Assay methods and compositions useful for measuring the transduction of an intracellular signal

• US 5,401,629 A
• Filed: 08/07/1990
• Issued: 03/28/1995
• 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): YARBROUGH, AMELIA
• Application Number:
  US07/563,751
• Time in Patent Office: 1,694 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: 435/6.16
• 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

Recombinant cells are provided which are useful for assaying compounds for their agonist or antagonist activity with respect to ion channels and/or cell surface-localized receptors. In addition, assay methods employing the invention recombinant cells are provided.

Claims

1. 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 the 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 the absence of the compound, or 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 cell surface protein is a surface receptor or ion channel;

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 constructs 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.

2. The method of claim 1, wherein said compound is an agonist or antagonist of said cell surface receptor or ion channel.

3. 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.

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

5. The method of claim 2, wherein said compound is an antagonist.

6. The method of claim 2, further comprising, prior to 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.

7. The method of claim 1, wherein said reporter gene construct includes a transcriptional regulatory region that includes a sequence of nucleotides that modulates transcription from said promoter in the absence of said cell surface protein or compound.

8. The method of claim 1, wherein the transcriptional control element includes a promoter selected from the group consisting of the c-fos gene promoter, the vasoactive intestinal peptide gene promoter, the somatostatin gene promoter, the proenkephalin gene promoter, the phosphoenolpyruvate carboxykinase gene promoter and the nerve growth factor-1 A gene promoter.

9. The method of claim 1, wherein the reporter gene is selected from the group consisting of the gene encoding bacterial chloramphenicol acetyltransferase, the gene encoding firefly luciferase, the gene encoding bacterial luciferase, and the gene encoding alkaline phosphatase.

10. The method of claim 1, wherein the transcriptional control region includes at least one regulatory element selected from the group consisting of serum responsive elements, cyclic adenosine monophosphate responsive elements, and elements responsive to intracellular calcium ion levels.

11. The method of claim 1, wherein the cell surface receptor is the Type 1 human muscarinic receptor, the promoter is the c-fos promoter and the reporter gene is the gene encoding bacterial chloramphenicol acetyltransferase.

12. The method of claim 1, wherein the cell surface receptor is selected from the group consisting of muscarinic receptors, neuronal nicotinic acetylcholine receptors, gamma aminobutyric acid receptors, glutamate receptors, adrenergic receptors, dopamine receptors, nerve growth factor receptors, and serotonin receptors.

13. The method of claim 1, wherein the ion channel is a calcium channel, potassium ion channel, or a sodium ion channel.

14. The method of claim 6, wherein said compound is an antagonist.