Figure 5.

Detection of protein-protein interactions by real-time BRET¹ assays comparing C-terminally truncated and nontruncated receptors. Kinetics data comparing V2R-wild type (A) and the three different mutants V2R-R137C (B), V2R-R137L (C), and V2R-R137H (D) and corresponding C-terminally truncated receptors V2R-trunc (A), V2R-R137C-trunc (B), V2R-R137L-trunc (C), and V2R-R137H-trunc (D) were generated by monitoring the interaction with β-arrestin 2/Venus. The Rluc8 substrate coelenterazine h was added immediately before real-time measurements at 37 C. The transiently cotransfected HEK293 cells were assayed before and after treatment with ligand (AVP final concentration of 1 μm) or vehicle (PBS). BRET signals are expressed as fluorescence/luminescence without background subtraction as described in Materials and Methods. Data shown are mean ± sem of four independent experiments.