articles

β-arrestin 2 regulates Aβ generation and γ-secretase

activity in Alzheimer’s disease
Amantha Thathiah1,2, Katrien Horré1,2, An Snellinx1,2, Elke Vandewyer1,2, Yunhong Huang1,2,
Marta Ciesielska1,2, Gerdien De Kloe1,3, Sebastian Munck1,2 & Bart De Strooper1,2

b-arrestins are associated with numerous aspects of G protein–coupled receptor (GPCR) signaling and regulation and accordingly
influence diverse physiological and pathophysiological processes. Here we report that b-arrestin 2 expression is elevated in two
independent cohorts of individuals with Alzheimer’s disease. Overexpression of b-arrestin 2 leads to an increase in amyloid-b
(Ab) peptide generation, whereas genetic silencing of Arrb2 (encoding b-arrestin 2) reduces generation of Ab in cell cultures and
© 2013 Nature America, Inc. All rights reserved.

in Arrb2−/− mice. Moreover, in a transgenic mouse model of Alzheimer’s disease, genetic deletion of Arrb2 leads to a reduction
in the production of Ab40 and Ab42. Two GPCRs implicated previously in Alzheimer’s disease (GPR3 and the b2-adrenergic
receptor) mediate their effects on Ab generation through interaction with b-arrestin 2. b-arrestin 2 physically associates with the
Aph-1a subunit of the g-secretase complex and redistributes the complex toward detergent-resistant membranes, increasing the
catalytic activity of the complex. Collectively, these studies identify b-arrestin 2 as a new therapeutic target for reducing amyloid
pathology and GPCR dysfunction in Alzheimer’s disease.

Considerable insight into the genetic and molecular biological basis of
Alzheimer’s disease has yet to be translated into a new medication for
the disease. Removal of tau1,2 or the Aβ peptide3–5 from the brain by
various vaccination strategies, inhibition or modulation of the β- and
γ-secretases, which cleave the β-amyloid precursor protein (APP) to
generate the Aβ peptide, or alterations in apolipoprotein E (APOE)
expression6 are the primary therapeutic avenues that are currently
pursued. Unfortunately, many therapeutic agents have failed at several
stages of development as a result of poor blood-brain barrier penetra-
tion or severe side effects3–5,7. Moreover, new findings suggest the
npg
the potential of Aβ-modulating agents in the early treatment of
Alzheimer’s disease3.
GPCRs, also called seven transmembrane receptors (7TMRs),
comprise the largest family of membrane proteins12 and are the most
common target for therapeutic drugs13. Over 370 nonsensory GPCRs
have been identified14, of which more than 90% are expressed in the
brain, where they have important roles in cognition, mood, appe-
tite, pain and synaptic transmission15. In the context of Alzheimer’s
disease, GPCRs are associated with multiple stages of APP proteo­
lysis, including modulation of processing of APP by the α-, β- and

need for very early symptomatic or presymptomatic treatment, which
will require the identification of alternative disease-modifying targets
to safely modulate the pathogenesis of Alzheimer’s disease3–5,7.
Central to many Aβ-directed therapeutic strategies is the
γ-secretase complex, which is a multimeric aspartyl protease com-
posed of four subunits: presenilin 1 or 2 (PS1 or PS2), nicastrin (NCT),
APH-1A or APH-1B and PEN 2 (ref. 8). The majority of cases of
early onset familial Alzheimer’s disease are attributed to mutations
in the PS1 (Presenilin 1), PS2 (Presenilin 2) and APP genes9, pro-
viding the rationale for the therapeutic targeting of the proteins
they encode. However, familial Alzheimer’s disease accounts for
less than 0.5% of all Alzheimer’s disease cases10. The vast major-
ity of late-onset Alzheimer’s disease cases are sporadic and include
environmental and genetic risk factors, among which the ε4 allele of
APOE accounts for more than 25% of the genetic risk. Interestingly,
APOE4 also seems to modulate Aβ accumulation11, strengthening
γ-secretases and the regulation of Aβ degradation and Aβ-mediated
toxicity16; however, the signaling mechanisms mediating these effects
remain at best only partially understood, and the hypothesized regula-
tion of the γ-secretase complex by GPCRs remains unexplained.
From a drug discovery perspective, GPCRs are very versatile
targets. Drugs that directly target a GPCR have been classically
described as either agonists or antagonists for G protein signaling.
The binding of an agonist to a GPCR promotes a conformational
change that results in the activation of receptor-associated hetero­
trimeric G proteins and consequent downstream signaling. However,
a small family of multifunctional GPCR regulatory or adaptor pro-
teins known as the β-arrestins, which have an almost universal role
in facilitating traditional GPCR desensitization, is also capable
of initiating distinct, independent signaling events17. These signals
are often both spatially and temporally different from the classic
G protein signals and result in unique cellular and physiological or

1Vlaams Instituut voor Biotechnologie (VIB) Center for the Biology of Disease, Leuven, Belgium. 2Center for Human Genetics and Leuven Institute for Neuroscience

and Disease (LIND), University of Leuven (KU Leuven), Leuven, Belgium. 3Neuroscience Department, Johnson & Johnson Pharmaceutical Research and
Development, Janssen Pharmaceutica, Beerse, Belgium. Correspondence should be addressed to A.T. (amantha.thathiah@cme.vib.kuleuven.be) or
B.D.S. (bart.destrooper@cme.vib-kuleuven.be).

Received 17 September; accepted 7 November; published online 2 December 2012; doi:10.1038/nm.3023

nature medicine  VOLUME 19 | NUMBER 1 | JANUARY 2013 43

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a 3.0 β-arrestin 2 b 2.0 β-arrestin 1 c Vector d Vector e Aβ40 f

β-arrestin 2 β-arrestin 2 Aβ42

nonsilencing siRNA (Aβ)

2.0 2.0 1.5

Normalized to vector

Normalized to vector
** ** *

in
Normalized to

st
2.5 1.5 1.5

re
or
1.0
Fold change (relative to control)

ar
ct
Fold change (relative to control)

(Aβ40)

(Aβ42)

Ve

β-
1.5 1.0 1.0 **
2.0 0.5 ** sAPPtotal
0.5 0.5
*** *** ** ***
sAPPα
***
1.5 1.0

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5, le
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5, e
L- ehi 8
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8

β- ilen 2 s A

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45

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ar in
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g h i Non
silencing β-arrestin 2
Vector β-arrestin siRNA siRNA
L-685,458: – – + – – +
Non β-arrestin 2
Control Alzheimer’s Control Alzheimer’s β-arrestin 2
silencing β-arrestin 2 ADAM10
disease disease siRNA siRNA ADAM10
β-secretase
sAPPtotal β-secretase
NCT
sAPPα NCT
PS1-NTF
PS1-NTF
PS1-CTF
Figure 1  Expression of β-arrestin 2 is elevated in individuals with Alzheimer’s disease, PS1-CTF
APH-1A
and overexpression and silencing of β-arrestin 2 differentially regulate Aβ accumulation. APH-1A
PEN 2
(a,b) Expression of β-arrestin 2 (a) and β-arrestin 1 (b) assessed by quantitative PCR (qPCR) in PEN 2
APP-FL
brain samples from control individuals and individuals with Alzheimer’s disease. **P = 0.0089, APP-FL
APP-CTF
© 2013 Nature America, Inc. All rights reserved.

***P = 0.0002 by unpaired Student’s t test. n = 20 control individuals, n = 18 individuals APP-CTF

with Alzheimer’s disease. (c,d) Amounts of Aβ40 (c) and Aβ42 (d) assessed by ELISA in culture β-actin
β-actin
supernatants from HEK293-APP695 cells after overexpression of β-arrestin 2 and treatment
with the γ-secretase inhibitor L-685,458. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way analysis of variance (ANOVA) and Dunnett’s multiple
comparisons post test. n = 4 experiments performed in triplicate. Error bars, s.e.m. (e) Amounts of Aβ40 (black bars) and Aβ42 (red bars) determined
by ELISA after silencing of β-arrestin 2. **P < 0.01 by ANOVA and Dunnett’s post test. n = 5–6 experiments performed in triplicate. Error bars, s.e.m.
(f,g) Amounts of total soluble APP (sAPPtotal) and the α-secretase–mediated cleavage product of APP (sAPPα) from HEK293-APP695 cell culture
supernatants determined by immunoblot analysis after overexpression (f) or silencing (g) of β-arrestin 2. (h) Expression of ADAM10, β-secretase,
the γ-secretase complex, APP-FL and APP-CTF evaluated by Immunoblot analysis after overexpression of β-arrestin 2 and treatment with L-685,458.
(i) Expression of ADAM10, β-secretase, the γ-secretase complex, APP-FL and APP-CTF evaluated by immunoblot analysis after silencing of β-arrestin 2.

pathophysiological consequences18 that can be exploited for the effectively abolished by the addition of L-685,458, a highly selective
therapeutic development of G protein− or β-arrestin–biased drugs. γ-secretase inhibitor22. Furthermore, the release of Aβ40 and Aβ42
Differential regulation of β-arrestins is implicated in type 2 diabetes was not diminished in cells with reduced β-arrestin 1 expression
and psychiatric disorders19,20, for which pharmacological manipula- (Supplementary Fig. 1c–e). In contrast, silencing of β-arrestin 2
tion of selective β-arrestin–dependent complexes may provide thera- efficiently suppressed Aβ secretion to 50% below that of control
peutic benefits21. As mediators of GPCR desensitization, trafficking transfected cells (Fig. 1e). Furthermore, the amounts of total soluble
and cell signaling, the β-arrestins could provide a putative basis to APP and the α-secretase–mediated cleavage product of APP
understand GPCR dysfunction in Alzheimer’s disease. Nevertheless, (Fig. 1f,g) and expression of the α- and β-secretases were unchanged
npg

no study so far has documented a role for β-arrestins in Alzheimer’s after expression or silencing of β-arrestin 2 (Fig. 1h,i), indicating
disease progression. that the modulation of Aβ generation by β-arrestin 2 occurs down-
stream of β-secretase activity. Expression of the NCT, PS1, APH-1A
RESULTS and PEN 2 subunits of the γ-secretase complex was also unaffected
b-arrestin 2 in Alzheimer’s disease and effects on Ab generation by expression or silencing of β-arrestin 2 (Fig. 1h,i). Notably,
We compared the expression of β-arrestins 1 and 2 in samples from accumulation of the APP C-terminal fragment (APP-CTF), which is
the hippocampus and entorhinal cortex of autopsied human brains typically observed after a reduction in γ-secretase activity, was only
with Alzheimer’s disease (N = 18) and age-matched control subjects modestly detectable, raising the question of how β-arrestin 2 affects
(N = 20). Levels of β-arrestin 2 mRNA were significantly increased Aβ generation (Fig. 1h,i).
(Fig. 1a), whereas levels of β-arrestin 1 mRNA were decreased, in To assess the physiological relevance of genetic silencing of
the Alzheimer’s disease samples compared to the control samples β-arrestins 1 and 2, we isolated embryonic neuronal cultures from
(Fig. 1b). We confirmed these observations in an independent group wild-type Arrb1+/+ (encoding endogeneous β-arrestin 1), Arrb2+/+,
of Braak-staged human brain samples (Braak 0–2 compared to Braak Arrb1−/− (ref. 23) and Arrb2−/− (ref. 24) mice (Fig. 2a,b). The amounts
5–6) (Supplementary Fig. 1a,b). Although there are limitations in of endogenous Aβ40 and Aβ42 were substantially reduced in Arrb2−/−
using postmortem tissue, such as mRNA degradation and cellular but not Arrb1−/− neurons, demonstrating that β-arrestin 2 is involved
alterations during disease progression that complicate the interpreta- endogenously in the modulation of neuronal Aβ production.
tion of results, these data suggest that β-arrestins 1 and 2 are differen-
tially regulated in brain areas affected in Alzheimer’s disease. GPCRs regulate Ab generation through b-arrestin 2
We next determined whether the expression of β-arrestins 1 and β-arrestins have traditionally been associated with the termination
2 directly affects Aβ generation in a cellular context. Expression of of GPCR signaling and receptor desensitization 25,26. In parallel,
β-arrestin 2 in the HEK293-APP695 cell line led to a significant β-arrestins can initiate a second set of G protein–independent signals,
increase in Aβ40 and Aβ42 release (Fig. 1c,d), an effect that was resulting in receptor desensitization, endocytosis and various cellular

44 VOLUME 19 | NUMBER 1 | JANUARY 2013  nature medicine

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a b c d e Arrb2+/+ Arrb2–/–
Aβ40 Aβ40 Ad-GFP Ad-GFP
1.5

GPR3
GPR3
1.5 ** * 1.5 0.3

GFP
GFP
Aβ42 Aβ42 Ad-GPR3 Ad-GPR3
NS NS ** * ** **
Normalized value

Normalized value

Aβ42 (ng ml–1)

1.0 Nct

Aβ40 (ng ml–1)

1.0 1.0 0.2
Ps1-CTF
NS Pen 2
NS
0.5 0.5 0.5 0.1 App-FL
App-CTF
GPR3
β-actin
+
–

+
–

–
+
–
–

+
–
–

–
–/

–/
+/

+/

–/
+/
+/
–/

+/
–/
+/

+/

–/
+/
1

1
Ar 1

Ar 1

2
2
2

2
Ar b2

Ar b2
Ar b2

Ar 2

2
2
rb

rb
rb

rb

rb
rb
rb

rb
rb

rb
rb
r

r
r
Ar

Ar

Ar
Ar
Ar

Ar

Ar
Ar
Figure 2  Endogenous Aβ generation is reduced in β-arrestin 2–deficient mice. (a) Amounts of Aβ40 and Aβ42 measured by ELISA in culture supernatant
samples from primary cortical neuronal cultures from wild-type Arrb1+/+ and Arrb1−/− embryonic day 14 fetal mice. Data are normalized to the amount
of Aβ secreted from the wild-type cultures (Arrb1+/+ or Arrb2+/+, respectively). NS, not significant by unpaired Student’s t test. n = 19 Arrb1+/+,
n = 20 Arrb1−/−. Error bars, s.e.m. (b) Amounts of Aβ40 and Aβ42 measured in Arrb2+/− and Arrb2−/− primary neuronal cultures. *P < 0.05, **P < 0.01
by ANOVA and Dunnett’s post test. n = 11 wild-type Arrb2+/+, n = 18 Arrb2+/−, n = 20 Arrb2−/−. Error bars, s.e.m. (c,d) Endogenous release of Aβ40 (c)
and Aβ42 (d) measured in culture supernatant samples from primary cortical neuronal cultures transduced with Ad-GPR3 (red bars) or Ad-GFP control
(black bars). **P < 0.01, NS, not significant by ANOVA and Dunnett’s post test. n = 18 Arrb2+/+, n = 18 Arrb2−/−. Error bars, s.e.m. (e) Expression of
the γ-secretase complex components in primary cortical neuronal cultures, App-FL and App-CTF determined by immunoblot analysis in the absence of
β-arrestin 2 or after transduction with GPR3.
© 2013 Nature America, Inc. All rights reserved.

responses17,27,28. Thus far, two GPCRs have been associated with the in the release of Aβ40 and Aβ42 (Supplementary Fig. 2c). We used
γ-secretase–mediated processing of APP and Aβ generation: GPR3 APP-C99, which is directly cleaved by the γ-secretase to yield Aβ40
(ref. 29) and the β2-adrenergic receptor (β2-AR)30. In addition, the and Aβ42, in these assays, further demonstrating that the β-arrestin
δ-opioid receptor has been implicated in modulation of the β-secretase– 2–mediated effect on Aβ generation is downstream of the α- and
mediated and subsequent γ-secretase–mediated proteolysis of APP31. β-secretases. This result is similar to the previously described effects of
All three GPCRs seem to modulate Aβ generation independently of GPR3 on APP processing29. β2-AR has also been reported to modulate
G protein signaling29–31. Consequently, we hypothesized that the γ-secretase–mediated Aβ release30. Therefore, we used the CHO-K1
β-arrestins could regulate Aβ generation through modulation of their cell line, which stably expresses β-arrestin 2 and the β2-AR, to perform
interaction with these specific GPCRs. We used the CHO-K1 cell line, a similar series of experiments. Silencing of β-arrestin 2 expression
which stably expresses β-arrestin 2 and GPR3, to monitor the direct in this cell line resulted in reduced interaction between β-arrestin 2
interaction between β-arrestin 2 and GPR3 using the PathHunter and the β2-AR after treatment with the β2-AR agonist isoproterenol as
technology32 (Supplementary Fig. 2a). In this cell line, silencing of monitored with the PathHunter assay (Supplementary Fig. 2d), and
β-arrestin 2 expression (Supplementary Fig. 2b) led to a reduction a reduction in Aβ generation (Supplementary Fig. 2e,f).

a b c Aβ40
d e Aβ40
npg

15,000 ** 4.0 Aβ42 10,000 2.5 Aβ42

Aβ generation normalized to C99

Aβ generation normalized to C99

**
*** 8,000 2.0 * ** ** *
Extracellular
3.0 ***
10,000
domain 6,000 1.5
* **
RLU

RLU

2.0
4,000 1.0
Plasma 5,000
membrane
1.0
2,000 0.5

Intracellular
domain
D + e
+ 9
99

D + ne
+ 9
9
D + ne
+ 9
99

se + ne
e 99
99

rin + ne
G T C +C 9
3 R al 9
rin + e
+ 9
99
3 R3 lon
Y 9

Y 9
G G C9 C9

Y 9

e C9
PR G 99 9
se 3 on
e C9
R C
C

3 R3 alo
R C

3 R3 alo
R C
C

3 R3 alo
rin C
C

se 3 lo

C
PR P a

3 PR 9 a
+
G G 99

PR P 9

PR P 9

PR P 9
G T G C9

G T G C9

PR G 9
W C

G T C

P
T

T
W

Figure 3  The C-terminal domain of GPR3 modulates the interaction with β-arrestin 2 and Aβ generation. (a) Schematic representation of GPR3.
The single-letter amino acid residue codes are used. The DRY putative binding site for G proteins is indicated in red and was mutated to AAY (GPR3
DRY/AAY mutant). Putative phosphorylation sites for G protein-coupled receptor kinases (GRKs) in the cytoplasmic tail are indicated in red. These
serine residues were mutated to alanine to obtain the GPR3 serine mutant that does not interact with β-arrestin 2. (b) Interaction of WT GPR3 and the
GPR3 DRY/AAY mutant (GPR3 DRY) with β-arrestin 2 in the CHO-K1 β-arrestin 2 cell line measured with the PathHunter assay as relative light units
(RLU). (c) Generation of Aβ40 (black bars) and Aβ42 (red bars) measured by ELISA in CHO-K1 β-arrestin 2 cells that express APP-C99 (C99) alone,
WT GPR3 + APP-C99 or the GPR3 DRY/AAY mutant + APP-C99. (d) Interaction of WT GPR3 and the GPR3 serine mutant with β-arrestin 2 in the
CHO-K1 β-arrestin 2 cell line measured with the PathHunter assay. (e) Generation of Aβ40 (black bars) and Aβ42 (red bars) measured by ELISA in
CHO-K1 β-arrestin 2 cells that express APP-C99 alone, WT GPR3 + APP-C99 or the GPR3 serine mutant + APP-C99. *P < 0.05, **P < 0.01,
***P < 0.001 by ANOVA and Dunnett’s post test. Data are the mean ± s.e.m. of four to six independent experiments performed in triplicate.

nature medicine  VOLUME 19 | NUMBER 1 | JANUARY 2013 45

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a Vehicle Lactacystin b c d

(normalized to nonsilencing siRNA)

2.0 Vehicle Lactacystin 2.0
ed
** ***

ed
+/+ –/– +/+ –/–

(normalized to Arrb2+/+)
APP-CTF accumulation

APP-CTF accumulation
Arrb2 Arrb2 Arrb2 Arrb2
g

g
in

in
1

2
ct

ct
nc

nc
fe

fe
in

in

in

in
1.5 1.5
st

st

st

st
ns

ns
le

le
re Nct

re

re

re
si

si
ra

ra
ar

ar

ar

ar
on

on
nt

nt
NS
β-

β-

β-

β-
U

N
Ps1-NTF
NCT 1.0 1.0
PS1-NTF Ps1-CTF

PS1-CTF 0.5 0.5

Pen 2
PEN 2
App-FL
APP-FL

–
App-CTF

re 1 NA
2 NA

+/

–/
N
β- rest siR

2
in R
R

2
APP-CTF

rb
rb
st si
si
β-actin

Ar
Ar
ar ing
ar in
β-actin

β- nc
le
si
on
N
JC-8
e Vector β-arrestin 2 f Vector g Input Vector β-arrestin 2 h JC-8 + X
2.5 β-arrestin 2 4
***

Normalized intensity
Normalized intensity (to vector)
* *

β-arrestin 2
Fractions:
10
11
12
13

10
11
12
13
1
2
3
4
5
6
7
8
9

1
2
3
4
5
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7
8
9

JC-8 + X

JC-8 + X

(to vector)
NCT 2.0

Vector
* *

JC-8

JC-8
β-arrestin 2 2
PS1-NTF 1.5 ***
PS1-CTF 1
Raft PS1-CTF
APH-1A 1.0
PEN 2

or

2
Non-raft PS1-CTF

in
ct
Caveolin-1 0.5

st
Ve

re
© 2013 Nature America, Inc. All rights reserved.

GM130

ar
β-
EEA1
T
TF

2
C

-1

N
C
N

PE
1-

AP
PS

Figure 4  β-arrestin 2 and the active γ-secretase complex are enriched in DRMs. (a) Immunoblot analysis of the γ-secretase complex and APP-FL
in HEK293-APP695 cells after silencing β-arrestin 1 or 2, treatment with lactacystin or both. (b) Quantification of APP-CTF accumulation after
lactacystin treatment normalized to nonsilencing siRNA. n = 3 independent experiments performed in triplicate. (c) Immunoblot analysis of the
γ-secretase complex and App-FL in mouse neuronal cultures after treatment with vehicle or lactacystin. (d) Quantification of APP-CTF accumulation
after lactacystin treatment normalized to Arrb2+/+ neuronal cultures. n = 3 independent experiments with four to six brain cultures per experiment.
(e) Sucrose gradient fractionation of HEK293 cells transfected with empty vector or β-arrestin 2–GFP complementary DNA (cDNA). Equivalent volumes
were assessed by immunoblot using antibodies that recognize γ-secretase complex subunits, β-arrestin 2 or the organelle-specific markers GM130
(cis-Golgi), EEA1 (early endosomes) and Caveolin-1 (caveolae). (f) Quantification of the expression of the γ-secretase complex subunits in pooled
fractions 3 and 4 from HEK293 cells transfected with empty vector or β-arrestin 2–GFP cDNA normalized to Caveolin 1 expression. n = 4–6
independent experiments. (g) Immunoblot analysis using a PS1-CTF–specific antibody of pooled raft and non-raft fractions incubated with JC-8
or JC-8 + L-685,458 (JC-8 + X) followed by photoaffinity crosslinking. (h) Quantification of the expression of the PS1-CTF in the raft and non-raft
fractions normalized to input. n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant by ANOVA and the Dunnett’s
post test. Error bars, s.e.m.

To genetically establish the requirement of β-arrestin 2 for the cellular responses33–35 (Fig. 3a). The GPR3 DRY/AAY mutant did
npg

modulation of Aβ generation by GPR3, we transduced wild-type
Arrb2+/+ and Arrb2−/− neuronal cultures with a GPR3 adenoviral
vector (Ad-GPR3). Notably, expression of Ad-GPR3 in the Arrb2−/−
neuronal cultures failed to increase Aβ generation, in contrast to the
elevation in Aβ generation observed in Arrb2+/+ neuronal cultures
(Fig. 2c,d). After GPR3 transduction, GPR3 expression was equiva-
lent in the Arrb2+/+ and Arrb2−/− neuronal cultures, and expression
of the γ-secretase complex components and APP was unaffected by
the absence of β-arrestin 2 or transduction with GPR3 (Fig. 2e).
Consistent with our previous work29, overexpression of GPR3 resulted
in a modest decrease in the amount of APP-CTF, whereas deficiency
of Arrb2 tended to increase accumulation of APP-CTF (Fig. 2e).
The C terminus of GPR3 is required for enhanced Ab generation
We further investigated the mechanism of the β-arrestin 2–mediated
effect on Aβ generation and γ-secretase activation by generating
mutations in GPR3 that alter either G protein activation or β-arrestin
recruitment29. Alanine substitution of the first two amino acids of
a conserved motif in the second intracellular loop of GPR3 (the
AspArgTyr (DRY) motif mutated to AlaAlaTyr (AAY), termed here
the GPR3 DRY/AAY mutant) renders a GPCR incapable of activating
G proteins while retaining its ability to activate β-arrestin–mediated
not activate G protein signaling, as measured by reduced intracel-
lular cyclic AMP (cAMP) generation (Supplementary Fig. 3). The
expression and cellular distribution of the GPR3 DRY/AAY mutant
were similar to those of wild-type (WT) GPR3 (data not shown),
establishing the involvement of this conserved motif in the activation
of GPR3. Notably, mutation of the DRY motif also led to an increase
in β-arrestin 2 recruitment to GPR3 (Fig. 3b) and release of Aβ40
and Aβ42 (Fig. 3c).
We then mutated conserved serine residues in the C terminus of
GPR3, which could serve as putative binding sites for β-arrestin 2
(Fig. 3a). The GPR3 serine mutant was defective in its ability to inter-
act with β-arrestin 2, as measured with the PathHunter complementa-
tion assay (Fig. 3d). The expression and cellular distribution of the
GPR3 serine mutant and WT GPR3 were similar (data not shown).
Mutation of these serine residues also resulted in a reduction in
the generation of Aβ40 and Aβ42 (Fig. 3e), providing validation that
β-arrestin 2 recruitment to GPR3 is required for Aβ generation.
Localization of b-arrestin 2 and the g-secretase complex in DRMs
The experiments described above indicate that the effect of β-arrestin 2
on Aβ generation is downstream of cleavage of APP by the α- and
β-secretases. Previous observations also indicated that both GPR3

46 VOLUME 19 | NUMBER 1 | JANUARY 2013  nature medicine

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a IP: Ps1-CTF IP: Aph-1a

c Input Bound
e IP: Aph-1a IP: β-arrestin 2
Input Unbound Bound Input Unbound Bound Input Bound Input Bound

2
tin
Ps1 Control Ps1 Control Aph-1a Control Aph-1a Control

2 –/–

2 –/–

2 –/–

2 –/–
s
l

3 –/–

3 –/–

3 –/–

3 –/–
tro

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rb

rb

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pr

pr

pr
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β-

Ar

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Ps1-NTF Nct
Nct
Ps1-CTF Aph-1a
Ps1-NTF
Aph-1a β-arrestin 2
Pen 2 Ps1-CTF

β-arrestin 2 Aph-1a

β-arrestin 2

b IP: Ps1-CTF IP: Aph-1a

d Input Bound
f IP: Aph-1a IP: β-arrestin 2
Input Unbound Bound Input Unbound Bound Input Bound Input Bound

2
in
st
Ps1 Control Ps1 Control Aph-1a Control Aph-1a Control

l
tro

/–

/–
re

3 –/–

3 –/–
–/

–/
2 –

2 –
–/

–/
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on

2
3

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Nct IP:

rb

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pr

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Ps1-NTF Nct Nct
Ps1-CTF Aph-1a
Ps1-NTF
Aph-1a β-arrestin 2
Ps1-CTF
Pen 2
β-arrestin 2 Aph-1a
© 2013 Nature America, Inc. All rights reserved.

β-arrestin 2

Figure 5  β-arrestin 2 interacts with the Aph-1a subunit of the γ-secretase complex. (a,b) Immunoprecipitation (IP) of cell lysates from the N2a
neuroblastoma cell line after extraction in 1% CHAPSO-containing (a) or 1% TX-100–containing (b) buffer with antibodies to Ps1-CTF (left), Aph-1a
(B80.3; right) or negative control antibodies and immunoblot analysis with the indicated antibodies to the γ-secretase complex subunits or β-arrestin 2.
(c,d) Immunoprecipitation of cell lysates from the N2a neuroblastoma cell line after extraction in 1% CHAPSO-containing (c) or 1% TX-100–containing
(d) buffer with β-arrestin 2–specific or negative control antibodies and immunoblot analysis with the indicated antibodies to the γ-secretase complex
subunits or β-arrestin 2. (e,f) Immunoprecipitation of cortical brain samples from WT, Gpr3−/− and Arrb2−/− mice after extraction in 1% CHAPSO-
containing (e) or 1% TX-100–containing (f) buffer with antibodies to Aph-1a (B80.3; left) or β-arrestin 2 (right) and immunoblot analysis with the
indicated antibodies to the γ-secretase complex subunits or β-arrestin 2.

and β2-AR affect γ-secretase–mediated proteolysis of APP29,30. Classic
inhibition of γ-secretase activity36 leads to accumulation of APP-CTF;
however, genetic silencing of β-arrestin 2 leads to only a limited or
barely observable accumulation of APP-CTF (Figs. 1i and 4). To
determine whether β-arrestin 2 affects turnover of the APP-CTF,
we treated HEK293-APP695 cells (after silencing β-arrestin 2) and
Arrb2−/− neuronal cultures with the proteasome inhibitor lactacystin
npg
We then determined whether increased localization of the indi-
vidual γ-secretase subunits in DRMs also coincided with the presence
of a more active γ-secretase complex in DRMs. JC-8 is a photoreactive,
biotinylated derivative of the highly specific and potent transition-
state analog inhibitor L-685,458 (ref. 43) that only binds the cata-
lytically active γ-secretase complex44. We combined DRM fractions
2–4 and the non–lipid raft fractions (pooled fractions 9–12) from

and found an accumulation of the APP-CTF under these conditions
(Fig. 4a–d), suggesting that downregulation of β-arrestin 2 leads not
only to inhibition of γ-secretase activity but also to increased APP-
CTF turnover through the proteasome. Given the complexity of the
signaling pathways modulated by the β-arrestins, an effect on Aβ
generation at multiple levels is not surprising. Nevertheless, from a
therapeutic point of view, stimulation of APP-CTF turnover could
be advantageous given that accumulation of the APP-CTF correlates
with cognitive deficits in mice37.
Previous studies indicated that GPCRs, the γ-secretase complex
and Aβ generation are localized in DRMs29,38–42. Therefore, we
assessed the distribution of the γ-secretase complex subunits and
β-arrestin 2 in lipid raft and non–lipid raft domains by differential
flotation after sucrose density gradient centrifugation. The γ-secretase
complex subunits co-distributed in low-density fractions 3 and 4.
Notably, GPR3 (ref. 29) and β-arrestin 2 also accumulated in
DRMs (Fig. 4e). Moreover, overexpression of β-arrestin 2 (Fig. 4e,f),
led to an enrichment of the γ-secretase subunits NCT, PS1-CTF,
APH-1A and PEN 2 in the detergent-resistant, buoyant fractions
(Fig. 4f). In contrast, silencing of β-arrestin 2 led to an appreciable
decrease in the distribution of the γ-secretase complex in DRMs
(Supplementary Fig. 4a,b).
empty vector–transfected control and β-arrestin 2–transfected cells.
We normalized the fractions for γ-secretase expression and incubated
these fractions with JC-8. We used unlabeled L-685,458 as a control
to compete with the biotinylated inhibitor and photoactivation to
crosslink the biotinylated inhibitor to the active γ-secretase complex45.
Subsequent recovery of the biotinylated polypeptides revealed that the
photoprobe readily labeled the PS1-CTF in the lipid raft DRM frac-
tions (Fig. 4g). We were not able to detect labeling in the non–lipid
raft fractions (Fig. 4g). Overexpression of β-arrestin 2 resulted in a
twofold to threefold increase of the active γ-secretase complex pool
in the DRMs (Fig. 4h).
b-arrestin 2 interacts with the Aph-1a g-secretase subunit
To determine whether β-arrestin 2 physically associates with
the γ-secretase complex, we performed coimmunoprecipitation exper-
iments in untransfected N2a neuroblastoma cells. The γ-secretase
subunits Nct, Ps1, Aph-1a and Pen 2 coimmunoprecipitated with
β-arrestin 2 in a 3-[(3-cholamidopropyl) dimethylammonio]-
2-hydroxy-1-propanesulfonate (CHAPSO)-containing buffer (Fig. 5a),
which maintains the integrity of the intact γ-secretase complex46,47.
In contrast, the detergent Triton X-100 (TX-100) dissociates the
γ-secretase complex48. In the presence of TX-100, β-arrestin 2 only

nature medicine  VOLUME 19 | NUMBER 1 | JANUARY 2013 47

 Articles
a b
APP/PS1 Arrb2+/+
+/–
APP/PS1 Arrb2–/–
APP/PS1 Arrb2
(to APP/PS1 Arrbb2+/+)
(to APP/PS1 Arrbb2+/+)
1.5
Aβ42 normalized value
Aβ40 normalized value
1.0
**
0.5 * 0.5
+ ***
s
pu
te
or
am
C
oc
p
ip
ip
H
Figure 6  β-arrestin 2 contributes to Aβ generation in an Alzheimer’s
disease transgenic mouse model. (a,b) Hippocampal and cortical
concentrations of soluble Aβ40 (a) and Aβ42 (b) in 3-month-old APP/PS1
transgenic mice crossed with wild-type Arrb2+/+, Arrb2+/− or Arrb2−/− mice
determined by ELISA. *P < 0.05 relative to Arrb2+/+, +P < 0.01 relative
to Arrb2+/+ for hippocampal Aβ40; **P < 0.001 relative to Arrb2+/+,
***P < 0.0001 relative to Arrb2+/+ for cortical Aβ40; *P < 0.05 relative
to Arrb2+/+, +P < 0.01 relative to Arrb2+/+ for hippocampal Aβ42;
**P < 0.005 relative to Arrb2+/+,++P < 0.001 relative to Arrb2+/+ for
cortical Aβ42 by ANOVA and Dunnett’s post test. n = 6 independent
female mice per cross. Error bars, s.e.m. (c) Immunoblot of the expression
© 2013 Nature America, Inc. All rights reserved.
of the γ-secretase complex components and APP in brain samples from
APP/PS1 Arrb2+/+, APP/PS1 Arrb2+/− and APP/PS1 Arrb2−/− mice.
coimmunoprecipitated with Aph-1a (Fig. 5b). In the reciprocal
assay, the interaction between β-arrestin 2 and the intact γ-secretase
complex was preserved in a CHAPSO-containing buffer (Fig. 5c);
however, β-arrestin 2 only coimmunoprecipitated with the Aph-1a
subunit (Fig. 5d) in the presence of a TX-100 solubilization
buffer. We confirmed this interaction in vivo, showing that in the
presence of either CHAPSO (Fig. 5e) or TX-100 detergent (Fig. 5f),
β-arrestin 2 coimmunoprecipitated with the Aph-1a subunit of
the γ-secretase complex in extracts of cortical brain samples from WT
but not Gpr3−/− or Arrb2−/− mice. Furthermore, we observed colocali-
zation of β-arrestin and APH-1A after coexpression of β-arrestin 2
and APH-1A in HeLa cells (Supplementary Fig. 5). Collectively,
these data suggest that expression of β-arrestin 2 regulates Aβ gen-
eration through interaction with the γ-secretase complex and redis-
npg
tribution and accumulation of the active γ-secretase complex in
DRM domains.
Genetic deletion of b-arrestin 2 reduces Ab generation
We then established the in vivo consequence of the absence of
β-arrestin 2 on Aβ generation in the APP/PS1 transgenic mouse
model for Alzheimer’s disease. This model coexpresses two familial
Alzheimer’s disease–linked mutations, APP with the KM670/671NL
‘Swedish’ mutation and PS1 with the L166P mutation49. Both het-
erozygosity and complete genetic ablation of β-arrestin 2 expression
resulted in a marked reduction in Aβ40 and Aβ42 generation in the
hippocampus and cortex of 3-month-old APP/PS1 Arrb2+/− and
APP/PS1 Arrb2−/− mice (Fig. 6a,b) with no effects on the expression
of NCT, PS1 or APP-FL (Fig. 6c), providing in vivo evidence for
the involvement of endogenous β-arrestin 2 in Aβ generation in an
Alzheimer’s disease mouse model.
DISCUSSION
β-arrestins are scaffolding proteins that are intimately involved
in numerous aspects of GPCR signaling and regulation. Here we
present evidence implicating this class of proteins in the regulation
of γ-secretase proteolytic activity and Aβ generation, with potential
48 VOLUME 19 | NUMBER 1 | JANUARY 2013  nature medicine
c implications for the pathogenesis of Alzheimer’s disease. β-arrestin 2
–
+/
+/
–/
APP/PS1 Arrb2+/+
2
2
2
induces the redistribution of an inactive γ-secretase complex
rb
rb
rb
+/–
APP/PS1 Arrb2
Ar
Ar
Ar
APP/PS1 Arrb2–/–
1
1
1
toward a DRM-associated, active γ-secretase pool through direct
PS
PS
PS
P/
P/
P/
1.5
interaction with Aph-1a. We corroborated the overexpression studies
AP
AP
AP
NCT
1.0
PS1-NTF
with several loss-of-function experiments that confirmed the overall
* ** PS1-CTF effects of β-arrestin 2 on γ-secretase–mediated Aβ generation.
++ APP-FL Our data also suggest additional mechanisms, including increased
+ APP-CTF APP-CTF turnover, by which APP metabolism is affected by
β-actin
β-arrestin 2 downregulation.
x
pu
te
or
am
This study provides insight into the mechanism of γ-secretase regu-
C
oc
lation by GPR3 (ref. 29) and β2-AR30, two GPCRs that have previously
been implicated in the pathogenesis of Alzheimer’s disease. Most nota-
bly, we have determined that β-arrestins are aberrantly expressed in
the brain of individuals with Alzheimer’s disease and genetic deletion
of β-arrestin 2 reduces the accumulation of endogenous mouse Aβ.
Furthermore, in an Alzheimer’s disease mouse model, we have shown
that β-arrestin 2 is also involved in Aβ generation, which provides
evidence for the therapeutic potential of β-arrestins in Alzheimer’s
disease. Ligands that show bias for either G protein–mediated
(G protein–biased) or β-arrestin–mediated (β-arrestin–biased) sig-
naling are being intensively investigated because they could selectively
promote beneficial signaling and even block or negate detrimental or
unwanted actions of receptor activation (for example, side effects, toxicity
or tolerance). Recently, the GPCR M3-muscarinic receptor–dependent
regulation of learning and memory has been shown to require receptor
phosphorylation and β-arrestin recruitment independent of G protein
signaling50. These data suggest that the development of biased ligands
could be beneficial for both learning and memory and, potentially, the
treatment of cognitive disorders such as Alzheimer’s disease.
Arrb2−/− mice develop normally in the absence of an apparent
Notch-deficiency phenotype. Instead, they show increased analge-
sia in response to morphine24 because of misregulated internali-
zation and desensitization of the µ-opioid receptor51. The current
study indicates that APP-CTF does not accumulate in APP/PS1
Arrb2−/− mice, which has been suggested to occur after treatment
with certain γ-secretase inhibitors37. Therefore, a physiologically
relevant regulatory mechanism of the modulation of Aβ generation
by the γ-secretase, potentially mediated through β-arrestin 2, could
be beneficial in preventing the adverse side effects associated with
direct γ-secretase inhibition, such as interference with Notch signal-
ing52 or APP-CTF accumulation37. As it becomes increasingly evident
that presymptomatic and/or very early symptomatic treatments are
necessary to prevent the onset of dementia, the work here suggests
a previously unexplored avenue involving β-arrestin 2 inhibition for
therapeutic intervention and prevention in Alzheimer’s disease.
Methods
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary information is available in the online version of the paper.
Acknowledgments
We are grateful to R.J. Lefkowitz and S. Ahn (Duke University Medical Center,
Durham, North Carolina, USA) for the generous gift of the β-arrestin 2 wild-type
and knockout mouse embryonic fibroblasts, the Arrb1−/− and Arrb2−/− mice,
the β-arrestin 2–GFP-Flag cDNA and helpful discussion. We thank M. Jucker
(University of Tübingen, Germany) for the gift of APP/PS1 transgenic mice. We
greatly appreciate the kind gift of human control and Alzheimer’s disease brain
samples from K. Bossers and D.F. Swaab (Netherlands Institute for Neuroscience,
Amsterdam, The Netherlands) and C. Troakes (the London Neurodegenerative
Diseases Brain Bank, London, UK). We thank M. Mercken (Johnson & Johnson

Pharmaceuticals Research and Development, Beerse, Belgium) for the antibodies
to Aβ. We are grateful to Y. Li (Memorial Sloan Kettering Cancer Center, New
York, USA) for the kind initial gift of JC-8. This work was supported by a Mentored
New Investigator Research grant from the Alzheimer’s Association to A.T., the
Fund for Scientific Research Flanders, KU Leuven, a Methusalem grant from
the KU Leuven and the Flemish government, and the Foundation for Alzheimer
Research (SAO/FRMA) to B.D.S. B.D.S. is the Arthur Bax and Anna Vanluffelen
chair for Alzheimer’s disease.
AUThOR CONTRIBUTIONS
A.T. and B.D.S. designed the experiments and wrote the manuscript. A.T., K.H.,
A.S., E.V. and Y.H. conducted the experiments. M.C. conducted the qPCR
experiments. G.D.K. synthesized JC-8. S.M. conducted the immunofluorescence
image analysis.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the online
version of the paper.
Published online at http://www.nature.com/doifinder/10.1038/nm.3023. 32. Olson, K.R. & Eglen, R.M. β galactosidase complementation: a cell-based
Reprints and permissions information is available online at http://www.nature.com/
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 ONLINE METHODS
Reagents. Unless otherwise indicated, chemicals were purchased from
Sigma-Aldrich.
Antibodies and compounds. Rabbit polyclonal antibodies to human PS1-NTF
(B14.5, 1:1,000), mouse PS1-NTF (B19.2, 1:5,000), APH-1AL (B80.3, 1:1,000),
PEN 2 (B126.2, 1:1,000) and the APP C terminus (B63.5, 1:5,000) and the
monoclonal antibody 9C3 (1:3,000) directed against the C terminus of NCT
have been previously described and were generated in house53,54. ADAM10
was detected using a polyclonal antiserum (B42.1, 1:1,000) generated against
the 17 C-terminal amino acid residues of ADAM10 and was generated in house.
Antibodies to the following were purchased: Flag (M2, Sigma, 1:1,000), 22C11
(Chemicon, 1:1,000), 6E10 (Sigma-Aldrich, 1:1,000), β-secretase (D10E5,
Cell Signaling, 1:1,000), PK (DiscoveRx, 1:500), β-arrestin (BD Biosciences,
1:500), β-arrestin 2 (Cell Signaling, 1:250), β-arrestin 1/2 (Santa Cruz, 1:500),
Myc (9E10, 1:1,000), PS1-NTF (MAB1563, Chemicon), PS1-CTF (MAB5232,
Chemicon), hemagglutinin (HA) (3F10, Roche), caveolin-1 (Santa Cruz),
calnexin, GM130 and EEA1 (Transduction lab) and β-actin (Sigma). L-685,458
was purchased from Calbiochem.
Plasmid construction. The human GPR3 plasmid was purchased from
DiscoveRx. All mutations in GPR3 were generated with the XL Site-Directed
Mutagenesis Kit (Stratagene) and confirmed by DNA sequence analysis. The
© 2013 Nature America, Inc. All rights reserved.
β-arrestin 2–GFP-Flag plasmid was the kind gift of R.J. Lefkowitz (Duke
University Medical Center, Durham, North Carolina, USA).
Cell lines. The CHO-K1, CHO-K1 GPR3 and CHO-K1 ADRB2 β-arrestin cell
lines were purchased from DiscoveRx. The WT HEK293, N2a and HeLa cell
lines were purchased from American Type Culture Collection.
Mice. The Arrb1−/− and Arrb2−/− mice (C57BL/6J background) were kindly
provided by R.J. Lefkowitz (Duke University Medical Center, Durham, North
Carolina, USA). Wild-type C57BL/6J mice or littermate mice were used as
controls for all of the experiments. The APP/PS1 transgenic mice (C57BL/6J
background) were the kind gift of M. Jucker (University of Tübingen, Tübingen,
Germany)49. The APP/PS1 transgenic mice were crossed with Arrb2+/− and
Arrb2−/− mice to obtain APP/PS1 Arrb2+/− and APP/PS1 Arrb2−/− mice,
respectively. Only female mice were used for the studies. The mouse studies
were approved by ethical committees of Leuven University and UZ Leuven
(LA1210231).
qPCR. qPCR was performed with the SYBR Green PCR Master Mix (Exiqon)
npg
and the LightCycler 480 Real-Time PCR System (Roche Applied Science)
following the manufacturer’s protocol. The primers for human β-arrestin 1
used were 5′-TGTTGAGGGAAGGTGCCAACCG-3′ and 5′-GATGCAAGA
TCTCCCAACAGGCCG-3′. The primers for human β-arrestin 2 used were
5′-CCTGTAGATGGCGTGGTGCTTG-3′ and 5′-CCAGGTCTTCACGGCCA
TAGCG-3′. The housekeeping primers used were to hypoxanthine guanine
phosphoribosyl transferase (HPRT) (5′-TGACACTGGCAAAACAATGCA-3′
and 5′-GGTCCTTTTCACCAGCAAGCT-3′), ribosomal protein L13a (RPL13A)
(5′-CCTGGAGGAGAAGAGGAAAGAGA-3′ and 5′-TTGAGGACCTCTG
TGTATTTGTCAA-3′) and β2 microglobulin (β2M) (5′-TGCTGTCTCCA
TGTTTGATGTATC-3′ and 5′-TCTCTGCTCCCCACCTCTAAGT-3′).
Neuronal cultures. Primary mouse cortical or hippocampal cultures were estab-
lished from the brains of embryonic day 14 or 17 fetal mice, respectively, as
previously described55. Briefly, the dissected brain cortices or hippocampi were
suspended in HBSS supplemented with 0.25% trypsin and incubated at 37 °C
for 15 min. The tissues were then transferred to HBSS supplemented with 10%
(v/v) horse serum and dissociated by repeated trituration. The dispersed cells
were counted and plated on poly-d-lysine–coated six-well cell culture plates
in Neurobasal medium supplemented with B27 (Invitrogen). Forty-eight to
72 h after plating, the cells were transduced with the recombinant adenoviral
vectors for 24 h. The infection medium was replaced with fresh Neurobasal
medium, and cells were maintained in culture for an additional 24 h. Cell culture
supernatants were collected, centrifuged for 10 min at 800g and used for ELISA
nature medicine
­ easurements. Cells were harvested in 1× PBS containing complete protease
m
inhibitors (Roche), centrifuged at 800g for 10 min and lysed in 150 mM NaCl,
50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4,
and 1% TX-100 supplemented with complete protease inhibitors for 30 min at
4 °C. After centrifugation at 16,000g for 15 min, the cleared cell extracts were
separated on 4–12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose
membranes, blocked and probed with the indicated antibodies for western blot
analysis. Immunodetection was performed using horseradish peroxidase (HRP)-
coupled secondary antibodies (Bio-Rad) and the chemiluminescent detection
reagent Renaissance (PerkinElmer Life Sciences).
Patient samples. Frozen hippocampal and entorhinal cortex tissue samples
were obtained from the London Neurodegenerative Diseases Brain Bank. These
samples were pathologically confirmed but not further categorized according
to Braak stage. The second cohort of patient samples consisted of snap-frozen
human medial frontal gyrus brain samples obtained from the Netherlands Brain
Bank, Amsterdam (NBB). For these samples, individual, neuropathological
reports, including Braak staging for neurofibrillary changes (NFC) and neuritic
plaques56, and clinical reports were available. For each of the six Braak stages,
seven individuals were included. Furthermore, seven individuals without any
NFC pathology were included as Braak stage 0. Samples were matched as closely
as possible for sex, age, postmortem interval, cerebrospinal fluid pH and APOE
genotype. Only samples with a relatively high RNA integrity number (>7) were
included. Tissue dissection was performed as previously described57. Total RNA
was isolated from both the London and the Netherlands Brain Bank samples
using a combination of TRIzol-based and mirVana RNA isolation methods.
Briefly, samples were homogenized in ice-cold TRIzol (Life Technologies). After
phase separation by the addition of chloroform, the aqueous phase was mixed
with an equal volume of 70% RNase-free ethanol. Samples were then applied to
a mirVana filter cartridge (Ambion/Life Technologies) and processed according
to the manufacturer’s instructions. RNA yields and purities were determined
using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies).
RNA integrity was determined by the RNA integrity number as measured by
the Agilent 2100 bioanalyzer (Agilent Technologies). All brain samples were col-
lected according to legislation and ethical boards of the respective Brain Banks.
The human study was evaluated and approved by ethical committees of Leuven
University and UZ Leuven (ML5919).
Ab ELISA screen. Ninety-six-well plates were coated and incubated overnight
with the capture antibodies previously described29 and detected using the HRP-
labeled detection antibodies29. Brains of the mice with the respective genotypes
of the ages indicated (±2 weeks) were dissected after transcardial perfusion with
ice-cold PBS. The hippocampus and the cerebral cortex were removed separately
and homogenized in Tissue Protein Extraction reagent (Pierce) supplemented
with complete protease inhibitor and phosphatase inhibitor tablets (Roche
Applied Science). The homogenized samples were briefly sonicated to shear the
DNA and centrifuged at 4 °C for 1 h at 100,000g. The supernatant was used for
immunoblot analysis and Aβ ELISA measurements. Alternately, the Aβ peptides
were allowed to accumulate in the absence of serum in the culture supernatants
from HEK293, HEK293-APP695, CHO-K1 GPR3 β-arrestin or CHO-K1 ADRB2
β-arrestin cells and neuronal cultures for 16–18 h before Aβ40 and Aβ42 ELISA
analysis of the culture supernatant samples.
siRNA-mediated knockdown experiments. The CHO-K1 GPR3 β-arrestin,
CHO-K1 ADRB2 β-arrestin or WT HEK293 cell lines were transfected with
siRNA directed against β-arrestin 1 or β-arrestin 2 or with control siRNA
using GeneSilencer (Genlantis) according to the manufacturer’s instruc-
tions as described27. Chemically synthesized, double-stranded siRNAs with
19-nucleotide duplex RNA and 2-nucleotide 3′-dTdT overhangs were pur-
chased from Qiagen. The siRNA sequences targeting human β-arrestin 1
and β-arrestin 2 were 5′-AAAGCCUUCUGCGCGGAGAAU-3′ and 5′-AAG
GACCGCAAAGUGUUUGUG-3′, corresponding to positions 439–459 and
148–168 relative to the start codon, respectively. A nonsilencing RNA duplex
(5′-AAUUCUCCGAACGUGUCACGU-3′) was used as a control. Briefly, the
cells were seeded on six-well plates at a cell density of 250,000 cells per well. One
day after seeding, the cells were transfected with the siRNAs. Two days after
doi:10.1038/nm.3023
 seeding, the cells were infected with an adenoviral vector expressing enhanced
GFP (eGFP) or APP-C99. On day 5, the medium was refreshed with serum-free
medium, and the cells were allowed to accumulate Aβ peptides in the condi-
tioned medium for 16–18 h before analysis of the culture supernatant samples
with the ELISA described above.
b-arrestin assay. The CHO-K1 GPR 3 β-arrestin or CHO-K1 ADRB2 β-arrestin
cell lines were seeded on 96-well plates at a cell density of 20,000 cells per well.
One day after seeding, cells were transfected with the siRNAs as described above.
Two days after seeding, the cells were infected with an adenoviral vector express-
ing eGFP or APP-C99. On day 5, the medium was refreshed with serum-free
medium, and the cells were allowed to accumulate Aβ peptides in the condi-
tioned medium for 24 h before analysis with the PathHunter β-arrestin assay
from DiscoveRx according to the manufacturer’s protocol.
Coimmunoprecipitation experiments. N2a cells were seeded in 10-cm plates
at a density of 2,000,000 cells per dish. Cell lysates were prepared 48 h after seed-
ing in PBS with 1% TX-100 or 1% CHAPSO and protease inhibitors (Complete
Protease Inhibitor Cocktail Tablets, Roche). Cell lysates were precleared with
protein G sepharose for 1 h at 4 °C followed by centrifugation at 10,000g for
15 min. Immunoprecipitations were conducted overnight at 4 °C using the
appropriate antibodies or a negative control antibody (9E10). The beads were
washed four times with PBS with 1% TX-100 or 1% CHAPSO and once with
© 2013 Nature America, Inc. All rights reserved.
PBS. Proteins were eluted with 1× lithium dodecyl sulfate (LDS) loading buffer.
Microsomal membranes were prepared from fresh or frozen postmortem mouse
cortical brain samples first by homogenization using a Teflon homogenizer in
0.5 M sucrose PKM buffer (100 mM potassium phosphate, 5 mM MgCl2 and
3 mM KCl, pH 6.5) followed by centrifugation for 10 min at 8,000 r.p.m. The
protein concentration was determined by the Bradford dye-binding procedure
(Bio-Rad), and the samples were centrifuged at 100,000g for 1 h. The mem-
brane pellets were each solubilized with the addition of an equal volume of
buffer containing 2% CHAPSO (Pierce) and incubated on ice for 1 h. After
centrifugation at 100,000g for 1 h, the immunoprecipitation was performed as
previously described58.
Endogenous cAMP assessment. CHO-K1 β-arrestin cells were transfected with
empty vector, WT GPR3 or the GPR3 mutant cDNA constructs (X-tremeGENE
HP, Roche). Forty-eight hours after transfection, the culture medium was replaced
with serum-free DMEM, and the cells were treated with 100 µM forskolin
in the presence or absence of 25 µM IBMX, a phosphodiesterase inhibitor, for
30–45 min. Intracellular cAMP levels were then measured in cells using a spe-
cific cAMP assay kit (R&D Systems) according to the manufacturer’s protocol.
npg
Subcellular fractionation. WT HEK293 cells were rinsed twice with ice-cold 1×
PBS, solubilized in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (25 mM
doi:10.1038/nm.3023
MES, pH 6.5, and 150 mM NaCl) containing 1% CHAPSO and supplemented
with a complete protease inhibitor cocktail. Cells were lysed by sequential pas-
sage through 18-gauge (five times) and 26-gauge (ten times) needles and then
placed on ice for 1 h. After the removal of insoluble material by centrifugation
at 15,000g for 15 min, the lysates were adjusted to a 45% sucrose concentration
and transferred to ultracentrifugation tubes. A discontinuous sucrose gradient
was prepared by layering 35% and 5% sucrose in MES buffer. Samples were
centrifuged at 100,000g for 16–18 h. Thirteen 960-µl fractions were collected
from the top of the gradient and used for western blot analysis.
Photoaffinity probe and crosslinking. JC-8 was synthesized according to a
previously described procedure43. Fractions 2–4 and 9–12 from the subcellular
fractionation experiments described above were combined, and the crosslinking
studies were performed as previously described45.
Structured illumination microscopy and immunofluorescence. HeLa cells
were plated on glass coverslips and transfected with β-arrestin 2–GFP and APH-
1A. Twenty-four hours after transfection, the cells were fixed (4% paraformalde-
hyde, 10 min), blocked (2% BSA, 2% FBS and 1% gelatin in PBS supplemented
with 5% serum), incubated with primary antibody (4 °C, overnight), washed
(PBS), incubated with fluorophore-conjugated secondary antibody (room tem-
perature, 1 h), rinsed and mounted in Mowiol-containing medium. The second-
ary antibodies were conjugated with the following fluorophores: Alexa-546 and
-647. Images were captured with a structured illumination microscope (Elyra
S1 (Carl Zeiss, Jena, Germany) equipped with a 63× oil objective lens and a
1.4 numerical aperture (NA) and an Andor iXon 885 EMCCD camera) that
is capable of revealing information beyond the diffraction barrier that limits
conventional widefield and confocal microscopes. β-arrestin 2 and APH-1A
were imaged at resolutions of ~110 nm and ~134 nm, respectively.
Statistical analyses. The data are presented as means ± s.e.m. The data were
analyzed using two-tailed Student’s t tests. ANOVA was performed with the
GraphPad Prism 5 software.
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predicts a ring structure for presenilins. Neuron 32, 579–589 (2001).
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neurons via an autophagic degradative pathway. J. Cell Biol. 166, 1041–1054
(2004).
55. Cai, H. et al. BACE1 is the major β-secretase for generation of Aβ peptides by
neurons. Nat. Neurosci. 4, 233–234 (2001).
56. Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes.
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57. Bossers, K. et al. Concerted changes in transcripts in the prefrontal cortex precede
neuropathology in Alzheimer’s disease. Brain 133, 3699–3723 (2010).
58. Hébert, S.S. et al. Coordinated and widespread expression of γ-secretase in vivo:
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