Conformational assays to detect binding to G protein-coupled receptors

• US 20030129649A1
• Filed: 08/21/2001
• Published: 07/10/2003
• Est. Priority Date: 04/24/2001
• Status: Abandoned Application

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Kobilka, Brian K., Lee, Tae Weon, Ghanouni, Pejman
• Application Number:
  US09/935,061
• Publication Number:
  US 20030129649A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The present invention provides methods and compositions for detection of compounds that have activity in modulating G protein-coupled receptor (GPCR) activity, e.g., agonists, and antagonists. The detection method is based upon detection of a conformational change in a GPCR upon interaction with a ligand. Conformational change of the GPCR upon ligand interaction can be accomplished by modifying the GPCR to have a bound detectable label so that ligand interaction results in a conformational change in the GPCR that is detected by a change in detectable signal from the detectable label. Conformational change of the GPCR upon ligand interaction can also be detected by detecting a change in the accessibility of a protease cleavage site to protease cleavage, where the protease cleavage site is naturally-occurring in the GPCR or introduced into the GPCR. The conformational assays of the invention provide for high-throughput screening,

Claims

1. A method for identifying an agent having activity agonist activity for a G protein-coupled receptor (GPCR), the method comprising:

contacting a G protein-coupled receptor (GPCR) with a candidate agent, the GPCR having a conformationally sensitive detectable probe positioned on or within a conformationally sensitive third intracellular loop of the GPCR; and

detecting a detectable signal of the conformationally sensitive detectable probe;

wherein detection of a change in the detectable signal in the present of the candidate agent indicates the candidate agent has agonist binding activity for the GPCR.

2. The method of claim 1, wherein the conformationally sensitive intracellular loop is a third intracellular loop of the GPCR and wherein the conformationally sensitive detectable probe is a detectable label attached to one or more amino acid residues within the third intracellular loop of the GPCR so that a conformational change in the GPCR due to agonist activity of the candidate agent causes a change in the detectable signal of the detectable label.

3. The method of claim 2, wherein the detectable label is a fluorescent probe.

4. The method of claim 2, wherein the detectable label is attached to an amino acid residue corresponding to amino acid residue at position 265 in a β

2-adrenergic receptor.

5. The method of claim 1, wherein the conformationally sensitive detectable probe is a protease cleavage site. within the GPCR so that a conformational change in the GPCR changes the accessibility of the protease cleavage site to protease cleavage, and the detectable signal is a protease cleavage product.

6. The method of claim 5, wherein the protease cleavage product is an N-terminal fragment of the GPCR.

7. The method of claim 5, wherein the protease cleavage product is an C-terminal fragment of the GPCR.

8. The method of claim 4, wherein the detectable probe comprises two protease cleavage sties within the third intracellular domain of the GPCR, the cleavage sites flanking an epitope tag, wherein a conformational change due to agonist activity changes the accessibility of the protease cleavage site to protease cleavage, and the detectable signal is a polypeptide of the epitope tag released by protease cleavage of the two cleavage sites.

9. The method of claim 1, wherein the GPCR is immobilized by attachment to a support.

10. The method of claim 9, wherein the GPCR is attached to the support by binding of an N-terminal portion to the support.

11. The method of claim 9, wherein the GPCR is attached to the support by binding of an C-terminal portion to the support.

12. The method of claim 1, wherein the GPCR is in a membrane.

13. The method of claim 5, wherein the GPCR is expressed in a eukaryotic host cell.

14. An apparatus for detecting a ligand having agonist activity for a G protein-coupled receptor, the apparatus comprising:

a G protein-coupled receptor (GPCR) with a candidate agent, the GPCR having a conformationally sensitive detectable probe positioned on or within a third intracellular loop of the GPCR; and

a immobilization phase in which the GPCR is positioned.

15. The apparatus of claim 14, wherein the conformationally sensitive detectable probe is a detectable label attached to one or more amino acid residues within the third intracellular loop of the GPCR so that a conformational change in the GPCR due to agonist activity of the candidate agent causes a change in the detectable signal of the detectable label.

16. The apparatus of claim 15, wherein the detectable label is a fluorescent probe.

17. The apparatus of claim 15, wherein the detectable label is attached to an amino acid residue corresponding to amino acid residue at position 265 in a β

2-adrenergic receptor.

18. The apparatus of claim 14, wherein the conformationally sensitive detectable probe is a protease cleavage site. within the GPCR so that a conformational change in the GPCR changes the accessibility of the protease cleavage site to protease cleavage, and the detectable signal is a protease cleavage product.

19. The apparatus of claim 14, wherein the detectable probe comprises two protease cleavage sties within the third intracellular domain of the GPCR, the cleavage sites flanking an epitope tag, wherein a conformational change due to agonist activity renders the cleavage sites accessible to protease cleavage, and the detectable signal is a polypeptide of the epitope tag released by protease cleavage of the two cleavage sites.