Insights into the Basal Activity and Activation Mechanism of the β1 Adrenergic Receptor Using Native Mass Spectrometry

  • Agni F. M. Gavriilidou
  • Hanna Hunziker
  • Daniel Mayer
  • Ziva Vuckovic
  • Dmitry B. Veprintsev
  • Renato Zenobi
Research Article


In the absence of orthosteric ligands, most G protein-coupled receptors (GPCRs) exist in an equilibrium of different conformational states. This equilibrium is shifted by an agonist towards the active state or by an inverse agonist towards the inactive state. The basal activity of the receptor, and its ability to activate intracellular signaling pathways, is defined by the probability that a fraction of the receptor adopts the active state in the absence of ligand. Despite breakthroughs in native MS of membrane proteins, GPCR-transducing complexes have not been studied by this approach until very recently. Here, we investigated different conformational states of the turkey β1 adrenergic receptor (tβ1AR) in complex with two transducing partners: a G protein mimicking nanobody, Nb80, and an engineered truncated Gs protein (miniGs), in the presence of the full agonist isoprenaline by native MS. Interestingly, complex formation with both transducing partners was also observed in the absence of agonist, and allowed us to quantify basal activity of tβ1AR. We followed the stepwise disassembly of the transducing complexes by increasing the concentration of the inverse agonist S32212 in the presence of a constant concentration of isoprenaline. This allowed us to determine the relative binding affinity of S32212 in comparison to isoprenaline by native MS. Our approach provides a fast and sensitive way to detect complexes, study their stability in the presence of different ligands, and determine relative ligand affinities. Native mass spectrometry thus has the potential to become a useful tool to screen for orthosteric and allosteric GPCR drugs.

Graphical Abstract


Native electrospray ionization mass spectrometry G-coupled protein receptors 



We thank David Sykes for critical reading of the manuscript, Chris Tate for sharing the sequences for the miniG proteins, and Jan Stayer and Brian Kobilka for sharing the plasmid for the Nb80. We thank the Swiss National Science Foundation (grants no. 200020_159929 and 200020_178765 to RZ and 31003A_159748 and CRSII3_141898 to DBV) for financial support of this research.


  1. 1.
    Hopkins, A.L., Groom, C.R.: The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 (2002)CrossRefPubMedGoogle Scholar
  2. 2.
    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)CrossRefPubMedGoogle Scholar
  3. 3.
    Gilman, A.G.: G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615–649 (1987)CrossRefPubMedGoogle Scholar
  4. 4.
    Arun, K.S., Kunhong, X., Lefkowitz, R.J.: Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem. Sci. 36, 457–469 (2011)CrossRefGoogle Scholar
  5. 5.
    Milligan, G.: Constitutive activity and inverse agonists of G protein-coupled receptors: a current perspective. Mol. Pharmacol. 64, 1271–1276 (2003)CrossRefPubMedGoogle Scholar
  6. 6.
    Neubig, R.R., Spedding, M., Kenakin, T., Christopoulos, A.: International Union of Pharmacology Committee on receptor nomenclature and drug classification. XXXVIII. Update on terms and symbols in quantitative pharmacology. Pharmacol. Rev. 55, 597–606 (2003)CrossRefPubMedGoogle Scholar
  7. 7.
    Khan, S.M., Sleno, R., Gora, S., Zylbergold, P., Laverdure, J.-P., Labbe, J.-C., Miller, G.J., Hebert, T.E.: The expanding roles of G subunits in G protein-coupled receptor signaling and drug action. Pharmacol. Rev. 65, 545–577 (2013)CrossRefPubMedGoogle Scholar
  8. 8.
    Dupré, D.J., Robitaille, M., Rebois, R.V., Hébert, T.E.: The role of Gβγ subunits in the organization, assembly, and function of GPCR signaling complexes. Annu. Rev. Pharmacol. Toxicol. 49, 31–56 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kenakin, T. P.: Pharmacology in drug discovery: understanding drug response (1st ed.). Elsevier Academic Press, Amsterdam, Boston (2012)CrossRefGoogle Scholar
  10. 10.
    Kenakin, T. P.: A pharmacology primer: techniques for more effective and strategic drug discovery (4th ed.). Elsevier Academic Press, Amsterdam, Boston (2014)Google Scholar
  11. 11.
    Loo, J.A.: Studying noncovalent protein complexes by electrospray ionization mass spectrometry. Mass Spectrom. Rev. 16, 1–23 (1997)CrossRefPubMedGoogle Scholar
  12. 12.
    Hernández, H., Robinson, C.V.: Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat. Protoc. 2, 715–726 (2007)CrossRefPubMedGoogle Scholar
  13. 13.
    Laganowsky, A., Reading, E., Hopper, J.T.S., Robinson, C.V.: Mass spectrometry of intact membrane protein complexes. Nat. Protoc. 8, 639–651 (2013)CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Barrera, N.P., Di Bartolo, N., Booth, P.J., Robinson, C.V.: Micelles protect membrane complexes from solution to vacuum. Science. 321, 243–246 (2008)CrossRefPubMedGoogle Scholar
  15. 15.
    Barrera, N.P., Isaacson, S.C., Zhou, M., Bavro, V.N., Welch, A., Schaedler, T.A., Seeger, M.A., Miguel, R.N., Korkhov, V.M., van Veen, H.W., Venter, H., Walmsley, A.R., Tate, C.G., Robinson, C.V.: Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nat. Methods. 6, 585–587 (2009)CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Marcoux, J., Wang, S.C., Politis, A., Reading, E., Ma, J., Biggin, P.C., Zhou, M., Tao, H., Zhang, Q., Chang, G., Morgner, N., Robinson, C.V.: Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc. Natl. Acad. Sci. 110, 9704–9709 (2013)CrossRefPubMedGoogle Scholar
  17. 17.
    Leney, A.C., McMorran, L.M., Radford, S.E., Ashcroft, A.E.: Amphipathic polymers enable the study of functional membrane proteins in the gas phase. Anal. Chem. 84, 9841–9847 (2012)CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hopper, J.T.S., Sokratous, K., Oldham, N.J.: Charge state and adduct reduction in electrospray ionization-mass spectrometry using solvent vapor exposure. Anal. Biochem. 421, 788–790 (2012)CrossRefPubMedGoogle Scholar
  19. 19.
    Rose, R.J., Damoc, E., Denisov, E., Makarov, A., Heck, A.J.R.: High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat. Methods. 9, 1084–1086 (2012)CrossRefPubMedGoogle Scholar
  20. 20.
    Yen, H., Hopper, J.T.S., Liko, I., Allison, T.M., Zhu, Y., Wang, D., Stegmann, M., Mohammed, S., Wu, B., Robinson, C.V.: Ligand binding to a G protein – coupled receptor captured in a mass spectrometer. Sci. Adv. 3, e1701016 (2017). CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Yen, H.-Y., Hoi, K.K., Liko, I., Hedger, G., Horrell, M.R., Song, W., Wu, D., Heine, P., Warne, T., Lee, Y., Carpenter, B., Plückthun, A., Tate, C.G., Sansom, M.S.P., Robinson, C.V.: PtdIns(4,5)P 2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling. Nature. 559, 423–427 (2018)CrossRefPubMedGoogle Scholar
  22. 22.
    Warne, T., Serrano-Vega, M.J., Baker, J.G., Moukhametzianov, R., Edwards, P.C., Henderson, R., Leslie, A.G.W., Tate, C.G., Schertler, G.F.X.: Structure of a β1-adrenergic G-protein-coupled receptor. Nature. 454, 486–491 (2008)CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Miller, J.L., Tate, C.G.: Engineering an ultra-thermostable β 1 -adrenoceptor. J. Mol. Biol. 413, 628–638 (2011). CrossRefPubMedGoogle Scholar
  24. 24.
    Isogai, S., Deupi, X., Opitz, C., Heydenreich, F.M., Tsai, C.-J., Brueckner, F., Schertler, G.F.X., Veprintsev, D.B., Grzesiek, S.: Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature. 314, 1–17 (2016)Google Scholar
  25. 25.
    Rasmussen, S.G.F., Choi, Fung, Pardon, Casarosa, Chae, Devree, Rosenbaum, Thian, Kobilka, Schnapp, A., Konetzki, Sunahara, R.K., Gellman, Pautsch, Steyaert, J., Weis, K.: Structure of a nanobody-stabilized active state of the β(2) adrenoceptor. Nature. 469, 175–180 (2011)CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    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)PubMedPubMedCentralGoogle Scholar
  27. 27.
    Carpenter, B., Nehmé, R., Warne, T., Leslie, A.G.W., Tate, C.G.: Structure of the adenosine A 2A receptor bound to an engineered G protein. Nature. 536, 104–107 (2016). CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Devree, B.T., Mahoney, J.P., Vélez-ruiz, G.A., Rasmussen, S.G.F., Kuszak, A.J., Edwald, E., Fung, J., Manglik, A., Masureel, M., Du, Y., Matt, R.A., Pardon, E., Steyaert, J., Kobilka, B.K., Roger, S.K.: Allosteric coupling from G protein to the agonist binding pocket in GPCRs. Nature. 535, 182–186 (2016)CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Department of Chemistry and Applied BiosciencesETH ZurichZurichSwitzerland
  2. 2.OMass Technologies Ltd The Schrodinger BuildingOxfordUK
  3. 3.Laboratory of Biomolecular ResearchPaul Scherrer InstituteVilligenSwitzerland
  4. 4.Department of BiologyETH ZurichZurichSwitzerland
  5. 5.Centre of Membrane Proteins and Receptors (COMPARE)University of Birmingham and University of NottinghamMidlandsUK
  6. 6.School of Life SciencesUniversity of NottinghamNottinghamUK

Personalised recommendations