Extended Data Fig. 2: Pharmacology of the A1R construct and rationally chosen mutations.
From: Structure of the adenosine-bound human adenosine A1 receptor–Gi complex
a, b, Competition between the antagonist [3H]DPCPX and either unlabelled DPCPX (a) or ADO (b), in membranes expressing HA–Flag–3C-A1R-3C-8×His construct in the absence or presence of wild-type (WT) Gi2 heterotrimer or dominant-negative (DN) Gi2 heterotrimer. Data are normalized to [3H]DPCPX binding in the absence of unlabelled competitor, with nonspecific binding determined in the presence of 1 µM of the antagonist, SLV320. c, ADO-mediated binding of [35S]GTPγS as a measure of G-protein activation by the HA–Flag–3C-A1R-3C-8×His construct in High Five cells expressing receptor alone, or together with either wild-type or dominant-negative Gi2 heterotrimer. d, e, [3H]DPCPX competition assays (d) or inhibition of forskolin-stimulated cAMP accumulation (e), at the wild-type human A1R or two key alanine substitution mutations stably expressed in CHOFlpIn cells. f, Changes in agonist (NECA) affinity (Ki) from the experiments shown in d. g, Changes in NECA signalling efficacy corrected for receptor expression (τc), determined from the experiments shown in e. Parameter estimates are the mean ± s.e.m. determined from 3 (a–c) or 6–48 (d–g) independent experiments performed in duplicate. ****P < 0.0001 (compared with wild type; one-way analysis of variance (ANOVA), Dunnett’s post hoc test). Data for wild-type and N159A are replotted from Nguyen et al28.
