Structure of an endosomal signaling GPCR–G protein–β-arrestin megacomplex
Authors: Chuan Hong
Li-Yin Huang
John Little 4th
Oliver B Clarke
Fadi Samaan
Jacob P Mahoney
Henrik Molina
Anthony H Nguyen
Jan Steyaert
Tara Marcink
Søren Heissel
Danya Ben-Hail
Zhiheng Yu
Alex R B Thomsen
Sarah Triest
Xin Chen
Rick Huang
Yong Zi Tan
Thomas J Cahill 3rd
Amedee des Georges
Venkata P Dandey
Ali Masoudi
Robert J Lefkowitz
Roger Sunahara
Journal: Nature Structural & Molecular Biology (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: G-protein-coupled receptors; GPCR-G protein-β-arr megaplex; cryo-electron microscopy structure; human G protein; bovine β-arr
Affiliations: Department of Medicine, Duke University Medical Center, Durham, NC, USA., Department of Biochemistry, Duke University Medical Center, Durham, NC, USA., Department of Surgery, Columbia University Irving Medical Center, New York, NY, USA., Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA., Department of Pediatrics, Columbia University Irving Medical Center, New York, NY, USA., Center for Host-Pathogen Interaction, Columbia University Irving Medical Center, New York, NY, USA., Department of Anesthesiology, Columbia University Irving Medical Center, New York, NY, USA., Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY, USA., The Irving Center for Clinical and Translational Research, Columbia University Irving Medical Center, New York, NY, USA., Proteomics Resource Center, The Rockefeller University, New York, NY, USA., Structural Biology Initiative, CUNY Advanced Science Research Center, New York, NY, USA., The National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA., Department of Biochemistry and Molecular Biophysics, Columbia University Irving Medical Center, New York, NY, USA., Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA., Department of Structural Biology, Stanford University, Stanford, CA, USA., Structural Biology Brussels, Vrije Universiteit Brussels, Brussels, Belgium., Structural Biology Research Center, Vlaams Instituut voor Biotechnologie, Brussels, Belgium., School of Pharmaceutical Engineering and Life Sciences, Changzhou University, Changzhou, China., Department of Pharmacology, University of California, San Diego, La Jolla, CA, USA., Structural Biology Initiative, CUNY Advanced Science Research Center, New York, NY, USA. ., Department of Chemistry and Biochemistry, City College of New York, New York, NY, USA. ., Biochemistry and Chemistry PhD Programs, Graduate Center, City University of New York, New York, NY, USA. ., Department of Medicine, Duke University Medical Center, Durham, NC, USA. ., Department of Biochemistry, Duke University Medical Center, Durham, NC, USA. ., Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA. .

Abstract:  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. 

Retracted: No

PubPeer comments:
 This is a very disappointing paper; there is no evidence that a complex containing all these components ever exists under normal physiological conditions OR has any physiological relevance. The complex is completely artificial, stabilized by the V2R tail fusion, 2 nanobodies and an Fab fragment. As such it should be viewed as either completely specious or, at best, an interesting piece of phenomenology. I am drawing attention the lack of evidence for biological relevance of this paper because it has now been recommended on the post-publication peer review site, Faculty Opinions (formerly F1000): 10.3410 f.736929268.793575111 . This is concerning, as the long-standing paradigm that signalling at opioid receptors that was biased away from arrestin signalling would provide clinical benefit while sparing on-target side effects led to a substantial waste in research effort and commercial investment and after almost 20 years has only now begun to be dispelled (see: 10.1111 bph.15004) The standing in the field of the senior authors of this paper may place undue confidence in this new observation, but without confirmation for the underlying biology this phenomenological observation needs to be viewed with much scepticism. , 
 #1 Our unknown critic makes these three points: 1.tu201cWe present no evidence in the paper that this complex forms in cells and is therefore of any physiological relevance.u201d We agree -we presented no such evidence because the purpose of the paper was to structurally illustrate in detail how an active GPCR simultaneously binds and accommodates both a G protein, which engages the receptor core, and a u03b2-arrestin (u03b2arr) bound to the GPCR C-terminal phosphorylated tail. This much we show unequivocally by cryo-EM, which serves as the main contribution of the paper. The physiological role of this GPCRu2013G proteinu2013u03b2arr megaplex has been explicitly demonstrated by our previous publication (Thomsen et al, 2016, Cell), which we prominently cited throughout this structure paper, and is also referenced by the Faculty Opinions referral. This 2016 paper shows extensive cellular data including BRET, confocal microscopy, and real-time cAMP measurement assays that is supported by in vitro evidence that said megaplexes not only form physiologically, but are also capable of activating G proteins. The megaplex explains a physiological observation made by a number of labs over the last decade that certain GPCRs that bind u03b2arr exceptionally well continue to stimulate G protein-signaling after they have been internalized into endosomes (Calebiro et al., 2009, PloS Biol; Feinstein et al., 2013, JBC; Mullershausen et al., 2009, Nat Chem Bio; Wehbi et al., 2013, PNAS). The significance of the megaplex in this mode of sustained signaling has been well demonstrated by multiple labs for various GPCRs (Wehbi et al., 2013, PNAS; Jensen et al., 2017, Sci Transl Med; Yarwood et al., 2017, PNAS; Wanka et al., 2018, Cell Signal). 2. u201cThe complex is completely artificial, stabilized by the V2R tail fusion, 2 nanobodies and a Fab fragmentu201d. Our critic is apparently completely unfamiliar with the structural literature on GPCRs. Virtually all of the many dozens of u201cactive stateu201d structures of GPCRs, either alone or in complex with G proteins or u03b2arr are stabilized by various antibodies-e.g. Nanobodies and or Fabs (often more than one) as well as other protein engineering tricks. While of course everyone would love to be able to solve such structures of these receptors and complexes in their native state this is not currently possible and the present approaches while not perfect nonetheless provide our first views of these structures. 3. The relevance of the criticu2019s final gratuitous and disconnected comment about the clinical utility or lack thereof of biased opioid receptor ligands to the paper under discussion is completely lost on us. , 
 On point 1. Prof. Lefkowitz main argument is that his 2016 Cell paper (Thomsen et al.) has physiological data for existence of the  megaplex . On this point I agree with Polyommatus pljushtchi, that this does not constitute physiological support for this  megaplex . All the experiments in Thomsen et al. are conducted in HEK293 cells using transfection of exogenous DNA. No doubt there are some aspects of a HEK293 cell that reflect normal physiology, however these are a transformed cell-line with substantial and variable anueploidy and all experiments in this paper involve heterologous expression of some sort of fusion protein. None of this is physiological. At best one might call this cell based, but certainly not physiological. On point 2, while it is true that structural data in the GPCR field does use engineering  tricks to maintain stability, this does not detract from the primary criticism by Polyommatus pljushtchi that there is a substantial amount of artificial engineering in order to get this  megaplex to hang together for structural studies. In the previous paper by Thomsen et al. there is preliminary cryo-EM data on this megaplex where the position of the b-arrestin appears to be where the the plasma membrane would lie, to me this further justifies the skepticism articulated by Polyommatus pljushtchi. On point 3, I don t know, but suspect this is a reference to the long-running dogma (promulgated by Prof. Lefkowitz among many others), that bias away from b-arrestin signalling may be a clinical route for improved safety of opioid analgesics. While this is a bit obtuse, I think the point here is that dogma based on phenomenology that is promoted by powerful leaders in the field can be damaging and hold back progress. If this is what was meant, then I agree. Without actual physiological support, the concept of a  megaplex is not particularly helpful. ,