PICK1 interacts with PACSIN to regulate AMPA
receptor internalization and cerebellar
Victor Anggonoa,b,c,d, Yeliz Koç-Schmitze, Jocelyn Widagdoa,b,d, Jan Kormanne, Annie Quanf, Chih-Ming Chena,
Phillip J. Robinsonf, Se-Young Choia,b,g, David J. Lindena, Markus Plomanne, and Richard L. Huganira,b,1
aThe Solomon H. Snyder Department of Neuroscience andbHoward Hughes Medical Institute, The Johns Hopkins University School of Medicine,
Baltimore, MD 21205;cClem Jones Centre for Ageing Dementia Research anddQueensland Brain Institute, The University of Queensland, Brisbane,
QLD 4072, Australia;eCenter for Biochemistry, Medical Faculty, University of Cologne, D-50931 Cologne, Germany;fCell Signalling Unit, Children’s
Medical Research Institute, The University of Sydney, Wentworthville, NSW 2145, Australia; andgDepartment of Physiology, Dental Research Institute,
Seoul National University School of Dentistry, Seoul 110-749, Republic of Korea
Contributed by Richard L. Huganir, July 9, 2013 (sent for review May 17, 2013)
The dynamic trafficking of AMPA receptors (AMPARs) into and out
of synapses is crucial for synaptic transmission, plasticity, learning,
and memory. The protein interacting with C-kinase 1 (PICK1) di-
rectly interacts with GluA2/3 subunits of the AMPARs. Although
the role of PICK1 in regulating AMPAR trafficking and multiple
forms of synaptic plasticity is known, the exact molecular mecha-
nisms underlying this process remain unclear. Here, we report
a unique interaction between PICK1 and all three members of
the protein kinase C and casein kinase II substrate in neurons
(PACSIN) family and show that they form a complex with AMPARs.
Our results reveal that knockdown of the neuronal-specific pro-
tein, PACSIN1, leads to a significant reduction in AMPAR internal-
ization following the activation of NMDA receptors in hippo-
campal neurons. The interaction between PICK1 and PACSIN1 is
regulated by PACSIN1 phosphorylation within the variable region
and is required for AMPAR endocytosis. Similarly, the binding of
PICK1 to the ubiquitously expressed PACSIN2 is also regulated by
the homologous phosphorylation sites within the PACSIN2-variable
region. Genetic deletion of PACSIN2, which is highly expressed in
Purkinje cells, eliminates cerebellar long-term depression. This defi-
cit can be fully rescued by overexpressing wild-type PACSIN2, but
not by a PACSIN2 phosphomimetic mutant, which does not bind
PICK1 efficiently. Taken together, our data demonstrate that the
interaction of PICK1 and PACSIN is required for the activity-depen-
dent internalization of AMPARs and for the expression of long-term
depression in the cerebellum.
excitatory synaptic transmission in the brain. AMPA re-
ceptors (AMPARs) are heterotetrameric assemblies of four
highly homologous subunits, GluA1–4, that are highly enriched
at synapses. The regulation of AMPAR density at synapses has
emerged as a key regulator of synaptic plasticity, which is thought
to be one of the key cellular components underlying learning and
memory (1, 2). In general, an increase in the number of synaptic
AMPARs leads to long-term potentiation (LTP), whereas the
removal of surface AMPARs by endocytosis results in long-term
depression (LTD) (1, 3). The trafficking of AMPARs into and
out of synapses is highly dynamic and is regulated by various
AMPAR-interacting proteins that bind to the cytoplasmic tail of
the receptors (1).
Protein interacting with C-kinase 1 (PICK1) interacts directly
with the C termini of the GluA2 and GluA3 subunits of AMPARs
through its postsynaptic density-95/discs-large/zona occludens-1
(PDZ) domain (4, 5). PICK1 has been shown to regulate the
surface expression, trafficking, and synaptic targeting of AMPARs
(6), and genetic deletion of PICK1 has revealed its important role
in several forms of synaptic plasticity, such as hippocampal and
cerebellar LTD, hippocampal LTP, Ca2+-permeable AMPAR
MPA-type glutamate receptors mediate most of the fast
plasticity, mGluR LTD, and homeostatic plasticity (7–14). PICK1
also contains a central box-dependent myc-interacting protein-1
(Bin)/amphiphysin/reduced viability to nutrient starvation-homology
(Rvs) (BAR) domain, which dimerizes to form a banana-shaped
crescent and binds to phospholipids (15). Many BAR-domain
proteins possess the ability to sense and/or generate curvature of
the plasma membrane and are believed to play key roles in en-
docytosis and vesicle trafficking (16). Expression of a PICK1
BAR-domain mutant that is deficient in lipid binding impairs both
hippocampal (15) and cerebellar LTD (8), suggesting a role for the
PICK1 BAR domain in mediating the internalization, recycling,
and/or intracellular retention of GluA2-containing AMPARs (1,
6). However, the molecular mechanisms by which PICK1 mediates
the trafficking of AMPARs are complex and remain unclear.
Protein kinase C and casein kinase II substrate in neurons
(PACSINs), also known as syndapins, belong to the feline sar-
coma-Cdc42 interacting protein 4 (Fes-CIP4) homology BAR
(F-BAR) subfamily within the BAR-domain superfamily of pro-
teins, which sense and/or generate lipid tubules with a larger and
shallower curvature than other BAR-domain proteins (17, 18).
PACSIN1 is a neuronal-specific protein that regulates activity-
dependent retrieval of synaptic vesicles in the presynaptic termi-
nals (19–22) as well as the endocytosis of the NR3A subunit of
NMDA receptors on the postsynaptic membrane (23). More
importantly, PACSIN1 has been implicated in various neurode-
generative disorders, including Huntington and Alzheimer’s
diseases (24, 25). PACSIN2 is ubiquitously expressed, whereas
the expression of PACSIN3 is restricted to the muscle, heart, and
lung (26). All three members of the PACSIN family of proteins
bind to endocytic machinery such as the large GTPase dyna-
min and the actin-modifier neural Wiskott-Aldrich syndrome
protein (N-WASP) through their conserved src-homology 3
(SH3) domains, potentially linking membrane trafficking and
actin cytoskeletal rearrangement (27).
In this study, we found that PICK1 interacts with PACSINs
and forms a complex with AMPARs. This interaction is regu-
lated by PACSIN phosphorylation and is required for NMDA-
induced AMPAR endocytosis and cerebellar LTD. Overall, our
data provide experimental evidence supporting the functional
Author contributions: V.A. and R.L.H. designed research; V.A., Y.K.-S., J.W., J.K., A.Q.,
C.-M.C., S.-Y.C., and D.J.L. performed research; M.P. contributed new reagents/analytic
tools; V.A., J.W., P.J.R., S.-Y.C., D.J.L., M.P., and R.L.H. analyzed data; and V.A. and R.L.H.
wrote the paper.
Conflict of interest statement: R.L.H. provides antibodies to Millipore Corporation for sale
and is entitled to a share of royalties received by Johns Hopkins University on sales of
products described in this article. This arrangement is managed under a licensing agree-
ment between Millipore Corporation and Johns Hopkins University, and the terms are
managed by Johns Hopkins University in accordance with its conflict-of-interest policies.
R.L.H. is a paid consultant to Millipore Corporation.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 20, 2013
| vol. 110
| no. 34www.pnas.org/cgi/doi/10.1073/pnas.1312467110
duality of presynaptic trafficking machinery in regulating post-
synaptic AMPAR trafficking and synaptic plasticity.
PACSIN Interacts with PICK1 and Forms a Complex with AMPARs. It is
well established that PICK1 plays an important role in multiple
forms of synaptic plasticity by modulating the subunit composi-
tion, trafficking, and surface expression of AMPARs (1). How-
ever, little is known about the detailed molecular mechanisms by
which PICK1 regulates AMPAR trafficking. To investigate this
we performed a yeast two-hybrid screen and discovered a unique
interaction between PICK1 and PACSIN3. To validate this in-
teraction, we first transfected HEK 293T cells with full-length
constructs encoding GFP, GFP-PACSIN1−3, and myc-PICK1,
either individually or in combination. GFP-PACSIN1−3, but not
GFP, coimmunoprecipitated with myc-PICK1 when coexpressed
in these cells and demonstrated that these coimmunoprecipita-
tions were abolished in the absence of myc-PICK1, confirming
the specificity of the interaction between PICK1 and all three
PACSIN proteins in vitro (Fig. 1A). This interaction was further
confirmed by the ability of recombinant GST-PACSIN1−3, but
not GST alone, to pull down PICK1 when incubated with total
lysates from HEK 293T cells that overexpressed Strep-tagged
PICK1 (Fig. 1B). Immunofluorescence labeling of cultured rat
hippocampal neurons with specific antibodies against PICK1 and
PACSIN1 revealed extensive colocalization in the perinuclear
region within the soma and, to a lesser extent, in the dendritic
shaft, indicating that endogenous PICK1 and PACSIN1 share
a similar subcellular distribution in neurons (Fig. 1C). Finally, to
determine if the interaction between PICK1 and PACSINs occurs
in vivo, we performed immunoprecipitation assays with total mouse
brain lysates using specific antibodies against PACSIN1, 2, and 3.
Western-blotting analysis revealed that PICK1 coimmunoprecipi-
tated with PACSIN1−3, but not with anti-matrilin 3 antibody, which
was used as a negative control (Fig. 1D).
To determine whether PACSIN associates with AMPARs, we
transfected HEK 293T cells with constructs encoding HA-
GluA2, myc-PICK1 and GFP-PACSIN1. Myc-PICK1 coimmu-
noprecipitated with HA-GluA2, but GFP-PACSIN1 only coim-
munoprecipitated with HA-GluA2 when myc-PICK1 was present
in the cells (Fig. 2A). These data suggest that PACSIN1 forms
a complex with GluA2 in vitro via PICK1 binding. Immunopre-
cipitation from total mouse brain extracts using specific anti-
bodies against PACSIN1, 2, and 3 revealed that GluA2/3
subunits of AMPARs are associated with all three PACSIN
proteins in vivo (Fig. 2B). Together, these results suggest that
PACSINs may play a role in the regulation of AMPAR traf-
ficking in neurons.
PACSIN1 Loss-of-Function Impairs Activity-Dependent AMPAR
Internalization. As all three PACSIN proteins bind endocytic
proteins and have been implicated in endocytosis (26), we hy-
pothesized that PACSIN might regulate AMPAR endocytosis in
neurons. To test this possibility, we first cotransfected full-length
PACSIN1−3 constructs with the GFP-GluA2 construct and ex-
amined the surface expression of AMPARs in hippocampal
neurons. This analysis revealed that overexpression of PACSINs
had no effect on the steady-state level of GFP-GluA2 surface
expression (surface/total GFP-GluA2 ratio, control, 1 ± 0.02;
PACSIN1, 0.97 ± 0.02; PACSIN2, 0.99 ± 0.03; PACSIN3, 0.94 ±
0.02) (Fig. S1). Next, we generated specific shRNAs against
PACSIN1, the major PACSIN isoform expressed in hippocampal
neurons (Fig. S2A). The PACSIN1 shRNA#1 efficiently reduced
endogenous PACSIN1 expression in neurons after 3 d of ex-
pression (Fig. 3A). Western-blotting analysis of lysates from
hippocampal neurons that were infected with lentiviral particles
expressing GFP and PACSIN1 shRNA revealed a near-complete
knockdown of PACSIN1 without affecting the level of PICK1
protein (Fig. 3B). Knockdown of PACSIN1 did not significantly
alter the dendritic morphology (Fig. S2 B and C) or the steady-
state level of myc-GluA2 surface expression in mature hippo-
campal neurons (surface/total myc-GluA2 ratio, control, 1 ±
0.06; sh#1, 1.14± 0.09) (Fig. 3 C and D). These data indicate
that PACSIN1 may not regulate AMPAR trafficking under
To investigate whether PACSIN1 regulates activity-dependent
trafficking of AMPARs, we used the fluorescence-based anti-
body-feeding assay with myc antibodies to measure the degree of
intracellular accumulation of endocytosed myc-GluA2 subunits
in live transfected hippocampal neurons. In control neurons that
were transfected with pSuper empty vector, intracellular accu-
mulation of myc-GluA2 could be observed 15 min after NMDA
treatment (50 μM NMDA + 1 μM tetrodotoxin) (Fig. 3E). The
staining for internalized receptors was specific, as no staining was
visible in nonpermeabilized neurons (Fig. S3). However, the
amount of internalized myc-GluA2 was significantly reduced in
PACSIN1 knockdown neurons compared with control neurons
(internalization index, pSuper, 1 ± 0.04; sh#1, 0.68 ± 0.03) (Fig.
3 E and F). The reduction in NMDA-induced myc-GluA2 in-
ternalization caused by PACSIN1 shRNA could be fully rescued
by the expression of an shRNA-resistant PACSIN1 rescue con-
struct in the hippocampal neurons (0.92 ± 0.05) (Fig. 3 E and F).
These results indicate that PACSIN1 regulates the activity-
dependent trafficking of AMPARs but not their trafficking under
basal conditions, demonstrating a unique role for PACSIN1 in
facilitating GluA2 endocytosis.
PICK1–PACSIN1 Interaction Is Required for NMDA-Induced AMPAR
Endocytosis. Having established a role of PACSIN1 in regulat-
ing AMPAR trafficking, we then asked whether its interaction
were transfected with GFP or GFP-PACSIN1−3 with or without myc-PICK1,
lysed, and immunoprecipitated with anti-myc antibody. Bound proteins
were subjected to Western blot analyses with anti-GFP and anti-myc anti-
bodies. Asterisk denotes IgG-heavy chain. (B) Total cell lysate containing
recombinant Strep-PICK1 was incubated with either GST or GST-PACSIN1−3
bound on glutathione-Sepharose beads. The binding of Strep-PICK1 was
visualized by Western blotting with HRP-conjugated Strep-tactin (Upper).
Equal amounts of bait proteins were used as shown by the Ponceau staining
of the membrane (Lower). (C) Endogenous PICK1 and PACSIN1 colocalize in
the perinuclear structure within the soma and in dendritic shafts of a cul-
tured hippocampal neuron as shown by immunostaining with anti-PICK1
and anti-PACSIN1 antibodies. Higher-magnification images are shown in the
bottom panels. (Scale bar, 50 μm.) (D) Total brain lysates were subjected to
immunoprecipitation assays with anti-PACSIN1−3 antibodies cross-linked on
protein-A agarose beads. Bound PICK1 was visualized by Western blot
analyses with two different anti-PICK1 antibodies. Matrilin3 (Mtrl3) coim-
munoprecipitation was included as a negative control.
PICK1 interacts with PACSIN in vitro and in vivo. (A) HEK 293T cells
Anggono et al.PNAS
| August 20, 2013
| vol. 110
| no. 34
with PICK1 was required to drive AMPAR endocytosis. To de-
fine the minimal region of PACSIN1 that interacts with PICK1,
we generated a series of GFP-PACSIN1 truncation constructs
and cotransfected them with myc-PICK1 into HEK 293T cells.
As expected, GFP-PACSIN1 full-length and GFP-PACSIN1 F-
BAR domain coimmunoprecipitated with myc-PICK1, suggest-
ing that PICK1 can interact with PACSIN1 through hetero-
dimerization of their BAR and F-BAR domains, respectively (Fig.
4 A and B, lanes 1–3). The interaction between PACSIN1 and
PICK1 was enhanced by the SH3-domain deletion (Fig. 4B, lane 4),
consistent with two earlier studies describing an intramolecular
interaction between the F-BAR and the SH3 domains that inhibits
PACSIN1 function (18, 28). Surprisingly, we also found that the
GFP-PACSIN1-ΔF-BAR domain [variable region (VAR) + SH3]
was able to bind myc-PICK1 robustly (Fig. 4B, lane 6). Further-
more, the GFP-PACSIN1–variable region alone, but not the SH3
domain, was sufficient to bind PICK1 (Fig. 4B, lanes 5 and 7),
establishing the PACSIN1-variable region as the minimal se-
quence required for PICK1 binding in addition to the conventional
BAR-domain interactions. Uniquely, the interaction between
PICK1 and PACSIN1 was independent of the asparagine-proline-
phenylalanine (NPF) motifs within the variable region, which are
known to mediate the interaction between PACSIN and Eps15-
homology-domain (EHD) proteins (Fig. S4) (29).
PACSIN1 is a phosphoprotein with 15 phosphorylation sites
identified through proteomics studies or candidate approaches
(30–35). Interestingly, seven of these phosphorylation sites are
clustered within the variable region of PACSIN1. To directly test
if PICK1 binding is modulated by PACSIN1 phosphorylation, we
generated a series of mutations on Ser-343, Ser-345, and Ser-346,
either individually or in combination, into Ala or Glu to prevent
or mimic phosphorylation on these sites. We chose these Ser
residues because they have been identified as in vivo phosphor-
ylation sites in the mouse brain (32, 34, 35). The PACSIN1-
variable region, either wild type (WT) or phosphomutants, was
expressed as a GST-fusion protein, immobilized on glutathione-
Sepharose and incubated with total brain extract. Western-blotting
analysis with specific antibodies against PICK1 revealed that the
binding of PICK1 to individual nonphosphorylatable (S to A)
and pseudophosphorylation (S to E) mutants of PACSIN1 had
no effect on PICK1 binding (Fig. 4C). In contrast, the binding of
cells were transfected with HA-GluA2, myc-PICK1, and GFP-PACSIN1, lysed,
and immunoprecipitated with anti-HA antibody. Bound proteins were blotted
with antibodies against HA, GFP, and myc. Asterisk denotes IgG-heavy chain.
(B) Total brain lysates were immunoprecipitated with anti-PACSIN1−3 anti-
bodies. Bound GluA2/3 was visualized by Western blot analyses with antibodies
against the GluA2/3 and GluA3 subunits of AMPARs. The immunoprecipitation
with GFP antibody was included as a negative control.
PACSIN forms a complex with GluA2 in vitro and in vivo. (A) HEK 293T
GluA2 internalization. (A) Hippocampal neurons
were cotransfected with pEGFP and a pSuper empty
vector, pSuper-PACSIN1-shRNA#1 or pRK5-shRNA#1-
HA-PACSIN1 rescue construct, fixed, and stained
with anti-PACSIN1 antibody. (Scale bar, 50 μm.) (B)
Cultured hippocampal neurons were infected with
lentivirus particles expressing GFP (FuGW), GFP and
PACSIN1-shRNA#1, or FuW-shRNA#1-myc-PACSIN1
rescue construct at days in vitro (DIV) 7. Neurons
were lysed at DIV15, and the efficacy of PACSIN1
knockdown was assessed by Western blot using
specific antibodies against PACSIN1. (C) Cultured
hippocampal neurons were transfected with either
empty pSuper vector (control) or shRNA#1 construct,
together with myc-GluA2. Representative images of
surface and total myc-GluA2 in a neuron from each
group are shown. (Scale bar, 50 μm.) (D) Quantifi-
cation of surface/total GluA2 ratio normalized to
the value of control neurons (n = 7–9 neurons per
group). (E) NMDA-induced internalization of myc-
GluA2 was measured by fluorescence-based anti-
body-feeding assay. (Scale bar, 50 μm.) (F) The in-
ternalization of myc-GluA2 was measured as the
ratio of internalized/total fluorescence (internalization
index), normalized to pSuper control. Data represent
mean ± SEM (ANOVA, ***P < 0.001 against control
neurons per group).
PACSIN1 knockdown reduces NMDA-induced
###P < 0.001 against rescued cells; n = 13–21
| www.pnas.org/cgi/doi/10.1073/pnas.1312467110 Anggono et al.
PICK1 to a PACSIN1 triple phosphomimetic mutant (tmE) was
markedly reduced, whereas PICK1 binding to the triple phospho-
mutant (tmA) remained intact (Fig. 4C). The interaction with EHD
protein, which binds to the PACSIN1-variable region, was mini-
mally affected, confirming the specificity of the pull-down assay
(Fig. 4C). This suggests that multiple phosphorylations of PACSIN1
on Ser-343, Ser-345, and Ser-346 may inhibit its interaction with
PICK1, but not with EHD proteins.
To determine the functional significance of the interaction
between PACSIN1 and PICK1 in regulating AMPAR trafficking,
we performed the antibody-feeding GluA2 internalization assay
by applying a molecular replacement strategy. We first generated
bicistronic constructs encoding shRNA#1 and HA-PACSIN1
shRNA-resistant cDNAs (either WT, tmA, or tmE) driven by H1
RNA polymerase III and CMV promoters, respectively. Trans-
fection of these constructs into primary hippocampal neurons allows
an efficient knockdown of endogenous PACSIN1 proteins and
simultaneous overexpression of exogenous PACSIN1 protein. The
NMDA-induced internalization of myc-GluA2 in neurons trans-
fected with HA-PACSIN1 tmA was indistinguishable from that in
neurons overexpressing HA-PACSIN1 WT (internalization index,
WT, 1 ± 0.04; tmA, 1.02 ± 0.04) (Fig. 4 D and E). However, there
was a significant decrease in intracellular myc-GluA2 follow-
ing NMDA stimulation in neurons that were transfected with
HA-PACSIN1 tmE, which had reduced binding with PICK1
(0.84 ± 0.03) (Fig. 4 D and E). These data demonstrate that PICK1
is a phosphorylation-regulated PACSIN1 partner and that their
interaction is required for efficient trafficking of AMPARs fol-
lowing NMDA receptor activation in hippocampal neurons.
PICK1–PACSIN2 Interaction Is Required for Cerebellar LTD. One of
the phenotypes of PICK1 knockout mice is the complete absence
of cerebellar LTD (8, 14). Cerebellar LTD is expressed in Pur-
kinje cells upon coactivation of parallel fibers and climbing fibers
of the cerebellar granule cells and the inferior olive, respectively.
In situ hybridization data from the Allen Brain Atlas show that
PACSIN2 is the major PACSIN protein expressed in the Pur-
kinje cells of the cerebellum (36). This was further confirmed by
our immunohistochemical analysis of the mouse brain stained
with specific antibodies against PACSIN2 (Fig. 5A and Fig. S5).
Our data demonstrated specific PACSIN2 staining in the soma
and dendrites of Purkinje cells in the cerebellum, whereas its
expression in the hippocampus was restricted to the CA3 and
dentate gyrus (Fig. S5). Given the functional interaction between
PACSIN1 and PICK1 in regulating AMPAR endocytosis in
cultured hippocampal neurons, we hypothesized that PACSIN2
might serve the same role in regulating AMPAR trafficking and
synaptic plasticity. No obvious abnormalities in cerebellar archi-
tecture were observed in hematoxylin- and eosin-stained brain
sections of PACSIN2 knockout mice (Fig. 5A). We then mea-
sured LTD evoked by glutamate/depolarization pairing in dis-
sociated cerebellar cultures derived from PACSIN2 knockout
mice (8). We found that the delivery of a depolarization stimulus
paired with a pulse of glutamate failed to induce LTD in PAC-
SIN2 knockout Purkinje cells (107 ± 7.5% of baseline at t = 17.5
min and 117 ± 8.6% of baseline at t = 40 min, n = 7) (Fig. 5B),
resembling the phenotype seen in PICK1 knockout neurons (8).
Gene gun-mediated transfection with full-length GFP-PACSIN2
WT construct produced a complete rescue of LTD (54 ± 7.4% of
baseline at t = 40 min, n = 8). Interestingly, the GFP-PACSIN1
construct failed to fully rescue LTD in PACSIN2 knockout neu-
rons (84 ± 6.5% of baseline at t = 40 min, n = 13). These results
demonstrate an essential role for PACSIN2 in the expression of
postsynaptic LTD in the cerebellum.
To investigate whether the interaction between PACSIN2 and
PICK1 is required for the expression of cerebellar LTD, we
attempted to identify a PACSIN2 mutant that decreases binding
with PICK1. A protein sequence alignment of the PACSIN1- and
PACSIN2-variable regions revealed that all three PACSIN1
phosphorylation sites that regulate PICK1 binding are com-
pletely conserved in PACSIN2 (Ser-372, Ser-386, and Ser-387)
(Fig. 5C). We therefore generated a series of PACSIN2 non-
phosphorylatable and phosphomimetic mutants on these con-
served Ser residues, as well as Ser-375, which has been shown to
be phosphorylated in HeLa cells (37). The binding affinity of
these mutants to PICK1 was tested by coimmunoprecipitation
assays in HEK 293T cell lysates expressing myc-PICK1 with ei-
ther WT or mutant GFP-PACSIN2. Most of these PACSIN2
mutants had no effect on binding to PICK1, except for the S372/
375A and S372/375/386/387E [quadruple glutamic acid mutant
(QmE)] mutants, which had significantly increased and decreased
binding to PICK1, respectively (Fig. 5 D and E). These data
suggest that, like the PACSIN1–PICK1 interaction, the binding
of PICK1 may also be regulated by PACSIN2 phosphorylation
status on the conserved Ser residues within the variable region.
Next, we transfected PACSIN2 knockout Purkinje cells with a
GFP-PACSIN2 QmE cDNA construct and found that LTD evoked
by glutamate/depolarization pairing was completely blocked (93 ±
7.9% of baseline at t = 40 min, n = 9) (Fig. 5B). In contrast, LTD
was rescued in GFP-PACSIN2 quadruple alanine mutant (QmA)-
transfected cultures (58 ± 6.9% of baseline at t = 40 min, n = 8)
(Fig. 5B). Neither PACSIN2 deletion nor any of the four rescue
constructs used herein produced attenuation of mGluR1 agonist-
evoked Ca2+mobilization or depolarization-evoked Ca2+influx as
measured with bis-fura-2 microfluorimetry. These results suggest
that PICK1 is a phosphorylation-regulated partner for PACSIN2
and that their interaction is required for cerebellar LTD.
dependent GluA2 internalization. (A) A schematic diagram of the domain
structure of PACSIN1 and various GFP-tagged PACSIN1 truncations used in
the study. (B) HEK 293T cells were cotransfected with myc-PICK1 and various
GFP-PACSIN1 constructs as shown in panel 4A, lysed, and immunoprecipi-
tated with anti-myc antibody. Bound proteins were subjected to Western
blot analyses with anti-GFP and anti-myc antibodies. (C) GST-PACSIN1-VAR
mutants were coupled to glutathione-Sepharose beads and incubated with
total rat brain lysates. Bound proteins were subjected to Western blot
analyses with anti-PICK1 and anti-EHD antibodies (Middle). Equal amounts
of bait proteins were used as shown by the Ponceau staining of the mem-
brane (Bottom). Representative blots from two independent experiments
are shown. (D) Cultured hippocampal neurons were transfected with PACSIN1
rescue constructs, either WT, tmA (S343A, S345A, and S346A), or tmE (S343E,
S345E, and S346E), together with myc-GluA2. NMDA-induced internalization
of myc-GluA2 was measured by fluorescence-based antibody-feeding assay.
(Scale bar, 50 μm.) (E) The internalization of myc-GluA2 was measured as the
ratio of internalized/total fluorescence (internalization index), normalized to
WT neurons. Data represent mean ± SEM (ANOVA, *P < 0.05 against WT cells,
#P < 0.05 against tmA cells; n = 15–17 neurons per group).
PICK1 binding to the PACSIN1-variable region is required for activity-
Anggono et al. PNAS
| August 20, 2013
| vol. 110
| no. 34
PACSINs have previously been implicated in endocytosis, in
particular the brain-specific PACSIN1, which interacts with dyna-
min to regulate activity-dependent bulk endocytosis in presynaptic
terminals (21, 22, 38–40). Although it is predominantly expressed
in presynaptic nerve terminals, our data indicate that PACSIN1
can also be found in the soma and dendrites, where it colocalizes
with PICK1 in mature hippocampal neurons. This is consistent
with a previous study reporting a postsynaptic role of PACSIN1
in mediating synaptic removal of NR3A-containing NMDA
receptors in developing hippocampal neurons (23). Our bio-
chemical data show that PACSIN1 also associates with AMPARs,
most likely through its interaction with PICK1. Indeed, specific
knockdown of PACSIN1 protein in neurons significantly reduced
the intracellular accumulation of GluA2 following NMDA receptor
activation. However, overexpression or knockdown of PACSIN1
had no effect on the steady-state surface expression of AMPARs,
suggesting that PACSIN1 regulates the activity-dependent inter-
nalization of AMPARs in hippocampal neurons. We propose that
PACSIN1 may serve as a scaffolding protein that links AMPARs
to the endocytic machinery, such as dynamin, and facilitate the
removal of AMPARs from the plasma membrane. In addition,
PACSIN1 may be involved in actin cytoskeleton remodeling and/or
in generating plasma-membrane curvature during endocytosis
through its interaction with N-WASP and its lipid-binding F-BAR
How might PICK1 and PACSIN1 work cooperatively to drive
AMPAR internalization in neurons? Both PICK1 and PACSIN1
share some structural features in that they are subjected to in-
tramolecular interactions, which allow them to exist in “open” or
“closed” conformations, and they can interact with actin cy-
toskeleton regulators, the Arp2/3 complex, and N-WASP, re-
spectively (18, 28, 41, 42). Our domain-mapping analysis showed
that the binding to PICK1 is mediated by the variable region in
PACSIN1, leaving the SH3 domain available to bind to endocytic
proteins and actin-reorganizing molecules. It is plausible that
when GluA2 binds PICK1 during the early stage of endocytosis,
PICK1 adopts an open conformation and inhibits the activity of
the Arp2/3 complex, leading to a reduction in plasma membrane
tension and an easing of membrane bending (41). This sub-
sequently leads to the recruitment of PACSIN1 in an open
conformational state when bound to N-WASP and/or dynamin.
At the endocytic sites, PACSIN1 may positively regulate the
internalization of AMPARs by one or all of the following me-
chanisms: generating plasma membrane curvature during the
invagination process through the F-BAR domain, facilitating
vesicle fission via its interaction with dynamin, or providing me-
chanical forces to propel vesicles away from the plasma mem-
brane by enhancing N-WASP-dependent actin polymerization.
The fact that PICK1 binding to PACSIN1 is modulated by the
phosphorylation status within the variable region indicates another
layer of regulation to the intricate process of AMPAR endocy-
tosis. Further studies will be necessary to address how phosphor-
ylation of these serine residues is regulated by synaptic activity.
Recently, PICK1 has been shown to associate with recycling
endosomes and inhibit the recycling of its binding partners from
the Rab11-positive endosomes toward the plasma membrane (43).
This model helps to explain the accelerated rate of GluA2 recycling
observed in PICK1 knockout or knockdown neurons (11, 44) and
the absence of LTD in PICK1 knockout mice (8, 10, 13, 14). In our
study, the intracellular accumulation of GluA2 measured 15 min
poststimulation reflects the balance between endocytosis of surface
receptors and the recycling of internalized receptors back to the
plasma membrane. Hence, we cannot rule out the possibility that
the decrease in internalized GluA2 in PACSIN1 knockdown neu-
rons is due to an impairment in AMPAR recycling. One of the
major interacting partners of PACSIN is EHD protein, which has
been shown to play a crucial role in endosomal recycling (29). In-
terestingly, EHD-1 has previously been implicated in the recycling
of AMPARs (45). This suggests that PACSIN may also be involved
in the intracellular endosomal trafficking of AMPARs via its in-
teraction with EHD-1. Regardless of whether PACSIN1 regulates
the trafficking of AMPARs at the steps of receptor internalization
and/or recycling, it requires efficient binding to PICK1.
Internalization of AMPARs is believed to be a postsynaptic
mechanism for the expression of LTD (1, 3, 46). Our data show
that the loss of PACSIN2 function in the Purkinje cells com-
pletely eliminates cerebellar LTD in the Purkinje cells. However,
given that PACSIN1 and PACSIN2 both bind PICK1, dynamin,
EHD-1, and N-WASP, it was surprising that PACSIN1 only
partially rescued the LTD phenotype in PACSIN2 knockout
neurons, suggesting a unique function of PACSIN2 in mediating
AMPAR trafficking and synaptic plasticity in Purkinje cells of
the cerebellum. Although there is a high degree of conservation
of the F-BAR and the SH3 domains between PACSIN1 and 2,
recent structural studies have provided evidence that the level of
membrane-sculpting activity and the extent of intramolecular
inhibition vary between these two proteins (47, 48). Despite this
structural difference, the binding of PICK1 is still regulated by
the conserved phosphorylation sites within the PACSIN2-variable
knockout mice, showing no gross abnormalities in the cytoarchitecture. The genotype in each case was confirmed by immunostaining with anti-PACSIN2
antibody. (Scale bar, 150 μm.) (B) Cerebellar LTD is abolished in Purkinje cells derived from PACSIN2 KO mice and is restored by acute transfection with the
PACSIN2 WT or phosphodeficient mutant, but not by the PACSIN2 phosphomimetic mutant or PACSIN1 WT. Following the acquisition of baseline responses to
iontophoretic pulses of glutamate, delivered at 0.05 Hz, LTD was induced in cerebellar cultures by a depolarization stimulus paired with an iontophoretic
glutamate pulse at t = 0 min (six repetitions, indicated by horizontal bar). Error bars indicate the SEM. (Scale bars, 1 s, 30 pA.) (C) The amino acid sequence
alignment of the PACSIN1- and PACSIN2-variable regions, highlighting the conservation of the phosphorylation sites between the two proteins. (D) HEK 293T
cells were transfected with myc-PICK1 and GFP-PACSIN2, either WT or phosphodeficient and phosphomimetic mutants, lysed, and immunoprecipitated with
anti-myc antibody. Bound proteins were blotted with anti-GFP and anti-myc antibodies. (E) The amount of myc-PICK1 bound to GFP-PACSIN2 mutants was
quantified by densitometry analysis of Western blots. Data represent mean ± SEM (ANOVA, *P < 0.05 and **P < 0.01 against PACSIN2-WT; n = 4).
PACSIN2 is required for the expression of cerebellar LTD. (A) Hematoxylin and eosin (HE)-stained sections of cerebellum from PACSIN2 WT and
| www.pnas.org/cgi/doi/10.1073/pnas.1312467110Anggono et al.
region. More importantly, the interaction between PACSIN2 and
PICK1 is absolutely required for cerebellar LTD.
In conclusion, we have identified a unique interaction between
PICK1 and the endocytic protein PACSIN and have shown that
their phosphorylation-regulated interaction is required for the
activity-dependent trafficking of GluA2-containing AMPARs and
the expression of cerebellar LTD. Our data also provide further
evidence supporting the functional duality of presynaptic trafficking
machinery, such as the lipid phosphatase, synaptojanin1 (49), and
the exocytic protein, complexin1 (50), in regulating the trafficking
of AMPARs on the postsynaptic membrane and synaptic plasticity.
Materials and Methods
Materials. The PACSIN1 shRNA targeting sequences were as described pre-
viously (22). The full details of DNA constructs and antibodies are described
in SI Materials and Methods.
GST Pull-Down and Immunoprecipitation Assays. GST-fusion proteins were ex-
pressed in Escherichia coli as described previously (21). GST pull-down and
immunoprecipitation assays were performed on total brain extracts or
transfected HEK 293T cells as described in SI Materials and Methods.
Hippocampal Neurons and Internalization Assays. Cultured neurons were
prepared from E18 rat pups as described previously (44). For the NMDA-
induced internalization assays, neurons were transfected with lipofectamine
and subjected to antibody-feeding assays as described in SI Materials
and Methods. All experimental procedures with mice and rats were
approved by the Animal Care and Use Committee of the Johns Hopkins
University School of Medicine and the University of Queensland.
Electrophysiology. Cerebellar LTD recordings from the cultured Purkinje cells
were performed as described in SI Methods.
ACKNOWLEDGMENTS. We thank Richard Johnson, Yi Lin Yu, Devorah
VanNess, and Jeffrey Hanks-Thompson for technical assistance and Rowan
Tweedale for helpful comments on this manuscript. This work was sup-
ported by National Institutes of Health Grants NS36715 (to R.L.H.) and
MH51106 (to D.J.L.), the Deutsche Forschungsgemeinschaft (PL233/3)
and the Köln Fortune program of the Medical Faculty of the University
of Cologne (M.P.), and the John T. Reid Charitable Trusts (V.A.) V.A. was
supported by fellowships from the International Human Frontier Science
Program (LT00399/2008-L) and the Australian National Health and Medical
Research Council (477108). R.L.H. is an Investigator of the Howard Hughes
1. Anggono V, Huganir RL (2012) Regulation of AMPA receptor trafficking and synaptic
plasticity. Curr Opin Neurobiol 22(3):461–469.
2. Shepherd JD, Huganir RL (2007) The cell biology of synaptic plasticity: AMPA receptor
trafficking. Annu Rev Cell Dev Biol 23:613–643.
3. Malinow R, Malenka RC (2002) AMPA receptor trafficking and synaptic plasticity.
Annu Rev Neurosci 25:103–126.
4. Dev KK, Nishimune A, Henley JM, Nakanishi S (1999) The protein kinase C alpha
binding protein PICK1 interacts with short but not long form alternative splice var-
iants of AMPA receptor subunits. Neuropharmacology 38(5):635–644.
5. Xia J, Zhang X, Staudinger J, Huganir RL (1999) Clustering of AMPA receptors by the
synaptic PDZ domain-containing protein PICK1. Neuron 22(1):179–187.
6. Hanley JG (2008) PICK1: A multi-talented modulator of AMPA receptor trafficking.
Pharmacol Ther 118(1):152–160.
7. Gardner SM, et al. (2005) Calcium-permeable AMPA receptor plasticity is mediated by
subunit-specific interactions with PICK1 and NSF. Neuron 45(6):903–915.
8. Steinberg JP, et al. (2006) Targeted in vivo mutations of the AMPA receptor subunit
GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression.
9. Clem RL, Anggono V, Huganir RL (2010) PICK1 regulates incorporation of calcium-perme-
able AMPA receptors during cortical synaptic strengthening. J Neurosci 30(18):6360–6366.
10. Volk L, Kim CH, Takamiya K, Yu Y, Huganir RL (2010) Developmental regulation of
protein interacting with C kinase 1 (PICK1) function in hippocampal synaptic plasticity
and learning. Proc Natl Acad Sci USA 107(50):21784–21789.
11. Anggono V, Clem RL, Huganir RL (2011) PICK1 loss of function occludes homeostatic
synaptic scaling. J Neurosci 31(6):2188–2196.
12. Jo J, et al. (2008) Metabotropic glutamate receptor-mediated LTD involves two in-
teracting Ca(2+) sensors, NCS-1 and PICK1. Neuron 60(6):1095–1111.
13. Terashima A, et al. (2008) An essential role for PICK1 in NMDA receptor-dependent
bidirectional synaptic plasticity. Neuron 57(6):872–882.
14. Xia J, Chung HJ, Wihler C, Huganir RL, Linden DJ (2000) Cerebellar long-term de-
pression requires PKC-regulated interactions between GluR2/3 and PDZ domain-
containing proteins. Neuron 28(2):499–510.
15. Jin W, et al. (2006) Lipid binding regulates synaptic targeting of PICK1, AMPA re-
ceptor trafficking, and synaptic plasticity. J Neurosci 26(9):2380–2390.
16. Gallop JL, McMahon HT (2005) BAR domains and membrane curvature: Bringing your
curves to the BAR. Biochem Soc Symp 72:223–231.
17. Itoh T, et al. (2005) Dynamin and the actin cytoskeleton cooperatively regulate
plasma membrane invagination by BAR and F-BAR proteins. Dev Cell 9(6):791–804.
18. Wang Q, et al. (2009) Molecular mechanism of membrane constriction and tubulation
mediated by the F-BAR protein Pacsin/Syndapin. Proc Natl Acad Sci USA 106(31):
19. Qualmann B, Roos J, DiGregorio PJ, Kelly RB (1999) Syndapin I, a synaptic dynamin-
binding protein that associates with the neural Wiskott-Aldrich syndrome protein.
Mol Biol Cell 10(2):501–513.
20. Plomann M, et al. (1998) PACSIN, a brain protein that is upregulated upon differ-
entiation into neuronal cells. Eur J Biochem 256(1):201–211.
21. Anggono V, et al. (2006) Syndapin I is the phosphorylation-regulated dynamin I
partner in synaptic vesicle endocytosis. Nat Neurosci 9(6):752–760.
22. Clayton EL, et al. (2009) The phospho-dependent dynamin-syndapin interaction triggers
activity-dependent bulk endocytosis of synaptic vesicles. J Neurosci 29(24):7706–7717.
23. Pérez-Otaño I, et al. (2006) Endocytosis and synaptic removal of NR3A-containing
NMDA receptors by PACSIN1/syndapin1. Nat Neurosci 9(5):611–621.
24. Modregger J, DiProspero NA, Charles V, Tagle DA, Plomann M (2002) PACSIN 1 in-
teracts with huntingtin and is absent from synaptic varicosities in presymptomatic
Huntington’s disease brains. Hum Mol Genet 11(21):2547–2558.
25. Takano M, et al. (2013) Proteomic analysis of the hippocampus in Alzheimer’s disease
model mice by using two-dimensional fluorescence difference in gel electrophoresis.
Neurosci Lett 534:85–89.
26. Modregger J, Ritter B, Witter B, Paulsson M, Plomann M (2000) All three PACSIN isoforms
bind to endocytic proteins and inhibit endocytosis. J Cell Sci 113(24):4511–4521.
27. Quan A, Robinson PJ (2013) Syndapin-a membrane remodelling and endocytic F-BAR
protein. FEBS J, 10.1111/febs.12343.
28. Rao Y, et al. (2010) Molecular basis for SH3 domain regulation of F-BAR-mediated
membrane deformation. Proc Natl Acad Sci USA 107(18):8213–8218.
29. Braun A, et al. (2005) EHD proteins associate with syndapin I and II and such inter-
actions play a crucial role in endosomal recycling. Mol Biol Cell 16(8):3642–3658.
30. Quan A, et al. (2012) Phosphorylation of syndapin I F-BAR domain at two helix-capping
motifs regulates membrane tubulation. Proc Natl Acad Sci USA 109(10):3760–3765.
31. Schael S, et al. (2013) Casein kinase 2 phosphorylation of protein kinase C and casein
kinase 2 substrate in neurons (PACSIN) 1 protein regulates neuronal spine formation.
J Biol Chem 288(13):9303–9312.
32. Huttlin EL, et al. (2010) A tissue-specific atlas of mouse protein phosphorylation and
expression. Cell 143(7):1174–1189.
33. Tweedie-Cullen RY, Reck JM, Mansuy IM (2009) Comprehensive mapping of post-
translational modifications on synaptic, nuclear, and histone proteins in the adult
mouse brain. J Proteome Res 8(11):4966–4982.
34. Goswami T, et al. (2012) Comparative phosphoproteomic analysis of neonatal and
adult murine brain. Proteomics 12(13):2185–2189.
35. Strochlic TI, et al. (2012) Identification of neuronal substrates implicates Pak5 in
synaptic vesicle trafficking. Proc Natl Acad Sci USA 109(11):4116–4121.
36. Lein ES, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain.
37. Chen RQ, et al. (2009) CDC25B mediates rapamycin-induced oncogenic responses in
cancer cells. Cancer Res 69(6):2663–2668.
38. Anggono V, Robinson PJ (2007) Syndapin I and endophilin I bind overlapping proline-
rich regions of dynamin I: Role in synaptic vesicle endocytosis. J Neurochem 102(3):
39. Andersson F, Jakobsson J, Löw P, Shupliakov O, Brodin L (2008) Perturbation of
syndapin/PACSIN impairs synaptic vesicle recycling evoked by intense stimula-
tion. J Neurosci 28(15):3925–3933.
40. Koch D, et al. (2011) Proper synaptic vesicle formation and neuronal network activity
critically rely on syndapin I. EMBO J 30(24):4955–4969.
41. Rocca DL, Martin S, Jenkins EL, Hanley JG (2008) Inhibition of Arp2/3-mediated actin
polymerization by PICK1 regulates neuronal morphology and AMPA receptor endo-
cytosis. Nat Cell Biol 10(3):259–271.
42. Kessels MM, Qualmann B (2002) Syndapins integrate N-WASP in receptor-mediated
endocytosis. EMBO J 21(22):6083–6094.
43. Madsen KL, Thorsen TS, Rahbek-Clemmensen T, Eriksen J, Gether U (2012) Protein
interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction
partners sorted to Rab11-dependent slow recycling pathway. J Biol Chem 287(15):
44. Lin DT, Huganir RL (2007) PICK1 and phosphorylation of the glutamate receptor
2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-
induced internalization. J Neurosci 27(50):13903–13908.
45. Park M, Penick EC, Edwards JG, Kauer JA, Ehlers MD (2004) Recycling endosomes
supply AMPA receptors for LTP. Science 305(5692):1972–1975.
46. Wang YT, Linden DJ (2000) Expression of cerebellar long-term depression requires
postsynaptic clathrin-mediated endocytosis. Neuron 25(3):635–647.
47. Plomann M, Wittmann JG, Rudolph MG (2010) A hinge in the distal end of the PACSIN 2
F-BAR domain may contribute to membrane-curvature sensing. J Mol Biol 400(2):129–136.
48. Goh SL, Wang Q, Byrnes LJ, Sondermann H (2012) Versatile membrane deformation
potential of activated pacsin. PLoS ONE 7(12):e51628.
49. Gong LW, De Camilli P (2008) Regulation of postsynaptic AMPA responses by syn-
aptojanin 1. Proc Natl Acad Sci USA 105(45):17561–17566.
50. Ahmad M, et al. (2012) Postsynaptic complexin controls AMPA receptor exocytosis
during LTP. Neuron 73(2):260–267.
Anggono et al.PNAS
| August 20, 2013
| vol. 110
| no. 34
Anggono et al. 10.1073/pnas.1312467110
SI Materials and Methods
DNA Constructs and Antibodies. The PACSIN1 shRNA-targeting
sequences were as follows: sh#1, 5′-GCGCCAGCTCATCGA-
GAAA-3′ and sh#2, 5′-GCCAAGATCGAGAAGGCATAC-3′.
They were inserted into the pSuper vector system (Oligoengine)
as described previously (1). The efficiency and specificity of these
shRNA constructs were tested in HEK 293T cells overexpressing
GFP-PACSIN1−3. The best sequence (sh#1) was subsequently
cloned into the FuGW vector for lentiviral production and was
the one used throughout the study. The shRNA#1-resistant
rescue construct was generated by introducing five silent muta-
tions, namely 5′-GCGCCAACTTATTGAAAAG-3′, into pRK5-
HA-PACSIN1 using the overlapping PCR protocol (boldface and
underlining represent mutated bases in the sequences). A cas-
sette containing the H1 RNA polymerase III promoter and the
PACSIN1 shRNA#1 sequence was amplified from the pSuper
construct and cloned into the unique PciI restriction site within
the pRK5-HA-PACSIN1 rescue construct to create a bicis-
tronic pRK5-H1-shRNA#1-CMV-HA-PACSIN1 rescue construct,
which was used in all molecular replacement experiments. Various
PACSIN1 domains and truncations were amplified by standard
PCR and subcloned into pGEX4T-1 or pEGFP-C3 vectors.
PACSIN1 and PACSIN2 point mutants were generated using
the overlapping PCR protocol or the QuikChange site-directed
mutagenesis kit (Stratagene). Other plasmids encoding GFP-
PACSIN1−3, GST-PACSIN1−3, myc-GluA2, GFP-GluA2, and
myc-PICK1 have been reported previously.
Specific antibodies against PICK1 (JH2906), myc (9E10), GFP
(JH4030), PACSIN1, PACSIN2, PACSIN3, GluA2/3 (JH1760),
and GluA3 (JH4300) were generated in-house. The following
antibodies were purchased from commercial sources: rabbit poly-
clonal antibodies against myc (Cell Signaling Technology), EHD
(Santa Cruz Biotechnology), mouse monoclonal antibodies against
PICK1 (NeuroMab), HA (Covance), and β3-tubulin (Abcam).
HEK 293T Cell Transfection and Immunoprecipitation Assays. HEK
293T cells were grown in DMEM supplemented with 10% (vol/
vol) FBS, 2 mM Glutamax, 50 U/mL penicillin, and 50 μg/mL
streptomycin. Cells were transfected by calcium phosphate pre-
cipitation method and lysed 48 h later with ice-cold cell lysis
buffer [1% (vol/vol) Triton X-100, 1 mM EDTA, 1 mM EGTA,
50 mM NaF, and 5 mM Na-pyrophosphate in PBS] supplemented
with 1 μg/mL leupeptin, 0.1 μg/mL aprotinin, 1 μg/mL phenyl-
methanesulfonyl fluoride, and 1 μg/mL pepstatin. Cell lysates were
centrifuged at 17,000 × g for 20 min at 4 °C and cleared with
protein A-Sepharose beads. Precleared lysates were then incubated
with antibodies coupled to protein A-Sepharose overnight at 4 °C,
followed by four washes with ice-cold lysis buffer and elution in
2× SDS sample buffer. The immunoprecipitated proteins were
resolved by SDS/PAGE and visualized by Western blot analysis.
Protein–protein interactions in vivo were determined by immu-
noprecipitation assays in mouse brain lysates instead of HEK cell
lysates. Briefly, a total mouse brain extract was prepared by ho-
mogenizing brain tissue in 10 volumes of ice-cold lysis buffer
supplemented with protease inhibitors. The homogenate was
centrifuged twice at 75,600 g for 30 min at 4 °C and precleared
before incubation with antibodies coupled to protein A-Sepharose.
GST Pull-Down Assays. Various GST-PACSIN1-VAR recombinant
proteins were expressed in E. coli and purified using glutathione
(GSH)-Sepharose beads (GE Healthcare) according to the man-
ufacturer’s instructions. Immobilized recombinant proteins were
then incubated with total mouse brain homogenates at 4 °C for
1 h. Beads were washed extensively with ice-cold cell lysis buffer
and eluted in 2× SDS sample buffer. Bound proteins were re-
solved by SDS/PAGE and visualized by Western blot analysis.
Hippocampal Neuronal Culture, Transfection, and Immunostaining.
Hippocampal neurons from E18 rat pups were plated onto
poly-L-lysine–coated dishes or coverslips in Neurobasal growth
medium supplemented with 2% (vol/vol) B27, 2 mM Glutamax,
50 U/mL penicillin, 50 μg/mL streptomycin, and 5% (vol/vol)
FBS. Neurons were switched to serum-free Neurobasal medium
24 h postseeding and fed twice a week. They were transfected at
days in vitro (DIV) 14–15 using lipofectamine 2000 (Invitrogen)
and used 48–72 h later. Neurons were fixed with 4% (wt/vol)
paraformaldehyde/4% (wt/vol) sucrose in PBS for 15 min, per-
meabilized with 0.25% Triton X-100 in PBS, and incubated with
10% (wt/vol) BSA for 1 h. Neurons were then incubated with
anti-PICK1 (NeuroMab) and/or anti-PACSIN1 followed by Alexa-
488-conjugated goat anti-mouse and Alexa-568-conjugated goat
anti-rabbit secondary antibodies (Invitrogen). Images were col-
lected with a 63× oil-immersion objective on a Zeiss LSM510
confocal microscope for both green (PICK1) and red (PACSIN1)
channels. Series of optical sections were collected at 0.38 μm in-
tervals, and maximal-intensity projection was shown.
Surface-Staining and Antibody-Feeding Assays. Cultured hippocampal
neurons were transfected with various PACSIN knockdown or
overexpression constructs, together with myc-GluA2 or GFP-
GluA2 reporter constructs.SurfaceGluA2 was labeled with mouse
anti-myc or rabbit anti-GFP antibody live for 30 min at 4 °C before
fixation. Total myc-GluA2 was labeled with rabbit anti-myc anti-
body. The total and surface myc-GluA2 were then visualized by
Alexa-568-conjugated anti-rabbit and Alexa-488-conjugated anti-
mouse secondary antibodies. For GFP-GluA2, the surface and
totalreceptors were visualized by Alexa-568-conjugatedanti-rabbit
secondary antibody and the endogenousGFP signal.To determine
the amount of receptor internalization, surface myc-GluA2 was
by 15-min incubation with 50 μM NMDA + 1 μM tetrodotoxin to
induce receptor internalization. The remaining surface-myc anti-
body was stained with Alexa-568 secondary antibody under non-
permeabilizing conditions(surface), and internalized myc antibody
was labeled with Alexa-488 secondary antibody once neurons were
permeabilized (internalized). Total myc-GluA2 expression was
visualized with rabbit anti-myc primary antibody and Alexa-647
objective on a Zeiss LSM510 confocal microscope. Fluorescence
intensities were quantified using image J software (National In-
stitutes of Health) for surface, internalized, and total GluA2. Data
were expressed as the surface/total GluA2 ratio or as internalized/
total GluA2 (internalization index).
Immunohistochemistry.Mice were killed and perfused through the
heart with Ringer’s solution followed by 4% (wt/vol) para-
formaldehyde solution (in PBS). The brains were postfixed in 4%
(wt/vol) paraformaldehyde overnight at 4 °C. Seven-micrometer
sections were cut from paraffin blocks and stained with specific
antibodies against PACSIN2 and β3-tubulin.
Lentivirus Preparation. HEK 293T cells were transfected with
FUGW (5 μg), Δ8.9 (3.75 μg), and VSVG (2.5 μg) in OptiMEM
medium using 22.5 μL of lipofectamine 2000 per 10-cm dish.
Cells were continuously maintained in OptiMEM medium, and
Anggono et al. www.pnas.org/cgi/content/short/13124671101 of 4
culture supernatants were collected twice at 24 and 48 h post-
transfection. Virus particles were concentrated by ultracentrifu-
gation at 25,000 rpm for 2 h in a Beckman SW28 rotor. Virus
particles were resuspended in Neurobasal medium and stored at
−80 °C until use.
Electrophysiology.Cerebella from E18 pups of PACSIN2 KO mice
were dissected, dissociated, and plated as described previously
(2). Cultures were generally transfected on DIV6-7 by particle-
mediated gene delivery, with recordings commencing 20–50 h
1. Clayton EL, et al. (2009) The phospho-dependent dynamin-syndapin interaction
triggers activity-dependent bulk endocytosis of synaptic vesicles. J Neurosci 29(24):
2. Steinberg JP, Huganir RL, Linden DJ (2004) N-ethylmaleimide-sensitive factor is
required for the synaptic incorporation and removal of AMPA receptors during
cerebellar long-term depression. Proc Natl Acad Sci USA 101(52):18,212–18,216.
were transfected with either pRK5 empty vector (control), pRK5-HA-PACSIN1, pRK5-myc-PACSIN2, or pRK5-HA-PACSIN3 together with GFP-GluA2 at DIV15. At
DIV17, surface GluA2 was labeled with rabbit anti-GFP antibody live for 30 min at 4 °C before fixation. HA-PACSIN1/3, myc-PACSIN2, and surface GluA2 were
visualized by immunostaining with anti-HA (Alexa-647), anti-myc (Alexa-647), and Alexa-568 anti-rabbit antibodies, respectively, whereas total GluA2 was
visualized by endogenous GFP signal. Representative images of a neuron from each group are shown. (Scale bar, 50 μm.) (B) Quantification of surface/total
GluA2 ratio normalized to the value of control neurons (n = 30–37 neurons per group).
Overexpression of PACSIN proteins does not affect basal surface expression of GluA2 in hippocampal neurons. (A) Cultured hippocampal neurons
Anggono et al. www.pnas.org/cgi/content/short/13124671102 of 4
PACSIN3 in HEK 293T cells. HEK 293T cells were cotransfected with pSuper, pSuper-PACSIN1-sh#1, or pSuper-PACSIN1-sh#2 together with either pEGFP, pEGFP-
PACSIN1, pEFGP-PACSIN2, or pEGFP-PACSIN3 for 48 h and lysed. Cell lysates were subjected to Western blot analyses with anti-GFP and anti-α-tubulin anti-
bodies. (B) PACSIN1 knockdown does not affect dendritic complexity in mature neurons. Cultured hippocampal neurons were transfected with either pSuper
empty vector (control), pSuper-PACSIN1-shRNA#1, or pSuper-PACSIN1-shRNA#2 together with the GFP construct at DIV14. Neurons were fixed and imaged at
DIV17. The morphology of the neurons was visualized by endogenous GFP fluorescence. (Scale bar, 50 μm.) (C) Sholl analysis for control and PACSIN1
knockdown neurons. Concentric circles with increasing radii were superimposed on GFP fluorescence images of neurons, and the number of processes crossing
each radius was counted after thresholding. Data represent mean ± SEM (n = 10 neurons per group).
Characterization of PACSIN1-shRNA constructs. (A) PACSIN1 shRNA#1 and #2 do not knock down the expression of GFP, GFP-PACSIN2, and GFP-
at DIV15. At DIV17, surface GluA2 was labeled with rabbit anti-GFP antibody live for 30 min at 4 °C prior followed by 20-min incubation with 50 μM NMDA + 1 μM
tetrodotoxin to induce receptor internalization. The remaining surface myc antibody was stained with Alexa-568 secondary antibody under nonpermeabilizing
conditions (surface), and internalized myc antibody was labeled with Alexa-488 secondary antibody (internalized). Total myc-GluA2 expression was visualized with
rabbit anti-myc primary antibody and Alexa-647 secondary antibody. Representative images of a neuron stained under permeabilized (Upper) and non-
permeabilized (Lower) conditions are shown. (Scale bar, 50 μm.)
Validation of NMDA-induced myc-GluA2 internalization by antibody-feeding assays. Cultured hippocampal neurons were transfected with myc-GluA2
Anggono et al. www.pnas.org/cgi/content/short/13124671103 of 4
Fig. S4. Download full-text
HA-PACSIN1 constructs, lysed, and immunoprecipitated with anti-myc antibody. Bound proteins were subjected to Western blot analyses with anti-HA and
anti-myc antibodies. The NPF-motif mutant contains the Asn substitution to Asp at positions 364 and 376.
PICK1 interacts with PACSIN1 independent of the NPF motifs within the variable region. HEK 293T cells were transfected with myc-PICK1 and various
osections of the mouse cerebellum (Upper) and hippocampus (Lower) using anti-PACSIN2 (red), the neuronal marker anti-β3-tubulin (green), and the nuclear
marker DAPI (blue). (Scale bar, 150 μm.) Note that the cerebellum cryosection presented is identical to the one presented in Fig. 5A.
PACSIN2 is expressed in Purkinje cells of the mouse cerebellum. Immunohistochemistry analyses of PACSIN2 expression were performed on cry-
Anggono et al. www.pnas.org/cgi/content/short/13124671104 of 4