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- Title
Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer.
- Authors
Audet, Martin; Lagacé, Monique; Silversides, David W.; Bouvier, Michel
- 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 Β2-adrenergic receptor fused to Renilla luciferase (Β2AR-Rluc) and Βarrestin-2 fused to a green fluorescent protein (GFP2-Βarr2) was generated. The fusion proteins were found to be functional in the transgenic animals and the Β2AR-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 Β2AR-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 Β2AR-expressing tissues. Stimulation with a Β-adrenergic agonist led to a significant dose- and time-dependent increase in BRET, which reflected ligand-promoted recruitment of Βarr2 to the receptor. Our study demonstrates that BRET can be used to monitor the dynamic regulation of proteinprotein interactions in cells derived from transgenic mice.
- Subjects
ADRENERGIC receptors; SEA pansies; GREEN fluorescent protein; BIOLUMINESCENCE; PROTEIN-protein interactions
- Publication
FASEB Journal, 2010, Vol 24, Issue 8, p2829
- ISSN
0892-6638
- Publication type
Article
- DOI
10.1096/fj.09-144816