G protein-coupled receptor gene and methods of use therefor
AssigneesChildren's Medical Center Corporation, Millennium Pharmaceuticals, Inc., The Brigham and Women's Hospital, Inc.
InventorsCraig J. Gerard, Norma P. Gerard, Charles R. Mackay, Paul D. Ponath, Theodore W. Post, Shixin Qin
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Abstract
The present invention relates to isolated and/or recombinant nucleic acids which encode a mammalian (e.g., human) receptor protein designated C-C Chemokine Receptor 3 (CKR-3) or Eos L2, and to proteins or polypeptides, referred to herein as isolated, recombinant mammalian CKR-3 receptors. The invention further relates to recombinant nucleic acid constructs, comprising a nucleic acid which encodes a receptor protein of the present invention or a portion thereof, and to host cells comprising such constructs, useful for the production of recombinant CKR-3 receptors or polypeptides.
Claims87
An isolated nucleic acid encoding a C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion binds eotaxin, and wherein said isolated nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:
- 3 or a portion thereof comprising the coding sequence;
- c) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- d) the complement of any one of a) through c);
- and
- e) the RNA counterpart of any one of a) through d), wherein U is substituted for T.
An isolated nucleic acid, wherein said nucleic acid encodes a polypeptide selected from the group consisting of:
- a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:2;
- b) a polypeptide having the amino acid sequence set forth in SEQ ID NO:4;
- c) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6;
- and
- d) a functional portion of any one of a) through c), wherein said functional portion binds a ligand selected from the group consisting of RANTES and MCP-3, and mediates chemotaxis or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca2+]i) in response to ligand binding.
An isolated recombinant construct comprising a nucleic acid which encodes a C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion binds eotaxin, and wherein said nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:
- 3 or a portion thereof comprising the coding sequence;
- c) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- d) the complement of any one of a) through c);
- and
- e) the RNA counterpart of any one of a) through d), wherein U is substituted for T.
An isolated recombinant construct comprising a nucleic acid which encodes a C-C chemokine receptor 3, wherein said C-C chemokine receptor 3 binds eotaxin and said nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:
- 3 or a portion thereof comprising the coding sequence;
- c) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- d) the complement of any one of a) through c);
- and
- e) the RNA counterpart of any one of a) through d), wherein U is substituted for T.
An isolated recombinant construct comprising a nucleic acid, wherein said nucleic acid encodes a polypeptide selected from the group consisting of:
- a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:2;
- b) a polypeptide having the amino acid sequence set forth in SEQ ID NO:4;
- c) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6;
- and
- d) a functional portion of any one of a) through c), wherein said functional portion binds a ligand selected from the group consisting of RANTES and MCP-3, and mediates chemotaxis or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca2+]i) in response to ligand binding.
An isolated nucleic acid encoding a polypeptide comprising a human C-C chemokine receptor 3 or functional portion thereof, wherein said functional portion binds a ligand selected from the group consisting of RANTES and MCP-3, and mediates chemotaxis or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca2+]i) in response to ligand binding, and wherein said receptor or portion thereof is encoded by a sequence selected from the group consisting of:
- a) SEQ ID NO:1 or a portion thereof;
- and
- b) SEQ ID NO:3 or a portion thereof.
An isolated nucleic acid encoding a polypeptide comprising a C-C chemokine receptor 3, said nucleic acid comprising a sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4.
A host cell comprising a recombinant nucleic acid encoding a polypeptide comprising a human C-C chemolcine receptor 3 or functional portion thereof, wherein said human C-C chemokine receptor 3 or functional portion binds eotaxin, wherein said nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:
- 3 or a portion thereof comprising the coding sequence;
- c) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- d) the complement of any one of a) through c);
- and
- e) the RNA counterpart of any one of a) through d), wherein U is substituted for T;
- and wherein said host cell is not in a transgenic animal.
A host cell comprising a recombinant nucleic acid encoding a polypeptide comprising a human C-C chemokine receptor 3, said nucleic acid comprising a sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4, wherein said host cell is not in a transgenic animal.
An isolated nucleic acid encoding a C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion binds eotaxin and wherein said isolated nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:5 or a portion thereof comprising the coding sequence;
- c) the complement of a) or b);
- and
- d) the RNA counterpart of any one of a) through c), wherein U is substituted for T.
An isolated nucleic acid, wherein said nucleic acid encodes a polypeptide selected from the group consisting of:
- a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:2;
- b) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6;
- and
- c) a functional portion of a) or b), wherein said functional portion binds a ligand selected from the group consisting of RANTES and MCP-3, and mediates chemotaxis or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca2+]i) in response to ligand binding.
An isolated nucleic acid encoding a protein comprising a C-C chemokine receptor 3, said nucleic acid comprising a sequence encoding the amino acid sequence of SEQ ID NO:2.
An isolated nucleic acid encoding a protein comprising a C-C chemokine receptor 3 or functional portion thereof, wherein said functional portion binds eotaxin and wherein said receptor or functional portion thereof is encoded by SEQ ID NO:1 or a portion thereof.
An isolated recombinant construct comprising a nucleic acid which encodes a C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion binds eotaxin and wherein said recombinant nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:5 or a portion thereof comprising the coding sequence;
- c) the complement of any one of a) or b);
- and
- d) the RNA counterpart of any one of a) through c), wherein U is substituted for T.
An isolated recombinant construct comprising a nucleic acid that encodes a polypeptide selected from the group consisting of:
- a) a polypeptide having the amino acid sequence set forth in SEQ ID NO:2;
- b) a polypeptide having the amino acid sequence set forth in SEQ ID NO:6;
- and
- c) a functional portion of a) or b), wherein said functional portion binds a ligand selected from the group consisting of RANTES and MCP-3, and mediates chemotaxis or a rapid and transient increase in the concentration of cytosolic free calcium ([Ca2+]i) in response to ligand binding.
A host cell comprising a recombinant nucleic acid encoding a polypeptide comprising a human C-C chemokine receptor 3 or functional portion thereof, wherein said human C-C chemokine receptor 3 or functional portion binds eotaxin, wherein said nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having The sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- c) the complement of a) or b);
- and
- d) the RNA counterpart of any one of a) through c), wherein U is substituted for T;
- and wherein said host cell is not in a transgenic animal.
A host cell comprising a recombinant nucleic acid encoding a polypeptide comprising a human C-C chemokine receptor 3, said nucleic acid comprising a sequence encoding the amino acid sequence of SEQ ID NO:2, wherein said host cell is not in a transgenic animal.
An isolated nucleic acid encoding a C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion binds eotaxin, and wherein said isolated nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to the EcoRI-NotI insert of A31-pcDNA3 (ATCC Accession No. PTA-2689).
An isolated recombinant construct comprising a nucleic acid encoding a C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion binds eotaxin, and wherein said isolated nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to the EcoRI-NotI insert of A31-pcDNA3 (ATCC Accession No. PTA-2689).
An isolated nucleic acid encoding a human C-C chemokine receptor 3 or functional portion thereof encoded by the EcoRI-NotI insert of A31-pcDNA3 (ATCC Accession No. PTA-2689) or portion thereof, wherein said functional portion binds eotaxin.
An isolated recombinant construct comprising a nucleic acid which encodes a human C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion is encoded by the EcoRI-NotI insert of A31-pcDNA3 (ATCC Accession No. PTA-2689) or portion thereof, and said functional portion binds eotaxin.
A host cell comprising a recombinant nucleic acid encoding a human C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion is encoded by the EcoRI-NotI insert of A31-pcDNA3 (ATCC Accession No. PTA-2689) or portion thereof, and said functional portion binds eotaxin, wherein said host cell is not in a transgenic animal.
An isolated nucleic acid encoding a C-C chemokine receptor 3, wherein said C-C chemokine receptor 3 binds eotaxin and said nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:
- 3 or a portion thereof comprising the coding sequence;
- c) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- d) the complement of any one of a) through c);
- and
- e) the RNA counterpart of any one of a) through d), wherein U is substituted for T.
A host cell comprising a recombinant nucleic acid encoding a C-C chemokine receptor 3, wherein said C-C chemokine receptor 3 binds eotaxin and said nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0.5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO 3 or a portion thereof comprising the coding sequence;
- c) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- d) the complement of any one of a) through c);
- and
- e) two RNA counterpart of any one of a) through d), wherein U is substituted for T;
- and wherein said host cell is not in a transgenic animal.
An isolated nucleic acid encoding a C-C chemokine receptor 3 or a functional portion thereof, wherein said C-C chemokine receptor 3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said C-C chemokine receptor 3 or functional portion thereof binds a ligand selected from the group consisting of RANTES and MCP-3.
An isolated recombinant construct comprising a nucleic acid that encodes a polypeptide comprising a C-C chemokine receptor 3 or a functional portion thereof, wherein said C-C chemokine receptor 3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said C-C chemokine receptor 3 or functional portion thereof binds a ligand selected from the group consisting of RANTES and MCP-3.
A host cell comprising a recombinant nucleic acid encoding a polypeptide comprising a C-C chemokine receptor 3 or a functional portion thereof, wherein said C-C chemokine receptor 3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said C-C chemokine receptor 3 or functional portion thereof binds a ligand selected from the group consisting of RANTES and MCP-3, wherein said host cell is not in a transgenic animal.
An isolated nucleic acid encoding a C-C chemokine receptor 3 or a functional portion thereof, wherein said C-C chemokine receptor 3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said C-C chemokine receptor 3 or functional portion thereof binds eotaxin.
An isolated recombinant construct comprising a nucleic acid that encodes a polypeptide comprising a C-C chemokine receptor 3 or a functional portion thereof, wherein said C-C chemokine receptor 3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said C-C chemokine receptor 3 or functional portion thereof binds eotaxin.
A host cell comprising a recombinant nucleic acid encoding a polypeptide comprising a C-C chemokine receptor 3 or a functional portion thereof, wherein said C-C chemokine receptor 3 has at least about 90% amino acid sequence identity with SEQ ID NO:2 or SEQ ID NO:6, and said C-C chemokine receptor 3 or functional portion thereof binds eotaxin, wherein said host cell is not in a transgenic animal.
A host cell comprising a recombinant nucleic acid encoding a C-C chemokine receptor 3 or functional portion thereof, wherein said C-C chemokine receptor 3 or functional portion binds eotaxin, wherein said isolated nucleic acid hybridizes under conditions of 6×SSC containing 5×Denhardt's solution, 10% (w/v) dextran sulfate, 2% SDS and sheared salmon sperm DNA (100 μg/mL) at 65° C. and wash conditions of 0.2×SSC, 0-5% SDS at 65° C. to a second nucleic acid selected from the group consisting of:
- a) a nucleic acid having the sequence set forth in SEQ ID NO:
- 1 or a portion thereof comprising the coding sequence;
- b) a nucleic acid having the sequence set forth in SEQ ID NO:
- 3 or a portion thereof comprising the coding sequence;
- c) a nucleic acid having the sequence set forth in SEQ ID NO:
- 5 or a portion thereof comprising the coding sequence;
- d) the complement of any one of a) through c);
- and
- e) the RNA counterpart of any one of a) through d), wherein U is substituted for T;
- and wherein said host cell is not in a transgenic animal.
Dependencies are inferred from the claim text.