FIGURE 1.
Expression of N-terminally tagged A2A receptor constructs in HEK293 cells. A, schematic representation of the differently tagged receptors employed; G2S-N-TAP (total size 176 amino acids, 19.2 kDa) and the G2S-C-TAP (194 amino acids, 21.3 kDa) contain two protein G moieties and a streptavidin binding protein (SBP) fused to the N terminus and C terminus, respectively. G2S-N-TAP-A2AR-YFP contains, in addition, a YFP moiety at the C terminus. FS2-N-TAP (45 amino acids, 4.6 kDa) contains a FLAG-epitope and two Strep-Tactin moieties at the N terminus of the receptor. B, membranes (5–10 μg/assay) prepared from HEK293 cells stably expressing the indicated receptor (G2S-N- A2AR, G2S-N-TAP-A2AR-YFP, and FS2-N-A2AR) were incubated with increasing concentrations of the antagonist radioligand [3H]ZM241385. Data are means from duplicate determinations in a representative experiment. Two additional clones per construct were characterized. C, HEK293 cells (4 × 105) stably expressing the indicated receptor (G2S-N-A2AR, G2S-N-TAP-A2AR-YFP, and FS2-N-A2AR) were incubated with [3H]adenine to metabolically label their adenine nucleotide pool. Accumulation of [3H]cAMP was measured after stimulation with the indicated concentrations of CGS21680. Data are means ± S.D. (error bars) from three independent experiments. One representative clone per construct is shown.