Methods of assaying receptor activity

• US 7,138,240 B2
• Filed: 05/09/2002
• Issued: 11/21/2006
• Est. Priority Date: 06/05/1997
• Status: Expired due to Term

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Zhang, Jie, Caron, Marc G., Barak, Lawrence S., Ferguson, Stephen S.
• Primary Examiner(s): Nickol, Gary B.
• Assistant Examiner(s): BASI, NIRMAL SINGH
• Application Number:
  US10/141,725
• Publication Number:
  US 20060188944A1
• Time in Patent Office: 1,657 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2333/91 Transferases (2.)
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Abstract

Described are methods of detecting G-protein coupled receptor (GPCR) activity in vivo and in vitro; methods of assaying GPCR activity; and methods of screening for GPCR ligands, G Protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process.

Claims

1. A method of automatically screening compounds for an effect on G protein coupled receptor (GPCR) pathway activity in a cell under test conditions in response to exposure to a test compound comprising:

a. providing cells including a detectably labeled cellular component involved in the desensitization pathway of a GPCR, wherein said cellular component is an arrestin, said cells over-expressing said GPCR;

b. providing a tool for at least one of detecting or measuring said detectably labeled cellular component;

c. providing a computer for controlling said tool for at least one of detecting or measuring said detectably labeled cellular component;

d. exposing the cells to one or more test compounds;

e. detecting or measuring translocation of said detectably labeled cellular component involved in the desensitization of a GPCR with said tool, said computer controlling said tool, wherein the detectably labeled cellular component is translocated from the cytosol of a cell to the membrane edge of said cell; and

f. wherein said detecting or measuring translocation indicates an effect on said protein coupled receptor (GPCR) pathway activity.

2. The method of claim 1, wherein said translocation is measured by comparing a cell before and after exposure to a test compound.

3. The method of claim 1, wherein said translocation is measured by comparison of a cell after exposure to a test compound to a pre-established parameter.

4. The method of claim 1, wherein said detectably labeled cellular component is inherently fluorescent in vivo.

5. The method of claim 1, wherein each cell is exposed to more than one test compound.

6. The method of claim 1, wherein the test compound is a GPCR agonist.

7. The method of claim 1, wherein the cellular component involved in the desensitization pathway is labeled with an immunochemically detectable molecule.

8. The method of claim 1, wherein the cellular component includes at least one of a β

arrestin.

9. The method of claim 1, wherein at least two of the steps may be automated.

10. The method of claim 1, wherein the detecting or measuring translocation of the detectably labeled cellular component includes measuring translocation to at least one of a cell membrane or cytoplasm of the cell.