Membrane molecule indicator compositions and methods

• US 6,596,499 B2
• Filed: 11/29/2001
• Issued: 07/22/2003
• Est. Priority Date: 11/30/2000
• Status: Expired due to Fees

• Current Assignee: Netherlands Vaccine Institute
• Original Assignee: The Netherlands Cancer Institute
• Inventors: Jalink, Kees
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/997,956
• Publication Number:
  US 20020106714A1
• Time in Patent Office: 600 Days
• Field of Search: 435/7.1, 435/7.8, 435/7.9, 435/252.3, 435/320.1, 435/325, 436/86, 436/172, 536/23.5
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5076 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

The invention provides membrane molecule indicators, including polypeptides, encoding nucleic acid molecules and cells containing such polypeptides and nucleic acid molecules. The invention membrane molecule indicators are characterized in that fluorescence resonance energy transfer (FRET) between a donor fluorescent domain and an acceptor fluorescent domain indicates a property of the membrane molecule. Also provided are methods of using the invention membrane molecule indicators to determine a property of a membrane molecule, and to identify compounds that modulates a property of a membrane molecule.

Claims

1. A phosphatidylinositol 4,5-bisphosphate (PIP2) indicator, said indicator comprising:

(a) a first polypeptide comprising;

(i) a pleckstrin homology (PH) domain; and

(ii) a donor fluorescent domain

(b) a second polypeptide comprising;

(i) a pleckstrin homology (PH) domain; and

(ii) an acceptor fluorescent domain;

wherein fluorescence resonance energy transfer (FRET) between said donor domain and said acceptor domain indicates PIP2 levels.

2. The indicator of claim 1, wherein said PH domain is a PLCδ

1 or PLCβ

PH domain.

3. The indicator of claim 1, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

4. The indicator of claim 1, wherein said donor fluorescent domain is a CFP.

5. The indicator of claim 1, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

6. The indicator of claim 1, wherein said acceptor fluorescent domain is a YFP.

7. A cell comprising the indicator of claim 1.

15. A method of indicating PIP2 levels in a cell, comprising:

(a) providing a cell containing the PIP2 indicator of claim 1; and

(b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain,

wherein FRET between said donor domain and said acceptor domain indicates PIP2 levels in the cell.

16. The method of claim 15, wherein said PH domain is a PLCδ

1 or PLCβ

PH domain.

17. The method of claim 15, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

18. The method of claim 15, wherein said donor fluorescent domain is a CFP.

19. The method of claim 15, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

20. The method of claim 15, wherein said acceptor fluorescent domain is a YFP.

21. The method of claim 15, wherein said cell recombinantly expresses a G-protein coupled receptor.

22. A method of identifying a compound that modulates PIP2 levels in a cell, comprising:

(a) contacting a cell containing the PIP2 indicator of claim 1 with one or more test compounds; and

(b) determining FRET between said donor fluorescent domain and said acceptor fluorescent domain following said contacting,

wherein increased or decreased FRET following said contacting indicates that said test compound is a compound that modulates PIP2 levels in the cell.

23. The method of claim 22, wherein said PH domain is a PLCδ

1 or PLCβ

PH domain.

24. The method of claim 22, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

25. The method of claim 22, wherein said donor fluorescent domain is a CFP.

26. The method of claim 22, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

27. The method of claim 22, wherein said acceptor fluorescent domain is a YFP.

28. The method of claim 22, wherein said contacting is by administration of said test compound to the exterior of said cell.

29. The method of claim 22, wherein said contacting is by recombinant expression of said test compound in said cell.

30. The method of claim 22, wherein said cell recombinantly expresses a G-protein coupled receptor.

8. A nucleic acid kit, the nucleic acid molecule components of which encode a PIP2 indicator, said indicator comprising:

(a) a first polypeptide comprising;

(i) a PH domain; and

(ii) a donor fluorescent domain

(b) a second polypeptide comprising;

(i) a PH domain; and

(ii) an acceptor fluorescent domain;

wherein fluorescence resonance energy transfer (FRET) between said donor domain and said acceptor domain indicates PIP2 levels.

9. The kit of claim 8, wherein said PH domain is a PLCδ

1 or PLCβ

PH domain.

10. The kit of claim 8, wherein said donor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

11. The kit of claim 8, wherein said donor fluorescent domain is a CFP.

12. The kit of claim 8, wherein said acceptor fluorescent domain is selected from the group consisting of a GFP and a dsRED.

13. The kit of claim 8, wherein said acceptor fluorescent domain is a YFP.

14. A cell expressing the nucleic acid molecule components of the kit of claim 8.