Claims
- 1. A purified nucleic acid molecule encoding a rhesus melanocortin 4 receptor protein which comprises the nucleotide sequence
- 2. A purified DNA molecule encoding rhesus melanocortin 4 receptor protein wherein said DNA molecule encodes a protein comprising the amino acid sequence
- 3. An expression vector for the expression of a rhesus MC-4R protein in a recombinant host cell wherein said expression vector comprises a DNA molecule which encodes the amino acid sequence of claim 2.
- 4. An expression vector of claim 3 which is a eukaryotic expression vector.
- 5. An expression vector of claim 3 which is a prokaryotic expression vector.
- 6. A host cell which expresses a recombinant rhesus MC-4R protein wherein said host cell contains the expression vector of claim 3.
- 7. A host cell which expresses a recombinant rhesus MC-4R protein wherein said host cell contains the expression vector of claim 4.
- 8. A host cell which expresses a recombinant rhesus MC-4R protein wherein said host cell contains the expression vector of claim 5.
- 9. A host cell of claim 6 wherein said rhesus MC-4R protein is overexpressed from said expression vector.
- 10. A host cell of claim 7 wherein said rhesus MC-4R protein is overexpressed from said expression vector.
- 11. A host cell of claim 8 wherein said rhesus MC-4R protein is overexpressed from said expression vector.
- 12. A subcellular membrane fraction obtained from the host cell of claim 9 which contains recombinant rhesus MC-4R protein.
- 13. A subcellular membrane fraction obtained from the host cell of claim 10 which contains recombinant rhesus MC-4R protein.
- 14. A subcellular membrane fraction obtained from the host cell of claim 11 which contains recombinant rhesus MC-4R protein.
- 15. A purified DNA molecule encoding a rhesus melanocortin 4 receptor protein which consists of the nucleotide sequence
- 16. A purified DNA molecule which encodes a MC-4R protein which consists of the amino acid sequence
- 17. The purified DNA molecule of claim 15 which consists of a nucleotide sequence from nucleotide 17 to nucleotide 1015 of SEQ ID NO: 1.
- 18. An expression vector for the expression of a rhesus MC-4R protein in a recombinant host cell wherein said expression vector comprises a DNA molecule of claim 17.
- 19. An expression vector of claim 18 which is a eukaryotic expression vector.
- 20. An expression vector of claim 18 which is a prokaryotic expression vector.
- 21. A host cell which expresses a recombinant rhesus MC-4R protein wherein said host cell contains the expression vector of claim 19.
- 22. A host cell which expresses a recombinant rhesus MC-4R protein wherein said host cell contains the expression vector of claim 20.
- 23. A host cell which expresses a recombinant rhesus MC-4R protein wherein said host cell contains the expression vector of claim 21.
- 24. A subcellular membrane fraction obtained from the host cell of claim 22 which contains recombinant rhesus MC-4R protein.
- 25. A subcellular membrane fraction obtained from the host cell of claim 23 which contains recombinant rhesus MC-4R protein.
- 26. A process for the expression of a rhesus MC-4R protein in a recombinant host cell, comprising:
(a) transfecting the expression vector of claim 3 into a suitable host cell; and, (b) culturing the host cells of step (a) under conditions which allow expression of the rhesus MC-4R protein from the expression vector.
- 27. A purified rhesus melanocortin 4 receptor protein which comprises the amino acid sequence as follows:
- 28. A purified rhesus melanocortin 4 receptor protein which consists of the amino acid sequence as set forth in SEQ ID NO:2.
- 29. A method for determining whether a substance is capable of binding to rhMC-4R comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of rhMC-4R in the cells; (b) exposing the test cells to the substance; (c) measuring the amount of binding of the substance to rhMC-4R; and, (d) comparing the amount of binding of the substance to rhMC-4R in the test cells with the amount of binding of the substance to control cells that have not been transfected with rhMC-4R.
- 30. A method for determining whether a substance is capable of activating rhMC-4R comprising:
(a) providing test cells by transfecting cells with an expression vector that directs the expression of rhMC-4R in the cells; (b) exposing the test cells to the substance; (c) measuring the amount of accumulated intracellular cAMP; and, (d) comparing the amount of cAMP in the test cells in response to the substance with the amount of cAMP in test cells that have not been exposed to the substance.
- 31. A method of identifying a substance which modulates MC-4R receptor activity, comprising:
(a) combining a test substance in the presence and absence of a MC-4R receptor protein wherein said MC-4R receptor protein comprises the amino acid sequence as set forth in SEQ ID NO:2; and, (b) measuring and comparing the effect of the test substance in the presence and absence of the MC-4R receptor protein.
- 32. A method for determining whether a substance is a potential agonist or antagonist of MC-4R comprising:
(a) transfecting or transforming cells with an expression vector of claim 3 that directs expression of MC-4R in the cells, resulting in test cells; (b) allowing the test cells to grow for a time sufficient to allow MC-4R to be expressed; (c) exposing the cells to a labeled ligand of MC-4R in the presence and in the absence of the substance; and, (d) measuring the binding of the labeled ligand to MC-4R; where if the amount of binding of the labeled ligand is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of MC-4R.
- 33. A method for determining whether a substance is capable of binding to MC-4R comprising:
(a) transfecting or transforming cells with an expression vector of claim 3 that directs the expression of MC-4R in the cells, resulting in test cells; (b) exposing the test cells to the substance; (c) measuring the amount of binding of the substance to MC-4R; and, (d) comparing the amount of binding of the substance to MC-4R in the test cells with the amount of binding of the substance to control cells that have not been transfected with MC-4R; wherein if the amount of binding of the substance is greater in the test cells as compared to the control cells, the substance is capable of binding to MC-4R.
- 34. A method for determining whether a substance is capable of binding to MC-4R comprising:
(a) transfecting or transforming cells with an expression vector of claim 3 that directs the expression of MC-4R in the cells, resulting in test cells; (b) preparing membranes containing MC-4R from the test cells and exposing the membranes to a ligand of MC-4R under conditions such that the ligand binds to the MC-4R in the membranes; (c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance; (d) measuring the amount of binding of the ligand to the MC-4R in the membranes in the presence and the absence of the substance; and, (e) comparing the amount of binding of the ligand to MC-4R in the membranes in the presence and the absence of the substance where a decrease in the amount of binding of the ligand to MC-4R in the membranes in the presence of the substance indicates that the substance is capable of binding to MC-4R.
- 35. A method for determining whether a substance is capable of binding to MC-4R comprising:
(a) transfecting or transforming cells with an expression vector of claim 3 that directs the expression of MC-4R in the cells, resulting in test cells; (b) preparing membranes containing MC-4R from the test cells and exposing the membranes from the test cells to the substance; (c) measuring the amount of binding of the substance to the MC-4R in the membranes from the test cells; (d) comparing the amount of binding of the substance to MC-4R in the membranes from the test cells with the amount of binding of the substance to membranes from control cells that have not been transfected with MC-4R, where if the amount of binding of the substance to MC-4R in the membranes from the test cells is greater than the amount of binding of the substance to the membranes from the control cells, then the substance is capable of binding to MC-4R.
- 36. A method of identifying agonists of MC-4R comprising:
(a) transfecting or transforming cells with a first expression vector of claim 3 which directs expression of MC-4R and a second expression vector which directs the expression of a promiscuous G-protein, resulting in test cells; (b) exposing the test cells to a substance that is a suspected agonist of MC-4R; and, (c) measuring the level of inositol phosphates in the cells; where an increase in the level of inositol phosphates in the cells as compared to the level of inositol phosphates in the cells in the absence of the suspected agonist indicates that the substance is an agonist of MC-4R.
- 37. A method of identifying antagonists of MC-4R comprising:
(a) transfecting or transforming cells with a first expression vector of claim 3 which directs expression of MC-4R and a second expression vector which directs the expression of a promiscuous G-protein, resulting in test cells; (b) exposing the test cells to a substance that is an agonist of MC-4R; (c) subsequently or concurrently to step (b), exposing the test cells to a substance that is a suspected antagonist of MC-4R; and, (d) measuring the level of inositol phosphates in the cells; where a decrease in the level of inositol phosphates in the cells in the presence of the suspected antagonist as compared to the level of inositol phosphates in the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of MC-4R.
- 38. A method of identifying antagonists of MC-4R as recited in claim 37 wherein the first and second expression vectors of step (a) are replaced with a single expression vector which expresses a chimeric MC-4R protein fused at its C-terminus to a promiscuous G-protein.
- 39. An antibody that binds specifically to MC-4R protein wherein the MC-4R receptor protein comprises the amino acid sequence as set forth in SEQ ID NO:2.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Serial No. 60/107,721, filed Nov. 9, 1998, which is hereby incorporated by reference.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60107721 |
Nov 1998 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09831206 |
May 2001 |
US |
Child |
10373355 |
Feb 2003 |
US |