Generation of Adenosine Receptor Constructs

The generation of NLuc-A₃-receptor (Stoddart et al., 2015 (link)) and SNAP-A₃-receptor (Stoddart et al., 2014 (link)) pcDNA3.1 plasmids has been described previously. We generated NLuc-labeled adenosine receptor constructs by amplifying the full length sequence of NLuc luciferase (as provided by Promega Corporation in the pNL1.1 vector) and fusing it in frame with the membrane signal sequence of the 5HT_3A receptor within pcDNA3.1(+) to yield sig-NLuc. We then fused the full-length human sequence of the adenosine A3-receptor (obtained from Missouri S&T cDNA Resource Centre; www.cdna.org; GenBank: AY136749) with the methionine start signal removed, to the 3′ end of the sig-NLuc in pcDNA3.1(+). This gave the construct designated as NLuc-A₃ receptor. To generate N-terminal SNAP-tagged adenosine A₃ receptor, the methionine start signal was removed from cDNA encoding the full length A₃-receptor and subcloned into a pcDNA3.1-zeo (+) vector containing the 5HT₃-receptor-derived signal sequence followed by the SNAP Tag sequence (New England Biolabs, Ipswich, MA, USA).