298 | Nature | Vol 579 | 12 March 2020
Article
highlights the importance of the membrane environment in these
processes.
Structural determination of M2R–βarr1
The stabilization of GPCR–β-arrestin complexes has been historically
difficult because of both the need for receptor phosphorylation and
the low strength of the β-arrestin–7TM interaction. To reconstitute a
functional complex we chose M2R, a family A GPCR that regulates car-
diac function^13 , because βarr1 has a relatively strong interaction with its
7TM core^14. To ensure homogeneous phosphorylation, we used sortase
to enzymatically ligate a synthetic phosphopeptide (pp) derived from
the C terminus of vasopressin 2 receptor (V2R) onto the C terminus of
M2R, forming M2Rpp^14 (Extended Data Fig. 1a). Although wild-type M2R
lacks a C terminus with phosphorylatable residues and is phosphoryl-
ated within ICL3, βarr1 enhances the affinity of agonists for M2Rpp to
the same extent as for natively phosphorylated M2R^14 ,^15. To enhance
the stability of βarr1 we generated a minimal cysteine variant truncated
at residue 393 (βarr1(MC-393)), which displayed the same allosteric
coupling to M2R as did the wild type (Extended Data Fig. 1b). Hereafter,
βarr1 refers to this variant unless otherwise specified.
Initially we characterized the ability of βarr1 to bind M2Rpp in
n-dodecyl-β-d-maltoside (DDM-M2Rpp) detergent. βarr1 and the anti-
body fragment Fab30, which stabilizes the βarr1–V2Rpp interaction,
co-immunoprecipitate with the receptor only when it is phosphorylated
(Extended Data Fig. 1c). Because βarr1 binding to the 7TM core of the
receptor enhances agonist binding^14 ,^15 , we monitored this interaction
using radioligand competition binding assays to measure agonist affin-
ity. The affinity of the agonist iperoxo for DDM-M2Rpp is increased by
the positive allosteric modulator LY2119620 but not by βarr1 (Fig. 1b). By
contrast, when M2Rpp is reconstituted into nanodiscs (HDL-M2Rpp)—
which provide a more physiological-like lipid bilayer—both LY2119620
and βarr1 increase the affinity of iperoxo for the receptor (Fig. 1b). The
importance of the lipid bilayer in βarr1–receptor binding was further
tested by labelling the βarr1 finger loop—which inserts into the 7TM
core—with the probe monobromobimane (mBr), the fluorescence of
which increases in hydrophobic environments. For these experiments
we used a labelled mutant with a valine-to-cysteine substitution at posi-
tion 70 (V70C); this variant was shown to still couple to the 7TM core
of M2Rpp by radioligand binding experiments (Extended Data Fig. 1b).
We observed an agonist-dependent increase in the fluorescence of
bimane with HDL-M2Rpp but not with DDM-M2Rpp (Fig. 1c, Extended–12 –10 –8 –60.000.250.500.751.001.25log[Iperoxo, M](^3) [H]NMS
speci c bindingM2RM2R + arr1 DDM a –12 –10 –8 –6 0.00 0.25 0.50 0.75 1.00 1.25 Nanodisc (HDL) GTP GDP βarr Extracellular GPCR Intracellular L G_ a
GRK
b
d
L L L L
βarr
c
90º
M2R
βarr1
Nanodisc
(MSP1D1E3)
C domain
N domain
V2Rpp
V2Rpp
V2Rpp
C domain
N domain
Nanodisc
(MSP1D1E3)
C domain
M2R + LY211
0.00 440460480500520
0.25
0.50
0.75
1.00
1.25
1.50
HDL Ctl
HDL-M2Rpp
DDM Ctl
Relative uor DDM-M2Rpp
escence
Wavelength (nm)
βarr
90º
log[Iperoxo, M]
Fig. 1 | Recruitment of βarr1 by M2R in a native lipid environment. a, Ligand
(L)-induced conformational changes in GPCRs lead to heterotrimeric G-protein
activation (GTP hydrolysis) and subsequent GPCR-kinase-mediated receptor
phosphorylation. The initial binding of β-arrestin (βarr) to phosphorylated
receptors leads to its coupling to the transmembrane (TM) bundle, sterically
occluding further G-protein binding. b, βarr1 allosterically enhances the
affinity of iperoxo to HDL-M2Rpp but not to DDM-M2Rpp, as determined by
competition radioligand binding assays. The positive allosteric modulator
LY211960 (LY211) enhanced iperoxo affinity regardless of the reconstitution
environment. Data are the mean of three independent experiments with error
bars representing s.e.m. * indicates significance compared to control (Ctl)
(one-way ANOVA). logIC 50 values: DDM-Ctl, −7.10 ± 0.09; DDM-βarr1,
−7.03 ± 0.06; DDM-LY211, −8.36 ± 0.04 (P < 0.0001); HDL-Ctl, −7.49 ± 0.08; HDL-
βarr1 −8.29 ± 0.08 (P < 0.0007); HDL-LY211, −9.17 ± 0.05 (P < 0.0001). c, H D L-
M2Rpp, but not DDM-M2Rpp, enhances the f luorescence of bimane in the
finger loop of βarr1 (red star, inset). Curves represent the difference in spectra
obtained with an antagonist (atropine) and an agonist (iperoxo). Data
represent the means of three independent experiments. d, Orthogonal views
of the cryo-EM density map of the HDL-M2Rpp–βarr1 complex coloured by
subunit (orange, M2Rpp; teal, βarr1; grey/white, HDL particle). The orange-
coloured density on βarr1 corresponds to the phosphorylated C-terminal
peptide (V2Rpp) ligated to the receptor. The nanodisc density, omitted in the
middle panel for clarity, was generated earlier in image processing before high-
resolution refinement of the M2Rpp–βarr1 complex.