A Dual Luciferase Multiplexed High-Throughput Screening Platform for Protein-Protein Interactions

Abstract

To study the biology of regulators of G-protein signaling (RGS) proteins and to facilitate the identification of small molecule modulators of RGS proteins, the authors recently developed an advanced yeast 2-hybrid (YTH) assay format for GαZand RGS-Z1. Moreover, they describe the development of a multiplexed luciferase-based assay that has been successfully adapted to screen large numbers of small molecule modulators of protein-protein interactions. They generated and evaluated 2 different luciferase reporter gene systems for YTH interactions, a Gal4 responsive firefly luciferase reporter gene and a Gal4 responsive Renilla luciferase reporter gene. Both the firefly and Renilla luciferase reporter genes demonstrated a 40-to 50-fold increase in luminescence in strains expressing interacting YTH fusion proteins versus negative control strains. Because the firefly and Renilla luciferase proteins have different substrate specificity, the assays were multiplexed. The multiplexed luciferase-based YTH platform adds speed, sensitivity, simplicity, quantification, and efficiency to YTH high-throughput applications and therefore greatly facilitates the identification of small molecule modulators of protein-protein interactions as tools or potential leads for drug discovery efforts.

References

1. Neer EJ: Heterotrimeric G proteins: organizers of transmembrane signals. Cell 1995;80:249-257.

2. Hamm HE: The many faces of G protein signaling. J Biol Chem 1998;273:669-672.

3. Bourne HR, Sanders DA, McCormick F: The GT Pase superfamily. A conserved switch for diverse cell functions. Nature 1990;348:125-132.

4. Dohlman HG, Thorner J: RGS proteins and signaling by heterotrimeric G proteins. J Biol Chem 1997;272:3871-3874.

5. Kehrl JH: Heterotrimeric G protein signaling: roles in immune function and fine-tuning by RGS proteins. Immunity 1998;8:1-10.

6. Arshavsky VY, Pugh EN Jr: Lifetime regulation of G protein-effector complex: emerging importance of RGS proteins. Neuron 1998;20:11-14.

7. De Vries L, Gist Farquhar M: RGS proteins: more than just GAPs for heterotrimeric G proteins. Trends Cell Biol 1999;9:138-144.

8. Hepler JR: Emerging roles for RGS proteins in cell signaling. Trends Pharm Sci 1999;20:376-382.

9. Zhong H, Neubig RR: Regulator of G protein signaling proteins: novel multifunctional drug targets. J Pharmacol Exp Ther 2001;297:837-845.

10.

10. Neubig RR, Siderovski DP: Regulators of G protein signaling as new central nervous system drug targets. Nat Rev Drug Disc 2002;11:187-197.

11.

11. Fong HK, Yoshimoto KK, Eversole-Cire P, et al: Identification of a GTP-binding protein α subunit that lacks an apparent ADP-ribosylation site for pertussis toxin. Proc Natl Acad Sci U S A 1988;85:3066-3070.

12.

12. Wang J, Ducret A, Tu Y, et al: RGS-Z1, a Gz-selective RGS protein in brain. Structure, membrane association, regulation by Gαz phosphorylation, and relationship to a Gz GT Pase-activating protein subfamily. JBiolChem 1998;273:26014-26025.

13.

13. Glick JL, Meigs TE, Miron A, et al: RGS-Z1, a Gz-selective regulator of G protein signaling whose action is sensitive to the phosphorylation state of Gzα. JBiolChem 1998;273:26008-26013.

14.

14. Yang J, Wu J, Kowalska MA, et al: Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs. Proc Natl Acad Sci U S A 2000;97:9984-9989.

15.

15. Wang Y, Ho G, Zhang JJ, et al: Regulator of G-protein signaling Z1 (RGS-Z1) interacts with Gαi subunits and regulates Gαi mediated cell signaling. JBiol Chem 2002;277:48325-48332.

16.

16. Fields S, Song O: A novel genetic system to detect protein-protein interactions. Nature 1989;340:245-246.

17.

17. Young K, Lin S, Sun L, et al: Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen. Nat Biotech 1998;16:946-950.

18.

18. de Wet JR, Wood KV, De Luca M, et al: Firefly luciferase gene: structure and expression in mammalian cells. MolCellBiol 1987;7:725-737.

19.

19. Vieites JM, Navarro-Garcia F, Perez-Diaz R, et al: Expression and in vivo determination of firefly luciferase as gene reporter in Saccharomyces cerevisiae. Yeast 1994;10:1321-1327.

20.

20. de Wet JR, Wood KV, Helsinki DR, et al: Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci USA 1985;82:7870-7873.

21.

21. Lorenz WW, Cormier MJ, O’Kane DJ, et al: Expression of the Renilla reniformis luciferase gene in mammalian cells. J Biolum Chemilum 1996;11:31-37.

22.

22. Parsons SJ, Rhodes SA, Connor HE, et al: Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors. Anal Biochem 2000;281:187-192.

23.

23. Young KH: Yeast two-hybrid: so many interactions, (in) so little time.... Biol Reprod 1998;58:302-311.

24.

24. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning. New York: CSHL Press, 1992.

25.

25. Ozenberger BA, Young KH: Functional interaction of ligands and receptors of the hematopoietic superfamily in yeast. Mol Endocrinol 1995;9:1321-1329.

26.

26. Liu J, Escher A: Improved assay sensitivity of an engineered secreted Renilla luciferase. Gene 1999;237:153-159.

27.

27. Rose MD, Winston R, Hieter P: Methods in Yeast Genetics. New York: CSHL Press, 1990.

28.

28. An WF, Bowlby MR, Betty M, et al: Modulation of A-type potassium channels by a family of calcium sensors. Nature 2000;403:553-556.

29.

29. SAS Institute Inc.: SAS/STAT User’s Guide, Version 8. Cary, NC: SAS Institute Inc., 1999.

30.

30. Lipinski CA, Lombardo F, Dominy BW, et al: Experimental and computational approaches to estimate solubility and permeability in drug discovery. Adv Drug Deliv Rev 1997;23:3-25.

31.

31. Ajay Walters WP, Murcko MA: Recognizing molecules with drug-like properties. Curr Opin Chem Biol 1999;3:384-387.