Nature | Vol 579 | 12 March 2020 | 297Article
Structure of the M2 muscarinic
receptor–β-arrestin complex in a lipid
nanodisc
Dean P. Staus1,2,1 0, Hongli Hu3,8,1 0, Michael J. Robertson3,4,1 0, Alissa L. W. Kleinhenz1,2,9,
Laura M. Wingler1,2, William D. Capel^1 , Naomi R. Latorraca3,5,6, Robert J. Lefkowitz1,2,7 ✉ &
Georgios Skiniotis3,4 ✉After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit
β-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes
receptor endocytosis^1. Additionally, β-arrestin directly regulates many cell signalling
pathways that can induce cellular responses distinct from that of G proteins^2. In
contrast to G proteins, for which there are many high-resolution structures in
complex with GPCRs, the molecular mechanisms underlying the interaction of
β-arrestin with GPCRs are much less understood. Here we present a cryo-electron
microscopy structure of β-arrestin 1 (βarr1) in complex with M2 muscarinic receptor
(M2R) reconstituted in lipid nanodiscs. The M2R–βarr1 complex displays a
multimodal network of flexible interactions, including binding of the N domain of
βarr1 to phosphorylated receptor residues and insertion of the finger loop of βarr1
into the M2R seven-transmembrane bundle, which adopts a conformation similar to
that in the M2R–heterotrimeric Go protein complex^3. Moreover, the cryo-electron
microscopy map reveals that the C-edge of βarr1 engages the lipid bilayer. Through
atomistic simulations and biophysical, biochemical and cellular assays, we show that
the C-edge is critical for stable complex formation, βarr1 recruitment, receptor
internalization, and desensitization of G-protein activation. Taken together, these
data suggest that the cooperative interactions of β-arrestin with both the receptor
and the phospholipid bilayer contribute to its functional versatility.Activation of GPCRs leads to heterotrimeric G-protein-mediated sig-
nalling that quickly returns to basal levels^1. This remarkably conserved
process of GPCR ‘desensitization’ (Fig. 1a) is mainly orchestrated by
two small families of proteins, GPCR kinases and arrestins (reviewed
in ref.^4 ). GPCR kinases phosphorylate agonist-bound GPCRs on their
carboxyl (C) terminus or intracellular loops (ICLs), leading to the
recruitment of arrestin (Fig. 1a). In humans, visual arrestin (arres-
tin-1) and X-arrestin (arrestin-4) are selectively expressed in the retina,
whereas the ubiquitously expressed β-arrestins 1 and 2 (also known
as arrestin-2 and arrestin-3, respectively) regulate the hundreds of
GPCRs that are found elsewhere. Arrestins comprise juxtaposed N- and
C-terminal seven-stranded β-sandwich domains with a central crest
of three loops (finger, middle and C-loops)^5 ,^6. After the phosphoryl-
ated GPCR C terminus engages the N domain of arrestin^7 ,^8 , conforma-
tional changes promote the binding of central crest elements to the
receptor seven-transmembrane (7TM) bundle, which sterically
blocks the coupling of G proteins^5. β-arrestins also act as adaptors for
endocytic machinery, thereby increasing receptor internalization^9.
As well as modulating desensitization, β-arrestins potentiate many
signalling pathways independently of G proteins^2. Notably, certain
‘biased’ GPCR agonists preferentially activate G-protein or β-arrestin
pathways; this could be exploited therapeutically to obtain more selec-
tive drugs^2 ,^10.
Numerous high-resolution structures of GPCR–G protein complexes
have been obtained, primarily by cryo-electron microscopy (cryo-EM)
(reviewed in ref.^11 ). However, to our knowledge, the only GPCR–arrestin
structure reported so far is a crystal structure of rhodopsin fused to a
constitutively active mutant of visual arrestin^8 ,^12. Therefore, knowledge
of the molecular framework of GPCR–arrestin interactions remains
limited—especially for the β-arrestins, which modulate the vast major-
ity of GPCRs. Here we report the cryo-EM structure of βarr1 in complex
with M2R in high-density lipoprotein (HDL) particles (lipid nanodiscs)
that mimic a native membrane. This structure provides new insights
into β-arrestin-mediated GPCR desensitization and signalling, andhttps://doi.org/10.1038/s41586-020-1954-0
Received: 11 August 2019
Accepted: 7 January 2020
Published online: 16 January 2020
Check for updates(^1) Department of Medicine, Duke University Medical Center, Durham, NC, USA. (^2) Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC, USA. (^3) Department of
Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA.^4 Department of Structural Biology, Stanford University School of Medicine, Stanford, CA,
USA.^5 Department of Computer Science, Stanford University, Stanford, CA, USA.^6 Biophysics Program, Stanford University, Stanford, CA, USA.^7 Department of Biochemistry, Duke
University Medical Center, Durham, NC, USA.^8 Present address: School of Life and Health Sciences, Kobilka Institute of Innovative Drug Discovery, The Chinese University of Hong Kong,
Shenzhen, China.^9 Present address: School of Medicine, University of Michigan, Ann Arbor, MI, USA.^10 These authors contributed equally: Dean P. Staus, Hongli Hu, Michael J. Robertson.
✉e-mail: [email protected]; [email protected]