Fig. 2: BnOCPA selectively activates Gob.
a cAMP accumulation in PTX-pre-treated CHO-K1-hA1R cells expressing PTX-insensitive Goa following co-stimulation with 1 μM forskolin and each agonist (1 nM–1 μM; n = 6 individual repeats). b as for a but cells were transfected with PTX-insensitive Gob (n = 6 individual repeats). Stimulation of cAMP production in a reflects BnOCPA’s activation of endogenous, PTX-resistant Gs by the A1R (see Supplementary Figs. 5 and 6 and29,153,154). c, d Heatmaps summarising Emax (c %) and potency (d pEC50; −log [agonist concentration] required for 50% inhibition of cAMP accumulation) for individual Gα subunit and β-arrestin1 and 2 activation by selective A1R agonists for the inhibition of forskolin-stimulated cAMP production. Data taken from: adenosine, CPA, BnOCPA Fig. 1, Supplementary Figs. 3, 6; NECA, Supplementary Fig. 3, 6; HOCPA, Supplementary Fig. 5. e Venn diagram of agonist interactions with individual Gαo/i subunits. f The inhibition of cAMP accumulation via A1R:Goa or A1R:Gob by adenosine, CPA, HOCPA and BnOCPA. Each data point represents a concentration of agonist from the data in Supplementary Figs. 5 and 6. Line of unity (broken grey line) represents no bias. Data presented as mean ± SEM. g Signalling bias of A1R-selective agonists for A1R-Goa and A1R-Gob (Δ(τ/KA)) was determined relative to the natural agonist adenosine using the change in (τ/KA) ratio. The values were calculated for all compounds at each individual G protein and the data was fitted globally to determine single values for τ and KA and then normalised to a reference agonist (adenosine). This approach, used by others155, precludes the provision of individual data points. Compared to adenosine, BnOCPA elicits no measurable response (NR) at Goa. h The TRUPATH assay for direct G protein activation reveals no preference between Goa and Gob by adenosine, CPA or HOCPA, but a significant >10-fold greater activation of Gob vs Goa by BnOCPA (two-tailed unpaired Student’s t-test; P = 0.0009; see also Supplementary Fig. 7a; n = 8 individual repeats for each agonist). i Adenosine/Goa-mediated inhibition of cAMP accumulation was antagonised by BnOCPA in a concentration-dependent manner (n = 3–4 individual repeats). j Example current traces produced by adenosine (10 µM) in control conditions or in the presence of intracellular Goa interfering peptide, scrambled Goa peptide or Gob interfering peptide (all at 100 µM). Scale bars measure 25 pA and 100 s. k Summary data of adenosine-induced outward current experiments. The mean amplitude of the outward current induced by adenosine (40.6 ± 2.2 pA, n = 16 cells) was significantly reduced (one-way ANOVA; F(3,37)=12.40, P = 9.22 × 10−6) to 20.9 ± 3.6 pA (n = 10 cells, P = 2.65 × 10−5) in 100 µM Goa interfering peptide. Neither the scrambled Goa peptide (Goa SCR; 43.4 ± 2.4 pA, n = 7 cells, P = 1) nor the Gob interfering peptide (39. 2 ± 2.7 pA, n = 8 cells, P = 1) reduced the amplitude of the adenosine-induced outward current compared to control, but each were significantly different from the Goa interfering peptide (P = 8.20 × 10−5; P = 8.86 × 10−4, respectively). Averaged data are presented as mean ± SEM. ****, P < 0.0001 relative to other groups. Source data are provided as a Source Data file.
