Construction of A1R and A2AR Fusion Proteins

The cDNA encoding the human A₁R tagged at its N-terminal tail with the O6-alkylguanine-DNA alkyltransferase (i.e., A₁R^SNAP) cloned in pRK5 vector (BD PharMingen, San Jose, CA, USA) was a gift from Prof. Jean-Philippe Pin (CNRS, Montpellier, France). Thus, to perform functional assays A_2AR^SNAP [24 (link)] and A₁R^SNAP were used. Also, A_2AR^RLuc and A₁R^YFP constructs [17 (link)] were used to perform classical BRET (Bioluminescence Resonance Energy Transfer) assays. Finally, to perform NanoBRET experiments with the MRS7396 fluorescent antagonist, we created an A_2AR NanoLuc sensor (A_2AR^NL). To this end, the cDNA encoding the human A_2AR was amplified by polymerase chain reaction from the pECFP-A_2AR vector using the primers: FA2AEco (5′-GCCGGAATTCCCCATCATGGGCTCCTCGGTGTAC-3′) and RA2ANot (5′-CGCGGCGGCCGCtcaggacactcctgctccatcctggg-3′). The amplified A_2AR insert was then cloned into the EcoRI/NotI sites of pNLF1-secN vector (Promega, Stockholm, Sweden) containing a hemagglutinin (HA) epitope tag. All the constructs were verified by DNA sequencing.