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302 | Nature | Vol 579 | 12 March 2020

Article

induces conformational changes in β-arrestin, which subsequently
promotes its coupling to the 7TM bundle of the receptor. Our find-
ings, supported by observations of the Rho–Arr1 complex^12 ,^19 and the
NTSR1–βarr1 complex^26 , expand this model to include a critical βarr1–
lipid interaction (Extended Data Fig. 9f ). In this three-site interaction
model, the recruitment of arrestin requires both GPCR phosphorylation
and an interaction of βarr1 with the plasma membrane (Fig. 5b), which
could increase the arrestin concentration at the cell membrane before
receptor activation. The C-edge–membrane interaction subsequently
enhances the binding of arrestin to the 7TM bundle (Fig. 4d) and the
desensitization of G-protein activation (Fig. 5a).
Whereas membrane anchoring may be a conserved property of
arrestins, the mechanisms that underlie membrane association could
be distinct. The C-edge loop buried in the nanodisc is found in visual
arrestin and the dominant form of βarr1, but not in βarr2 or an alternative
βarr1 splice variant. The binding site of phosphatidylinositol-4,5-bis-
phosphate (PtdIns(4,5)P 2 )—a phospholipid observed in the structure of
the NTSR1–βarr1 complex, described in a related study^26 —is conserved
in β-arrestins but not in retinal arrestins^12 ,^19 ,^27 ,^28 (Extended Data Fig. 7).
Accordingly, the variability of membrane compositions could provide
yet another level of regulation for GPCR desensitization, internalization
and signalling. Notably, βarr2 can stimulate MAP kinase signalling from
clathrin-coated structures after dissociation from activated GPCRs^29.
These and other recent data suggest that the lipid membrane stabilizes
the activation of β-arrestin when bound to, and even after dissocia-
tion from, the receptor^21 ,^29 –^31. Thus, as is evident from the structures
of βarr1 bound to M2Rpp or NTSR1^26 , a complex cooperative network
of low-affinity interactions involving both receptor and membrane
phospholipids endow arrestins with the necessary plasticity to variably
couple to hundreds of GPCRs.

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Flag–M2R

Flag–M2R

GFP–

`arr1 3

×D

GFP–

`arr1 WT

Unstimulated 5 min 30 min

a Iperoxo

WT 3 ×D

M2R inter

nalization (%)

βarr1

b

c

M2R-stimulated G-pr

otein

activation (GTP hydr

olysis)

––WT 3 ×D

+ Iperoxo

βarr1

0

1

2

3

4

5

6

0

10

20

30

40

50

Fig. 5 | The functionality of βarr1 depends on C domain–lipid interactions.
a, Activation of purified heterotrimeric Gi protein by iperoxo-stimulated HDL-
M2Rpp in vitro is reduced by wild-type βarr1 but not by βarr1(3×D). Data are the
mean of three independent experiments with error bars representing s.e.m.
P < 0.0001, one-way ANOVA compared to wild-type βarr1 plus iperoxo.
b, Iperoxo stimulation of Flag–M2R causes plasma membrane recruitment of
GFP–βarr1(WT) and subsequent receptor internalization, which is impaired by
the 3×D mutations, as demonstrated by confocal microscopy. βarr1, nuclei and
M2R are coloured green, blue, and red, respectively. Confocal microscopy
images are representative of three independent experiments. c, Quantification
of Flag–M2R internalization by f low cytometry in same cells as b. Data are the
mean of three independent experiments with error bars representing s.e.m.
P = 0.0008, unpaired two-sided t-test compared to wild-type βarr1.