Extended Data Fig. 6: PtdIns(4,5)P2 binds preferentially to β1AR in an active state and stabilizes β1AR coupled to mini-Gs and A2AR-mini-Gs complex.
From: PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling
a, A time-course experiment was performed to monitor the formation of active β1AR–mini-Gs complex. The coupling efficiency (percentage) was calculated from the relative intensity of peaks assigned to β1AR–mini-Gs coupling in the appropriate lipid-bound state. The plot indicates that mini-Gs coupling is enhanced by PtdIns(4,5)P2 when more than two lipid molecules are bound to the receptor. Error bars represent s.d. from at least three independent experiments. b, Plot of PMF for the interaction of mini-Gs with A2AR in the presence of PtdIns(4,5)P2 (green) or PS (grey). The PMF is calculated along a reaction coordinate (Δz) corresponding to the centre–centre separation of the mini-Gs and receptor proteins along the z axis (normal to the bilayer plane). The interaction of mini-Gs with the A2AR is stabilized in the presence of PtdIns(4,5)P2 by 50 ± 10 kJ mol−1 relative to PS. Error bars (which are <10 kJ mol−1) are from bootstrap sampling of the PMFs and therefore represent the ‘statistical’ errors in estimating the well depth from a given set of simulations and PMF calculation (n = 3 independent experiments). We therefore estimate a minimum error of ≤10 kJ mol−1. c, Mass spectra were recorded for a 1:1 equimolar mix of an inactive unliganded β1AR variant, E130W, and its unmodified active counterpart (co-purified with the agonist isoprenaline) in the presence of PI(4,5)P2. Lipid binding occurred on both receptors, but following normalization to account for differences in ionization efficiency, a clear preference for PtdIns(4,5)P2 binding to the active receptor was observed. Bars represent mean ± s.d.
