Methods of assaying receptor activity and constructs useful in such methods

• US 20040209308A1
• Filed: 02/24/2004
• Published: 10/21/2004
• Est. Priority Date: 06/05/1997
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Zhang, Jie, Caron, Marc G., Barak, Lawrence S., Ferguson, Stephen S.
• Granted Patent: US 7,572,888 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• 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 an arrestin protein and a detectable molecule, wherein the detectable molecule is capable of indicating intracellular translocation and/or distribution of the arrestin protein.

2. The conjugate according to claim 1 which is a fusion protein.

3. The conjugate of claim 1, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

4. A nucleic acid construct comprising an expression cassette, which construct comprises, in the 5′

to 3′

direction, a promoter and a nucleic acid segment operatively associated therewith, the nucleic acid segment comprising a sequence encoding a polypeptide comprising an arrestin protein and a detectable molecule, wherein said detectable molecule is capable of indicating intracellular translocation and/or distribution of the arrestin protein.

5. A nucleic acid construct according to claim 4, wherein the nucleic acid sequence encoding a detectable molecule encodes an optically detectable molecule, and optionally it encodes Green Fluorescent Protein.

6. A nucleic acid construct according to claim 4, including any one or more of the following features:

(i) the construct further comprises a plasmid;

(ii) the construct is a DNA or RNA construct; and

(iii) the promoter is an arrestin promoter.

7. A host cell containing the nucleic acid construct of claim 4.

8. A host cell comprising a nucleic acid molecule, the nucleic acid molecule comprising, in the 5′

to 3′

direction, a promoter operable in the host cell, a nucleic acid sequence encoding an arrestin protein and a nucleic acid sequence encoding a detectable molecule, the encoding nucleic acid sequences being operably linked to the promoter wherein the nucleic acid molecule expresses an arrestin protein and wherein the detectable molecule is capable of indicating intracellular translocation and/or distribution of the arrestin protein.

9. The cell according to claim 8, wherein the cell:

(i) is a mammalian cell, and optionally is a HEK-293 cell or a COS cell;

or

(ii) is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells.

10. A substrate having deposited thereon the host cell of claim 8.

11. The substrate of claim 10, wherein the substrate is made of a material selected from glass, plastic, ceramic, semiconductor, silica, fiber optic, diamond, biocompatible monomer, and biocompatible polymer materials.

12. A method of assessing G protein coupled receptor (GPCR) pathway activity under test conditions, the test condition optionally being the presence in a test cell of a test kinase or a test G-protein or is exposure of the test cell to a test ligand or is co-expression in the test cell of a second receptor, the method comprising:

(a) providing a test cell that expresses a GPCR, and contains a conjugate comprising an arrestin protein and a visually detectable molecule;

(b) exposing the test cell to a known GPCR agonist under test conditions; and

then

(c) detecting translocation of the detectable molecule from the cytosol of the test cell to the membrane edge of the test cell;

wherein the translocation of the detectable molecule in the test cell indicates activation of the GPCR pathway.

13. A method for screening an arrestin protein or fragment thereof for the ability to bind to a phosphorylated GPCR, comprising:

(a) providing a cell that;

(i) expresses a GPCR; and

(ii) contains a conjugate comprising a test arrestin protein and a visually detectable molecule;

(b) exposing the cell to a known GPCR agonist; and

then

(c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;

wherein translocation of the detectable molecule in the test cell indicates arrestin protein binding to the phosphorylated GPCR.

14. A method for screening a test compound for G protein coupled receptor (GPCR) agonist activity, comprising:

(a) providing a cell expressing a GPCR and containing a conjugate comprising an arrestin protein and a visually detectable molecule;

(b) exposing the cell to the test compound, the test compound optionally being in aqueous solution; and

then

(c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell, the cells optionally being deposited on a substrate;

wherein movement of the detectable molecule from the cytosol to the membrane edge of the cell after exposure of the cell to the test compound indicates GPCR agonist activity of the test compound.

15. The method according to claim 14 wherein the cell expresses any one or more of the following:

(i) a known GPCR;

(ii) an unknown GPCR;

(iii) an odorant GPCR;

(iv) a β

-adrenergic GPCR.

16. A method according to claim 14, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

17. The method according to claim 14, wherein the cell:

(i) is a mammalian cell, and optionally is a HEK-293 cell or a COS cell;

or

(ii) is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells.

18. The method of claim 14, wherein the cell normally expresses a GPCR or optionally the cell has been transformed to express a GPCR not normally expressed by such a cell.

19. A method of screening a sample solution for the presence of an agonist to a G protein coupled receptor (GPCR);

comprising;

(a) providing a cell expressing a GPCR and containing a conjugate, the conjugate comprising an arrestin protein and a visually detectable molecule;

(b) exposing the cell to a sample solution; and

then

(c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;

wherein movement of the detectable molecule from the cytosol to the membrane edge of the cell after exposure of the cell to the sample solution indicates the sample solution contains an agonist for a GPCR expressed in the cell.

20. The method of claim 19, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

21. The method of claim 19, wherein the cell:

(i) is a mammalian cell, and optionally is a HEK-293 cell or a COS cell;

or.

(ii) is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells.

22. The method of claim 19, wherein the cell expresses any one or more of the following:

(i) a known GPCR;

(ii) an unknown GPCR;

(iii) an odorant GPCR;

(iv) a β

-adrenergic GPCR.

23. A method of screening a cell for the presence of a G protein coupled receptor (GPCR);

comprising;

(a) providing a test cell, said test cell containing a conjugate comprising an arrestin protein and a visually detectable molecule;

(b) exposing the test cell to a solution containing a GPCR agonist; and

(c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;

wherein movement of the detectable molecule from the cytosol to the membrane edge of the test cell after exposure of the test cell to the GPCR agonist indicates that the test cell contains a GPCR.

24. A method according to claim 23, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

25. A method of screening a plurality of cells for those cells which contain a G protein coupled receptor (GPCR), the cells optionally being contained in tissue or an organ, comprising:

(a) providing a plurality of test cells, said test cells containing a conjugate comprising an arrestin protein and a visually detectable molecule;

(b) exposing the test cells to one, or a plurality of, known GPCR agonist(s); and

(c) detecting those cells in which the detectable molecule is translocated from the cytosol of the cell to the membrane edge of the cell;

wherein movement of the detectable molecule from the cytosol to the membrane edge of a cell after exposure to the GPCR agonist indicates that the cell contains a GPCR for that known GPCR agonist

26. A method according to claim 25, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

27. The use of a GPCR-translocatable beta-arrestin protein coupled to a detectable molecule to screen a test compound for GPCR agonist function, by detecting for intracellular translocation from the cell cytosol to the membrane edge of the cell of the arrestin protein responsive to exposure of the cell to the test compound.

28. A method of screening a test compound for G protein coupled receptor (GPCR) antagonist activity, comprising:

(a) providing a cell expressing a GPCR, and containing a conjugate, the conjugate comprising an arrestin protein and a visually detectable molecule;

(b) exposing the cell to a test compound;

(c) exposing the cell to a GPCR agonist; and

(d) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;

the cells optionally being deposited on a substrate;

wherein exposure to the agonist occurs at the same time as, or subsequent to, exposure to the test compound, and wherein movement of the detectable molecule from the cytosol to the membrane edge of the cell after exposure of the cell to the test compound indicates that the test compound is not a GPCR antagonist.

29. The method of claim 28, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

30. The method of claim 28, wherein the cell:

(i) is a mammalian cell, and optionally is a HEK-293 cell or a COS cell;

or

(ii) is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells.

31. The method of claim 28, wherein the cell expresses any one or more of the following:

(i) a known GPCR;

(ii) an unknown GPCR;

(iii) an odorant GPCR;

(iv) a β

-adrenergic GPCR.

32. A method of screening a test compound for G protein coupled receptor (GPCR) antagonist activity, the test compound optionally being in aqueous solution, comprising:

(a) providing a cell expressing a GPCR and containing a conjugate, the conjugate comprising an arrestin protein and a visually detectable molecule;

(b) exposing the cell to a GPCR agonist so that translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell occurs;

(c) exposing the cell to a test compound, the cells optionally being deposited on a substrate;

where exposure to the agonist occurs prior to exposure to the test compound, and wherein movement of the detectable molecule from the membrane edge of the cell to the cytosol after exposure of the cell to the test compound indicates that the test compound has a GPCR antagonist activity.

33. The method of claim 32, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

34. The method of claim 32, wherein the cell:

(i) is a mammalian cell, and optionally is a HEK-293 cell or a COS cell;

or

(ii) is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells.

35. The use of a GPCR-translocatable beta-arrestin protein coupled to a detectable molecule to screen a test compound for GPCR antagonist function by detecting for translocation from the membrane edge of the cell to the cell cytosol of the beta arrestin protein responsive to exposure of the cell to the test compound.

36. A substrate having deposited thereon a plurality of cells, said cells expressing a GPCR and a labelled arrestin protein, wherein the label is capable of indicating intracellular translocation and/or distribution of the arrestin.

37. The method of claim 36, wherein the detectable molecule is an optically detectable molecule, and optionally it is Green Fluorescent Protein.

38. The method of claim 36, wherein the cell:

(i) is a mammalian cell, and optionally is a HEK-293 cell or a COS cell;

or

(ii) is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells.

39. The method of claim 36, wherein the cell expresses any one or more of the following:

(i) a known GPCR;

(ii) an unknown GPCR;

(iii) an odorant GPCR;

(iv) a β

-adrenergic GPCR.

40. The substrate of claim 36, wherein the substrate is made of a material selected from glass, plastic, ceramic, semiconductor, silica, fiber optic, diamond, biocompatible monomer, and biocompatible polymer materials.

41. A conjugate comprising a β

-arrestin protein and a detectable molecule, wherein the detectable molecule is capable of indicating intracellular translocation and/or distribution of the β

-arrestin protein.