Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer
- PMID:20335229
- DOI: 10.1096/fj.09-144816
Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer
Abstract
Monitoring the dynamics of protein-protein interactions in their natural environment remains a challenge. Resonance energy transfer approaches represent a promising avenue to directly probe these interactions in real time. The present study aims at establishing a proof of principle that bioluminescence resonance energy transfer (BRET) can be used to study the regulation of protein-protein interaction in cells from transgenic animals. A transgenic mouse line coexpressing the beta(2)-adrenergic receptor fused to Renilla luciferase (beta(2)AR-Rluc) and beta arrestin-2 fused to a green fluorescent protein (GFP2-beta arr2) was generated. The fusion proteins were found to be functional in the transgenic animals and the beta(2)AR-Rluc maintained pharmacological properties, comparable to that of the native receptor. Sufficiently high luminescence signal was generated to allow detection of BRET in testis cells where the beta(2)AR-Rluc transgene was expressed at levels significantly higher than that of the endogenous receptor in this tissue but remain within physiological range when compared with other beta(2)AR-expressing tissues. Stimulation with a beta-adrenergic agonist led to a significant dose- and time-dependent increase in BRET, which reflected ligand-promoted recruitment of beta arr2 to the receptor. Our study demonstrates that BRET can be used to monitor the dynamic regulation of protein-protein interactions in cells derived from transgenic mice.
Similar articles
- Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M.Angers S, et al.Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3684-9. doi: 10.1073/pnas.97.7.3684.Proc Natl Acad Sci U S A. 2000.PMID:10725388Free PMC article.
- The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS).Bertrand L, Parent S, Caron M, Legault M, Joly E, Angers S, Bouvier M, Brown M, Houle B, Ménard L.Bertrand L, et al.J Recept Signal Transduct Res. 2002 Feb-Nov;22(1-4):533-41. doi: 10.1081/rrs-120014619.J Recept Signal Transduct Res. 2002.PMID:12503639
- Development of a BRET2 screening assay using beta-arrestin 2 mutants.Vrecl M, Jorgensen R, Pogacnik A, Heding A.Vrecl M, et al.J Biomol Screen. 2004 Jun;9(4):322-33. doi: 10.1177/1087057104263212.J Biomol Screen. 2004.PMID:15191649
- New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors.Pfleger KD, Eidne KA.Pfleger KD, et al.Pituitary. 2003;6(3):141-51. doi: 10.1023/b:pitu.0000011175.41760.5d.Pituitary. 2003.PMID:14974443Review.
- Monitoring interactions between G-protein-coupled receptors and beta-arrestins.Pfleger KD, Dalrymple MB, Dromey JR, Eidne KA.Pfleger KD, et al.Biochem Soc Trans. 2007 Aug;35(Pt 4):764-6. doi: 10.1042/BST0350764.Biochem Soc Trans. 2007.PMID:17635143Review.
Cited by
- Genetically encoded tools for in vivo G-protein-coupled receptor agonist detection at cellular resolution.Kroning KE, Wang W.Kroning KE, et al.Clin Transl Med. 2022 Dec;12(12):e1124. doi: 10.1002/ctm2.1124.Clin Transl Med. 2022.PMID:36446954Free PMC article.Review.
- Evolution of BRET Biosensors from Live Cell to Tissue-Scale In vivo Imaging.De A, Jasani A, Arora R, Gambhir SS.De A, et al.Front Endocrinol (Lausanne). 2013 Sep 23;4:131. doi: 10.3389/fendo.2013.00131.Front Endocrinol (Lausanne). 2013.PMID:24065957Free PMC article.Review.
- Genetically encoded fluorescent biosensors for live-cell visualization of protein phosphorylation.Oldach L, Zhang J.Oldach L, et al.Chem Biol. 2014 Feb 20;21(2):186-97. doi: 10.1016/j.chembiol.2013.12.012. Epub 2014 Jan 30.Chem Biol. 2014.PMID:24485761Free PMC article.Review.
- Fundamentals of protein interaction network mapping.Snider J, Kotlyar M, Saraon P, Yao Z, Jurisica I, Stagljar I.Snider J, et al.Mol Syst Biol. 2015 Dec 17;11(12):848. doi: 10.15252/msb.20156351.Mol Syst Biol. 2015.PMID:26681426Free PMC article.Review.
- Moonlighting proteins and protein-protein interactions as neurotherapeutic targets in the G protein-coupled receptor field.Fuxe K, Borroto-Escuela DO, Romero-Fernandez W, Palkovits M, Tarakanov AO, Ciruela F, Agnati LF.Fuxe K, et al.Neuropsychopharmacology. 2014 Jan;39(1):131-55. doi: 10.1038/npp.2013.242. Epub 2013 Sep 6.Neuropsychopharmacology. 2014.PMID:24105074Free PMC article.Review.
Publication types
MeSH terms
Substances
Related information
Grants and funding
LinkOut - more resources
Full Text Sources
Molecular Biology Databases
Research Materials