Structure of an endosomal signaling GPCR–G protein–β-arrestin megacomplex (original) (raw)

Data availability

Cryo-EM maps corresponding to the consensus megaplex reconstruction as well as the signal-subtracted β2V2R–Gs and β-arr1–V2T subcomplexes have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-9377, EMD-9376 and EMD-9375, respectively. Atomic coordinates for the β2V2R–Gs and β-arr1–V2T subcomplexes have been deposited in the Protein Data Bank (PDB) with accession codes PDB 6NI3 and PDB 6NI2, respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015298. Source data for Extended Data Figs. 1c and 1d are available with the paper online. Other data that support the findings of this study are available from the corresponding authors upon request.

References

1. Reiter, E., Ahn, S., Shukla, A. K. & Lefkowitz, R. J. Molecular mechanism of beta-arrestin-biased agonism at seven-transmembrane receptors. Annu. Rev. Pharmacol. Toxicol. 52, 179–197 (2012).
   Article CAS PubMed Google Scholar
2. Lefkowitz, R. J., Stadel, J. M. & Caron, M. G. Adenylate cyclase-coupled beta-adrenergic receptors: structure and mechanisms of activation and desensitization. Annu. Rev. Biochem. 52, 159–186 (1983).
   Article CAS PubMed Google Scholar
3. Rajagopal, S., Rajagopal, K. & Lefkowitz, R. J. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. Drug Discov. 9, 373–386 (2010).
   Article CAS PubMed PubMed Central Google Scholar
4. Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S. & Caron, M. G. Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J. Biol. Chem. 274, 32248–32257 (1999).
   Article CAS PubMed Google Scholar
5. Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G. & Barak, L. S. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J. Biol. Chem. 275, 17201–17210 (2000).
   Article CAS PubMed Google Scholar
6. Calebiro, D. et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol. 7, e1000172 (2009).
   Article PubMed PubMed Central CAS Google Scholar
7. Ferrandon, S. et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat. Chem. Biol. 5, 734–742 (2009).
   Article CAS PubMed PubMed Central Google Scholar
8. Feinstein, T. N. et al. Noncanonical control of vasopressin receptor type 2 signaling by retromer and arrestin. J. Biol. Chem. 288, 27849–27860 (2013).
   Article CAS PubMed PubMed Central Google Scholar
9. Thomsen, A. R. B. et al. GPCR-G Protein-beta-Arrestin Super-Complex Mediates Sustained G Protein Signaling. Cell 166, 907–919 (2016).
   Article CAS PubMed PubMed Central Google Scholar
10. Shukla, A. K. et al. Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature 512, 218–222 (2014).
    Article CAS PubMed PubMed Central Google Scholar
11. Cahill, T. J. 3rd et al. Distinct conformations of GPCR-beta-arrestin complexes mediate desensitization, signaling, and endocytosis. Proc. Natl Acad. Sci. USA 114, 2562–2567 (2017).
    Article CAS PubMed PubMed Central Google Scholar
12. Wehbi, V. L. et al. Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gbetagamma complex. Proc. Natl Acad. Sci. USA 110, 1530–1535 (2013).
    Article CAS PubMed PubMed Central Google Scholar
13. Jimenez-Vargas, N. N. et al. Protease-activated receptor-2 in endosomes signals persistent pain of irritable bowel syndrome. Proc. Natl Acad. Sci. USA 115, E7438–E7447 (2018).
    Article CAS PubMed PubMed Central Google Scholar
14. Wanka, L., Babilon, S., Kaiser, A., Morl, K. & Beck-Sickinger, A. G. Different mode of arrestin-3 binding at the human Y1 and Y2 receptor. Cell Signal 50, 58–71 (2018).
    Article CAS PubMed Google Scholar
15. Rasmussen, S. G. et al. Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).
    Article CAS PubMed PubMed Central Google Scholar
16. Cherezov, V. et al. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).
    Article CAS PubMed PubMed Central Google Scholar
17. Ballesteros, J. A. & Weinstein. H. in Methods in Neurosciences Vol. 25 (ed. Sealfon, S. C.) 366–428 (Elsevier, 1995).
18. Ring, A. M. et al. Adrenaline-activated structure of beta2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013).
    Article CAS PubMed PubMed Central Google Scholar
19. Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469, 175–180 (2011).
    Article CAS PubMed PubMed Central Google Scholar
20. Shukla, A. K. et al. Structure of active beta-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497, 137–141 (2013).
    Article CAS PubMed PubMed Central Google Scholar
21. Fredericks, Z. L., Pitcher, J. A. & Lefkowitz, R. J. Identification of the G protein-coupled receptor kinase phosphorylation sites in the human beta2-adrenergic receptor. J. Biol. Chem. 271, 13796–13803 (1996).
    Article CAS PubMed Google Scholar
22. Lally, C. C., Bauer, B., Selent, J. & Sommer, M. E. C-edge loops of arrestin function as a membrane anchor. Nat. Commun. 8, 14258 (2017).
    Article CAS PubMed PubMed Central Google Scholar
23. Zhou, X. E. et al. Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors. Cell 170, 457–469 (2017). e13.
    Article CAS PubMed PubMed Central Google Scholar
24. Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G. & Lefkowitz, R. J. beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248, 1547–1550 (1990).
    Article CAS PubMed Google Scholar
25. Lohse, M. J. et al. Receptor-specific desensitization with purified proteins. Kinase dependence and receptor specificity of beta-arrestin and arrestin in the beta 2-adrenergic receptor and rhodopsin systems. J. Biol. Chem. 267, 8558–8564 (1992).
    Article CAS PubMed Google Scholar
26. Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015).
    Article CAS PubMed PubMed Central Google Scholar
27. Liang, Y. L. et al. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118–123 (2017).
    Article CAS PubMed PubMed Central Google Scholar
28. Zhang, Y. et al. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248–253 (2017).
    Article CAS PubMed PubMed Central Google Scholar
29. Le Gouill, C., Innamorati, G. & Birnbaumer, M. An expanded V2 receptor retention signal. FEBS Lett. 532, 363–366 (2002).
    Article PubMed Google Scholar
30. Innamorati, G., Sadeghi, H. M., Tran, N. T. & Birnbaumer, M. A serine cluster prevents recycling of the V2 vasopressin receptor. Proc. Natl Acad. Sci. USA 95, 2222–2226 (1998).
    Article CAS PubMed PubMed Central Google Scholar
31. Mayer, D. et al. Distinct G protein-coupled receptor phosphorylation motifs modulate arrestin affinity and activation and global conformation. Nat. Commun. 10, 1261 (2019).
    Article PubMed PubMed Central CAS Google Scholar
32. Liang, Y. L. et al. Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor. Nature 561, 492–497 (2018).
    Article CAS PubMed PubMed Central Google Scholar
33. Liang, Y. L. et al. Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex. Nature 555, 121–125 (2018).
    Article CAS PubMed Google Scholar
34. Irannejad, R. et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013).
    Article CAS PubMed Google Scholar
35. Yang, M., He, R. L., Benovic, J. L. & Ye, R. D. beta-Arrestin1 interacts with the G-protein subunits beta1gamma2 and promotes beta1gamma2-dependent Akt signalling for NF-kappaB activation. Biochem. J. 417, 287–296 (2009).
    Article CAS PubMed Google Scholar
36. Gaudet, R., Bohm, A. & Sigler, P. B. Crystal structure at 2.4 angstroms resolution of the complex of transducin betagamma and its regulator, phosducin. Cell 87, 577–588 (1996).
    Article CAS PubMed Google Scholar
37. Lodowski, D. T., Pitcher, J. A., Capel, W. D., Lefkowitz, R. J. & Tesmer, J. J. Keeping G proteins at bay: a complex between G protein-coupled receptor kinase 2 and Gbetagamma. Science 300, 1256–1262 (2003).
    Article CAS PubMed Google Scholar
38. Whorton, M. R. & MacKinnon, R. X-ray structure of the mammalian GIRK2-betagamma G-protein complex. Nature 498, 190–197 (2013).
    Article CAS PubMed PubMed Central Google Scholar
39. Noble, A. J. et al. Routine single particle CryoEM sample and grid characterization by tomography. eLife 7, e34257 (2018).
    Article PubMed PubMed Central Google Scholar
40. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
    Article PubMed Google Scholar
41. Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).
    Article CAS PubMed PubMed Central Google Scholar
42. Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).
    Article CAS PubMed Google Scholar
43. Tegunov, D. & Cramer, P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat. Methods 16, 1146–1152 (2019).
    Article CAS PubMed PubMed Central Google Scholar
44. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
    Article PubMed PubMed Central Google Scholar
45. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
    Article CAS PubMed Google Scholar
46. Vilas, J. L. et al. MonoRes: Automatic and Accurate Estimation of Local Resolution for Electron Microscopy Maps. Structure 26, 337–344 (2018). e4.
    Article CAS PubMed Google Scholar
47. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 6, 213–221 (2010).
    Article CAS Google Scholar
48. Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738 (2010).
    Article CAS PubMed PubMed Central Google Scholar
49. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
    Article CAS PubMed Google Scholar
50. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
    Article CAS PubMed PubMed Central Google Scholar
51. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
    Article CAS PubMed Google Scholar
52. Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
    Article CAS PubMed Google Scholar
53. Marrink, S. J. & Mark, A. E. Molecular view of hexagonal phase formation in phospholipid membranes. Biophys. J. 87, 3894–3900 (2004).
    Article CAS PubMed PubMed Central Google Scholar
54. Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).
    Article CAS PubMed Google Scholar
55. Pronk, S. et al. GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 29, 845–854 (2013).
    Article CAS PubMed PubMed Central Google Scholar
56. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 27–28 (1996).
    Article Google Scholar
57. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).
    Article CAS PubMed Google Scholar
58. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
    Article CAS PubMed Google Scholar
59. Olsen, J. V. & Mann, M. Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. Proc. Natl Acad. Sci. USA 101, 13417–13422 (2004).
    Article CAS PubMed PubMed Central Google Scholar
60. MacLean, B. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010).
    Article CAS PubMed PubMed Central Google Scholar

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Acknowledgements

We thank Q. Lennon, J. Bisson and J. Taylor for excellent administrative support, W. Capel for technical support, Y. Zhang and G. Skiniotis for help with initial sample screening, C.-R. Liang, L.-L. Gu and J.-M. Shan for synthesizing BI-167107, T. Wang and the CUNY Advanced Science Research Center Imaging Facility for help with sample screening and data collection, the lab of K. Gardner at the CUNY Advanced Science Research Center for providing various general lab reagents and equipment during initial sample screening, M. Walters, M. DeLong, M. Plue, T. Milledge, D. Capel and X. Jiang at Duke University for technical support and discussion, D. Lyumkis, D. Tegunov, W. Rice, E. Eng, L. Kim, M. Kopylov and A. Cheng for help with tilted data collection and processing and S. Houston and B. Plouffe for helpful discussion. This work received support from NIH grants (nos. T32GM007171 and F30HL149213 to A.H.N; F30HL129803 to T.J.C.; T32GM007767 to J.P.M.; R35GM133598 to A.d.G. and R01HL016037 to R.J.L.); HHMI Medical Research Fellowship to A.H.N.; the Danish Council for Independent Research & Lundbeck Foundation (DFF-5053-00136 and R172-2014-1468 to A.R.B.T.); American Heart Association Innovative Project Award (no. 19IPLOI34760706 to A.d.G); Institut de Recherche Servier (no. 18021932 to A.d.G. and D.B.-H.); and American Heart Association Predoctoral Fellowship (no. 13PRE17110027 to J.P.M.). Some of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (grant no. SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (grant no. GM103310) with additional support from Agouron Institute (grant no. F00316) and the NIH (grant no. OD019994). R.J.L. is an HHMI Investigator.

Author information

Author notes

1. Jacob P. Mahoney
   Present address: Department of Structural Biology, Stanford University, Stanford, CA, USA
2. These authors contributed equally: Anthony H. Nguyen, Alex R. B. Thomsen, Thomas J. Cahill III.

Authors and Affiliations

1. Department of Medicine, Duke University Medical Center, Durham, NC, USA
   Anthony H. Nguyen, Alex R. B. Thomsen, Thomas J. Cahill III, Li-Yin Huang, Ali Masoudi & Robert J. Lefkowitz
2. Department of Biochemistry, Duke University Medical Center, Durham, NC, USA
   Anthony H. Nguyen, Thomas J. Cahill III, John Little IV & Robert J. Lefkowitz
3. Department of Surgery, Columbia University Irving Medical Center, New York, NY, USA
   Alex R. B. Thomsen
4. Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA
   Rick Huang, Chuan Hong & Zhiheng Yu
5. Department of Pediatrics, Columbia University Irving Medical Center, New York, NY, USA
   Tara Marcink
6. Center for Host-Pathogen Interaction, Columbia University Irving Medical Center, New York, NY, USA
   Tara Marcink
7. Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA
   Oliver B. Clarke
8. Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY, USA
   Oliver B. Clarke
9. The Irving Center for Clinical and Translational Research, Columbia University Irving Medical Center, New York, NY, USA
   Oliver B. Clarke
10. Proteomics Resource Center, The Rockefeller University, New York, NY, USA
    Søren Heissel & Henrik Molina
11. Structural Biology Initiative, CUNY Advanced Science Research Center, New York, NY, USA
    Danya Ben-Hail, Fadi Samaan & Amedee des Georges
12. The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA
    Venkata P. Dandey & Yong Zi Tan
13. Department of Biochemistry and Molecular Biophysics, Columbia University Irving Medical Center, New York, NY, USA
    Yong Zi Tan
14. Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA
    Jacob P. Mahoney & Roger Sunahara
15. Structural Biology Brussels, Vrije Universiteit Brussels, Brussels, Belgium
    Sarah Triest & Jan Steyaert
16. Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium
    Sarah Triest
17. School of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou, China
    Xin Chen
18. Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA
    Roger Sunahara
19. Department of Chemistry and Biochemistry, City College of New York, New York, NY, USA
    Amedee des Georges
20. Biochemistry and Chemistry PhD Programs, Graduate Center, City University of New York, New York, NY, USA
    Amedee des Georges
21. Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA
    Robert J. Lefkowitz

Authors

1. Anthony H. Nguyen
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2. Alex R. B. Thomsen
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3. Thomas J. Cahill III
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4. Rick Huang
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5. Li-Yin Huang
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6. Tara Marcink
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7. Oliver B. Clarke
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8. Søren Heissel
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9. Ali Masoudi
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10. Danya Ben-Hail
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11. Fadi Samaan
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12. Venkata P. Dandey
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15. Jacob P. Mahoney
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Contributions

A.H.N., A.R.B.T., T.J.C. III, A.d.G. and R.J.L. conceived the project and designed experimental approaches. A.H.N., A.R.B.T., T.J.C. III, L.-Y.H., A.M., J.P.M., J.L. IV and R.S. purified protein for cryo-EM structural determination. A.H.N. prepared cryo-EM samples with contributions from V.P.D. A.H.N., A.R.B.T., T.J.C. III, D.B.-H., F.S. and A.d.G. performed initial sample screenings. R.H. and Z.Y. performed cryo-EM imaging with contributions from C.H. A.H.N. processed cryo-EM data with input from A.d.G. and Y.Z.T. A.H.N. and O.B.C. built the atomic models. S.T. and J.S. raised nanobody 32 and X.C. synthesized BI-167107. S.H. and H.M. performed LC–MS/MS experiments and data analyses. T.M. performed coarse-grained molecular dynamics analysis. A.R.B.T. performed real-time cellular cAMP measurement experiments. A.H.N., A.R.B.T., T.J.C. III, A.d.G. and R.J.L. interpreted the data and wrote the manuscript. R.J.L. and A.d.G. were responsible for project supervision and management.

Corresponding authors

Correspondence toAmedee des Georges or Robert J. Lefkowitz.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Sample preparation and purification of the megaplex.

a, Schematic illustration of the purification and in vitro formation procedure of the megaplex. b, Size exclusion chromatogram of the precursor β2V2R–βarr1–Fab30 complex. c, SDS-PAGE gel of the β2V2R–βarr1–Fab30 complex after purification by size exclusion chromatography. d, SDS-PAGE gel of the megaplex after in vitro formation and M1 anti-Flag purification. For c-d, M denotes molecular weight (kDa) marker. Uncropped gel images for Extended Data Fig. 1c,d are provided as Source Data.

Source data

Extended Data Fig. 2 Nanobody 32 (Nb32) stabilizes the megaplex.

a, Representative micrograph and 2D class averages of megaplex samples prepared without nanobody 32 (Nb32), displaying a small percentage of megaplexes. b, Same as in a, but with a megaplex sample prepared with Nb32.

Extended Data Fig. 4

Data processing workflow for all datasets of the megaplex.

Extended Data Fig. 5 Megaplex consensus reconstruction.

a, Representative 2D class averages of the consensus megaplex reconstruction. b-c, The megaplex reconstruction is shown at high (0.115) threshold (b), and low (0.05) threshold (c). The T4L and flexible portion of the V2T appears at a lower threshold. The atomic models of the components, derived from signal subtracted reconstructions, are fitted to the consensus reconstruction. Densities for the flexible V2T and steric clash between the β2V2R and Nb32 are denoted by black circles.

Extended Data Fig. 6 Orientational distribution and resolution measurements of the megaplex.

a–d, orientational distribution (a), FSC curves indicating overall resolution (FSC = 0.143) (b), 3D-FSC to assess directional resolution anisotropy (c), and local resolution measurements (d) of the megaplex consensus reconstruction. e–j, orientational distribution (e), FSC curves indicating overall resolution (FSC = 0.143) (f), 3D-FSC to assess directional resolution anisotropy (g), map-to-model FSC and sphericity (h), local resolution measurements (i), and map-to-model FSC curve (j) of the β2V2R–Gs reconstruction. k–p, same as e–j, but for the βarr1–V2T reconstruction.

Extended Data Fig. 7 Representative densities in black mesh of various protein components.

Representative densities, from the 3.8 Å β2V2R–Gs and 4.0 Å βarr1–V2T structures, of the β2V2R, Gs subunits, and βarr1.

Extended Data Fig. 8 Representative density of the β2V2R–Gs portion of the megaplex, and comparison against other active β2AR structures.

a, Comparison of the binding pose of BI-167107 (BI) in the megaplex against three other available BI-bound β2AR structures. BI is colored green. b, Representative density showing contacts between the β2V2R and Gs in the megaplex. c, The BI binding pocket within the megaplex, accompanied by EM density for all residues within 5 Å of the ligand.

Extended Data Fig. 9 Interaction between Fab30, V2T and protein stabilizers.

a, b, Interface between βarr1 and V2T with either Nb32 (a) or Fab30 (b). Interface residues are labeled.

Extended Data Fig. 10 Verification of observed phosphorylation sites on the V2T.

a, Cryo-EM density for the six phosphorylated residues on the V2T. b, Localization probabilities of eight potential sites of phosphorylation on the V2T assessed by LC-MS/MS. A trypsin-digested fragment of the V2T is displayed. Bolded residues are phosphorylation sites observed in the cryo-EM map. Residues in red were not observed in the map, and yellow-highlighted residues were phosphorylated in both unstimulated and BI-stimulated receptors.

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Nguyen, A.H., Thomsen, A.R.B., Cahill, T.J. et al. Structure of an endosomal signaling GPCR–G protein–β-arrestin megacomplex.Nat Struct Mol Biol 26, 1123–1131 (2019). https://doi.org/10.1038/s41594-019-0330-y

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• Received: 10 August 2019
• Accepted: 09 October 2019
• Published: 18 November 2019
• Issue Date: December 2019
• DOI: https://doi.org/10.1038/s41594-019-0330-y