SEQ ID NO: IP12008: 5'-GCAAGGTAGGCACAGGTCATCACAGTGG-3' SEQ ID NO: IP12936: 5'-ATAAGCTTCTCCCTGGCCCTTAATAAATGAC-3' SEQ ID NO: IP12937: 5'-AGGAATTCAGACAGACAGGGGCAAAGTTG-3' Example 1b. Cloning of genomic DNA fragments containing the complete IGS3 coding sequence.
The complete coding sequence of IGS3 was obtained via hybridization screening of a human genomic library. A human genomic DNA library (Clontech #HL1067j), constructed in the lambda EMBL3 SP6/T7 phage vector was screened by hybridization using an IGS3 specific probe. This probe was derived from a 130 by PCR fragment amplified from the plasmid (which contained an identical insert as HNT1370) using IGS3 specific primers IP11969 (SEQ ID NO: 6) and IP12008 (SEQ ID NO: 7) (Fig.1 ). The 130 by fragment was purified from gel using the Qiaex-IIT"" purification kit (Qiagen) and radiolabelled via random primed incorporation of [a 32P]dCTP to a specific activity of > 109 cpm/ug using the Prime-It II
kitT"~ (Stratagene) according to the instructions provided by the supplier. Aproximately 550,000 plaques were screened with the 130 by probe according to the Lambda Library User Manual of Clontech (PT1010-1 ). Three positive clones (~,-IGS3.1, ~-IGS3.3 and 7~-IGS3.5) were plaque-purified and recombinant phage DNA was prepared from small-scale liquid cultures as described by Maniatis et al. (Sambrook, J. et al. Molecular Cloning: A Laboratory Manual Second Edition [1989], CSH
Laboratory Press).
Sequence analysis of the recombinant phage DNA using IGS3 specific primers showed that the inserts of all 3 lambda clones contained a long open reading frame encoding a novel putative (intron-less) GPCR of 330 amino acids (the postulated start of translation was preceded by an in-frame stop codon). The IGS3 coding sequence was subcloned into a plasmid vector after PCR amplification. PCR reactions were carried out on the isolated 7~-IGS3.1, 7~-IGS3.3 and 7~-IGS3.5 phage DNA (500 ng) with the IP12936 (SEO ID NO: 8) and IP12937 (SEQ
ID NO: 9) oligonucleotide primers using the ExpandT"" High Fidelity PCR system (Boehringer). PCR
reaction tubes were heated at 95°C for 2 min and then subjected to 35 cycles of denaturation (95°C, 30 sec), annealing (58°C, 30 sec) and extension (72°C, 1 min). There was a final elongation step at 72°C (10 min). A ~ 1,200 by PCR product was purified from gel and ligated into the pGEM-T vector. The recombinant DNA was then used to transform E.coli bacterial strain DHSaF'. This yielded bacterial clones HB4971, HB4972 (both subcloned from 7~-IGS3.1 ), HB4973 and HB4974 (both subcloned from ~-IGS3.3) and HB4975 and HB4976 (both subcloned from ~-IGS3.5). The inserts of all plasmid clones were completely sequenced. A meld of all sequence data yielded a consensus sequence, which confirmed the existence of a long open reading frame of 330 amino acids that encoded a putative novel GPCR
receptor (IGS3) (Fig.1 ). The consensus cDNA and protein sequence of IGS3 are presented here as IGS3DNA
(SEO ID NO: 1 ) and IGS3PROT (SEQ ID NO: 2) respectively. Homology searches of DNA
databanks with the IGS3DNA sequence showed one EST sequence (accession n° AF003828) which partially overlapped with IGS3DNA at the 3' end (Fig.1 ).
The bacterial strain harboring plasmid HNT4971 (containing the IGS3DNA
sequence) was recloned after replating on LB agar plates containing 100 Ng ampicillin/ml and deposited both in the Innogenetics N.V. strain list (ICCG4319) and at the "Centraalbureau voor Schimmelculturen (CBS)" in Baarn, The Netherlands (accession n°
102196). Plasmid DNA was prepared from the recloned isolate and the insert was resequenced and found to be identical to the IGS3DNA sequence.
PCT
Original (for SUBMISSION) - printed on 15.09.2000 04:00:52 PM
0-1 Form - PCT/R0/134 (EASY) Indications Relating to Deposited Microorganisms) or Other Biological Material (PCT Rule l3bis) 0-1-1 Prepared using PCT-EASY Version 2 . 90 (updated 15.12.1999) onal Aoolication No.
0-~Aoolicant's or a4ent's file reference I SPW99 . 07 1 The indications made below relate to the deposited microorganisms) or other biological material referred to in the description on:
1-1 page 33 1-2 line 3>
1-3 Identification of Deposit 1-3-1Name of depositary Centraalbureau voor Schimmelcultures institution 1-3-2Address of depositaryOOSterStraat 1, PostbuS 273 , NL-3740 AG
institution Baarn, Netherlands 1-3-3Date of deposit 15 September 1999 (15.09.1999) 1-3-4Accession Number CBS 102196 1~t AdditionallndicationsNONE
1-5 Designated States all designated States for which Indications are Made 1-6 Separate Furnishing rjONE
of Indications These indications will be submitted to the International Bureau later FOR RECEIVING OFFICE USE ONLY
0-4 This form was received with the international application:
(yes or no) 0-4-1 Authorized officer FOR INTERNATIONAL BUREAU USE ONLY
0-5 ~ This form was received by the international Bureau on' 0-5-1 Authorized officer WO 01/19983 3r~ PCT/EP00/09116 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Duphar International Research B.V. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
POStbUS 900 issued pursuant to Rule 7.1 by the identified at the bottom of this page The Netherlands name and address of depositor I. IDENTIFICATION OF THE MICROORGANISM
Identification reference given by Accession number given by the the 7EPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:
E. coli DH5 alpha F' pGEM-ThIGS3 CBS 102196 II. SCIENTIFIC DESCRIPTION AND/OR
PROPOSED TAXONOMIC DESIGNATION
The microorganism identified under I above was accompanied by:
a scientific description a proposed taxonomic designation (mark with a cross where applicable) III. RECEIPT AND ACCEPTANCE
This International Depositary accepts the microorganism identified under I above, which received by it on 15-09-99 (date dd-mm-yy of the original deposit) IV. RECEIPT OF REQUEST FOR CONVERSION
The microorganism identified under I above was received by this International Depositary Authority on notappliC2ble (date dd-mm-yy of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on notappliCable (date dd-mm-yy of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY
Name : Centraalbureau voor Schimmelculturessignature ( s ) of person ( s ) having the power to represent the International Depositary Authorit or_of authorized official(s):
Address: posterstraat 1 P.O. Box 273 3740 AG BAARN Mrs F.B. Snippe- laus Dr J. Stalper The Netherlands Date (cLd-mm-yy) : 17-09-99 o ~ ~
.
1 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
Form BP/4 (sole page) CBS/9107 BUDAPEST TREATY ON THE INTERNATIONAL
RECOGNITION OF THE DEPOSIT OF MICROORGANISMS
FOR THE PURPOSES OF PATENT PROCEDURE
INTERNATIONAL FORM
Duphar International Research B.V. VIABILITY STATEMENT
POStIJUS 900 issued pursuant to Rule 10.2 by the The Netherlands identified on the following page name and address of the party to whom the viability statement is issued I. DEPOSITOR II. IDENTIFICATION OF THE MICROORGANISM
Name: Duphar International B.V. Accession number given by the Research INTERNATIONAL DEPOSITARY AUTHORITY:
Address: POStbUS 90O
1380 DA WEESP Date (dd-mm-yy) of the deposit or of the The Netherlands transfer: 1 III. VIABILITY STATEMENT
The viability of the microorganismidentified under II
above was tested on 17-09-99 2 . On that date (dd-mm-yy), the said microorganism was n3 viable ~
r--,3 no longer viable lIndicate the date of the original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).
2 In the cases referred to in Rule 10.2(a)(ii) and (iii), refer to the most recent viability test.
Mark with a cross the applicable box.
Form BP/9 (first page) WO 01/19983 3~ PCT/EP00/09116 i IV. CONDITIONS UNDER WHICH THE VIABILITY HAS BEEN PERFORMED 4 I
V. INTERNATIONAL DEPOSITARY AUTHORITY
Name : Centraalbureau voor Schimmelcultures signature ( s ) of person ( s ) having the power to represent the International Depositary Authorit~of authorized official(s):
Addres s : posterstraat 1 ,~ ..--P.O. Box 273 3740 AG BAARN Mrs F.B. Snippe-Claus Dr .~ ers -The Netherlands Date (dd-mm-yy) : 17-09-99 p ~, R ftrSe~.
4Fi11 in if the information has been requested and if the results of the test were negative.
Form BP/9 (second and last page) WO 01/19983 ~ ~6 PCT/EP00/09116 SEQUENCE LISTING
<110> SOLVAY PHARMACEUTICALS B.V.
<120> Novel Human G-Protein coupled Receptor <130> SPW 99.07 <140>
<141>
<160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 1176 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (149)..(1138) <400> 1 ttaatctctt caagcctctg atttcctctc ctgtaaaaca ggggcggtaa ttaccacata 60 acaggctggt catgaaaatc agtgaacatg cagcaggtgc tcaagtcttg tttttgtttc 120 caggggcacc agtggaggtt ttctgagc atg gat cca acc acc ccg gcc tgg 172 Met Asp Pro Thr Thr Pro Ala Trp gga aca gaa agt aca aca gtg aat gga aat gac caa gcc ctt ctt ctg 220 Gly Thr Glu Ser Thr Thr Val Asn Gly Asn Asp Gln Ala Leu Leu Leu ctt tgt ggc aag gag acc ctg atc ccg gtc ttc ctg atc ctt ttc att 268 Leu Cys Gly Lys Glu Thr Leu Ile Pro Val Phe Leu Ile Leu Phe.Ile gcc ctg gtc ggg ctg gta gga aac ggg ttt gtg ctc tgg ctc ctg ggc 316 Ala Leu Val Gly Leu Val Gly Asn Gly Phe Val Leu Trp Leu Leu Gly ttc cgc atg cgc agg aac gcc ttc tct gtc tac gtc ctc agc ctg gcc 364 Phe Arg Met Arg Arg Asn Ala Phe Ser Val Tyr Val Leu Ser Leu Ala ggg gcc gac ttc ctc ttc ctc tgc ttc cag att ata aat tgc ctg gtg 412 Gly Ala Asp Phe Leu Phe Leu Cys Phe Gln Ile Ile Asn Cys Leu Val WO 01/19983 2~6 PCT/EP00/09116 tac ctc agt aac ttc ttc tgt tcc atc tcc atc aat ttc cct agc ttc 460 Tyr Leu Ser Asn Phe Phe Cys Ser Ile Ser Ile Asn Phe Pro Ser Phe ttc acc act gtg atg acc tgt gcc tac ctt gca ggc ctg agc atg ctg 508 Phe Thr Thr Val Met Thr Cys Ala Tyr Leu Ala Gly Leu Ser Met Leu agc acc gtc agc acc gag cgc tgc ctg tcc gtc ctg tgg ccc atc tgg 556 0 Ser Thr Val Ser Thr Glu Arg Cys Leu Ser Val Leu Trp Pro Ile Trp tat cgc tgc cgc cgc ccc aga cac ctg tca gcg gtc gtg tgt gtc ctg 604 Tyr Arg Cys Arg Arg Pro Arg His Leu Ser Ala Val Val Cys Val Leu ctc tgg gcc ctg tcc cta ctg ctg agc atc ttg gaa ggg aag ttc tgt 652 Leu Trp Ala Leu Ser Leu Leu Leu Ser Ile Leu Glu Gly Lys Phe Cys ggc ttc tta ttt agt gat ggt gac tct ggt tgg tgt cag aca ttt gat 700 Gly Phe Leu Phe Ser Asp Gly Asp Ser Gly Trp Cys Gln Thr Phe Asp ttc atc act gca gcg tgg ctg att ttt tta ttc atg gtt ctc tgt ggg 748 Phe Ile Thr Ala Ala Trp Leu Ile Phe Leu Phe Met Val Leu Cys Gly tcc agt ctg gcc ctg ctg gtc agg atc ctc tgt ggc tcc agg ggt ctg 796 Ser Ser Leu Ala Leu Leu Val Arg Ile Leu Cys Gly Ser Arg Gly Leu cca ctg acc agg ctg tac ctg acc atc ctg ctc aca gtg ctg gtg ttc 844 Pro Leu Thr Arg Leu Tyr Leu Thr Ile Leu Leu Thr Val Leu Val Phe ctc ctc tgc ggc ctg ccc ttt ggc att cag tgg ttc cta ata tta tgg 892 Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Trp Phe Leu Ile Leu Trp atc tgg aag gat tct gat gtc tta ttt tgt cat att cat cca gtt tca 940 Ile Trp Lys Asp Ser Asp Val Leu Phe Cys His Ile His Pro Val Ser gtt gtc ctg tca tct ctt aac agc agt gcc aac ccc atc att tac ttc 988 Val Val Leu Ser Ser Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr Phe ttc gtg ggc tct ttt agg aag cag tgg cgg ctg cag cag ccg atc ctc 1036 Phe Val Gly Ser Phe Arg Lys Gln Trp Arg Leu Gln Gln Pro Ile Leu 2g5 290 295 aag ctg get ctc cag agg get ctg cag gac att get gag gtg gat cac 1084 Lys Leu Ala Leu Gln Arg Ala Leu Gln Asp Ile Ala Glu Val Asp His WO 01/19983 3~6 PCT/EP00/09116 agt gaa gga tgc ttc cgt cag ggc acc ccg gag atg tcg aga agc agt 1132 Ser Glu Gly Cys Phe Arg Gln Gly Thr Pro Glu Met Ser Arg Ser Ser ctg gtg tagagatgga cagcctctac ttccatcaga tatatgtg 1176 Leu Val <210> 2 <211> 330 <212> PRT
<213> Homo Sapiens <400> 2 Met Asp Pro Thr Thr Pro Ala Trp Gly Thr Glu Ser Thr Thr Val Asn Gly Asn Asp Gln Ala Leu Leu Leu Leu Cys Gly Lys Glu Thr Leu Ile Pro ValPhe LeuIleLeu PheIleAla LeuValGlyLeu ValGlyAsn Gly PheVal LeuTrpLeu LeuGlyPhe ArgMetArgArg AsnAlaPhe Ser ValTyr ValLeuSer LeuAlaGly AlaAspPheLeu PheLeuCys Phe GlnIle IleAsnCys LeuValTyr LeuSerAsnPhe PheCysSer Ile SerIle AsnPhePro SerPhePhe ThrThrValMet ThrCysAla Tyr LeuAla GlyLeuSer MetLeuSer ThrValSerThr GluArgCys Leu SerVal LeuTrpPro IleTrpTyr ArgCysArgArg ProArgHis Leu SerAla ValValCys ValLeuLeu TrpAlaLeuSer LeuLeuLeu Ser IleLeu GluGlyLys PheCysGly PheLeuPheSer AspGlyAsp WO 01/19983 4~6 PCT/EP00/09116 Ser Gly Trp Cys Gln Thr Phe Asp Phe Ile Thr Ala Ala Trp Leu Ile Phe Leu Phe Met Val Leu Cys Gly Ser Ser Leu Ala Leu Leu Val Arg Ile Leu Cys Gly Ser Arg Gly Leu Pro Leu Thr Arg Leu Tyr Leu Thr Ile Leu Leu Thr Val Leu Val Phe Leu Leu Cys Gly Leu Pro Phe Gly Ile Gln Trp Phe Leu Ile Leu Trp Ile Trp Lys Asp Ser Asp Val Leu Phe Cys His Ile His Pro Val Ser Val Val Leu Ser Ser Leu Asn Ser Ser Ala Asn Pro Ile Ile Tyr Phe Phe Val Gly Ser Phe Arg Lys Gln Trp Arg Leu Gln Gln Pro Ile Leu Lys Leu Ala Leu Gln Arg Ala Leu Gln Asp Ile Ala Glu Val Asp His Ser Glu Gly Cys Phe Arg Gln Gly Thr Pro Glu Met Ser Arg Ser Ser Leu Val <210> 3 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Degenerated primers <220>
<221> variation <222> (21) <223> A,C,G or T
<220>
<221> variation <222> (24) <223> A,C,G or T
<400> 3 ctgcactacc acgtgctcws nytngc 26 WO 01/19983 5~6 PCT/EP00/09116 <210> 4 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Degenerated primers <220>
<221> variation <222> (21) <223> A,C,G or T
<220>
<221> variation <222> (27) <223> C or Inosine <400> 4 gggtggcaga tggccarrya nckytcnb 28 <210> 5 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Degenerated primers <220>
<221> variation <222> (25) <223> A,C,G or T
<400> 5 gtggcagatg gccaggcagc grtcnrcrct d 31 <210> 6 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 6 ggggccgact tcctcttcct ctgcttcc 28 <210> 7 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400>
gcaaggtagg cacaggtcat cacagtgg 28 <210> 8 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 8 ataagcttct ccctggccct taataaatga c 31 <210> 9 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 9 aggaattcag acagacaggg gcaaagttg 29
Claims (24)
1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding the IGS3 polypeptide according to SEQ ID NO:
2;
b) a nucleotide sequence encoding the polypeptide encoded by the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), in particular a nucleotide sequence corresponding to the SEQ ID NO: 1;
c) a nucleotide sequence having at least 80 % (preferably at least 90%) sequence identity over its entire length to the nucleotide sequence of (a) or (b);
d) a nucleotide sequence which is complimentary to the nucleotide sequence of (a) or (b) or (c).
a) a nucleotide sequence encoding the IGS3 polypeptide according to SEQ ID NO:
2;
b) a nucleotide sequence encoding the polypeptide encoded by the DNA insert contained in the deposit no. CBS 102196 at the Centraalbureau voor Schimmelcultures at Baarn (The Netherlands), in particular a nucleotide sequence corresponding to the SEQ ID NO: 1;
c) a nucleotide sequence having at least 80 % (preferably at least 90%) sequence identity over its entire length to the nucleotide sequence of (a) or (b);
d) a nucleotide sequence which is complimentary to the nucleotide sequence of (a) or (b) or (c).
2. The polynucleotide of claim 1 wherein said polynucleotide comprises the nucleotide sequence contained in SEQ ID NO:1 encoding the IGS3 polypeptide of SEQ ID
NO:2.
NO:2.
3. The polynucleotide of claim 1 wherein said polynucleotide comprises a nucleotide sequence that is at least 80% identical to that of SEQ ID NO:1 over its entire length.
4. The polynucleotide of claim 3 which is the polynucleotide of SEQ ID NO:1.
5. The polynucleotide of claim 1-4 which is DNA or RNA.
6. A DNA or RNA molecule comprising an expression vector, wherein said expression vector is capable of producing an IGS3 polypeptide comprising an amino acid sequence, which has at least 80% identity with the polypeptide of SEQ ID NO:2 when said expression vector is present in a compatible host cell.
7. A host cell comprising the expression vector of claim 6.
8. A host cell according to claim 7 which is a yeast cell
9. A host call according to claim 7 which is an animal cell
10. IGS3 receptor membrane preparation derived from a cell according to claim 7-9.
11. A process for producing an IGS3 polypeptide comprising culturing a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture.
12. A process for producing a cell which produces an IGS3 polypeptide thereof comprising transforming or transfecting a cell with the expression vector of claim 6 such that the cell, under appropriate culture conditions, is capable of producing an IGS3 polypeptide.
13. An IGS3 polypeptide comprising an amino acid sequence which is at least 80% identical to the amino acid sequence of SEQ ID NO:2 over its entire length.
14. The polypeptide of claim 13 which comprises the amino acid sequence of SEQ
ID NO:2.
ID NO:2.
15. An antibody immunospecific for the IGS3 polypeptide of SEQ ID NO:2 or a Variant thereof.
16. A method for the treatment of a subject suffering from a disease related to expression or activity of the IGS3 polypeptide receptor of claim 13, in need of enhanced activity or expression of the IGS3 polypeptide receptor of claim 13, comprising:
(a) administering to the subject a therapeutically effective amount of an agonist to said receptor; and/or (b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the IGS3 polypeptide of SEQ ID NO:2 over its entire length; or a nucleotide sequence complementary to said nucleotide sequence in a form so as to effect production of said receptor activity in vivo.
(a) administering to the subject a therapeutically effective amount of an agonist to said receptor; and/or (b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence that has at least 80% identity to a nucleotide sequence encoding the IGS3 polypeptide of SEQ ID NO:2 over its entire length; or a nucleotide sequence complementary to said nucleotide sequence in a form so as to effect production of said receptor activity in vivo.
17. A method for the treatment of a subject suffering from a disease related to expression or activity of the IGS3 polypeptide receptor of claim 13, having need to inhibit activity or expression of the IGS3 polypeptide receptor of claim 13, comprising:
(a) administering to, the subject a therapeutically effective amount of an antagonist to said receptor; and/or (b) administering to the subject a polynucleotide that inhibits the expression of the nucleotide sequence encoding said receptor; and/or (c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said receptor for its ligand.
(a) administering to, the subject a therapeutically effective amount of an antagonist to said receptor; and/or (b) administering to the subject a polynucleotide that inhibits the expression of the nucleotide sequence encoding said receptor; and/or (c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said receptor for its ligand.
18. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of the IGS3 polypeptide of claim 13 in a subject comprising:
(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said IGS3 polypeptide in the genome of said subject; and/or (b) analyzing for the presence or amount of the IGS3 polypeptide expression in a sample derived from said subject.
(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said IGS3 polypeptide in the genome of said subject; and/or (b) analyzing for the presence or amount of the IGS3 polypeptide expression in a sample derived from said subject.
19. A method for identifying agonists to the IGS3 polypeptide of claim 13 comprising:
(a) contacting a cell which produces a IGS3 polypeptide with a test compound;
and (b) determining whether the test compound effects a signal generated by activation of the IGS3 polypeptide.
(a) contacting a cell which produces a IGS3 polypeptide with a test compound;
and (b) determining whether the test compound effects a signal generated by activation of the IGS3 polypeptide.
20. An agonist identified by the method of claim 19.
21. A method for identifying antagonists to the IGS3 polypeptide of claim 13 comprising:
(a) contacting a cell which produces a IGS3 polypeptide with an agonist; and (b) determining whether the signal generated by said agonist is diminished in the presence of a candidate compound.
(a) contacting a cell which produces a IGS3 polypeptide with an agonist; and (b) determining whether the signal generated by said agonist is diminished in the presence of a candidate compound.
22. An antagonist identified by the method of claim 21.
23. A recombinant host cell produced by a method of claim 12 or a membrane thereof expressing an IGS3 polypeptide.
24. A method of creating a genetically modified non-human animal comprising the steps of:
a) ligating the coding portion of a polynucleotide consisting essentially of a nucleic acid sequence encoding a protein having the amino acid sequence SEQ ID NO:
2 to a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in said animal; or b) engineering the coding portion of a polynucleotide consisting essentially of a nucleic acid sequence encoding a protein having the amino acid sequence SEQ
ID NO: 2 and reintroducing said sequence in the genome of said animal in such a way that the endogenous gene alleles encoding a protein having the amino acid sequence SEQ ID NO: 2 are fully or partially inactivated.
a) ligating the coding portion of a polynucleotide consisting essentially of a nucleic acid sequence encoding a protein having the amino acid sequence SEQ ID NO:
2 to a regulatory sequence which is capable of driving high level gene expression or expression in a cell type in which the gene is not normally expressed in said animal; or b) engineering the coding portion of a polynucleotide consisting essentially of a nucleic acid sequence encoding a protein having the amino acid sequence SEQ
ID NO: 2 and reintroducing said sequence in the genome of said animal in such a way that the endogenous gene alleles encoding a protein having the amino acid sequence SEQ ID NO: 2 are fully or partially inactivated.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NL1013062 | 1999-09-16 | ||
EP99203014 | 1999-09-16 | ||
NL1013062 | 1999-09-16 | ||
EP99203014.8 | 1999-09-16 | ||
PCT/EP2000/009116 WO2001019983A1 (en) | 1999-09-16 | 2000-09-15 | Human g-protein coupled receptor |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2383177A1 true CA2383177A1 (en) | 2001-03-22 |
Family
ID=26153370
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002383177A Abandoned CA2383177A1 (en) | 1999-09-16 | 2000-09-15 | Human g-protein coupled receptor |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP1220914A1 (en) |
JP (1) | JP2004500039A (en) |
CN (1) | CN1379816A (en) |
AU (1) | AU7777400A (en) |
CA (1) | CA2383177A1 (en) |
HK (1) | HK1044567A1 (en) |
IL (1) | IL148515A0 (en) |
WO (1) | WO2001019983A1 (en) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1242454A2 (en) * | 1999-12-28 | 2002-09-25 | PHARMACIA & UPJOHN COMPANY | G protein-coupled receptors |
JP2003528634A (en) * | 2000-03-29 | 2003-09-30 | ピーイー コーポレーション (エヌワイ) | Isolated human G protein-coupled receptors, nucleic acid molecules encoding human GPCR proteins, and uses thereof |
US20030092035A1 (en) | 2000-05-04 | 2003-05-15 | Anderson David J. | Pain signaling molecules |
US7510845B2 (en) | 2000-05-04 | 2009-03-31 | California Institute Of Technology | Assay employing G protein-coupled receptor expressed in dorsal root ganglia |
US7691604B1 (en) | 2000-05-04 | 2010-04-06 | California Institute Of Technology | MRG polypeptides and nucleic acids encoding the same |
AU2001265717A1 (en) * | 2000-06-20 | 2002-01-02 | Euroscreen S.A. | A recombinant cell line expressing gpcrx11 as a functional receptor validated byangiopeptin and useful for screening of agonists and antagonists |
AU2001269463A1 (en) * | 2000-07-07 | 2002-01-21 | Takeda Chemical Industries Ltd. | Novel g protein-coupled receptor protein and dna thereof |
CN100406891C (en) | 2001-06-27 | 2008-07-30 | 詹森药业有限公司 | EPF receptor assays, compounds and therapeutic compsns. |
US20030096751A1 (en) * | 2001-08-20 | 2003-05-22 | Ramanathan Chandra S. | G-protein coupled receptor polynucleotides and methods of use thereof |
EP1340979A3 (en) * | 2002-02-27 | 2004-02-04 | Pfizer Limited | Neuropeptide receptor and uses thereof |
EP3909998A3 (en) | 2005-04-22 | 2022-03-16 | Mitsubishi Chemical Corporation | Biomass-resource-derived polyester and production process thereof |
JP6374409B2 (en) * | 2014-01-27 | 2018-08-15 | 株式会社日立製作所 | Method for analyzing reaction solution after nucleic acid amplification reaction, analysis device, and reaction solution processing device after nucleic acid amplification reaction |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9223084D0 (en) * | 1992-11-04 | 1992-12-16 | Imp Cancer Res Tech | Compounds to target cells |
-
2000
- 2000-09-15 CA CA002383177A patent/CA2383177A1/en not_active Abandoned
- 2000-09-15 CN CN00814366A patent/CN1379816A/en active Pending
- 2000-09-15 WO PCT/EP2000/009116 patent/WO2001019983A1/en not_active Application Discontinuation
- 2000-09-15 IL IL14851500A patent/IL148515A0/en unknown
- 2000-09-15 AU AU77774/00A patent/AU7777400A/en not_active Abandoned
- 2000-09-15 JP JP2001523754A patent/JP2004500039A/en active Pending
- 2000-09-15 EP EP00967696A patent/EP1220914A1/en not_active Withdrawn
-
2002
- 2002-08-22 HK HK02106162.7A patent/HK1044567A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
JP2004500039A (en) | 2004-01-08 |
HK1044567A1 (en) | 2002-10-25 |
IL148515A0 (en) | 2002-09-12 |
WO2001019983A1 (en) | 2001-03-22 |
AU7777400A (en) | 2001-04-17 |
CN1379816A (en) | 2002-11-13 |
EP1220914A1 (en) | 2002-07-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20020106655A1 (en) | Human GPCR proteins | |
US5874243A (en) | OLRCC15 receptor | |
JP2002512781A (en) | G protein-coupled 7TM receptor (AXOR-1) | |
AU764310B2 (en) | Human G-protein coupled receptor | |
US6010877A (en) | cDNA clone HE8CS41 that encodes a novel 7-transmembrane receptor | |
CA2383177A1 (en) | Human g-protein coupled receptor | |
AU779993B2 (en) | Novel human G-protein coupled receptor | |
US7459292B2 (en) | Polynucleotides and expression system of a novel human G-protein coupled receptor | |
US5994098A (en) | Human 7-TM receptor similar to murine frizzled-6 gene | |
WO2002044212A2 (en) | Human g-protein coupled receptor and uses thereof | |
US20040072327A1 (en) | Human g-protein coupled receptor and uses thereof | |
US20040191797A1 (en) | Human G-protein coupled receptor | |
EP1334191A2 (en) | Human g-protein coupled receptor and uses thereof | |
AU782817B2 (en) | Novel human G protein coupled receptor proteins and polynucleotides encoding the same | |
EP0875568A1 (en) | Novel human neurotensin receptor type 2 and splice variants thereof | |
US6570003B1 (en) | Human 7TM proteins and polynucleotides encoding the same | |
JPWO2002088355A1 (en) | Novel guanosine triphosphate binding protein-coupled receptor PLACE 600002312 and its gene, and production and use thereof | |
WO1999053054A1 (en) | Axor4 g-protein-coupled receptor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |