G-protein coupled receptor and uses therefor

• US 20030022211A1
• Filed: 06/07/2002
• Published: 01/30/2003
• Est. Priority Date: 06/07/2001
• Status: Abandoned Application

• Current Assignee: Wyeth (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Blatcher, Maria, Bates, Brian Gaither, Paulsen, Janet Elizabeth
• Application Number:
  US10/166,221
• Publication Number:
  US 20030022211A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor

Abstract

The present invention is based on the identification of a G-protein coupled receptor (GPCR) that is expressed predominantly in the brain and placenta and nucleic acid molecules that encoded the GPCR, which is referred to herein as the hCAR protein and hCAR gene respectively (for human Constitutively Active Receptor). Based on this identification, the present invention provides: (1) isolated hCAR protein; (2) isolated nucleic acid molecules that encode an hCAR protein; (3) antibodies that selectively bind to the hCAR protein; (4) methods of isolating allelic variants of the hCAR protein and gene; (5) methods of identifying cells and tissues that express the hCAR protein/gene; (6) methods of identifying agents and cellular compounds that bind to the hCAR protein; (7) methods of identifying agents that modulate the expression of the hCAR gene; and (8) methods of modulating the activity of the hCAR protein in a cell or organism.

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid which encodes a protein comprising the amino acid sequence of SEQ ID NO:

2.

2. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.

3. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous protein or a fragment of a heterologous protein.

4. A host cell which contains the nucleic acid molecule of claim 1.

5. The host cell of claim 4 which is a mammalian host cell.

6. A non-human mammalian host cell containing the nucleic acid molecule of claim 1.

20. A method for producing a protein selected from the group consisting of:

a) a protein comprising the amino acid sequence of SEQ ID NO;

2;

b) a fragment of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the fragment comprises at least 15 contiguous amino acids of a transmembrane or extracellular region of SEQ ID NO;

2; and

c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO;

1 under stringent conditions;

the method comprising the step of culturing the host cell of claim 4 under conditions in which the nucleic acid molecule is expressed.

7. An isolated nucleic acid molecule comprising SEQ ID NO:

1, or a degenerate variant thereof.

9. A nucleic acid according to claim 7 further comprising a single nucleotide polymorphism.

10. A nucleic acid according to claim 9 wherein the polymorphism is selected from the those listed in Table 3.

8. An isolated nucleic acid molecule comprising the coding region of SEQ ID NO:

1.

11. An isolated nucleic acid molecule comprising a nucleic acid which would hybridize under stringent conditions to a nucleic acid comprising a non-coding region of the hCAR gene or the complement of said non-coding region.

12. A gene encoding a hCAR protein.

13. An isolated hCAR protein comprising the amino acid sequence of SEQ ID NO:

2.

16. The protein of claim 13 further comprising heterologous amino acid sequences.

17. An antibody which selectively binds to a protein of claim 13.

14. An isolated peptide comprising an extracellular domain of the hCAR protein.

15. A peptide according to claim 14 comprising a sequence selected from the group consisting of SEQ ID NOs 4, 5, 6, and 7.

18. An antibody which selectively binds to a peptide according to claim 14.

19. An antibody which selectively binds to a peptide according to claim 15.

21. A method for detecting the presence of a protein selected from the group consisting of:

a) a protein comprising the amino acid sequence of SEQ ID NO;

2;

b) a fragment of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the fragment comprises at least 15 contiguous amino acids of a transmembrane or extracellular region of SEQ ID NO;

2; and

c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO;

1 under stringent conditions;

the method comprising the steps of;

i) contacting the sample with a compound which selectively binds to the protein; and

ii) determining whether the compound binds to the protein in the sample.

22. The method of claim 21, wherein the compound which binds to the protein is an antibody.

23. A kit comprising reagents used for the method of claim 21, wherein the reagents comprise a compound which selectively binds to a protein selected from the group consisting of:

a) a protein comprising the amino acid sequence of SEQ ID NO;

2;

b) a fragment of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the fragment comprises at least 15 contiguous amino acids of a transmembrane or extracellular region of SEQ ID NO;

2; and

C) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO;

1 under stringent conditions.

24. A method for detecting the presence of a nucleic acid molecule selected from the group consisting of:

a) a nucleic acid molecule which encodes a protein comprising the amino acid sequence of SEQ ID NO;

2;

b) a nucleic acid molecule which encodes a fragment of a protein comprising the amino acid of SEQ ID NO;

2, wherein the fragment comprises at least 15 contiguous amino acids of a transmembrane or extracellular region of SEQ ID NO;

2; and

c) a nucleic acid molecule which encodes a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO;

1 under stringent conditions;

in a sample, the method comprising the steps of;

i) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and

ii) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.

25. The method of claim 24, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

26. A kit comprising reagents used for the method of claim 24, wherein the reagents comprise a compound which selectively hybridizes to a nucleic acid molecule selected from the group consisting of:

a) a nucleic acid molecule which encodes a protein comprising the amino acid sequence of SEQ ID NO;

2;

b) a nucleic acid molecule which encodes a fragment of a protein comprising the amino acid of SEQ ID NO;

2, wherein the fragment comprises at least 15 contiguous amino acids of a transmembrane or extracellular region of SEQ ID NO;

2; and

c) a nucleic acid molecule which encodes a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO;

1 under stringent conditions.

27. A method for identifying a compound which binds to a protein selected from the group consisting of:

a) a protein comprising the amino acid sequence of SEQ ID NO;

2;

b) a fragment of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the fragment comprises at least 15 contiguous amino acids of a transmembrane or extracellular region of SEQ ID NO;

2; and

c) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO;

1 under stringent conditions;

the method comprising the steps of;

i) contacting the protein, or a cell expressing the protein with a test compound; and

ii) ii) determining whether the protein binds to the test compound.

28. The method of claim 27, wherein the binding of the test compound to the protein is detected by a method selected from the group consisting of:

a) detection of binding by direct detecting of test compound/protein binding;

b) detection of binding using a competition binding assay; and

c) detection of binding using an assay for hCAR activity.

29. A method for modulating the activity of a protein selected from the group consisting of:

a) a protein comprising the amino acid sequence of SEQ ID NO;

2; and

b) a naturally occurring allelic variant of a protein comprising the amino acid sequence of SEQ ID NO;

2, wherein the protein is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO;

1 under stringent conditions, the method comprising the step of contacting a cell expressing the protein with a compound which binds to the protein in a sufficient concentration to modulate the activity of the protein.

30. A method for the treatment of a patient having need of the inhibition of hCAR activity such treatment comprising administering to the patient a therapeutically effective amount of an antibody which binds to an extracellular portion of HCAR.

31. A transgenic or chimeric nonhuman animal comprising the nucleic acid of SEQ ID NO:

1.

32. The animal of claim 31 wherein the transgene is under the control of a regulatable expression system.

33. A knockout nonhuman animal wherein at least one allele of the HCAR gene has been functionally disrupted.

34. A knockout nonhuman animal wherein at least one allele of the HCAR gene can be functionally disrupted by the induction of the Cre gene.

35. A knockout according to claim 34 wherein the Cre gene is under the control of a tissue specific promoter.

36. A knockout according to claim 34 wherein the Cre gene is under the control of a developmentally specific promoter.

37. A knockout according to claim 34 wherein the Cre gene is under the control of an inducible promoter.

38. A method for inhibiting expression of the HCAR gene comprising providing to a cell double stranded ribonucleic acid complementary to a portion of the HCAR gene wherein said ribonucleic acid comprises about 600 base pairs.

39. A method of inhibiting expression of the HCAR gene in a cell comprising providing said cell with an antisense nucleic acid.

40. A method of producing a hCAR protein comprising the amino acid sequence of SEQ ID NO:

2 which method comprises hCAR gene activation.

41. A method of producing a hCAR protein comprising the amino acid sequence of SEQ ID NO:

2 which method comprises operably linking a heterologous regulatory sequence to the hCAR gene in a cell such that the cell expresses the hCAR protein.