Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Identification of endogenous lipids, preferential binding of PI(4,5)P2, molecular dynamics simulation and site-directed mutagenesis define intracellular PtdIns(4,5)P2-binding hotspots.
Fig. 2: Selectivity of G-protein coupling and the presence of endogenous lipids on coupled receptors.
Fig. 3: The effect of PtdIns(4,5)P2 on coupling to mini-Gs, and comparison with PS, Nb6B9 and mini-Gi.

Similar content being viewed by others

References

  1. Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schiöth, H. B. & Gloriam, D. E. Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discov. 16, 829–842 (2017).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  2. Nehmé, R. et al. Mini-G proteins: Novel tools for studying GPCRs in their active conformation. PLoS One 12, e0175642 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Zocher, M., Zhang, C., Rasmussen, S. G., Kobilka, B. K. & Muller, D. J. Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor. Proc. Natl Acad. Sci. USA 109, E3463–E3472 (2012).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  4. Dawaliby, R. et al. Allosteric regulation of G protein-coupled receptor activity by phospholipids. Nat. Chem. Biol. 12, 35–39 (2016).

    Article  PubMed  CAS  Google Scholar 

  5. Gault, J. et al. High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods 13, 333–336 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Yen, H. Y. et al. Ligand binding to a G protein-coupled receptor captured in a mass spectrometer. Sci. Adv. 3, e1701016 (2017).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  7. Egloff, P. et al. Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc. Natl Acad. Sci. USA 111, E655–E662 (2014).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  8. Inagaki, S., Ghirlando, R. & Grisshammer, R. Biophysical characterization of membrane proteins in nanodiscs. Methods 59, 287–300 (2013).

    Article  PubMed  CAS  Google Scholar 

  9. Hedger, G. & Sansom, M. S. P. Lipid interaction sites on channels, transporters and receptors: Recent insights from molecular dynamics simulations. BBA-Biomembranes 1858, 2390–2400, (2016).

    Article  PubMed  CAS  Google Scholar 

  10. Schlinkmann, K. M. et al. Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations. Proc. Natl Acad. Sci. USA 109, 9810–9815 (2012).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  11. Carpenter, B., Nehmé, R., Warne, T., Leslie, A. G. W. & Tate, C. G. Structure of the adenosine A2A receptor bound to an engineered G protein. Nature 536, 104–107 (2016).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  12. Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  13. Hansen, S. B., Tao, X. & MacKinnon, R. Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2. Nature 477, 495–498 (2011).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  14. Roux, B. The calculation of the potential of mean force using computer simulations. Comput. Phys. Commun. 91, 275–282 (1995).

    Article  ADS  CAS  Google Scholar 

  15. Domański, J., Hedger, G., Best, R. B., Stansfeld, P. J. & Sansom, M. S. P. Convergence and sampling in determining free energy landscapes for membrane protein association. J. Phys. Chem. B 121, 3364–3375 (2017).

    Article  PubMed  CAS  Google Scholar 

  16. Roth, C. B., Hanson, M. A. & Stevens, R. C. Stabilization of the human β2-adrenergic receptor TM4–TM3–TM5 helix interface by mutagenesis of Glu1223.41, a critical residue in GPCR structure. J. Mol. Biol. 376, 1305–1319 (2008).

    Article  PubMed  CAS  Google Scholar 

  17. Che, T. et al. Structure of the nanobody-stabilized active state of the kappa opioid receptor. Cell 172, 55–67 e15, (2018).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  18. Miller-Gallacher, J. L. et al. The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor. PLoS One 9, e92727 (2014).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  19. Injin, B. & Hee-Jung, C. Structural features of β2 adrenergic receptor: crystal structures and beyond. Mol. Cells 38, 105–111 (2015).

    Article  CAS  Google Scholar 

  20. Venkatakrishnan, A. J. et al. Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region. Nature 536, 484–487 (2016).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  21. Zheng, Y. et al. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540, 458–461 (2016).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  22. Liu, X. et al. Mechanism of intracellular allosteric β2AR antagonist revealed by X-ray crystal structure. Nature 548, 480–484 (2017).

    Article  ADS  PubMed  CAS  PubMed Central  Google Scholar 

  23. Gavi, S., Shumay, E., Wang, H. Y. & Malbon, C. C. G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation. Trends Endocrinol. Metab. 17, 48–54 (2006).

    Article  PubMed  CAS  Google Scholar 

  24. Scott, D. J., Kummer, L., Egloff, P., Bathgate, R. A. D. & Plückthun, A. Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies. BBA-Biomembranes 1838, 2817–2824, (2014).

    Article  PubMed  CAS  Google Scholar 

  25. Carpenter, B. & Tate, C. G. Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation. Protein Eng. Des. Sel. 29, 583–594 (2016).

    PubMed  PubMed Central  CAS  Google Scholar 

  26. Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  27. Warne, T. et al. The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 469, 241–244 (2011).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  28. Warne, T., Serrano-Vega, M. J., Tate, C. G. & Schertler, G. F. Development and crystallization of a minimal thermostabilised G protein-coupled receptor. Protein Expr. Purif. 65, 204–213 (2009).

    Article  PubMed  CAS  Google Scholar 

  29. Warne, T., Chirnside, J. & Schertler, G. F. Expression and purification of truncated, non-glycosylated turkey beta-adrenergic receptors for crystallization. Biochim. Biophys. Acta 1610, 133–140 (2003).

    Article  PubMed  CAS  Google Scholar 

  30. Carpenter, B. & Tate, C. G. Expression and Purification of Mini G Proteins from Escherichia coli. Bio Protoc. 7, (2017).

  31. Ring, A. M. et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  32. Hillenbrand, M., Schori, C., Schoppe, J. & Pluckthun, A. Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution. Proc. Natl Acad. Sci. USA 112, E1181–E1190 (2015).

    Article  ADS  PubMed  CAS  PubMed Central  Google Scholar 

  33. Laganowsky, A., Reading, E., Hopper, J. T. S. & Robinson, C. V. Mass spectrometry of intact membrane protein complexes. Nat. Protocols 8, 639–651 (2013).

    Article  PubMed  CAS  Google Scholar 

  34. Chen, P. S., Toribara, T. Y. & Warner, H. Microdetermination of phosphorus. Anal. Chem. 28, 1756–1758 (1956).

    Article  CAS  Google Scholar 

  35. Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Bird, S. S., Marur, V. R., Sniatynski, M. J., Greenberg, H. K. & Kristal, B. S. Lipidomics profiling by high resolution LC–MS and HCD fragmentation: focus on characterization of mitochondrial cardiolipins and monolysocardiolipins. Anal. Chem. 83, 940–949 (2011).

    Article  PubMed  CAS  Google Scholar 

  37. Bechara, C. et al. A subset of annular lipids is linked to the flippase activity of an ABC transporter. Nat. Chem. 7, 255–262 (2015).

    Article  PubMed  CAS  Google Scholar 

  38. Egloff, P., Deluigi, M., Heine, P., Balada, S. & Pluckthun, A. A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli. Protein Expr. Purif. 108, 106–114 (2015).

    Article  PubMed  CAS  Google Scholar 

  39. Sobott, F., Hernández, H., McCammon, M. G., Tito, M. A. & Robinson, C. V. A tandem mass spectrometer for improved transmission and analysis of large macromolecular assemblies. Anal. Chem. 74, 1402–1407 (2002).

    Article  PubMed  CAS  Google Scholar 

  40. Šali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  PubMed  Google Scholar 

  41. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008).

    Article  PubMed  CAS  Google Scholar 

  42. de Jong, D. H. et al. Improved parameters for the Martini coarse-grained protein force field. J. Chem. Theory Comput. 9, 687–697 (2013).

    Article  PubMed  CAS  Google Scholar 

  43. Koldsø, H., Shorthouse, D., Hélie, J. & Sansom, M. S. P. Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers. PLOS Comput. Biol. 10, e1003911 (2014).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  44. Periole, X., Cavalli, M., Marrink, S. J. & Ceruso, M. A. Combining an elastic network with a coarse-grained molecular force field: structure, dynamics, and intermolecular recognition. J. Chem. Theory Comput. 5, 2531–2543 (2009).

    Article  PubMed  CAS  Google Scholar 

  45. Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A. & Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81, 3684–3690 (1984).

    Article  ADS  CAS  Google Scholar 

  46. Tironi, I. G., Sperb, R., Smith, P. E. & van Gunsteren, W. F. A generalized reaction field method for molecular dynamics simulations. J. Chem. Phys. 102, 5451–5459 (1995).

    Article  ADS  CAS  Google Scholar 

  47. Hess, B., Bekker, H., Berendsen, H. J. C. & Fraaije, J. G. E. M. LINCS: A linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997).

    Article  CAS  Google Scholar 

  48. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

    Article  PubMed  CAS  Google Scholar 

  49. Hedger, G., Sansom, M. S. P. & Koldsø, H. The juxtamembrane regions of human receptor tyrosine kinases exhibit conserved interaction sites with anionic lipids. Sci. Rep. 5, 9198 (2015).

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  50. Hedger, G., Shorthouse, D., Koldso, H. & Sansom, M. S. Free energy landscape of lipid interactions with regulatory binding sites on the transmembrane domain of the EGF receptor. J. Phys. Chem. B 120, 8154–8163 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Hub, J. S., de Groot, B. L. & van der Spoel, D. g_wham—A free weighted histogram analysis implementation including robust error and autocorrelation estimates. J. Chem. Theory Comput. 6, 3713–3720 (2010).

    Article  CAS  Google Scholar 

  52. Notredame, C., Higgins, D. G. & Heringa, J. T-coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302, 205–217 (2000).

    Article  PubMed  CAS  Google Scholar 

  53. Bond, C. S. & Schuttelkopf, A. W. ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr. D 65, 510–512 (2009).

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

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.

Reviewer information

Nature thanks A. Lee, C. Reynolds, J. Whitelegge and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Authors and Affiliations

Authors

Contributions

H.-Y.Y., K.K.H. and I.L. performed all mass spectrometry experiments on the GPCR, mini-GS and nanobody. D.W. performed lipidomics. G.H., M.R.H., W.S., and M.S.P.S. performed molecular dynamics simulations and analyses. P.H. purified the NTSR1 receptor in the apo state. T.W. purified β1AR, Y.L. purified A2AR and B.C. purified mini-GS. A.P., C.G.T., M.S.P.S. and C.V.R supervised the research and H.-Y.Y. and C.V.R. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Mark S. P. Sansom or Carol V. Robinson.

Ethics declarations

Competing interests

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.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Identification of lipids bound to NTSR1(HTGH4-ΔIC3B).

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.

Extended Data Fig. 2 Lipid-binding preference of NTSR1 and β1AR.

ae, 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

Extended Data Fig. 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 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.

Extended Data Fig. 5 Site-directed mutagenesis attenuates PtdIns(4,5)P2 binding to NSTR1.

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

Extended Data Fig. 7 Detection of nanobody coupling to β1AR.

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.

Extended Data Table 1 Lipidomics analysis of purified β1AR
Extended Data Table 2 Simulations run

Supplementary information

Supplementary Information

This file contains Supplementary Notes and References

Reporting Summary

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yen, HY., 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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-018-0325-6

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing