G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsβγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the β1 adrenergic receptor (β1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between β1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαiβγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.
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C.V.R. acknowledges an ERC Advanced Grant ENABLE (641317), an MRC Programme Grant (G1000819) and a Wellcome Trust Investigator Award (104633/Z/14/Z). Further support was provided by the MRC (G.H.), the Royal Society Newton International Fellowship (W.S.), and the BBSRC (M.S.P.S.; BB/L002558/1) and the Wellcome Trust (M.S.P.S.; 092970/Z/10/Z). This work used the ARCHER UK National Supercomputing Service (http://www.archer.ac.uk), supported by EPSRC. A.P. was funded by the Schweizerischer Nationalfonds Grant (31003A_153143). C.G.T. acknowledges the MRC (MC_U105197215), an ERC Advanced Grant EMPSI (339995) and funding from Heptares Therapeutics. We thank M. Hillenbrand for providing Sf9 cells.
Nature thanks A. Lee, C. Reynolds, J. Whitelegge and the other anonymous reviewer(s) for their contribution to the peer review of this work.
H.-Y.Y. and I.L. are founders and employees of OMass Technologies. C.V.R is a founder of and consultant for OMass Technologies.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Endogenous lipids bound to NTSR1(HTGH4-ΔIC3B), isolated from E. coli, are identified as PA following m/z selection in the mass spectrometry quadrupole of the NTSR1:lipid 11+ charge state (highlighted yellow) and collisional activation to dissociate PA and its homologues (m/z, 700–760 Da). b, Lipidomics analysis of purified NTSR1 with three technical replicates reveals peaks at low m/z. MS/MS spectra of the precursor ion (M-H-1) at m/z 699.32 highlighted yellow, leads to definitive fragment ions at m/z 281 and 417 consistent with the structure of PA (36:2). c, Analogous lipidomics analysis of purified β1AR from insect cells with three technical replicates. MS/MS spectra of the two [M-H-1] precursor ions (m/z 758.50 and 786.53) identified the lipids as PS (34:2) and PS (36:2) respectively with diagnostic fragments indicated.
a–e, The binding of NTSR1(HTGH4-ΔIC3B), measured by mass spectrometry (n = 3 independent experiments), to the phospholipids PA (a), PS (b), PI (c), PC (d) and DAG (e). The measurements were performed at different lipid concentrations (0 to 160 µM) and the percentages of individual lipid-binding peaks (sum of apo protein and all lipid adducts obtained in the region of the mass spectrum under study) were plotted against lipid concentrations in solution. The lipid-binding curves were deduced from fitting to one-site total binding. Values of s.d. were calculated from three independent replicate experiments at each concentration. The results show that NTSR1 interacts preferentially with anionic phospholipids (PA and PS), as no binding was observed for neutral (DAG) and zwitterionic (PC) lipids. f, g, Exogenous POPS (f) and PtdIns(4)P (g) were added to β1AR at different final concentrations (10 µM is shown here). Spectra were recorded for a range of lipid concentrations from 0 to 80 µM for PS and 0 to 20 µM for PtdIns(4)P. Peak intensities of the individual PtdIns(4)P-bound species were measured and plotted against lipid concentration to yield a relative affinity for one PtdIns(4)P binding (1×), two PtdIns(4)P molecules binding (2×) or three PtdIns(4)P molecules binding (3×); only the first PtdIns(4)P molecule binds with high affinity (see Fig. 1a). Data are mean ± s.d. from three independent experiments. Source data
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. Source data
Extended Data Fig. 4 NTSR1–PtdIns(4,5)P2 and β1AR–PtdIns(4,5)P2 interactions within CGMD simulations, and comparison of PtdIns(4,5)P2 contacts among different GPCRs.
a, Volumetric density surfaces showing the average spatial occupancy of PtdIns(4,5)P2 lipids around a crystal structure of NTSR1(TM86V-ΔIC3B) (PDB: 4BUO), which shares a greater sequence identity to the wild-type receptor (91%) than NTSR1(HTGH4-ΔIC3B) (86%), contoured to show the major PtdIns(4,5)P2-interaction sites. Density surfaces were calculated over 5 μs of CGMD (blue surface, n = 10 independent experiments), and 100 μs of CGMD (magenta, n = 1 experiment). The cytoplasmic side of NTSR1 structure is coloured from white (low PtdIns(4,5)P2 interaction) to red (high PtdIns(4,5)P2 interaction). Extending a simulation to 100 µs revealed no overall change in the patterns of PtdIns(4,5)P2 interaction. Less specific, and hence more dynamic, interaction was seen for the acyl chain moieties of PtdIns(4,5)P2, which yielded more diffuse probability densities. b, β1AR–PtdIns(4,5)P2 interactions within CGMD simulations. Contact patterns are shown for simulations containing 5% PtdIns(4,5)P2 in the lipid bilayer and thermostable β1AR (PDB: 2Y03, top), 10% PtdIns(4,5)P2 and thermostable β1AR (middle), and 10% PtdIns(4,5)P2 and β1AR(S68R) construct (bottom). In each case PtdIns(4,5)P2 contacts were calculated over 5 μs of CGMD (n = 10 independent experiments; error bars, s.d.), with each repeat simulation initiated from different random system configurations. c, PS and PtdIns(4,5)P2 contacts with NTSR1 as a function of residue position, for PC:PS membranes (top left), PC:PS:PtdIns(4,5)P2 membranes (top right), PC:PtdIns(4,5)P2 membrane (bottom left) and PC:PS:PtdIns(4,5)P2 (bottom right). The position of helices is denoted by horizontal grey bars. Lipid contact is calculated as the mean number of contacts between each residue and a given lipid species per frame, using a 6 Å distance cut-off. n = 3; error bars, s.d.. d, PtdIns(4,5)P2 contacts seen in CGMD simulations for nine class A GPCRs: histamine H1 receptor, PDB 3RZE; β1 adrenergic receptor, 2VT4; β2 adrenergic receptor, 2RH1; CB1 cannabinoid receptor, 5TGZ; M4 muscarinic acetylcholine receptor, 5DSG; adenosine A2A receptor, 3EML; dopamine D3 receptor, 3PBL; sphingosine 1-phosphate receptor, 3V2W; rhodopsin, 1F88. GPCR sequences are shown, with TM helices, intracellular loops (ICL) and H8 helices indicated by horizontal bars, and with amino acids coloured according to the mean number of contacts per simulation frame with the PtdIns(4,5)P2 molecules. Green boxes correspond to the high frequency of PtdIns(4,5)P2 interactions discussed in the main text for the TM1, TM4, and TM7/H8 motifs of NTSR1. Contacts were computed over 1 μs CGMD simulations (n = 3 independent experiments) for each GPCR, using a 6 Å cut-off. Sequences were aligned using T-Coffee52 and mapping of protein–lipid contact data onto the sequence alignment used ALINE53.
a, Schematic representation of the experimental protocol designed to combine mass spectrometry with mutagenesis to produce mutants of lower molecular mass than wild type, which, when incubated with PtdIns(4,5)P2, yield a direct readout of the effect of mutations in specific regions. b, PtdIns(4,5)P2 binding of NTSR1 mutants on residues that exhibit the highest frequency of PtdIns(4,5)P2 interaction in molecular dynamics simulation. Mutation of NTSR1(HTGH4-ΔIC3B) residues on TM1 (R46G, K47G and K48G (R43G, K44G and K45G in NTSR1(TM86-ΔIC3B); R91G, K92G, K93G in wild type)), TM4 (R138I, R140T, K142L and K143L (R135I, R137T, K139L and K140L in NTSR1(TM86-ΔIC3B); R183I, R185T, K187L and K188L in wild type)) and TM7-H8 (R316N (R311N in NTSR1(TM86-ΔIC3B); R377N in wild type)) attenuate PtdIns(4,5)P2 binding, and indicate that the TM4 interface is a preferential binding site over TM1 and TM7-H8 interfaces. Selection of residues for mutations was guided by molecular dynamics (Extended Data Fig. 4) and previous studies in which binding of a fluorescently labelled agonist, BODIPY neurotensin, to NTSR1, was screened and used to monitor efficient production, insertion, and folding10.
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.
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. Source data
Peaks in the mass spectrum assigned to Nb6B9 binding to β1AR to form an equimolar β1AR–Nb6B9 complex are highlighted in orange, and demonstrate complete complex formation, implying that nanobody has a higher affinity than mini-Gs for β1AR. n = 3 independent experiments.
Extended Data Fig. 8 Structural comparison of class A and class B GPCRs in complex with trimeric Gαβγ complexes.
The PtdIns(4,5)P2 contacts of the Gαs subunit observed in molecular dynamics simulations (green spheres) are highlighted on the structures of trimeric G-protein interactions with β2AR (PDB: 3SN6), the glucagon-like peptide-1 receptor (GLP-1) (PDB: 5VAI) and the calcitonin receptor (CTR) (PDB: 5UZ7). Basic residues on the interface adjacent to the cytoplasmic end of TM4 are highlighted as purple spheres. Lower panels show an expanded view, highlighting the conserved pattern of PtdIns(4,5)P2 bridging in class A GPCRs (β2AR and A2AR (Fig. 3e)), both of which have basic residues on TM4 (Lys140 and Arg107/111) that are not present in the class B GPCRs GLP-1R and CTR.
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Yen, H., Hoi, K.K., Liko, I. et al. PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling. Nature 559, 423–427 (2018). https://doi.org/10.1038/s41586-018-0325-6
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