Assay methods and compositions for detecting and evaluating the intracellular transduction of an extracellular signal

• US 5,436,128 A
• Filed: 01/27/1993
• Issued: 07/25/1995
• Est. Priority Date: 08/07/1990
• Status: Expired due to Fees

• Current Assignee: Merck & Co., Inc.
• Original Assignee: The SALK Institute Biotechnology/Industrial Associates Incorporated
• Inventors: Brust, Paul, Harpold, Michael M.
• Primary Examiner(s): Fleisher, Mindy B.
• Application Number:
  US07/962,238
• Time in Patent Office: 909 Days
• Field of Search: 435/6, 435/172.3, 435/240.1, 436/63, 436/94, 935/36, 935/39, 935/41
• US Class Current: 435/6.13
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G01N 33/9406 Neurotransmitters
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided.

Claims

1. A method for identifying extracellular signals that modulate cell surface protein-mediated activity, comprising:

comparing the difference in the amount of transcription of a reporter gens in a recombinant cell in the presence of the signal with the amount of transcription in the absence of said signal, or with the amount of transcription in the absence of the cell surface protein, wherein;

the recombinant cell contains a reporter gens construct and expresses the cell surface protein; and

transcription of the reporter gens is under the control of at least one transcriptional control element whose activity is regulated by the cell surface protein.

2. The method of claim 1, wherein said protein is a cell surface receptor or ion channel.

3. The method of claim 2, wherein said signal is an agonist or antagonist of said cell surface receptor or ion channel.

4. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 100 μ

M.

5. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 10 μ

M.

6. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 1 μ

M.

7. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 0.1 μ

M.

8. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 10 nM.

9. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 1 nM.

10. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 0.1 nM.

11. The method of claim 3, wherein the concentration of said agonist or antagonist is less than 0.01 nM.

12. The method of claim 1, wherein the amount of transcription is determined by measuring the amount of mRNA that is transcribed from said reporter gene.

13. The method of claim 1, wherein the amount of transcription is determined by measuring the amount of reporter gene protein that is produced.

14. The method of claim 3, wherein said signal is an antagonist.

15. The method of claim 14, further comprising, prior to or simultaneously with comparing the difference in the amount of transcription of the reporter gene, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby transcription of said reporter gene is induced.

16. The method of claim 1, wherein said reporter gene construct includes transcriptional regulatory sequences of nucleotides that modulate transcription from said transcriptional control element in the absence of said cell surface protein or extracellular signal.

17. The method of claim 16, wherein said transcriptional regulatory sequence is the c-fos gene intragenic regulatory element, whereby the level of transcription of said reporter gene in the absence of said cell surface protein or extracellular signal is less than in the absence of the the intragenic regulatory element.

18. The method of claim 1, wherein the cell surface protein is a cell surface receptor selected from:

muscarinic receptors, neuronal nicotinic acetylcholine receptors, gamma-aminobutyric acid receptors, glutamate receptors, adrenergic receptors, dopamine receptors, nerve growth factor receptors, or serotonin receptors.

19. The method of claim 1, wherein the cell surface protein is an ion channel selected from:

calcium ion channels, potassium ion channels, or sodrum ion channels.

20. The method of claim 1, wherein the transcriptional control element is a promoter selected from:

the c-fos gene promoter, the vasoactive intestinal peptide gene promoter, the somatostatin gene promoter, the proenkephalin promoter, the phosphoenolpyruvate carboxykinase gene promoter or the nerve growth factor-1 A gene promoter.

21. The method of claim 1, wherein the reporter gene is selected from:

the gene encoding bacterial chloramphenicol acetyltransferase, the gene encoding firefly luciferase, the gene encoding bacterial luciferase, the gene encoding β

-galactosidase or the gene encoding alkaline phosphatase.

22. The method of claim 1, wherein the gene construct includes at least one transcriptional regulatory sequence of nucleotides selected from the group consisting of serum responsive elements, cyclic adenosine monophosphate responsive elements, and elements responsive to intracellular calcium ion levels.

23. The method of claim 18, wherein said receptor is the Type 1 human muscarinic receptor, said transcriptional control element is the c-fos promoter and said reporter gene is the gene encoding bacterial chloramphenicol acetyltransferase.

35. The method of claim 1, wherein said cell surface protein is an ion channel and prior to comparing the difference in the amount of transcription the cell is depolarized.

36. The method claim 1, wherein said cell surface protein is an ion channel and said signal causes depolarization of the cell membrane.

43. The method of claim 15, wherein said compound is an antagonist.

44. The method of claim 1, wherein transcription is assessed by measuring the activity of the reporter gene protein.

45. The method of claim 1, wherein the cell is preincubated with the extracellular signal prior to comparing the difference in transcription.

46. The method of claim 45, wherein the cell is contacted with an agonist when comparing the difference in transcription.

24. A recombinant cell, comprising:

DNA that encodes a heterologous cell surface protein whose activity is modulated by extracellular signals; and

a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional control element that is regulated by said cell surface protein.

25. The cell of claim 24, wherein said cell is a PC12 cell or a COS-7 cell that has been modified by introduction of said DNA and reporter gene construct.

26. The cell of claim 25, wherein said reporter gene construct includes transcriptional regulatory sequences of nucleotides that modulate transcription from said promoter in the absence of said cell surface protein or extracellular signal.

27. The cell of claim 26, wherein said transcriptional regulatory sequence is the c-fos gene intragenic regulatory element, which is inserted downstream, with respect to the direction of transcription, from the transcription binding and initiation site of said promoter.

28. The cell of claim 27, wherein said reporter gene construct is FC4XP1FIRE or FC4XP2FIRE.

29. The cell of claim 27, wherein said reporter gene is fused to a sufficient portion of the c-fos gene first exon and intragenic region to reduce the level of transcription of said reporter gene in the absence of said cell surface protein or signal to levels that are substantially undetectable;

and

said c-fos gene intragenic regulatory element is inserted in reading frame at the 3'"'"' end of the first exon of said c-fos gene.

30. The recombinant cell of claim 24, wherein said cell surface proteins are receptors selected from:

muscarinic receptors, neuronal nicotinic acetylcholine receptors, gamma-aminobutyric acid receptors, glutamate receptors, adrenergic receptors,dopamine receptors, nerve growth factor receptors, or serotonin receptors.

31. The recombinant cell of claim 24, wherein said cell surface proteins are ion channels selected from:

calcium ion channels, potassium ion channels, or sodium ion channels.

32. The recombinant cell of claim 24, wherein said transcriptional control element is selected from:

the c-fos gene promoter, the vasoactive intestinal peptide gene promoter, the somatostatin gene promoter, the proenkephalin promoter, the phosphoenolpyruvate carboxykinase gene promoter, or the nerve growth factor-1 A gene promoter.

33. The recombinant cell of claim 24, wherein said reporter gene is selected from:

the gene encoding bacterial chloramphenicol acetyltransferase, the gene encoding firefly luciferase, the gene encoding bacterial luciferase, the gene encoding β

-galactosidase or the gene encoding alkaline phosphatase.

34. The recombinant cell of claim 30, wherein said receptor is Type 1 human muscarinic receptor, said transcriptional control element is the c-fos gene promoter and said reporter gene is the gene encoding bacterial chloramphenicol acetyltransferase.

37. A method for identifying compounds that modulate cell surface protein-mediated activity by detecting intracellular transduction of a signal generated upon interaction of the compound with a cell surface protein, comprising:

comparing the amount of transcription of a reporter gene or the amount of reporter gene product expressed in a first recombinant cell in the presence of the compound with the amount of transcription or product in a second recombinant cell; and

selecting compounds that change the amount of transcription of a reporter gene or the amount of reporter gene product expressed in the first recombinant cell in the presence of the compound compared to the amount of transcription or product in the absence of the compound, or compared to the amount of transcription or product in the second recombinant cell, wherein;

the first recombinant cell contains a reporter gene construct and expresses the cell surface protein;

the second recombinant cell is identical to the first recombinant cell, except that it does not express the cell surface protein; and

the reporter gene construct contains;

(a) a transcriptional control element that is responsive to the intracellular signal that is generated by the interaction of an agonist with the cell surface protein; and

(b) a reporter gene that encodes a detectable transcriptional or translational product and that is in operative association with the transcriptional control element. Transcription based assays that identify extracellular signals that modulate the activity of cell surface proteins are provided. Extracellular signals are identified by measuring the amount of transcription of a reporter gene in a recombinant cell that expresses the cell surface protein and contains DNA encoding the reporter gene under the transcriptional control of a promoter that is regulated, directly or indirectly, by the cell surface protein. The assays, provide a means for identifying potential pharmaceutical compounds that can be used to treat disease by virtue of their agonistic or antagonistic effects on the cell surface protein. Recombinant cells that express cell surface receptors and that contain reporter gene constructs that include transcriptional regulatory elements that are responsive to the activity of the cell surface receptors are also provided.

38. The method of claim 37, wherein the compound is an agonist or antagonist of said cell surface receptor or ion channel.

39. The method of claim 37, wherein the amount of transcription is determined by measuring the amount of mRNA that is transcribed from said reporter gene.

40. The method of claim 37, wherein the amount of transcription is determined by measuring the amount of reporter gene protein that is produced.

41. The method of claim 37, further comprising, prior to or simultaneously with comparing the difference in the amount of transcription of the reporter gene, contacting the recombinant cell with an agonist that activates said cell surface protein, whereby transcription of said reporter gene is induced.

42. The method of claim 41, wherein said compound is an antagonist.

47. The method of claim 37, wherein the cell is preincubated with the compound prior to comparing the difference in transcription.

48. The method of claim 47, wherein the cell is contacted with an agonist when comparing the difference in transcription.