Claims
- 1. A method to identify an agent that is an agonist of a mutant μ opioid receptor and an antagonist of a wild-type μ opioid receptor, comprising:
a) contacting one or more agents with i) a mutant μ opioid comprising at least two amino acid substitutions in one or more transmembrane regions of the receptor, or a portion of the mutant receptor which has substantially the same activity as the corresponding full-length mutant μ opioid receptor, wherein at least one substitution is in the fourth transmembrane region, and ii) a wild-type μ opioid receptor; and b) identifying an agent which is an agonist of the mutant μ opioid receptor and an antagonist of the wild-type μ opioid receptor.
- 2. A method to detect a mutant μ opioid receptor which is activated upon contact with an antagonist of a corresponding wild-type μ opioid receptor, comprising:
a) contacting an antagonist of a wild-type μ opioid receptor with a mutant μ opioid receptor comprising at least two amino acid substitutions in one or more transmembrane regions, or a portion of the mutant receptor which has substantially the same activity as the corresponding full-length mutant μ opioid receptor, wherein at least one substitution is in the fourth transmembrane region; and b) detecting or determining whether the antagonist is an agonist of the mutant μ opioid receptor.
- 3. The method of claim 1 or 2 wherein the mutant receptor has two amino acid substitutions.
- 4. The method of claim 1 or 2 wherein the mutant receptor comprises one amino acid substitution in the fourth transmembrane region and one amino acid substitution in the seventh transmembrane region.
- 5. The method of claim 1 or 2 wherein one amino acid substitution is at a position corresponding to position 196 in the fourth transmembrane region of the murine μ opioid receptor.
- 6. The method of claim 5 wherein the amino acid substitution is serine to leucine, alanine, glycine or phenylalanine.
- 7. The method of claim 5 wherein the amino acid substitution results in a mutant opioid receptor with the properties of one having a serine to leucine substitution at position 196.
- 8. The method of claim 1 or 2 wherein one amino acid substitution is at a position corresponding to position 327 in the seventh transmembrane region of the murine μ opioid receptor.
- 9. The method of claim 8 wherein the amino acid substitution results in a mutant opioid receptor with the properties of one having a threonine to alanine substitution at position 327.
- 10. The method of claim 8 wherein the amino acid substitution is threonine to alanine.
- 11. The method of claim 1 or 2 wherein one amino acid substitution is at a position corresponding to position 330 in the seventh transmembrane region of the murine μ opioid receptor.
- 12. The method of claim 11 wherein the amino acid substitution results in a mutant opioid receptor with the properties of one having a cysteine to serine substitution at position 330.
- 13. The method of claim 11 wherein the amino acid substitution is cysteine to serine.
- 14. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a mutant μ opioid receptor comprising two or more amino acid substitutions in one or more transmembrane regions of the receptor or encoding a portion of the mutant receptor which has substantially the same activity as the corresponding full-length mutant μ opioid receptor, wherein at least one substitution is in the fourth transmembrane region, wherein the mutant receptor is activated when contacted with an antagonist of a corresponding wild-type μ opioid receptor.
- 15. The nucleic acid molecule of claim 14 wherein the mutant receptor has two amino acid substitutions.
- 16. The nucleic acid molecule of claim 14 wherein the mutant receptor comprises one amino acid substitution in the fourth transmembrane region and one amino acid substitution in the seventh transmembrane domain.
- 17. The nucleic acid molecule of claim 14 wherein the mutant receptor comprises one amino acid substitution in the fourth transmembrane region and one substitution in the first transmembrane region.
- 18. The nucleic acid molecule of claim 14 wherein one amino acid substitution is at a position corresponding to position 196 in the murine μ opioid receptor.
- 19. The nucleic acid molecule of claim 18 wherein the amino acid substitution results in a mutant opioid receptor with the properties of one having a serine to leucine substitution at position 196.
- 20. The nucleic acid molecule of claim 18 wherein the amino acid substitution is serine to alanine, phenylalanine, glycine or leucine.
- 21. The nucleic acid molecule of claim 14 wherein one amino acid substitution is at a position corresponding to position 327 in the murine μ opioid receptor.
- 22. The nucleic acid molecule of claim 21 wherein the amino acid substitution results in a mutant opioid receptor with the properties of one having a threonine to alanine substitution at position 327.
- 23. The nucleic acid molecule of claim 21 wherein the amino acid substitution is threonine to alanine.
- 24. The nucleic acid molecule of claim 14 wherein one amino acid substitution is at a position corresponding to position 330 in the murine μ opioid receptor.
- 25. The nucleic acid molecule of claim 24 wherein the amino acid substitution results in a mutant opioid receptor with the properties of one having a cysteine to serine at position 330.
- 26. The nucleic acid molecule 24 wherein the amino acid substitution is cysteine to serine.
- 27. An expression cassette comprising a promoter operably linked to the nucleic acid molecule of claim 14.
- 28. The expression cassette of claim 27 wherein the promoter is expressed in neurons.
- 29. The expression cassette of claim 27 wherein the promoter is the μ opioid receptor promoter.
- 30. A vector comprising the expression cassette of claim 27.
- 31. The vector of claim 30 which is a viral vector.
- 32. A host cell augmented with the expression cassette of claim 27.
- 33. A method to inhibit pain, comprising: administering an amount of a composition comprising the expression cassette of claim 27 and an amount of an antagonist of a wild-type μ opioid receptor to a mammal effective to inhibit pain in the mammal.
- 34. The method of claim 33 wherein the mammal is a human.
- 35. The method of claim 33 wherein the expression cassette is administered to the spinal cord of the mammal.
- 36. The method of claim 33 wherein the composition comprises recombinant adenovirus comprising the expression cassette.
- 37. The method of claim 36 wherein about 1010 to 1014 recombinant adenovirus particles are administered.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of U.S. application Serial No. 60/389,862, filed on Jun. 18, 2002, the disclosure of which is incorporated by reference herein.
STATEMENT OF GOVERNMENT RIGHTS
[0002] The invention was made, at least in part, with grants from the Government of the United States of America (grants DA00564, DA01583, and DA07339 from the National Institute on Drug Abuse). The Government may have certain rights to the invention.
Provisional Applications (1)
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Number |
Date |
Country |
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60389862 |
Jun 2002 |
US |