Figure 3. The effect of PIP₂ on coupling to mini-G_s, and comparison with PS, a nanobody and mini-G_i.

a, A representative mass spectrum of the β₁AR:mini-G_s complex (n=3) in the presence of PIP₂ and the agonist isoprenaline with uncoupled β₁AR lipid bound states highlighted according to the colour coding (upper) and β₁AR:mini-G_s lipid bound states highlighted in the same spectrum (lower). b, A representative mass spectrum of the β₁AR:mini-G_s complex (n=3) in the presence of PS and the agonist isoprenaline. No appreciable difference can be attributed to PS binding between β₁AR and β₁AR:mini-G_s. c, Snapshots of steered MD simulations to pull mini-G_s away from A_2AR in the presence of PIP₂ (green) and PS (pink). Results reveal different binding modes of PIP₂ and PS to the receptor (outlined orange boxes). The interaction of mini-G_s with A_2AR is stabilised in the presence of PIP₂ by ~50 kJ/mol relative to PS (Extended Data Fig. 6b). d, A representative mass spectrum recorded following incubation of β₁AR with PIP₂, isoprenaline and a nanobody (Nb6B9) (0.3 molar ratio to receptor, n=3). e, PIP₂ contacts of A_2AR-miniG_s complex are shown on the receptor (purple) and miniG _s (Thr40, His41, Arg42, Lys216, Arg380; green), and juxtaposed to basic residues on β₂AR-Nb80 complex (purple). f, A representative mass spectrum of PIP₂ binding to mini-G_i(s) (n=3)_. No difference is detected +/- PIP_2. g, The intensity ratios of different lipid bound states to the apo state of receptor in the uncoupled/coupled state are plotted. The asterisk denotes a statistically significant difference (p < 0.05) calculated as one-way ANOVA with Dunnett’s multiple comparison test. Error bars represent standard deviations of the mean from three independent replicates.