Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR–G protein–β-arr ‘megaplex’. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (β2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.
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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.
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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.
The authors declare no competing interests.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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. 3 A procedure utilizing Warp and cryoSPARC for initial data processing and cleaning of one representative dataset (Dataset 2).
The same procedure was used on all dataset.
Data processing workflow for all datasets of the megaplex.
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.
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.
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.
a, b, Interface between βarr1 and V2T with either Nb32 (a) or Fab30 (b). Interface residues are labeled.
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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