thathiah 13 gpr3 arr2

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httpslidepdfcomreaderfullthathiah-13-gpr3-arr2 19

NATURE MEDICINE VOLUME 19 | NUMBER 1 | JANUARY 2013 43

A R T I C L E S

Considerable insight into the genetic and molecular biological basis of

Alzheimerrsquos disease has yet to be translated into a new medication forthe disease Removal of tau12 or the Aβ peptide3ndash5 from the brain by

various vaccination strategies inhibition or modulation of the β- andγ -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 currentlypursued Unfortunately many therapeutic agents have failed at severalstages of development as a result of poor blood-brain barrier penetra-

tion or severe side effects3ndash57 Moreover new findings suggest the

need for very early symptomatic or presymptomatic treatment whichwill require the identification of alternative disease-modifying targets

to safely modulate the pathogenesis of Alzheimerrsquos disease3ndash57Central 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 ofearly onset familial Alzheimerrsquos 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 proteinsthey encode However familial Alzheimerrsquos disease accounts forless than 05 of all Alzheimerrsquos disease cases10 The vast major-

ity of late-onset Alzheimerrsquos disease cases are sporadic and includeenvironmental and genetic risk factors among which the ε4 allele of

APOE accounts for more than 25 of the genetic risk InterestinglyAPOE4 also seems to modulate Aβ accumulation11 strengthening

the potential of Aβ-modulating agents in the early treatment of

Alzheimerrsquos disease3GPCRs also called seven transmembrane receptors (7TMRs)

comprise the largest family of membrane proteins12 and are the mostcommon 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 Alzheimerrsquosdisease GPCRs are associated with multiple stages of APP proteo-

lysis including modulation of processing of APP by the α- β- andγ -secretases and the regulation of Aβ degradation and Aβ-mediatedtoxicity 16 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 versatiletargets Drugs that directly target a GPCR have been classically

described as either agonists or antagonists for G protein signalingThe 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 Howevera 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 capableof initiating distinct independent signaling events17 These signals

are often both spatially and temporally different from the classicG 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 amp Johnson Pharmaceutical Research and

Development Janssen Pharmaceutica Beerse Belgium Correspondence should be addressed to AT (amanthathathiahcmevibkuleuvenbe) or

BDS (bartdestroopercmevib-kuleuvenbe)

Received 17 September accepted 7 November published online 2 December 2012 doi101038nm3023

β-arrestin 2 regulates Aβ generation and γ -secretaseactivity in Alzheimerrsquos disease

Amantha Thathiah12 Katrien Horreacute12 An Snellinx 12 Elke Vandewyer12 Yunhong Huang12Marta Ciesielska12 Gerdien De Kloe13 Sebastian Munck 12 amp Bart De Strooper12

b-arrestins are associated with numerous aspects of G proteinndashcoupled 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 Alzheimerrsquos 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

in Arrb2 minusminus mice Moreover in a transgenic mouse model of Alzheimerrsquos disease genetic deletion of Arrb2 leads to a reductionin the production of Ab40 and Ab42 Two GPCRs implicated previously in Alzheimerrsquos 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 Alzheimerrsquos disease



A R T I C L E S

44 VOLUME 19 | NUMBER 1 | JANUARY 2013 NATURE MEDICINE

pathophysiological consequences18 that can be exploited for the

therapeutic development of G proteinminus or β-arrestinndashbiased drugs

Differential regulation of β-arrestins is implicated in type 2 diabetes

and psychiatric disorders1920 for which pharmacological manipula-

tion of selective β-arrestinndashdependent complexes may provide thera-peutic benefits21 As mediators of GPCR desensitization trafficking

and cell signaling the β-arrestins could provide a putative basis to

understand GPCR dysfunction in Alzheimerrsquos disease Nevertheless

no study so far has documented a role for β-arrestins in Alzheimerrsquos

disease progression

RESULTS

b-arrestin 2 in Alzheimerrsquos disease and effects on Ab generation

We compared the expression of β-arrestins 1 and 2 in samples from

the hippocampus and entorhinal cortex of autopsied human brains

with Alzheimerrsquos disease (N = 18) and age-matched control subjects

(N = 20) Levels of β-arrestin 2 mRNA were significantly increased

(Fig 1a) whereas levels of β-arrestin 1 mRNA were decreased inthe Alzheimerrsquos disease samples compared to the control samples

(Fig 1b) We confirmed these observations in an independent group

of Braak-staged human brain samples (Braak 0ndash2 compared to Braak

5ndash6) (Supplementary Fig 1ab) Although there are limitations in

using postmortem tissue such as mRNA degradation and cellular

alterations during disease progression that complicate the interpreta-

tion of results these data suggest that β-arrestins 1 and 2 are differen-

tially regulated in brain areas affected in Alzheimerrsquos disease

We next determined whether the expression of β-arrestins 1 and

2 directly affects Aβ generation in a cellular context Expression of

β-arrestin 2 in the HEK293-APP695 cell line led to a significant

increase in Aβ40 and Aβ42 release (Fig 1cd) an effect that was

effectively abolished by the addition of L-685458 a highly selective

γ -secretase inhibitor22 Furthermore the release of Aβ40 and Aβ42

was not diminished in cells with reduced β-arrestin 1 expression

(Supplementary Fig 1cndashe) In contrast silencing of β-arrestin 2

efficiently suppressed Aβ secretion to 50 below that of controltransfected cells (Fig 1e) Furthermore the amounts of total soluble

APP and the α-secretasendashmediated cleavage product of APP

(Fig 1f g) and expression of the α- and β-secretases were unchanged

after expression or silencing of β-arrestin 2 (Fig 1hi) indicating

that the modulation of Aβ generation by β-arrestin 2 occurs down-

stream of β-secretase activity Expression of the NCT PS1 APH-1A

and PEN 2 subunits of the γ -secretase complex was also unaffected

by expression or silencing of β-arrestin 2 (Fig 1hi) Notably

accumulation of the APP C-terminal fragment (APP-CTF) which is

typically observed after a reduction in γ -secretase activity was only

modestly detectable raising the question of how β-arrestin 2 affects

Aβ generation (Fig 1hi)

To assess the physiological relevance of genetic silencing ofβ-arrestins 1 and 2 we isolated embryonic neuronal cultures from

wild-type Arrb1++ (encoding endogeneous β-arrestin 1) Arrb2++

Arrb1minusminus (ref 23) and Arrb2minusminus (ref 24) mice (Fig 2ab) The amounts

of endogenous Aβ40 and Aβ42 were substantially reduced in Arrb2minusminus

but not Arrb1minusminus neurons demonstrating that β-arrestin 2 is involved

endogenously in the modulation of neuronal Aβ production

GPCRs regulate Ab generation through b-arrestin 2

β-arrestins have traditionally been associated with the termination

of GPCR signaling and receptor desensitization2526 In parallel

β-arrestins can initiate a second set of G proteinndashindependent signals

resulting in receptor desensitization endocytosis and various cellular

a 30 β-arrestin 2

F o l d c h a n g e

( r e l a t i v e t o c o n t r o l ) 25

20

15

10

05

Control Alzheimerrsquos

disease

b β-arrestin 1

F o l d c h a n g e ( r e l a t i v e t o c o n t r o l )

20


disease

15

10

05

c

V

e h i c l

e

L - 6 8 5

4 5 8

V

e h i c l

e

L - 6 8 5

4 5 8

Vector

β-arrestin 2

N o r m a l i z e d t o v e c t o r

( A β 4 0

)

20

15

10

05

d

V e h i c l e

L - 6 8 5

4 5 8

V e h i c l e

L - 6 8 5

4 5 8

Vector

β-arrestin 2


( A β 4 2

) 15

20

10

05

e

β - a r r e s t i n 2 s

i R N A


i R N A

N o n s i l e

n c i n g

s i R N A

N o n s i l e

n c i n g

s i R N A

N o r m a l i z e d t o

n o n s i l e n c i n g s i R N A ( A β )

Aβ40Aβ42

10

15

05

f

V e c t o r

β - a r r e s t i n

sAPPtotal

sAPPα

PEN 2

APP-CTF

APP-FL

β-actin

g

β-arrestin 2

siRNA

Nonsilencing

siRNA

sAPPtotal

sAPPα

h

L-685458 ndash ndash + ndash ndash +

Vector β-arrestin

β-arrestin 2

ADAM10

β-secretase

NCT

PS1-NTF

PS1-CTF

APH-1A

PEN 2

APP-CTF

APP-FL

β-actin

i

β-arrestin 2

ADAM10

β-secretase

NCT

PS1-NTF

PS1-CTF

APH-1A

Nonsilencing

siRNAβ-arrestin 2

siRNA

Figure 1 Expression of β-arrestin 2 is elevated in individuals with Alzheimerrsquos disease

and overexpression and silencing of β-arrestin 2 differentially regulate Aβ accumulation

(ab) Expression of β-arrestin 2 (a) and β-arrestin 1 (b) assessed by quantitative PCR (qPCR) in

brain samples from control individuals and individuals with Alzheimerrsquos disease P = 00089

P = 00002 by unpaired Studentrsquos t test n = 20 control individuals n = 18 individuals

with Alzheimerrsquos disease (cd) Amounts of Aβ40 (c) and Aβ42 (d) assessed by ELISA in culture

supernatants from HEK293-APP695 cells after overexpression of β-arrestin 2 and treatment

with the γ -secretase inhibitor L-685458 P lt 005 P lt 001 P lt 0001 by one-way analysis of variance (ANOVA) and Dunnettrsquos multiple

comparisons post test n = 4 experiments performed in triplicate Error bars sem (e) Amounts of Aβ40 (black bars) and Aβ42 (red bars) determined

by ELISA after silencing of β-arrestin 2 P lt 001 by ANOVA and Dunnettrsquos post test n = 5ndash6 experiments performed in triplicate Error bars sem

(fg) Amounts of total soluble APP (sAPPtotal) and the α-secretasendashmediated 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-685458

(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



A R T I C L E S


responses172728 Thus far two GPCRs have been associated with the

γ -secretasendashmediated processing of APP and Aβ generation GPR3

(ref 29) and the β2-adrenergic receptor (β2-AR)30 In addition the

δ-opioid receptor has been implicated in modulation of theβ-secretasendash

mediated and subsequent γ -secretasendashmediated proteolysis of APP31

All three GPCRs seem to modulate Aβ generation independently of

G protein signaling29ndash31 Consequently we hypothesized that the

β-arrestins could regulate Aβ generation through modulation of their

interaction with these specific GPCRs We used the CHO-K1 cell line

which stably expresses β-arrestin 2 and GPR3 to monitor the direct

interaction between β-arrestin 2 and GPR3 using the PathHunter

technology 32 (Supplementary Fig 2a) In this cell line silencing of

β-arrestin 2 expression (Supplementary Fig 2b) led to a reduction

in the release of Aβ40 and Aβ42 (Supplementary Fig 2c) We used

APP-C99 which is directly cleaved by the γ -secretase to yield Aβ40

and Aβ42 in these assays further demonstrating that the β-arrestin

2ndashmediated effect on Aβ generation is downstream of the α- and

β-secretases This result is similar to the previously described effects of

GPR3 on APP processing29 β2-AR has also been reported to modulate

γ -secretasendashmediated Aβ release30 Therefore we used the CHO-K1

cell line which stably expresses β-arrestin 2 and the β2-AR to perform

a similar series of experiments Silencing of β-arrestin 2 expression

in this cell line resulted in reduced interaction between β-arrestin 2

and the β2-AR after treatment with the β2-AR agonist isoproterenol as

monitored with the PathHunter assay (Supplementary Fig 2d) and

a reduction in Aβ generation (Supplementary Fig 2ef )

bAβ40

Aβ42

A r r b 2

+ +

A r r b 2

+ +

A r r b 2 + ndash

A r r b 2 + ndash

A r r b 2 ndash ndash


15

10

05

N

o r m a l i z e d v a l u e

c

NS

A β 4 0

( n g m l ndash 1 )

15

10

05

A r r b 2 + +


Ad-GFP

Ad-GPR3

d

NS A β 4 2

( n g m l ndash 1 )

03

02

01

A r r b 2 + +

A r r b 2

ndash ndash

Ad-GFP

Ad-GPR3

e

G F P

G P R 3

G F P

G P R 3

Nct

Ps1-CTF

Pen 2

App-FL

App-CTF

GPR3

β-actin

Arrb2++

Arrb2ndashndash

15

aAβ40

Aβ42

10

NS NS

05

A r r b 1 + +


A r r b 1 + +


N


Figure 2 Endogenous Aβ generation is reduced in β-arrestin 2ndashdeficient mice (a) Amounts of Aβ40 and Aβ42 measured by ELISA in culture supernatant

samples from primary cortical neuronal cultures from wild-type Arrb1++ and Arrb1minus minus 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 Studentrsquos t test n = 19 Arrb1++

n = 20 Arrb1minus minus Error bars sem (b) Amounts of Aβ40 and Aβ42 measured in Arrb2 + minus and Arrb2 minus minus primary neuronal cultures P lt 005 P lt 001

by ANOVA and Dunnettrsquos post test n = 11 wild-type Arrb2 ++ n = 18 Arrb2 + minus n = 20 Arrb2 minus minus Error bars sem (cd) 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 lt 001 NS not significant by ANOVA and Dunnettrsquos post test n = 18 Arrb2 ++ n = 18 Arrb2 minus minus Error bars sem (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

a

Extracellular

domain

Plasma

membrane

Intracellular

domain

15000

b

10000

R L U

5000

C 9 9 a l o

n e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

10000

d

8000

R L U

4000

2000

6000

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

e

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

25

20

15

A β g

e n e r a t i o n n o r m a l i z e d t o C 9 9

05

10

Aβ40

Aβ42

c

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

40

30

20

A β g


10

Aβ40

Aβ42

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

DRYAAY 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 DRYAAY 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 DRYAAY 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 lt 005 P lt 001

P lt 0001 by ANOVA and Dunnettrsquos post test Data are the mean plusmn sem of four to six independent experiments performed in triplicate



A R T I C L E S


To genetically establish the requirement of β-arrestin 2 for the

modulation of Aβ generation by GPR3 we transduced wild-type

Arrb2++ and Arrb2minusminus neuronal cultures with a GPR3 adenoviral

vector (Ad-GPR3) Notably expression of Ad-GPR3 in the Arrb2minusminus

neuronal cultures failed to increase Aβ generation in contrast to the

elevation in Aβ generation observed in Arrb2++ neuronal cultures

(Fig 2cd) After GPR3 transduction GPR3 expression was equiva-

lent in the Arrb2++ and Arrb2minusminus 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 work 29

overexpression of GPR3 resultedin 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 2ndashmediated

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 DRYAAY mutant) renders a GPCR incapable of activating

G proteins while retaining its ability to activateβ-arrestinndashmediated

cellular responses33ndash35 (Fig 3a) The GPR3 DRYAAY mutant did

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 DRYAAY 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

Vehiclea

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

Lactacystin

NCT

PS1-NTF

PS1-CTF

PEN 2

APP-FL

APP-CTF

β-actin

cVehicle

Arrb2++

Arrb2ndashndash

Arrb2++

Arrb2ndashndash

Lactacystin

Nct

Ps1-NTF

Ps1-CTF

Pen 2

App-FL

App-CTF

β-actin

b

N o n s i l e

n c i n

g s i R

N A


1 s i R N A


2 s i R N A

20

15

10

05

A P P - C T F a c c u m u l a t i o n

( n o r m

a l i z e d t o n o n s i l e n c i n g s i R N A )

NS

d

A r r b 2 + +

A r r b 2 ndash

ndash

20

15

10

05


( n o r m a l i z e d t o A r r b 2 + + )

h

N o r m a l i z e d i n t e n s i t y

( t o v e c t o r )

4

3

JC-8

JC-8 + X

2

1

V e c t

o r

β

- a r r e

s t i n

2

f25

20

Vector

β-arrestin 2

15

10

N o

r m a l i z e d i n t e n s i t y ( t o v e c t o r )

05

N C T

P S 1 -

C T F

A P H

- 1 A

P E N

2

e Vector

Fractions

β-arrestin 2

NCT

β-arrestin 2

PS1-NTF

PS1-CTF

APH-1A

PEN 2

Caveolin-1

GM130

EEA1

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

g Input

Raft

Non-raft

Vector β-arrestin 2

V e c t o r

β - a r r e s t i n 2

J C - 8

J C - 8 + X

J C - 8

J C - 8 + X

PS1-CTF

PS1-CTF

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 2ndashGFP 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 2ndashGFP cDNA normalized to Caveolin 1 expression n = 4ndash6

independent experiments (g) Immunoblot analysis using a PS1-CTFndashspecific antibody of pooled raft and non-raft fractions incubated with JC-8

or JC-8 + L-685458 (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 lt 005 P lt 001 P lt 0001 NS not significant by ANOVA and the Dunnettrsquospost test Error bars sem



A R T I C L E S


andβ2-AR affect γ -secretasendashmediated proteolysis of APP2930 Classic

inhibition of γ -secretase activity 36 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) Todetermine whether β-arrestin 2 affects turnover of the APP-CTF

we treated HEK293-APP695 cells (after silencing β-arrestin 2) and

Arrb2minusminus neuronal cultures with the proteasome inhibitor lactacystin

and found an accumulation of the APP-CTF under these conditions

(Fig 4andashd) 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 complexand Aβ generation are localized in DRMs2938ndash42 Therefore we

assessed the distribution of the γ -secretase complex subunits and

β-arrestin 2 in lipid raft and nonndashlipid 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 4ef )

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 4ab)

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-685458 (ref 43) that only binds the cata-

lytically active γ -secretase complex44 We combined DRM fractions

2ndash4 and the nonndashlipid raft fractions (pooled fractions 9ndash12) from

empty vectorndashtransfected control and β-arrestin 2ndashtransfected cells

We normalized the fractions for γ -secretase expression and incubated

these fractions with JC-8 We used unlabeled L-685458 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 nonndashlipid

raft fractions (Fig 4g ) Overexpression of β-arrestin 2 resulted in a

twofold to threefold increase of the active γ -secretase complex poolin 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 complex4647

In contrast the detergent Triton X-100 (TX-100) dissociates the

γ -secretase complex48 In the presence of TX-100 β-arrestin 2 only

Input Bound Input Bound

IP Aph-1a

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

d

eIP β-arrestin 2

W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

Nct

Aph-1a

β-arrestin 2

a

Nct

Input InputUnbound UnboundBound Bound

Ps1 Ps1 Aph-1a Aph-1aControl Control Control

IP Ps1-CTF IP Aph-1a

Control

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2

Input Unbound Bound Input Unbound Bound

A ph -1 a A ph -1 aCon trol Con trol


Ps1 Ps1Control Con trol

b

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

c

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

f


IP Aph-1a IP β-arrestin 2

W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Aph-1a

β-arrestin 2

Figure 5 β-arrestin 2 interacts with the Aph-1a subunit of the γ -secretase complex (ab) Immunoprecipitation (IP) of cell lysates from the N2a

neuroblastoma cell line after extraction in 1 CHAPSO-containing (a) or 1 TX-100ndashcontaining (b) buffer with antibodies to Ps1-CTF (left) Aph-1a

(B803 right) or negative control antibodies and immunoblot analysis with the indicated antibodies to the γ -secretase complex subunits or β-arrestin 2

(cd) Immunoprecipitation of cell lysates from the N2a neuroblastoma cell line after extraction in 1 CHAPSO-containing (c) or 1 TX-100ndashcontaining

(d) buffer with β-arrestin 2ndashspecific or negative control antibodies and immunoblot analysis with the indicated antibodies to the γ -secretase complex

subunits or β-arrestin 2 (ef) Immunoprecipitation of cortical brain samples from WT Gpr3 minus minus and Arrb2 minus minus mice after extraction in 1 CHAPSO-

containing (e) or 1 TX-100ndashcontaining (f) buffer with antibodies to Aph-1a (B803 left) or β-arrestin 2 (right) and immunoblot analysis with the

indicated antibodies to the γ -secretase complex subunits or β-arrestin 2



A R T I C L E S


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 Gpr3minusminus or Arrb2minusminus mice Furthermore we observed colocali-

zation of β-arrestin and APH-1A after coexpression of β-arrestin 2and 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-

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 Alzheimerrsquos disease This model coexpresses two familial

Alzheimerrsquos diseasendashlinked mutations APP with the KM670671NL

lsquoSwedishrsquo mutation and PS1 with the L166P mutation49 Both het-

erozygosity and complete genetic ablation of β-arrestin 2 expressionresulted in a marked reduction in Aβ40 and Aβ42 generation in the

hippocampus and cortex of 3-month-old APP PS1 Arrb2+minus and

APP PS1 Arrb2minusminus mice (Fig 6ab) 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

Alzheimerrsquos 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

implications for the pathogenesis of Alzheimerrsquos disease β-arrestin 2

induces the redistribution of an inactive γ -secretase complex

toward a DRM-associated active γ -secretase pool through direct

interaction with Aph-1a We corroborated the overexpression studies

with several loss-of-function experiments that confirmed the overall

effects of β-arrestin 2 on γ -secretasendashmediated Aβ generation

Our data also suggest additional mechanisms including increased

APP-CTF turnover by which APP metabolism is affected byβ-arrestin 2 downregulation

This study provides insight into the mechanism of γ -secretase regu-

lation by GPR3 (ref 29) and β2-AR 30 two GPCRs that have previously

been implicated in the pathogenesis of Alzheimerrsquos disease Most nota-

bly we have determined that β-arrestins are aberrantly expressed in

the brain of individuals with Alzheimerrsquos disease and genetic deletion

of β-arrestin 2 reduces the accumulation of endogenous mouse Aβ

Furthermore in an Alzheimerrsquos 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 Alzheimerrsquos

disease Ligands that show bias for either G proteinndashmediated

(G proteinndashbiased) or β-arrestinndashmediated (β-arrestinndashbiased) 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 receptorndashdependent

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 Alzheimerrsquos disease

Arrb2minusminus 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 micro-opioid receptor51 The current

study indicates that APP-CTF does not accumulate in APP PS1

Arrb2minusminus mice which has been suggested to occur after treatmentwith 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 andor 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 Alzheimerrsquos 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

ACKNOWLEDGMENTSWe are grateful to RJ Lefkowitz and S Ahn (Duke University Medical CenterDurham North Carolina USA) for the generous gift of the β-arrestin 2 wild-typeand knockout mouse embryonic fibroblasts the Arrb1minusminus and Arrb2minusminus micethe β-arrestin 2ndashGFP-Flag cDNA and helpful discussion We thank M Jucker(University of Tuumlbingen Germany) for the gift of APP PS1 transgenic mice Wegreatly appreciate the kind gift of human control and Alzheimerrsquos disease brainsamples from K Bossers and DF Swaab (Netherlands Institute for NeuroscienceAmsterdam The Netherlands) and C Troakes (the London NeurodegenerativeDiseases Brain Bank London UK) We thank M Mercken (Johnson amp Johnson

15

a

10

05

H i p p

o c a m

p u s

C o r t e

x A β 4 0 n o r m a l i z e d v a l u e

( t o

A P P P S 1 A r r b b 2 + + )

+

H i p p

o c a m

p u s

C o r t e

x

APPPS1 Arrb2++

APPPS1 Arrb2+ndash

APPPS1 Arrb2ndashndash

b

15

10

05

A β 4 2 n o r m a l i z e d v a l u e

( t o

A P P P S 1 A r r b b 2 + + )

+

++

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


c

A P P P S

1 A r r b 2 + +

A P P P S

1 A r r b 2 + ndash

A P P P S

1 A r r b 2 ndash ndash

NCT

PS1-CTF

PS1-NTF

APP-FL

APP-CTF

β-actin

Figure 6 β-arrestin 2 contributes to Aβ generation in an Alzheimerrsquos

disease transgenic mouse model (ab) 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 + minus or Arrb2 minus minus mice

determined by ELISA P lt 005 relative to Arrb2 ++ +P lt 001 relative

to Arrb2 ++ for hippocampal Aβ40 P lt 0001 relative to Arrb2 ++

P lt 00001 relative to Arrb2 ++ for cortical Aβ40 P lt 005 relative

to Arrb2 ++ +P lt 001 relative to Arrb2 ++ for hippocampal Aβ42

P lt 0005 relative to Arrb2 ++++P lt 0001 relative to Arrb2 ++ for

cortical Aβ42 by ANOVA and Dunnettrsquos post test n = 6 independent

female mice per cross Error bars sem (c) Immunoblot of the expression

of the γ -secretase complex components and APP in brain samples fromAPP PS1 Arrb2 ++ APP PS1 Arrb2 + minus and APP PS1 Arrb2 minus minus mice



A R T I C L E S


Pharmaceuticals Research and Development Beerse Belgium) for the antibodiesto Aβ We are grateful to Y Li (Memorial Sloan Kettering Cancer Center NewYork USA) for the k ind initial gift of JC-8 This work was supported by a MentoredNew Investigator Research grant from the Alzheimerrsquos Association to AT theFund for Scientific Research Flanders KU Leuven a Methusalem grant fromthe KU Leuven and the Flemish government and the Foundation for AlzheimerResearch (SAOFRMA) to BDS BDS is the Arthur Bax and Anna Vanluffelenchair for Alzheimerrsquos disease

AUTHOR CONTRIBUTIONSAT and BDS designed the experiments and wrote the manuscript AT KHAS EV and YH conducted the experiments MC conducted the qPCRexperiments GDK synthesized JC-8 SM conducted the immunofluorescence

image analysis

COMPETING FINANCIAL INTERESTSThe authors declare competing financial interests details are available in the online version of the paper

Published online at httpwwwnaturecomdoifinder101038nm3023

Reprints and permissions information is available online at httpwwwnaturecom

reprintsindexhtml

1 Ballatore C Lee VM amp Trojanowski JQ Tau-mediated neurodegeneration

in Alzheimerrsquos disease and related disorders Nat Rev Neurosci 8 663ndash672

(2007)2 Goumltz J Ittner A amp Ittner LM Tau-targeted treatment strategies in Alzheimerrsquos

disease Br J Pharmacol 165 1246ndash1259 (2012)

3 Holtzman DM Goate A Kelly J amp Sperling R Mapping the road forward in

Alzheimerrsquos disease Sci Transl Med 3 114ps148 (2011)

4 Golde TE Schneider LS amp Koo EH Anti-aβ therapeutics in Alzheimerrsquos disease

the need for a paradigm shift Neuron 69 203ndash213 (2011)

5 Karran E Mercken M amp De Strooper B The amyloid cascade hypothesis for

Alzheimerrsquos disease an appraisal for the development of therapeutics Nat Rev

Drug Discov 10 698ndash712 (2011)

6 Cramer PE et al ApoE-directed therapeutics rapidly clear β-amyloid and reverse

deficits in AD mouse models Science 335 1503ndash1506 (2012)

7 Selkoe DJ Resolving controversies on the path to Alzheimerrsquos therapeutics

Nat Med 17 1060ndash1065 (2011)

8 De Strooper B Aph-1 Pen-2 and Nicastrin with Presenilin generate an active

γ -Secretase complex Neuron 38 9ndash12 (2003)

9 Bertram L Lill CM amp Tanzi RE The genetics of Alzheimer disease back to

the future Neuron 68 270ndash281 (2010)

10 Bekris LM Yu CE Bird TD amp Tsuang DW Genetics of Alzheimer disease

J Geriatr Psychiatry Neurol 23 213ndash227 (2010)

11 Morris JC et al APOE predicts amyloid-β but not tau Alzheimer pathology in

cognitively normal aging Ann Neurol 67 122ndash131 (2010)

12 Fredriksson R amp Schioth HB The repertoire of G-proteinndashcoupled receptors in

fully sequenced genomes Mol Pharmacol 67 1414ndash1425 (2005)

13 Gudermann T Nurnberg B amp Schultz G Receptors and G proteins as primary

components of transmembrane signal transduction Part 1 G-proteinndashcoupled

receptors structure and function J Mol Med 73 51ndash63 (1995)

14 Watson SAS The G Protein-Coupled Receptor Factors Book (Academic San Diego

1994)

15 Vassilatis DK et al The G proteinndashcoupled receptor repertoires of human and

mouse Proc Natl Acad Sci USA 100 4903ndash4908 (2003)

16 Thathiah A amp De Strooper B The role of G proteinndashcoupled receptors in the

pathology of Alzheimerrsquos disease Nat Rev Neurosci 12 73ndash87 (2011)

17 DeWire SM Ahn S Lefkowitz RJ amp Shenoy SK β-arrestins and cell signaling

Annu Rev Physiol 69 483ndash510 (2007)

18 Whalen EJ Rajagopal S amp Lefkowitz RJ Therapeutic potential of β-arrestinndash

and G proteinndashbiased agonists Trends Mol Med 17 126ndash139 (2011)

19 Beaulieu JM et al An Akt β-arrestin 2PP2A signaling complex mediatesdopaminergic neurotransmission and behavior Cell 122 261ndash273 (2005)

20 Luan B et al Deficiency of a β-arrestinndash2 signal complex contributes to insulin

resistance Nature 457 1146ndash1149 (2009)

21 Beaulieu JM et al A β-arrestin 2 signaling complex mediates lithium action on

behavior Cell 132 125ndash136 (2008)

22 Shearman MS et al L-685458 an aspartyl protease transition state mimic is

a potent inhibitor of amyloid β-protein precursor γ -secretase activity Biochemistry 39

8698ndash8704 (2000)

23 Conner DA et al β-arrestin1 knockout mice appear normal but demonstrate

altered cardiac responses to β-adrenergic stimulation Circ Res 81 1021ndash1026

(1997)

24 Bohn LM et al Enhanced morphine analgesia in mice lacking β-arrestin 2 Science

286 2495ndash2498 (1999)

25 Ferguson SS et al Role of β-arrestin in mediating agonist-promoted G proteinndash

coupled receptor internalization Science 271 363ndash366 (1996)

26 Lohse MJ Lefkowitz RJ Caron MG amp Benovic JL Inhibition of β-adrenergic

receptor kinase prevents rapid homologous desensitization of β 2-adrenergic

receptors Proc Natl Acad Sci USA 86 3011ndash3015 (1989)

27 Ahn S Shenoy SK Wei H amp Lefkowitz RJ Differential kinetic and spatialpatterns of β-arrestin and G proteinndashmediated ERK activation by the angiotensin II

receptor J Biol Chem 279 35518ndash35525 (2004)

28 Luttrell LM et al β-arrestinndashdependent formation of β2 adrenergic receptor-Src

protein kinase complexes Science 283 655ndash661 (1999)

29 Thathiah A et al The orphan G proteinndashcoupled receptor 3 modulates amyloid-β

peptide generation in neurons Science 323 946ndash951 (2009)

30 Ni Y et al Activation of β2-adrenergic receptor stimulates γ -secretase activity and

accelerates amyloid plaque formation Nat Med 12 1390ndash1396 (2006)

31 Teng L Zhao J Wang F Ma L amp Pei GA GPCRsecretase complex regulates

β- and γ -secretase specificity for Aβ production and contributes to AD pathogenesis

Cell Res 20 138ndash153 (2010)

32 Olson KR amp Eglen RM β galactosidase complementation a cell-based

luminescent assay platform for drug discovery Assay Drug Dev Technol 5 137ndash144

(2007)

33 Gaacuteborik Z et al The role of a conserved region of the second intracellular loop in

AT1 angiotensin receptor activation and signaling Endocrinology 144 2220ndash2228

(2003)

34 Shenoy SK et al β-arrestinndashdependent G proteinndashindependent ERK12 activation

by the β2 adrenergic receptor J Biol Chem 281 1261ndash1273 (2006)35 Wei H et al Independent β-arrestin 2 and G proteinndashmediated pathways for

angiotensin II activation of extracellular signal-regulated kinases 1 and 2 Proc

Natl Acad Sci USA 100 10782ndash10787 (2003)

36 De Strooper B et al Deficiency of presenilin-1 inhibits the normal cleavage of

amyloid precursor protein Nature 391 387ndash390 (1998)

37 Mitani Y et al Differential effects between γ -secretase inhibitors and modulators

on cognitive function in amyloid precursor proteinndashtransgenic and nontransgenic

mice J Neurosci 32 2037ndash2050 (2012)

38 Chini B amp Parenti M G-protein coupled receptors in lipid rafts and caveolae

how when and why do they go there J Mol Endocrinol 32 325ndash338 (2004)

39 Vetrivel KS et al Association of γ -secretase with lipid rafts in post-Golgi and

endosome membranes J Biol Chem 279 44945ndash44954 (2004)

40 Wada S et al γ -secretase activity is present in rafts but is not cholesterol-

dependent Biochemistry 42 13977ndash13986 (2003)

41 Wahrle S et al Cholesterol-dependent γ -secretase activity in buoyant cholesterol-

rich membrane microdomains Neurobiol Dis 9 11ndash23 (2002)

42 Yagishita S Morishima-Kawashima M Ishiura S amp Ihara Y Aβ46 is processed

to Aβ40 and A

β43 but not to A

β42 in the low density membrane domains

J Biol Chem 283 733ndash738 (2008)

43 Chun J Yin YI Yang G Tarassishin L amp Li YM Stereoselective synthesis of

photoreactive peptidomimetic γ -secretase inhibitors J Org Chem 69 7344ndash7347

(2004)

44 Chau DM Crump CJ Villa JC Scheinberg DA amp Li YM Familial Alzheimer

disease presenilin-1 mutations alter the active site conformation of γ -secretase

J Biol Chem 287 17288ndash17296 (2012)

45 Vetrivel KS et al Spatial segregation of γ -secretase and substrates in distinct

membrane domains J Biol Chem 280 25892ndash25900 (2005)

46 Li YM et al Presenilin 1 is linked with γ -secretase activity in the detergent

solubilized state Proc Natl Acad Sci USA 97 6138ndash6143 (2000)

47 Esler WP et al Activity-dependent isolation of the presenilinndashγ -secretase complex

reveals nicastrin and a γ substrate Proc Natl Acad Sci USA 99 2720ndash2725

(2002)

48 Fraering PC et al Detergent-dependent dissociation of active γ -secretase reveals

an interaction between Pen-2 and PS1-NTF and offers a model for subunit

organization within the complex Biochemistry 43 323ndash333 (2004)

49 Radde R et al Aβ42-driven cerebral amyloidosis in transgenic mice reveals early

and robust pathology EMBO Rep 7 940ndash946 (2006)50 Poulin B et al The M3-muscarinic receptor regulates learning and memory in

a receptor phosphorylationarrestin-dependent manner Proc Natl Acad Sci USA

107 9440ndash9445 (2010)

51 Bohn LM Gainetdinov RR Lin FT Lefkowitz RJ amp Caron MG Mu-opioid

receptor desensitization by β-arrestinndash2 determines morphine tolerance but not

dependence Nature 408 720ndash723 (2000)

52 De Strooper B et al A presenilin-1ndashdependent γ -secretasendashlike protease mediates

release of Notch intracellular domain Nature 398 518ndash522 (1999)



NATURE MEDICINE doi101038nm3023

ONLINE METHODSReagents Unless otherwise indicated chemicals were purchased from

Sigma-Aldrich

Antibodies and compounds Rabbit polyclonal antibodies to human PS1-NTF

(B145 11000) mouse PS1-NTF (B192 15000) APH-1AL (B803 11000)

PEN 2 (B1262 11000) and the APP C terminus (B635 15000) and the

monoclonal antibody 9C3 (13000) directed against the C terminus of NCT

have been previously described and were generated in house5354 ADAM10was detected using a polyclonal antiserum (B421 11000) 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 11000) 22C11

(Chemicon 11000) 6E10 (Sigma-Aldrich 11000) β-secretase (D10E5

Cell Signaling 11000) PK (DiscoveRx 1500) β-arrestin (BD Biosciences

1500) β-arrestin 2 (Cell Signaling 1250) β-arrestin 12 (Santa Cruz 1500)

Myc (9E10 11000) 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-685458

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

β-arrestin 2ndashGFP-Flag plasmid was the kind gift of RJ Lefkowitz (DukeUniversity 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 Arrb1minusminus and Arrb2minusminus mice (C57BL6J background) were kindly

provided by RJ Lefkowitz (Duke University Medical Center Durham North

Carolina USA) Wild-type C57BL6J mice or littermate mice were used as

controls for all of the experiments The APP PS1 transgenic mice (C57BL6J

background) were the kind gift of M Jucker (University of Tuumlbingen Tuumlbingen

Germany)49 The APP PS1 transgenic mice were crossed with Arrb2+minus and

Arrb2minusminus mice to obtain APP PS1 Arrb2+minus and APP PS1 Arrb2minusminus 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)

and the LightCycler 480 Real-Time PCR System (Roche Applied Science)

following the manufacturerrsquos protocol The primers for human β-arrestin 1

used were 5prime-TGTTGAGGGAAGGTGCCAACCG-3prime and 5prime-GATGCAAGA

TCTCCCAACAGGCCG-3prime The primers for human β-arrestin 2 used were

5prime-CCTGTAGATGGCGTGGTGCTTG-3prime and 5prime-CCAGGTCTTCACGGCCA

TAGCG-3prime The housekeeping primers used were to hypoxanthine guanine

phosphoribosyl transferase (HPRT) (5prime-TGACACTGGCAAAACAATGCA-3prime

and 5prime-GGTCCTTTTCACCAGCAAGCT-3 prime) ribosomal protein L13a (RPL13A)

(5prime-CCTGGAGGAGAAGAGGAAAGAGA-3 prime and 5prime-TTGAGGACCTCTG

TGTATTTGTCAA-3prime) and β2 microglobulin (β2M) (5prime-TGCTGTCTCCA

TGTTTGATGTATC-3prime and 5prime-TCTCTGCTCCCCACCTCTAAGT-3prime)

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 025 trypsin and incubated at 37 degC

for 15 min The tissues were then transferred to HBSS supplemented with 10

(vv) horse serum and dissociated by repeated trituration The dispersed cells

were counted and plated on poly-983140-lysinendashcoated 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 800 g and used for ELISA

measurements Cells were harvested in 1times PBS containing complete protease

inhibitors (Roche) centrifuged at 800 g for 10 min and lysed in 150 mM NaCl

50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 74

and 1 TX-100 supplemented with complete protease inhibitors for 30 min at

4 degC After centrifugation at 16000 g for 15 min the cleared cell extracts were

separated on 4ndash12 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 detectionreagent 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 (gt7) were

included Tissue dissection was performed as previously described57 Total RNAwas 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 (AmbionLife Technologies) and processed according

to the manufacturerrsquos 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 overnightwith 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 (plusmn2 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 degC for 1 h at 100000 g 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 16ndash18 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 manufacturerrsquos instruc-

tions as described27 Chemically synthesized double-stranded siRNAs with

19-nucleotide duplex RNA and 2-nucleotide 3prime-dTdT overhangs were pur-

chased from Qiagen The siRNA sequences targeting human β-arrestin 1

and β-arrestin 2 were 5prime-AAAGCCUUCUGCGCGGAGAAU-3 prime and 5prime-AAG

GACCGCAAAGUGUUUGUG-3prime corresponding to positions 439ndash459 and

148ndash168 relative to the start codon respectively A nonsilencing RNA duplex

(5prime-AAUUCUCCGAACGUGUCACGU-3 prime) was used as a control Briefly the

cells were seeded on six-well plates at a cell density of 250000 cells per well One

day after seeding the cells were transfected with the siRNAs Two days after



NATURE MEDICINEdoi101038nm3023

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 16ndash18 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 20000 cells per well

One day after seeding cells were transfected with the siRNAs as described aboveTwo 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


tioned medium for 24 h before analysis with the PathHunter β-arrestin assay

from DiscoveRx according to the manufacturerrsquos protocol

Coimmunoprecipitation experiments N2a cells were seeded in 10-cm plates

at a density of 2000000 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 degC followed by centrifugation at 10000 g for

15 min Immunoprecipitations were conducted overnight at 4 degC 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

PBS Proteins were eluted with 1times lithium dodecyl sulfate (LDS) loading bufferMicrosomal membranes were prepared from fresh or frozen postmortem mouse

cortical brain samples first by homogenization using a Teflon homogenizer in

05 M sucrose PKM buffer (100 mM potassium phosphate 5 mM MgCl2 and

3 mM KCl pH 65) followed by centrifugation for 10 min at 8000 rpm The

protein concentration was determined by the Bradford dye-binding procedure

(Bio-Rad) and the samples were centrifuged at 100000 g 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 100000 g for 1 h the immunoprecipitation was performed as

previously described58

Endogenous cAMP assessment CHO-K1β-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 microM forskolinin the presence or absence of 25 microM IBMX a phosphodiesterase inhibitor for

30ndash45 min Intracellular cAMP levels were then measured in cells using a spe-

cific cAMP assay kit (RampD Systems) according to the manufacturerrsquos protocol

Subcellular fractionation WT HEK293 cells were rinsed twice with ice-cold 1times

PBS solubilized in 2-(N -morpholino)ethanesulfonic acid (MES) buffer (25 mM

MES pH 65 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 15000 g 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 100000 g for 16ndash18 h Thirteen 960-microl 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 2ndash4 and 9ndash12 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 2ndashGFP 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 degC 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 (ElyraS1 (Carl Zeiss Jena Germany) equipped with a 63times oil objective lens and a

14 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 plusmn sem The data were

analyzed using two-tailed Studentrsquos t tests ANOVA was performed with the

GraphPad Prism 5 software

53 Annaert WG et al Interaction with telencephalin and the amyloid precursor protein

predicts a ring structure for presenilins Neuron 32 579ndash589 (2001)

54 Esselens C et al Presenilin 1 mediates the turnover of telencephalin in hippocampal

neurons via an autophagic degradative pathway J Cell Biol 166 1041ndash1054

(2004)55 Cai H et al BACE1 is the major β-secretase for generation of Aβ peptides by

neurons Nat Neurosci 4 233ndash234 (2001)

56 Braak H amp Braak E Neuropathological stageing of Alzheimer-related changes

Acta Neuropathol 82 239ndash259 (1991)

57 Bossers K et al Concerted changes in transcripts in the prefrontal cortex precede

neuropathology in Alzheimerrsquos disease Brain 133 3699ndash3723 (2010)

58 Heacutebert SS et al Coordinated and widespread expression of γ -secretase in vivo

evidence for size and molecular heterogeneity Neurobiol Dis 17 260ndash272 (2004)



A R T I C L E S


pathophysiological consequences18 that can be exploited for the

therapeutic development of G proteinminus or β-arrestinndashbiased drugs

Differential regulation of β-arrestins is implicated in type 2 diabetes

and psychiatric disorders1920 for which pharmacological manipula-

tion of selective β-arrestinndashdependent complexes may provide thera-peutic benefits21 As mediators of GPCR desensitization trafficking

and cell signaling the β-arrestins could provide a putative basis to

understand GPCR dysfunction in Alzheimerrsquos disease Nevertheless

no study so far has documented a role for β-arrestins in Alzheimerrsquos

disease progression

RESULTS

b-arrestin 2 in Alzheimerrsquos disease and effects on Ab generation

We compared the expression of β-arrestins 1 and 2 in samples from

the hippocampus and entorhinal cortex of autopsied human brains

with Alzheimerrsquos disease (N = 18) and age-matched control subjects

(N = 20) Levels of β-arrestin 2 mRNA were significantly increased

(Fig 1a) whereas levels of β-arrestin 1 mRNA were decreased inthe Alzheimerrsquos disease samples compared to the control samples

(Fig 1b) We confirmed these observations in an independent group

of Braak-staged human brain samples (Braak 0ndash2 compared to Braak

5ndash6) (Supplementary Fig 1ab) Although there are limitations in

using postmortem tissue such as mRNA degradation and cellular

alterations during disease progression that complicate the interpreta-

tion of results these data suggest that β-arrestins 1 and 2 are differen-

tially regulated in brain areas affected in Alzheimerrsquos disease

We next determined whether the expression of β-arrestins 1 and

2 directly affects Aβ generation in a cellular context Expression of

β-arrestin 2 in the HEK293-APP695 cell line led to a significant

increase in Aβ40 and Aβ42 release (Fig 1cd) an effect that was

effectively abolished by the addition of L-685458 a highly selective

γ -secretase inhibitor22 Furthermore the release of Aβ40 and Aβ42

was not diminished in cells with reduced β-arrestin 1 expression

(Supplementary Fig 1cndashe) In contrast silencing of β-arrestin 2

efficiently suppressed Aβ secretion to 50 below that of controltransfected cells (Fig 1e) Furthermore the amounts of total soluble

APP and the α-secretasendashmediated cleavage product of APP

(Fig 1f g) and expression of the α- and β-secretases were unchanged

after expression or silencing of β-arrestin 2 (Fig 1hi) indicating

that the modulation of Aβ generation by β-arrestin 2 occurs down-

stream of β-secretase activity Expression of the NCT PS1 APH-1A

and PEN 2 subunits of the γ -secretase complex was also unaffected

by expression or silencing of β-arrestin 2 (Fig 1hi) Notably

accumulation of the APP C-terminal fragment (APP-CTF) which is

typically observed after a reduction in γ -secretase activity was only

modestly detectable raising the question of how β-arrestin 2 affects

Aβ generation (Fig 1hi)

To assess the physiological relevance of genetic silencing ofβ-arrestins 1 and 2 we isolated embryonic neuronal cultures from

wild-type Arrb1++ (encoding endogeneous β-arrestin 1) Arrb2++

Arrb1minusminus (ref 23) and Arrb2minusminus (ref 24) mice (Fig 2ab) The amounts

of endogenous Aβ40 and Aβ42 were substantially reduced in Arrb2minusminus

but not Arrb1minusminus neurons demonstrating that β-arrestin 2 is involved

endogenously in the modulation of neuronal Aβ production

GPCRs regulate Ab generation through b-arrestin 2

β-arrestins have traditionally been associated with the termination

of GPCR signaling and receptor desensitization2526 In parallel

β-arrestins can initiate a second set of G proteinndashindependent signals

resulting in receptor desensitization endocytosis and various cellular

a 30 β-arrestin 2

F o l d c h a n g e

( r e l a t i v e t o c o n t r o l ) 25

20

15

10

05


disease

b β-arrestin 1

F o l d c h a n g e ( r e l a t i v e t o c o n t r o l )

20


disease

15

10

05

c

V

e h i c l

e

L - 6 8 5

4 5 8

V

e h i c l

e

L - 6 8 5

4 5 8

Vector

β-arrestin 2


( A β 4 0

)

20

15

10

05

d

V e h i c l e

L - 6 8 5

4 5 8

V e h i c l e

L - 6 8 5

4 5 8

Vector

β-arrestin 2


( A β 4 2

) 15

20

10

05

e


i R N A


i R N A

N o n s i l e

n c i n g

s i R N A

N o n s i l e

n c i n g

s i R N A

N o r m a l i z e d t o

n o n s i l e n c i n g s i R N A ( A β )

Aβ40Aβ42

10

15

05

f

V e c t o r


sAPPtotal

sAPPα

PEN 2

APP-CTF

APP-FL

β-actin

g

β-arrestin 2

siRNA

Nonsilencing

siRNA

sAPPtotal

sAPPα

h

L-685458 ndash ndash + ndash ndash +

Vector β-arrestin

β-arrestin 2

ADAM10

β-secretase

NCT

PS1-NTF

PS1-CTF

APH-1A

PEN 2

APP-CTF

APP-FL

β-actin

i

β-arrestin 2

ADAM10

β-secretase

NCT

PS1-NTF

PS1-CTF

APH-1A

Nonsilencing

siRNAβ-arrestin 2

siRNA

Figure 1 Expression of β-arrestin 2 is elevated in individuals with Alzheimerrsquos disease

and overexpression and silencing of β-arrestin 2 differentially regulate Aβ accumulation

(ab) Expression of β-arrestin 2 (a) and β-arrestin 1 (b) assessed by quantitative PCR (qPCR) in

brain samples from control individuals and individuals with Alzheimerrsquos disease P = 00089

P = 00002 by unpaired Studentrsquos t test n = 20 control individuals n = 18 individuals

with Alzheimerrsquos disease (cd) Amounts of Aβ40 (c) and Aβ42 (d) assessed by ELISA in culture

supernatants from HEK293-APP695 cells after overexpression of β-arrestin 2 and treatment

with the γ -secretase inhibitor L-685458 P lt 005 P lt 001 P lt 0001 by one-way analysis of variance (ANOVA) and Dunnettrsquos multiple

comparisons post test n = 4 experiments performed in triplicate Error bars sem (e) Amounts of Aβ40 (black bars) and Aβ42 (red bars) determined

by ELISA after silencing of β-arrestin 2 P lt 001 by ANOVA and Dunnettrsquos post test n = 5ndash6 experiments performed in triplicate Error bars sem

(fg) Amounts of total soluble APP (sAPPtotal) and the α-secretasendashmediated 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-685458

(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



A R T I C L E S




























bAβ40

Aβ42

A r r b 2

+ +

A r r b 2

+ +

A r r b 2 + ndash

A r r b 2 + ndash



15

10

05

N


c

NS

A β 4 0

( n g m l ndash 1 )

15

10

05

A r r b 2 + +


Ad-GFP

Ad-GPR3

d

NS A β 4 2

( n g m l ndash 1 )

03

02

01

A r r b 2 + +

A r r b 2

ndash ndash

Ad-GFP

Ad-GPR3

e

G F P

G P R 3

G F P

G P R 3

Nct

Ps1-CTF

Pen 2

App-FL

App-CTF

GPR3

β-actin

Arrb2++

Arrb2ndashndash

15

aAβ40

Aβ42

10

NS NS

05

A r r b 1 + +


A r r b 1 + +


N











a

Extracellular

domain

Plasma

membrane

Intracellular

domain

15000

b

10000

R L U

5000

C 9 9 a l o

n e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

10000

d

8000

R L U

4000

2000

6000

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

e

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

25

20

15

A β g


05

10

Aβ40

Aβ42

c

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

40

30

20

A β g


10

Aβ40

Aβ42













A R T I C L E S
































and Aβ42 (Fig 3c)













Vehiclea

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

Lactacystin

NCT

PS1-NTF

PS1-CTF

PEN 2

APP-FL

APP-CTF

β-actin

cVehicle

Arrb2++

Arrb2ndashndash

Arrb2++

Arrb2ndashndash

Lactacystin

Nct

Ps1-NTF

Ps1-CTF

Pen 2

App-FL

App-CTF

β-actin

b

N o n s i l e

n c i n

g s i R

N A


1 s i R N A


2 s i R N A

20

15

10

05


( n o r m


NS

d

A r r b 2 + +

A r r b 2 ndash

ndash

20

15

10

05



h


( t o v e c t o r )

4

3

JC-8

JC-8 + X

2

1

V e c t

o r

β

- a r r e

s t i n

2

f25

20

Vector

β-arrestin 2

15

10

N o


05

N C T

P S 1 -

C T F

A P H

- 1 A

P E N

2

e Vector

Fractions

β-arrestin 2

NCT

β-arrestin 2

PS1-NTF

PS1-CTF

APH-1A

PEN 2

Caveolin-1

GM130

EEA1

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

g Input

Raft

Non-raft


V e c t o r


J C - 8

J C - 8 + X

J C - 8

J C - 8 + X

PS1-CTF

PS1-CTF















A R T I C L E S
























































IP Aph-1a

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

d

eIP β-arrestin 2

W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

Nct

Aph-1a

β-arrestin 2

a

Nct




Control

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2





b

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

c

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

f



W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Aph-1a

β-arrestin 2











A R T I C L E S
















DRM domains













DISCUSSION
















































METHODS




15

a

10

05

H i p p

o c a m

p u s

C o r t e


( t o

A P P P S 1 A r r b b 2 + + )

+

H i p p

o c a m

p u s

C o r t e

x

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


b

15

10

05


( t o

A P P P S 1 A r r b b 2 + + )

+

++

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


c

A P P P S

1 A r r b 2 + +

A P P P S

1 A r r b 2 + ndash

A P P P S


NCT

PS1-CTF

PS1-NTF

APP-FL

APP-CTF

β-actin















A R T I C L E S




image analysis




reprintsindexhtml















Nat Med 17 1060ndash1065 (2011)















1994)
















8698ndash8704 (2000)



(1997)


286 2495ndash2498 (1999)



















(2007)



(2003)



















to Aβ40 and A

β43 but not to A





(2004)



J Biol Chem 287 17288ndash17296 (2012)







(2002)







107 9440ndash9445 (2010)










Sigma-Aldrich







































































































































































































A R T I C L E S




























bAβ40

Aβ42

A r r b 2

+ +

A r r b 2

+ +

A r r b 2 + ndash

A r r b 2 + ndash



15

10

05

N


c

NS

A β 4 0

( n g m l ndash 1 )

15

10

05

A r r b 2 + +


Ad-GFP

Ad-GPR3

d

NS A β 4 2

( n g m l ndash 1 )

03

02

01

A r r b 2 + +

A r r b 2

ndash ndash

Ad-GFP

Ad-GPR3

e

G F P

G P R 3

G F P

G P R 3

Nct

Ps1-CTF

Pen 2

App-FL

App-CTF

GPR3

β-actin

Arrb2++

Arrb2ndashndash

15

aAβ40

Aβ42

10

NS NS

05

A r r b 1 + +


A r r b 1 + +


N











a

Extracellular

domain

Plasma

membrane

Intracellular

domain

15000

b

10000

R L U

5000

C 9 9 a l o

n e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

10000

d

8000

R L U

4000

2000

6000

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

e

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

s e r i n e + C 9

9

25

20

15

A β g


05

10

Aβ40

Aβ42

c

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

C 9 9 a l o n

e

W T

G P R 3

+ C 9 9

G P R 3

D R Y

+ C 9 9

40

30

20

A β g


10

Aβ40

Aβ42













A R T I C L E S
































and Aβ42 (Fig 3c)













Vehiclea

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

Lactacystin

NCT

PS1-NTF

PS1-CTF

PEN 2

APP-FL

APP-CTF

β-actin

cVehicle

Arrb2++

Arrb2ndashndash

Arrb2++

Arrb2ndashndash

Lactacystin

Nct

Ps1-NTF

Ps1-CTF

Pen 2

App-FL

App-CTF

β-actin

b

N o n s i l e

n c i n

g s i R

N A


1 s i R N A


2 s i R N A

20

15

10

05


( n o r m


NS

d

A r r b 2 + +

A r r b 2 ndash

ndash

20

15

10

05



h


( t o v e c t o r )

4

3

JC-8

JC-8 + X

2

1

V e c t

o r

β

- a r r e

s t i n

2

f25

20

Vector

β-arrestin 2

15

10

N o


05

N C T

P S 1 -

C T F

A P H

- 1 A

P E N

2

e Vector

Fractions

β-arrestin 2

NCT

β-arrestin 2

PS1-NTF

PS1-CTF

APH-1A

PEN 2

Caveolin-1

GM130

EEA1

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

g Input

Raft

Non-raft


V e c t o r


J C - 8

J C - 8 + X

J C - 8

J C - 8 + X

PS1-CTF

PS1-CTF















A R T I C L E S
























































IP Aph-1a

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

d

eIP β-arrestin 2

W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

Nct

Aph-1a

β-arrestin 2

a

Nct




Control

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2





b

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

c

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

f



W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Aph-1a

β-arrestin 2











A R T I C L E S
















DRM domains













DISCUSSION
















































METHODS




15

a

10

05

H i p p

o c a m

p u s

C o r t e


( t o

A P P P S 1 A r r b b 2 + + )

+

H i p p

o c a m

p u s

C o r t e

x

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


b

15

10

05


( t o

A P P P S 1 A r r b b 2 + + )

+

++

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


c

A P P P S

1 A r r b 2 + +

A P P P S

1 A r r b 2 + ndash

A P P P S


NCT

PS1-CTF

PS1-NTF

APP-FL

APP-CTF

β-actin















A R T I C L E S




image analysis




reprintsindexhtml















Nat Med 17 1060ndash1065 (2011)















1994)
















8698ndash8704 (2000)



(1997)


286 2495ndash2498 (1999)



















(2007)



(2003)



















to Aβ40 and A

β43 but not to A





(2004)



J Biol Chem 287 17288ndash17296 (2012)







(2002)







107 9440ndash9445 (2010)










Sigma-Aldrich







































































































































































































A R T I C L E S
































and Aβ42 (Fig 3c)













Vehiclea

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

U n t r a

n s f e c t e d

N o n s i l e

n c i n

g


1


2

Lactacystin

NCT

PS1-NTF

PS1-CTF

PEN 2

APP-FL

APP-CTF

β-actin

cVehicle

Arrb2++

Arrb2ndashndash

Arrb2++

Arrb2ndashndash

Lactacystin

Nct

Ps1-NTF

Ps1-CTF

Pen 2

App-FL

App-CTF

β-actin

b

N o n s i l e

n c i n

g s i R

N A


1 s i R N A


2 s i R N A

20

15

10

05


( n o r m


NS

d

A r r b 2 + +

A r r b 2 ndash

ndash

20

15

10

05



h


( t o v e c t o r )

4

3

JC-8

JC-8 + X

2

1

V e c t

o r

β

- a r r e

s t i n

2

f25

20

Vector

β-arrestin 2

15

10

N o


05

N C T

P S 1 -

C T F

A P H

- 1 A

P E N

2

e Vector

Fractions

β-arrestin 2

NCT

β-arrestin 2

PS1-NTF

PS1-CTF

APH-1A

PEN 2

Caveolin-1

GM130

EEA1

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

g Input

Raft

Non-raft


V e c t o r


J C - 8

J C - 8 + X

J C - 8

J C - 8 + X

PS1-CTF

PS1-CTF















A R T I C L E S
























































IP Aph-1a

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

d

eIP β-arrestin 2

W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

Nct

Aph-1a

β-arrestin 2

a

Nct




Control

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2





b

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

c

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

f



W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Aph-1a

β-arrestin 2











A R T I C L E S
















DRM domains













DISCUSSION
















































METHODS




15

a

10

05

H i p p

o c a m

p u s

C o r t e


( t o

A P P P S 1 A r r b b 2 + + )

+

H i p p

o c a m

p u s

C o r t e

x

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


b

15

10

05


( t o

A P P P S 1 A r r b b 2 + + )

+

++

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


c

A P P P S

1 A r r b 2 + +

A P P P S

1 A r r b 2 + ndash

A P P P S


NCT

PS1-CTF

PS1-NTF

APP-FL

APP-CTF

β-actin















A R T I C L E S




image analysis




reprintsindexhtml















Nat Med 17 1060ndash1065 (2011)















1994)
















8698ndash8704 (2000)



(1997)


286 2495ndash2498 (1999)



















(2007)



(2003)



















to Aβ40 and A

β43 but not to A





(2004)



J Biol Chem 287 17288ndash17296 (2012)







(2002)







107 9440ndash9445 (2010)










Sigma-Aldrich







































































































































































































A R T I C L E S
























































IP Aph-1a

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

d

eIP β-arrestin 2

W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

Nct

Aph-1a

β-arrestin 2

a

Nct




Control

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2





b

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

Pen 2

β-arrestin 2

Input Bound

C o n t r

o l

β - a r r e s t i

n 2

IP

c

Nct

Ps1-NTF

Ps1-CTF

Aph-1a

β-arrestin 2

f



W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


W T

G p r 3 ndash ndash


Nct

Aph-1a

β-arrestin 2











A R T I C L E S
















DRM domains













DISCUSSION
















































METHODS




15

a

10

05

H i p p

o c a m

p u s

C o r t e


( t o

A P P P S 1 A r r b b 2 + + )

+

H i p p

o c a m

p u s

C o r t e

x

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


b

15

10

05


( t o

A P P P S 1 A r r b b 2 + + )

+

++

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


c

A P P P S

1 A r r b 2 + +

A P P P S

1 A r r b 2 + ndash

A P P P S


NCT

PS1-CTF

PS1-NTF

APP-FL

APP-CTF

β-actin















A R T I C L E S




image analysis




reprintsindexhtml















Nat Med 17 1060ndash1065 (2011)















1994)
















8698ndash8704 (2000)



(1997)


286 2495ndash2498 (1999)



















(2007)



(2003)



















to Aβ40 and A

β43 but not to A





(2004)



J Biol Chem 287 17288ndash17296 (2012)







(2002)







107 9440ndash9445 (2010)










Sigma-Aldrich







































































































































































































A R T I C L E S
















DRM domains













DISCUSSION
















































METHODS




15

a

10

05

H i p p

o c a m

p u s

C o r t e


( t o

A P P P S 1 A r r b b 2 + + )

+

H i p p

o c a m

p u s

C o r t e

x

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


b

15

10

05


( t o

A P P P S 1 A r r b b 2 + + )

+

++

APPPS1 Arrb2++

APPPS1 Arrb2+ndash


c

A P P P S

1 A r r b 2 + +

A P P P S

1 A r r b 2 + ndash

A P P P S


NCT

PS1-CTF

PS1-NTF

APP-FL

APP-CTF

β-actin















A R T I C L E S




image analysis




reprintsindexhtml















Nat Med 17 1060ndash1065 (2011)















1994)
















8698ndash8704 (2000)



(1997)


286 2495ndash2498 (1999)



















(2007)



(2003)



















to Aβ40 and A

β43 but not to A





(2004)



J Biol Chem 287 17288ndash17296 (2012)







(2002)







107 9440ndash9445 (2010)










Sigma-Aldrich







































































































































































































A R T I C L E S




image analysis




reprintsindexhtml















Nat Med 17 1060ndash1065 (2011)















1994)
















8698ndash8704 (2000)



(1997)


286 2495ndash2498 (1999)



















(2007)



(2003)



















to Aβ40 and A

β43 but not to A





(2004)



J Biol Chem 287 17288ndash17296 (2012)







(2002)







107 9440ndash9445 (2010)










Sigma-Aldrich









































































































































































































Sigma-Aldrich





































































































































































































thathiah 13 gpr3 arr2

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