Extended Data Fig. 3: Investigation of the phospholipid preferences of A2AR and NTSR1.
From: PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling
a, A representative mass spectrum of purified A2AR from three independent experiments revealed truncations of the N-terminal sequence (MPIM). The arrows between species indicate the mass differences corresponding to truncated amino acids (M, PI and M). b, A competitive binding assay (n = 3 independent experiments) in which A2AR was incubated with a mixture of lipids (PI, PtdIns(4)P, PI(4,5)P2, and PtdIns(3,4,5)P3) before mass spectrometry, indicated that PtdIns(4,5)P2 binds with a higher affinity than the other phospholipids to A2AR. c, The analogous competitive binding assay, in which NTSR1 was incubated with a mixture of lipids (PI, PtdIns(4)P, PI(4,5)P2 and PtdIns(3,4,5)P3) before mass spectrometry. Ratio to apo is plotted as a function of concentration and defined as the ratio of the intensity corresponding to individual PI phosphate adducts to the receptor in the apo state (inset). The same data analysis methods are used for Fig. 1b. PtdIns(4,5)P2 binds with a higher affinity than the other phospholipids to A2AR. Data are shown as mean ± s.d. from three independent replicates. d, A representative mass spectrum of A2AR (n = 3 independent experiments) used for preparation of the G-protein complex reveals lower abundance of PS and PI adducts prior to coupling to G proteins.
