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A simple method to generate stable cell lines for the analysis of transient protein-protein interactions
 
Emilia Elizabeth Savage, Denise Wootten, Arthur Christopoulos, Patrick Michael Sexton, and Sebastian George Barton Furness
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Figure 2.  Ligand-induced recruitment of ARRB2 & ARRB1 to AVPR2, GLP1R and CHRM1 receptors. (A), Stable flpIN CHO cell lines expressing AVPR2-RLuc8/ARRB2-Venus (green), CHRM1-RLuc8/ARRB2-Venus (orange), GLP1R-Rluc8/ARRB2-Venus (blue) or flpIN CHO cells transiently transfected with AVPR2/ARRB2-Venus (black) and stimulated with 1μM arginine vasopressin for AVPR2-RLuc8/ARRB2-Venus, 100μM acetylcholine for CHRM1-RLuc8/ARRB2-Venus or 100nM GLP-1(7–36)NH2 for GLP1R-Rluc8/ARRB2-Venus. Data shown are mean ± SEM from 3–4 independent experiments performed in triplicate over passages 17 to 35. Peak ligand induced milliBRET responses for ARRB2 recruitment are 87 ± 3.2 (AVPR2), 16.2 ± 3.3 (CHRM1), 24 ± 2.1 (GLP1R), and 71 ± 4 (AVPR2 transient), respectively. (B) and (C) are the same as A but with expanded y-axis shown for CHRM1-RLuc8/ARRB2-Venus (B) and GLP1R-Rluc8/ARRB2-Venus (C). (Click to enlarge)







In conclusion, we report a simplified scheme for generation of stable cell lines expressing both donor and acceptor fusion partners for protein-protein interaction studies by BRET. The incorporation of Gateway technology for the donor fusion makes this a useful tool for the development of cell lines for screening GPCRs and GPCR small molecule libraries for interactions with arrestin and other partner proteins. In addition, we have confirmed that this system also works with the distantly related Class C GPCR calcium sensing receptor (CaSR). The NheI and BsiWI sites flanking the acceptor protein allow facile replacement of these fusions. We have developed BIVISTI variants containing GRK acceptor fusions and have been able to demonstrate robust recruitment of four isoforms of these kinases to GLP1R. The fact that cell lines generated using this system are isogenic also makes it a useful tool to examine structure-activity relationships of receptor mutants for partner protein interactions. This is a simple and robust system that should be amenable to a wide variety of applications and will assist in improving assay-to-assay variation as well as the general shortcomings associated with transient transfection.

Acknowledgements

PMS and AC are Principal Research Fellows of the National Health and Medical Research Council of Australia (NHMRC). This work was funded by the NHMRC program grant #519461

Competing interests

The authors declare no competing interests.

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