Methods of assaying receptor activity and constructs useful in such methods

• US 7,572,888 B2
• Filed: 02/24/2004
• Issued: 08/11/2009
• Est. Priority Date: 06/05/1997
• Status: Expired due to Fees

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Zhang, Jie, Ferguson, Stephen S., Caron, Marc G., Barak, Lawrence S.
• Primary Examiner(s): FALK, ANNE MARIE
• Application Number:
  US10/785,841
• Publication Number:
  US 20040209308A1
• Time in Patent Office: 1,995 Days
• Field of Search: 435/7.1, 435/7.2, 530/350
• US Class Current: 530/350
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/0356 Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0276 Knock-out vertebrates
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• 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: C12N 15/8509 for producing genetically modified animals, e.g. transgenic

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. Constructs useful in such methods are described.

Claims

1. A conjugate comprising a β

-arrestin protein and a label, wherein said label is capable of indicating intracellular translocation and/or distribution of said β

-arrestin protein, wherein said β

-arrestin is capable of interacting with a G protein coupled receptor (GPCR).

2. The conjugate according to claim 1, wherein said conjugate is a fusion protein.

3. The conjugate of claim 1, wherein said label is an optically detectable label.

5. The conjugate according to claim 1, wherein said label is a fluorescent label.

6. The conjugate according to claim 1, wherein said label is a colorimetric label.

7. The conjugate according to claim 1, wherein said label is a radioactive label.

8. The conjugate according to claim 1, wherein said label is an electron-dense label.

9. The conjugate according to claim 5, wherein said fluorescent label is Green Fluorescent Protein (GFP).

4. A conjugate comprising a β

-arrestin-2 protein and a label, wherein said label is capable of indicating intracellular translocation and/or distribution of said β

-arrestin-2 protein, wherein said β

-arrestin-2 is capable of interacting with a G protein coupled receptor (GPCR).