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. 2018 Jul;559(7714):423-427.
doi: 10.1038/s41586-018-0325-6. Epub 2018 Jul 11.

PtdIns(4,5)P 2 Stabilizes Active States of GPCRs and Enhances Selectivity of G-protein Coupling

Free PMC article

PtdIns(4,5)P 2 Stabilizes Active States of GPCRs and Enhances Selectivity of G-protein Coupling

Hsin-Yung Yen et al. Nature. .
Free PMC article


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.

Conflict of interest statement

The authors declare competing interests: H.-Y.Y., and I.L. are founders and employees of OMass Technologies. C.V.R is a founder and consultant of OMass Technologies.


Extended Data Figure 1
Extended Data Figure 1. Identification of lipids bound to NTSR1 HTGH4-ΔIC3B.
a, Endogenous lipid bounds to NTSR1 HTGH4-ΔIC3B, isolated from E. coli, are identified as PA following m/z selection in the MS 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.
Extended Data Figure 2
Extended Data Figure 2. Lipid binding preference of NTSR1 and β1AR.
The binding of NTSR1 HTGH4-ΔIC3B, measured by mass spectrometry (n=3), to the phospholipids PA (a), PS (b), PI (c), PC (d) and DAG (e). The measurements were performed at different lipid concentration (0 to160 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 (GraphPad Prism software). Standard deviations of the mean 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. Exogenous POPS (f) and PI(4)P (g) were added to β1AR at different final concentrations (10 μM shown here). Spectra were recorded for a range of lipid concentrations from 0 – 80 μM for PS and 0 – 20 μM for PI(4)P. Peak intensities of the individual PI(4)P-bound species were measured and plotted against lipid concentration to yield a relative affinity for one PI(4)P binding (1x), two PI(4)P molecules binding (2x) or three PI(4)P molecules binding (3x); only the first PI(4)P molecule binds with high affinity (see Fig. 1a). Error bars represent the standard deviation of the mean between three independent experiments.
Extended Data Figure 3
Extended Data Figure 3. Investigation of the phospholipid preferences of A2AR and NTSR1.
a, A representative mass spectrum of purified A2AR from three independent experiments revealed truncations of the N-terminal sequence (MPIM). The arrow between each species refers to the mass differences corresponding to truncated amino acids (M, PI and M). b, A competitive binding assay (n=3) in which A2AR was incubated with a mixture of lipids (PI, PI(4)P, PI(4,5)P2, and PI(3,4,5)P3) prior to MS, indicated that PIP2 binds with a higher affinity than other phospholipids to A2AR. c, The analogous competitive binding assay in which NTSR1 was incubated with a mixture of lipids (PI, PI(4)P, PI(4,5)P2, and PI(3,4,5)P3) prior to MS. Ratio to apo is plotted as a function of concentration and defined as the intensity ratio of individual PIP adducts to the receptor in the apo state. The same data analysis methods are used for Fig. 1b. Results indicate that PIP2 binds with a higher affinity than other phospholipids to A2AR. Error bars represent standard deviation of the mean from three independent replicates. d, A representative mass spectrum of A2AR (n=3) used for preparation of the G protein complex reveals lower abundance of PS and PI adducts prior coupling to G proteins.
Extended Data Figure 4
Extended Data Figure 4. NTSR1- and β1AR-PIP2 interactions within CGMD simulations and comparison of PIP2 contacts over different GPCRs.
a, Volumetric density surfaces showing the average spatial occupancy of PIP2 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 PIP2 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 independent experiment). The cytoplasmic side of NTSR1 structure is colored from white (low PIP2 interaction) to red (high PIP2 interaction). Extending a simulation to 100 μs revealed no overall change in the patterns of PIP2 interaction. Less specific, and hence more dynamic, interaction was seen for the acyl chain moieties of PIP2, which yielded more diffuse probability densities. b, β1AR-PIP2 interactions within CGMD simulations. Contact patterns are shown for simulations containing 5% PIP2 in the lipid bilayer and thermostable β1AR (PDB: 2Y03, upper panel), 10% PIP2 and thermostable β1AR (middle panel), and 10% PIP2 and S68R β1AR construct (bottom panel). In each case PIP2 contacts were calculated over 5-μs of CGMD (n=10 independent experiments), with each repeat simulation initiated from different random system configurations. The std of the mean of lipid contact number is denoted by black error bars. c, PS and PIP2 contacts with NTSR1 as a function of residue position, for PC:PS membranes (top left), PC:PS:PIP2 membranes (top right), PC:PIP2 membrane (bottom left) and PC:PS:PIP2 (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. Error bars (black) denote the standard deviation of the mean between simulation repeats (n=3). d, PIP2 contacts seen in CG MD simulations for nine Class A GPCRs (3RZE = histamine H1 receptor; 2VT4 = β1 adrenergic receptor; 2RH1 = β2 adrenergic receptor; 5TGZ = CB1 cannabinoid receptor; 5DSG = M4 muscarinic acetylcholine receptor; 3EML = adenosine A2A receptor; 3PBL = dopamine D3 receptor; 3V2W = sphingosine 1-phosphate receptor; 1F88 = rhodopsin). The sequences of the GPCRs are shown, with the TM helices, intracellular loops (ICL) and H8 helices indicated by horizontal bars, and with amino acids coloured by their mean number of contacts per simulation frame with the PIP2 molecules. The three green boxes correspond to the high frequency of PIP2 interactions discussed in the main text for the NTSR1, for the TM1, TM4, and TM7/H8 motifs. Contacts were computed over 1 μs CG-MD simulations (n=3 independent experiments) for each GPCR, using a 6 Å cut-off. Sequences were aligned using T-Coffee and mapping of protein-lipid contact data onto the sequence alignment used ALINE.
Extended Data Figure 5
Extended Data Figure 5. Site-directed mutagenesis attenuates PIP2 binding to NSTR1.
a, Schematic representation of the experimental protocol designed to combine MS with mutagenesis to produce mutants of lower molecular mass than wild type which when incubated with PIP2 yield a direct readout of the effect of mutations in specific regions. b, PIP2-binding of NTSR1 mutants on residues that exhibit the highest frequency of PIP2 interaction in MD 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); red bar), TM4 (R138I, R140T, K142L and K143L (R135I, R137T, K139L and K140L in NTSR1 TM86-ΔIC3B; R183I, R185T, K187L and K188L in wild-type); orange bar) and TM7-H8 (R316N (R311N in NTSR1 TM86-ΔIC3B; R377N in wild-type); green bar) attenuate PIP2 binding, and indicates that the TM4 interface is a preferential binding site over TM1 and TM7-H8 interfaces. The selection of residues for mutations was guided by MD (Extended Data Figure 4) and previous studies wherein binding of a fluorescence-labeled agonist, BODIPY neurotensin, to NTSR1, was screened and used to monitor efficient production, insertion, and folding.
Extended Data Figure 6
Extended Data Figure 6. PIP2 binds preferentially to β1AR in an active state and stabilises β1AR coupled to mini-Gs and A2AR-mini-Gs complex.
a, A time course experiment was performed to capture the complex formation of mini-Gs and active β1AR as a function of time. The coupling efficiency (percentage) was calculated from the relative intensity of peaks assigned to the β1AR coupling to mini-Gs at the appropriate lipid-bound state. The plot indicates that mini-Gs coupling is enhanced by PIP2 when more than two lipids are bound to the receptor. Error bars represent standard deviations of the mean from at least three independent experiments. b, Plot of potential of mean force (PMF) for the interaction of mini-Gs with A2AR in the presence of PIP2 (green curve) and 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 stabilised in the presence of PIP2 by ~50 ± 10 kJ/mol relative to PS. The error bars on the figure (which are less than 10 kJ/mol) are from bootstrap sampling of the PMFs and thus are the ‘statistical’ errors in estimating the well depth from a given set of simulations/PMF calculation (n=3 independent experiments). A minimum error of <= ~10 kJ/mol is therefore estimated. 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 PIP2 binding to the active receptor was observed. (Error bars denote standard deviation of the mean between three independent experiments).
Extended Data Figure 7
Extended Data Figure 7. Detection of nanobody coupling to β1AR.
Mass spectral peaks assigned to the nanobody (Nb6B9) binding to β1AR to form a β1AR·Nb6B9 complex at an equimolar ratio are highlighted (orange) and demonstrate complete complex formation implying a higher affinity of the nanobody than mini-Gs for β1AR (N=3 independent experiments).
Extended Data Figure 8
Extended Data Figure 8. Structural comparison of class A and class B GPCRs in complex with trimeric Gαβγ complexes.
The PIP2 contacts of the Gαs subunit observed in MD simulation (green spheres) were highlighted on the structures of trimeric G interactions with β2-adrenergic receptor (β2AR) (PDB: 3SN6), the glucagon-like peptide-1 receptor (GLP-1) (PDB: 5VAI), and the calcitonin receptor (CTR) (PDB: 5UZ7). The basic residues on the interface adjacent to the cytoplasmic end of TM4 are also highlighted (purple spheres). Expansion indicates the conserved pattern of PIP2 bridging in class A GPCRs (β2AR and A2AR (Fig. 3e)) both of which have basic residues on TM4 (Lys140 and Arg107/111) which are not observed in class B GPCRs GLP-1R and CTR.
Figure 1
Figure 1. Identification of endogenous lipids, preferential binding of PI(4,5)P2, MD simulation and site-directed mutagenesis define intracellular PIP2 binding hotspots.
a, Charge states of β1AR (agonist free, 11+ to 14+ green) and adducts are observed bound to the receptor (red, orange). Peaks (highlighted yellow) are selected in the quadrupole and subjected to tandem MS. Phosphatidylserine (PS) and PI(4)P (PIP) were identified in the resulting mass spectra. Binding curves plotted against lipid concentration confirm the preferential binding of PI(4)P over PS. Error bars represent standard deviation of the mean from three independent replicates. b, Mass spectra of β1AR following incubation with an equimolar solution containing PI, PI(4)P, PI(4,5)P2, and PI(3,4,5)P3. Binding curves confirm favorable binding of PI(4,5)P2. Error bars represent standard deviation of the mean from three independent replicates. c, CGMD simulation for NTSR1 TM86V-ΔIC3B embedded in the lipid bilayer containing mixed PC and PIP2. Basic residues forming high interaction levels (green spheres) and PIP2 particle densities (purple surface) representing the most occupied regions (0.6 nm distance cutoff based on the radial distribution of CG particles). d, Mutation of residues in NTSR1 (TM86V-ΔIC3B) highlighted are: TM1 (R43G, K44G and K45G; red), TM4 (R135I, R137T, K139L and K140L; orange) and TM7-H8 (R311N; green). Inhibition of PIP2 binding is plotted from three independent experiments with standard deviation of the mean. Results indicate that mutations on the TM4 interface have a greater effect than the TM1 and TM7-H8 interfaces.
Figure 2
Figure 2. The selectivity of G-protein coupling and the presence of endogenous lipids on coupled receptors.
a, A representative mass spectrum of A2AR receptor coupled to a trimeric G-protein complex stabilised by a nanobody (Inset top left) from three independent experiments. Isolating and subjecting charge state 26+ (orange main figure) to collision-induced dissociation gives rise to subcomplexes and the liberated receptor with lipid adducts (highlighted orange). b, GTPase assays indicate an increase of GTP hydrolysis by active NTSR1coupled to trimeric Gαiβγ in the presence of PIP2 (*** denotes a statistically significant difference (p (0.0006) < 0.001) calculated with a t-test to compare the effect of PIP2 (one variable) on receptor-induced GTPase activation. Data points were overlaid and error bars represent standard deviations of the mean from three independent replicates. c, Schematic representation of the influence of lipids and agonists on the binding of mini-G proteins. d, Mass spectra of isoprenaline bound β1AR with three different mini-G subunits (mini-Gs, i(s),12). Enhanced coupling and lipid adducts are observed in the presence of Gs. (highlighted green top right). Error bars denote standard deviations of the mean from three independent replicates and each data point was overlaid.
Figure 3
Figure 3. The effect of PIP2 on coupling to mini-Gs, and comparison with PS, a nanobody and mini-Gi.
a, A representative mass spectrum of the β1AR:mini-Gs 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-Gs lipid bound states highlighted in the same spectrum (lower). b, A representative mass spectrum of the β1AR:mini-Gs 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-Gs. c, Snapshots of steered MD simulations to pull mini-Gs away from A2AR 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-Gs with A2AR 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 A2AR-miniGs 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-Gi(s) (n=3). No difference is detected +/- PIP2. 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.

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