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Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer.

Audet M, Lagacé M, Silversides DW, Bouvier M

Department of Biochemistry, Institute for Research in Immunology and Cancer, and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Québec, Canada.

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.

FASEB J. 2010;24(8):2829-38.

Pubmed ID: 20335229

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