NMDA Receptor-Mediated PIP5K Activation to...

Neuron

Article

NMDA Receptor-Mediated PIP5K Activationto Produce PI(4,5)P2 Is Essential for AMPAReceptor Endocytosis during LTDTakamitsu Unoki,1,5 Shinji Matsuda,2,3,5 Wataru Kakegawa,2,3,5 Ngo Thai Bich Van,1,6 Kazuhisa Kohda,2,3 Atsushi Suzuki,1

Yuji Funakoshi,1 Hiroshi Hasegawa,4 Michisuke Yuzaki,2,3,* and Yasunori Kanaho1,*1Department of Physiological Chemistry, Graduate School of Comprehensive Human Sciences and Institute of Basic Medical Sciences,

University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8571, Japan2Department of Physiology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan3Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan4Laboratory of Developmental Neuroscience, Initiative for the Promotion of Young Scientists’ Independent Research, Graduate School of

Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575, Japan5These authors contributed equally to this work6Present address: Department of Biotechnology, Faculty of Chemistry Engineering, DanangUniversity of Technology, 54Nguyen LuongBang

Street, Lien Chieu District, Danang City, Vietnam

*Correspondence: [email protected] (M.Y.), [email protected] (Y.K.)DOI 10.1016/j.neuron.2011.09.034

SUMMARY

NMDA receptor activation leads to clathrin-depen-dent endocytosis of postsynaptic AMPA receptors.Although this process controls long-term depression(LTD) induction in the hippocampus, how it is regu-lated by neuronal activities is not completely clear.Here, we show that Ca2+ influx through the NMDAreceptor activates calcineurin and protein phos-phatase 1 to dephosphorylate phosphatidylinositol4-phosphate 5-kinaseg661 (PIP5Kg661), the majorphosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-producing enzyme in the brain. Bimolecular fluores-cence complementation analysis revealed that thedephosphorylated PIP5Kg661 became associatedwith the clathrin adaptor protein complex AP-2at postsynapses in situ. NMDA-induced AMPAreceptor endocytosis and low-frequency stimula-tion-induced LTD were completely blocked by inhib-iting the association between dephosphorylatedPIP5Kg661 and AP-2 and by overexpression of akinase-dead PIP5Kg661 mutant in hippocampalneurons. Furthermore, knockdown of PIP5Kg661inhibited the NMDA-induced AMPA receptor endo-cytosis. Therefore, NMDA receptor activationcontrols AMPA receptor endocytosis during hippo-campal LTD by regulating PIP5Kg661 activity atpostsynapses.

INTRODUCTION

AMPA receptors mainly mediate fast excitatory neurotransmis-

sion at synapses located on dendritic spines of vertebrate

neurons. Alterations in neural activities induce long-lasting

changes in synaptic transmission, such as long-term depression

(LTD), which underlies certain aspects of learning and memory

(Bredt and Nicoll, 2003). When neuronal activities increase

moderately during LTD-inducing stimuli, postsynaptic AMPA

receptors are thought to be freed from their anchoring proteins,

to diffuse laterally to the endocytic zone, and to undergo clathrin-

dependent endocytosis (Bredt and Nicoll, 2003; Cognet et al.,

2006; Wang and Linden, 2000). Adaptor protein complex-2

(AP-2) binds to the AMPA receptor and plays a crucial role in re-

cruiting clathrin (Carroll et al., 1999; Lee et al., 2002; Man et al.,

2000). However, it has been unclear whether and how neuronal

activities regulate AP-2 and other components of the endocytic

machinery at postsynapses.

At presynaptic sites, elevated neuronal activity induces the

clathrin-mediated endocytosis of synaptic vesicles (SVs). The

membrane phospholipid phosphatidylinositol (4,5)-bisphos-

phate (PI(4,5)P2) plays a key role in recruiting AP-2 and several

components of the endocytic machinery to endocytic hot spots

at the presynaptic terminal (Ford et al., 2001; Gaidarov and

Keen, 1999; Itoh et al., 2001; Rohde et al., 2002). PI(4,5)P2 is

produced predominantly from phosphatidylinositol 4-phosphate

by phosphatidylinositol 4-phosphate 5-kinase (PIP5K) (Loijens

and Anderson, 1996). Of three PIP5Ka-g isozymes, PIP5Kg is

highly and predominantly expressed in the brain (Akiba et al.,

2002; Wenk et al., 2001) and has three splicing variants,

PIP5Kg635, PIP5g661, and PIP5Kg687 (Giudici et al., 2004; Ish-

ihara et al., 1996; Ishihara et al., 1998; Loijens and Anderson,

1996). The small GTPase ARF6 activates both PIP5Kg (Krauss

et al., 2003) and PIP5Ka (Honda et al., 1999). PIP5Kg661 is

also activated by talin (Morgan et al., 2004). In addition,

increased neuronal activity induces dephosphorylation of

PIP5Kg661 (Akiba et al., 2002; Wenk et al., 2001) by calcineurin,

which is activated by Ca2+ influx through voltage-gated Ca2+

channels (VDCCs) (Lee et al., 2005; Nakano-Kobayashi et al.,

2007). The dephosphorylated PIP5Kg661 becomes enzymati-

cally active by binding AP-2 at presynaptic endocytic spots

and produces PI(4,5)P2 to further recruit AP-2 and other

Neuron 73, 135–148, January 12, 2012 ª2012 Elsevier Inc. 135

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http://dx.doi.org/10.1016/j.neuron.2011.09.034

Figure 1. Expression of PIP5Kg661 in Hippocampal Neurons

(A and B) The expression of PIP5Kg splicing variants in mouse hippocampus (A) and cultured hippocampal neurons (B) at various developmental stages.

PIP5Kg635 (635), PIP5Kg661 (661), and PIP5Kg687 (687) overexpressed in HEK293T cells were loaded as controls. All samples were treated with the phos-

phatase l-PPase before SDS-PAGE to prevent the mobility shift by phosphorylation and were western blotted with anti-PIP5Kg antibody. E, embryonic day;

P, postnatal day; DIV, days in vitro.

(C) Localization of overexpressed GFP-PIP5Kg661 in cultured hippocampal neurons at 18 DIV. MAP2 was also stained. The region enclosed by the white square

is magnified in the right panels. Scale bars in the left and the right panels correspond to 10 mm and 1 mm, respectively.

(D and E) Immunocytochemical analysis of endogenous PIP5Kg661 and PSD-95 (D) or F-actin (E) in cultured hippocampal neurons at 18 DIV (D) or 20 DIV (E). The

dendritic segment enclosed by the white square in the left is magnified in the right panels. The arrows in the right panels indicate the colocalization of PIP5Kg661

(green) with PSD-95 or F-actin (red). Scale bars correspond in the left and right panels to 10 mm and 1 mm, respectively.

(F) Distribution of PIP5Kg661 in subcellular fractions of the mouse brain. After fractionation, PIP5Kg661 and the postsynaptic and presynaptic markers PSD-95

and synaptophysin, respectively, were detected by immunoblotting. H, Homogenates; P2, crude synaptosomal pellet; P3, lysed synaptosomal membrane

fraction; SV, synaptic vesicle; PSD1 and PSD2, postsynaptic density fractions unsolubilized with Triton X-100 from synaptosomal membranes and the PSD1

fraction, respectively; S1 and S2, supernatant extracted with Triton X-100 from synaptosomal membranes and the PSD1 fraction, respectively; 661, overex-

pressed PIP5Kg661 in HEK293T cells ; l-PPase, lambda protein phosphatase.

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Activation of PIP5K Is Essential for LTD

components of the early endocytic machinery (Nakano-Kobaya-

shi et al., 2007).

In this study, we examined whether and how the endocytic

machinery is regulated during low-frequency stimulation

(LFS)-induced LTD at postsynapses of pyramidal neurons in

the CA1 region of the mouse hippocampal slices. This form

of LTD depends on the activation of NMDA receptors and

protein phosphatases (Mulkey et al., 1993). We found that

Ca2+ influx through the NMDA receptor, but not through

VDCC, activated protein phosphatase 1 (PP1) and calcineurin

and dephosphorylated PIP5Kg661, which then bound to AP-2

at postsynapses. NMDA-induced AMPA receptor endocytosis

and the LFS-induced LTD were completely blocked by inhibit-

ing the association between PIP5Kg661 and AP-2 and by over-

expression of a kinase-dead PIP5Kg661 mutant in postsynaptic

neurons. These results suggest that NMDA receptor activation

dynamically controls early steps of the clathrin-mediated endo-

cytosis during hippocampal LTD by regulating the PIP5Kg661

activity.

136 Neuron 73, 135–148, January 12, 2012 ª2012 Elsevier Inc.

RESULTS

PIP5Kg661 Localizes to PostsynapsesOf the three splice variants of PIP5Kg, PIP5Kg661 was selec-

tively expressed in mouse hippocampus in vivo and in vitro

(Figures 1A and 1B). PIP5Kg661 expression was observed in

mouse hippocampus from approximately 2 weeks after birth

in vivo and in vitro, and it increased during late postnatal devel-

opment (Figures 1A and 1B).

To examine whether PIP5Kg661 is present on postsynapses,

we first analyzed cultured hippocampal neurons at 18 days

in vitro (DIV) by immunocytochemistry. When green fluorescent

protein (GFP)-tagged PIP5Kg661 (GFP-PIP5Kg661) was ex-

pressed in hippocampal neurons, the GFP signal was observed

in dendrites, which were immunopositive for microtubule-asso-

ciated protein 2 (MAP2), and in spines protruding from the

dendrites (Figure 1C). Like postsynaptic density 95 (PSD-95)

and filamentous actin (F-actin), which were concentrated in the

dendritic spines, endogenous PIP5Kg661 was enriched in

Figure 2. NMDA-Induced Dephosphorylation of PIP5Kg661 in Cultured Hippocampal Neurons

(A) Cultured hippocampal neurons were treated with (NMDA) or without (control) 50 mM NMDA for 10 min. Third lane shows a band of PIP5Kg661 dephos-

phorylated by l-PPase treatment of the lysate of hippocampal neurons.

(B and C) NMDA stimulation of hippocampal neurons induces the dephosphorylation of PIP5Kg661 in concentration- and time-dependent manners. Cultured

hippocampal neurons were treated with NMDA at the indicated concentrations for 10 min (B) or with 50 mM NMDA for the indicated times (C), and endogenous

PIP5Kg661 was detected by western blotting (top). Bottom panels show quantitative data for dephosphorylated PIP5Kg661 (mean ± SEM, n = 3). The ratio of

dephosphorylated/phosphorylated PIP5Kg661 intensity at 1 mM NMDA (B) or before NMDA stimulation (C) was defined as 1.0.

(D) Effects of pharmacological inhibitors on the dephosphorylation of PIP5Kg661 induced by 50 mM NMDA stimulation. Bottom shows the quantitative data for

dephosphorylated PIP5Kg661. The ratio of dephosphorylated/phosphorylated PIP5Kg661 intensity in control neurons was defined as 1.0. The inhibitors used

were 50 mM D-APV (NMDA receptor antagonist), 5 mM EGTA, 10 mM Cyclosporine A (CysA; Calcineurin inhibitor), 1 mM FK-520 (Calcineurin inhibitor), 1 mM

Okadaic acid (High Oka; PP1 and PP2A inhibitor), 10 nMOkadaic acid (LowOka; PP2A inhibitor), and 50 nMFostriecin (PP2A inhibitor). Student’s t test, *p < 0.05;

**p < 0.01; n = 3–7. Error bars indicate SEM.

(E) Effects of a cocktail of VDCC blockers on the KCl- and NMDA-induced dephosphorylation of PIP5Kg661. Cultured hippocampal neurons pretreated for 1 hr

with 2 mM TTX and a cocktail of VDCC blockers, which included 50 mM nimodipine, 500 nM u-conotoxin GVIA, and 1 mM u-agatoxin IVA, were stimulated with

50 mM KCl or 50 mM NMDA for 10 min. The ratio of dephosphorylated/phosphorylated PIP5Kg661 intensity in control neurons was defined as 1.0. (Student’s

t test, *p < 0.05; n = 3). Error bars indicate SEM.

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dendritic spine-like protrusions (see Figures S1A–S1E available

online). Endogenous PIP5Kg661 partially colocalized with

PSD-95 and F-actin (Figures 1D and 1E). Furthermore, immuno-

blot analysis of the subcellular fractions of adult mouse brain

showed PIP5Kg661 not only in the SV fraction, which was immu-

nonegative for PSD-95, but also in the PSD fractions, which were

immunonegative for an SV marker synaptophysin (Figure 1F).

Together, these results indicate that PIP5Kg661 localizes at least

in part to postsynapses.

PIP5Kg661 Is Dephosphorylated by NMDA-InducedCa2+ InfluxThe dephosphorylation of PIP5Kg661 by calcineurin plays an

essential role in the activity-dependent production of PI(4,5)P2

at presynapses (Lee et al., 2005; Nakano-Kobayashi et al.,

2007). To examinewhether PIP5Kg661 is also dephosphorylated

at postsynapses, we treated hippocampal neurons with NMDA,

which induces AMPA receptor endocytosis and LTD (Beattie

et al., 2000; Carroll et al., 1999; Lee et al., 2002; Lin et al.,

2000). To block action potential-induced VDCC activation at pre-

synapses, we included tetrodotoxin (TTX) in the culture medium.

Immunoblot analysis of the cell lysates with an anti-PIP5Kg

antibody revealed that an additional PIP5Kg661 band, which

migrated faster on electrophoresis gels, appeared after NMDA

treatment (Figure 2A). This band likely corresponds to the

dephosphorylated form of PIP5Kg661, because PIP5Kg661

migrated to the same position when the lysates were treated

with l-phosphatase before electrophoresis (Figure 2A). NMDA


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treatment increased the dephosphorylated form of PIP5Kg661

in a dose-dependent manner, with an EC50 of approximately

30 mM (Figure 2B). The dephosphorylation of PIP5Kg661 was

observed as early as 5 min and was saturated by 20 min after

50 mM NMDA treatment (Figure 2C). These results indicate that

PIP5Kg661 is mostly phosphorylated at the basal level and is

rapidly dephosphorylated upon NMDA treatment. The concen-

tration and duration of NMDA treatment were similar to those

used previously to induce AMPA receptor endocytosis in

cultured neurons (Beattie et al., 2000; Carroll et al., 1999; Lee

et al., 2002; Lin et al., 2000).

To examine the molecular mechanism responsible for the

NMDA-induced dephosphorylation of PIP5Kg661, we treated

hippocampal neurons with various pharmacological reagents.

The NMDA antagonist D-APV or the Ca2+ chelator EGTA

completely blocked the NMDA-induced dephosphorylation of

PIP5Kg661 (Figure 2D), demonstrating that Ca2+ entry through

the NMDA receptor is essential for this process. Calcineurin

inhibitors, cyclosporine A and FK-520, partially inhibited the

NMDA-induced dephosphorylation of PIP5Kg661 (Figure 2D).

In contrast, the dephosphorylation of PIP5Kg661 was almost

completely blocked by a high concentration (1 mM) of okadaic

acid (Figure 2D), which inhibits both PP1 and protein phospha-

tase 2A (PP2A). Because a low concentration (10 nM) of

okadaic acid and fostriecin, which specifically inhibit PP2A

(Bialojan and Takai, 1988; Boritzki et al., 1988), were ineffective

(Figure 2D), the NMDA-induced dephosphorylation of

PIP5Kg661 was probably mediated by PP1 and partially by cal-

cineurin. These results were in contrast to the dominant role of

calcineurin in the high-KCl-induced dephosphorylation of

PIP5Kg661 at presynapses (Nakano-Kobayashi et al., 2007).

Indeed, the NMDA-induced dephosphorylation of PIP5Kg661

was not inhibited by a cocktail of Ca2+ channel blockers (Fig-

ures 2E and S2). Therefore, the direct calcium influx through

NMDA receptors likely activates a specific pathway that

involves PP1 and calcineurin and dephosphorylates PIP5Kg661

at postsynapses.

NMDA Receptor Activation Triggers an Interactionof PIP5Kg661 with AP-2The AP-2 subunit b2 adaptin was previously shown to interact

directly with the dephosphorylated form of PIP5Kg661 in vitro

(Nakano-Kobayashi et al., 2007). To examine whether NMDA

treatment induces the interaction of PIP5Kg661 with AP-2 in

neurons, we performed a coimmunoprecipitation assay using

cultured hippocampal neurons. AP-2 subunits (a and b adaptins)

were coimmunoprecipitated with PIP5Kg661 upon NMDA treat-

ment (Figure 3A). Furthermore, immunocytochemical analysis of

hippocampal neurons expressing hemagglutinin (HA)-tagged

PIP5Kg661 and FLAG-tagged b2 adaptin revealed that as early

as 5 min after NMDA application, colocalization of HA and

FLAG immunoreactivities was detected and saturated by

10 min in the dendrites (Figure S3).

To examine whether this interaction occurs at postsynapses,

we performed a bimolecular fluorescence complementation

(BiFC) assay using N- and C-terminal subfragments of Venus,

which were fused to the ear domain of b2 adaptin (VN-b2 ear)

and wild-type PIP5Kg661 (VC-PIP5K-WT), respectively. In this


assay, interaction of fused proteins mediates the reconstitution

of Venus, resulting in efficient fluorescence emission (Kerppola,

2006). Time-lapse imaging of hippocampal neurons expressing

these fusion proteins showed Venus fluorescent puncta appear-

ing along the neurites, approximately 2 min after NMDA

treatment (Figure 3B and Movie S1). Changes in fluorescence

intensity were detected faster than those reported for full chro-

mophore maturation in BiFC studies (Kerppola, 2006), but

several studies of interaction between various proteins have re-

ported rapid changes in fluorescence intensity in response to

stimuli (Guo et al., 2005; MacDonald et al., 2006; Schmidt

et al., 2003). Furthermore, immunocytochemical analysis con-

firmed that most Venus puncta corresponded to spines on

MAP2-positive dendrites (Figure 3C) and colocalized with

PSD-95 and F-actin (Figures 3D and 3E). As a negative control,

we expressed N- and C-terminal fragments of Venus fused to

proteins that do not interact with each other in hippocampal

neurons. The combination of VN-tagged glutathione S-trans-

ferase (GST) and VC, VN-b2 ear, and VC, or VN-GST and

VC-PIP5K-WT gave rise to diffuse background fluorescence

throughout dendrites and never showed punctate fluorescence

(Figure S4A). These results indicate that NMDA receptor activa-

tion triggers the interaction of PIP5Kg661 with AP-2 at postsy-

napses in hippocampal neurons.

The phosphorylation of Ser at position 645 of PIP5Kg661

blocks its interaction with b2 adaptin (Nakano-Kobayashi

et al., 2007). To examine whether such a dephosphorylation-

dependent interaction occurs at postsynapses, we used the

phosphomimetic mutant of PIP5Kg661, VC-PIP5K-S645E, in

which Glu replaced Ser at 645. The BiFC assay using VN-b2

ear and VC-PIP5K-S645E revealed no Venus fluorescent

puncta after NMDA treatment in hippocampal neurons (Figures

3C and 3G). When dephosphorylation was inhibited by FK520

(1 mM) or okadaic acid (1 mM), the NMDA-induced formation of

Venus fluorescent puncta was significantly reduced in neurons

expressing VN-b2 ear and VC-PIP5K-WT (Figures 3F and 3G).

Because Ser645 of PIP5Kg661 is phosphorylated by Cdk5

(Lee et al., 2005), we performed the BiFC assay in the pres-

ence of a Cdk5 inhibitor olomoucine. The NMDA-induced

formation of Venus fluorescent puncta in neurons expressing

VN-b2 ear and VC-PIP5K-WT was significantly increased by

olomoucine (Figures S4B and S4C). Together, these results

confirm that the emergence of Venus fluorescent puncta re-

flects specific interaction between b2 adaptin and PIP5Kg661.

They also indicate that the NMDA-induced dephosphorylation

of PIP5Kg661 at Ser 645 plays an essential role in its associ-

ation with AP-2 at postsynapses. In contrast, immunoblot

analysis of the cell lysates at 5 min after NMDA application

showed that only approximately 35% of PI5Kg661 was de-

phosphorylated compared to the maximum dephosphorylation

level (Figure 2C). The rapid emergence of the BiFC signal may

be caused by the high sensitivity of fluorescence detection

(Kerppola, 2009) in spines, whereas the immunoblot assay

reflects total endogenous PIP5Kg661. The dephosphorylated

form of PIP5Kg661 was mostly observed in the membrane

fractions, such as SV and PSD (Figure 1F), probably because

it is more tightly associated with the plasma membrane

via AP-2.

Figure 3. NMDA-Induced Interaction of Dephosphorylated PIP5Kg661 with AP-2 in Hippocampal Neurons

(A) Coimmunoprecipitation of AP-2 with PIP5Kg661 from NMDA-stimulated hippocampal neurons. After cultured hippocampal neurons were treated with 50 mM

NMDA for 10 min, the endogenous PIP5Kg661 was immunoprecipitated with the anti-PIP5Kg antibody, and the coimmunoprecipitated AP-2 subunits were

detected by anti-a and -b adaptin antibodies. The same result was obtained in four independent experiments.

(B) BiFC assay for the spatiotemporal interaction of PIP5Kg661 with the b2 adaptin ear domain. Cultured hippocampal neurons expressing VN-b2 ear and

VC-PIP5K-WT, respectively, were stimulated with 50 mMNMDA and were observed for up to 3 min. The dendritic region enclosed by a white square is magnified

in the right panels. Scale bars represent 5 mm.

(C) Failure of the phosphomimetic PIP5Kg661 mutant to interact with b2 adaptin. Cultured hippocampal neurons coexpressing VN-b2 ear with VC-PIP5K-WT or

VC-PIP5K-S645E were treated with 50 mMNMDA for 5 min and were monitored for the Venus signal. Neurons were also immunostained for MAP2. The dendritic

regions enclosed by white squares are magnified in the bottom panels. Arrowheads indicate the Venus puncta. Scale bar represents 10 mm.

(D and E) Interaction of PIP5Kg661with b2 adaptin at spines. Cultured hippocampal neurons coexpressing VN-b2 ear and VC-PIP5K-WTwere treatedwith NMDA

and were monitored for the Venus signal. Neurons were also immunostained for PSD-95 (D) or F-actin (E). Scale bar represents 10 mm.

(F) Suppression of the PIP5Kg661 interaction with b2 adaptin by phosphatase inhibitors. After pretreatment with 1 mM FK-520 or 1 mM Okadaic acid for 1 hr,

cultured hippocampal neurons coexpressing VN-b2 ear and VC-PIP5K-WT were treated with NMDA, monitored for the Venus signals, and stained for MAP2.

Scale bar represents 10 mm.

(G) Quantitative data derived from the results shown in (C) and (F). The number of Venus puncta was normalized to the dendrite length. Student’s t test, *p < 0.05;

**p < 0.001; n = 11–30. Error bars indicate SEM.

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Interaction of Dephosphorylated PIP5Kg661with AP-2 Is Required for NMDA-Induced AMPAReceptor EndocytosisAssociation with AP-2 activates PIP5Kg661, leading to the pro-

duction of PI(4,5)P2 in vitro (Nakano-Kobayashi et al., 2007);

PI(4,5)P2 triggers further accumulation of AP-2 and other endo-

cytic components at presynapses. Thus, we hypothesized that

the NMDA-induced association of PIP5Kg661 with AP-2 (Fig-

ure 3) plays an essential role in NMDA-induced AMPA receptor

endocytosis at postsynapses. To test this hypothesis, we ex-

pressed the GluA2 subunit tagged with HA at its extracellular N

terminus in cultured hippocampal neurons. As reported previ-

ously (Beattie et al., 2000; Carroll et al., 1999; Ehlers, 2000),

the surface HA-GluA2, which is mainly localized to the somato-

dendritic domain (Matsuda et al., 2008), rapidly decreased

following NMDA stimulation (Figures 4A and 4E). When the

GFP-tagged C-terminal fragment of PIP5Kg661 (PIP5K-CT-

WT) was expressed, the NMDA-induced reduction of surface

AMPA receptors was blocked (Figures 4B and 4E). Similarly,

the expression of the GFP-tagged dephosphomimetic mutant


Figure 4. Interaction of PIP5Kg661 with AP-2 Is Essential for the NMDA-Induced AMPA Receptor Endocytosis

(A–D) Effects of phosphomimetic and dephosphomimetic C-terminal peptides of PIP5Kg661 on the NMDA-dependent endocytosis of surface GluA2. Cultured

hippocampal neurons expressing GFP (A), GFP-tagged C terminus of wild-type PIP5Kg661 (PIP5K-CT-WT) (B), dephosphomimetic PIP5K-CT (PIP5K-CT-

S645A) (C), and phosphomimetic PIP5K-CT (PIP5K-CT-S645E) (D) were treated with 50 mMNMDA for 10 min and stained for surface HA-GluA2 (red). After Triton

X treatment, the neurons were stained for the total HA-GluA2 (blue). The dendritic regionsmarked bywhite squares aremagnified in the bottom panels. Scale bars

represent 10 mm.

(E) Quantification of the NMDA-induced endocytosis of surface GluA2. Data are represented as the ratio of surface HA-GluA2/total HA-GluA2 staining intensity.

The ratio in control neurons was defined as 1.0. Student’s t test, *p < 0.005 compared with the control for each condition; n = 8–15 cells. Error bars indicate SEM.

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of the PIP5Kg661 C-terminal fragment (PIP5K-CT-S645A), in

which Ala replaced Ser 645, blocked NMDA-induced AMPA

receptor endocytosis (Figures 4C and 4E). In contrast, expres-

sion of GFP-tagged phosphomimetic mutant of the PIP5Kg661

C-terminal fragment (PIP5K-CT-S645E) failed to do so (Figures

4D and 4E). GST pull-down assays confirmed that wild-type

and PIP5K-CT-S645A, but not PIP5K-CT-S645E, bound to b2

adaptin (Figure S5A). These results suggest that overexpression

of the wild-type and dephosphomimetic C-terminal fragment of

PIP5Kg661 interferes with the interaction between endogenous

PIP5Kg661 and AP-2, thereby inhibiting the NMDA-induced

AMPA receptor endocytosis.

The C terminus of PIP5Kg661 is also reported to bind to the m2

adaptin of AP-2 (Bairstow et al., 2006) and talin (Di Paolo et al.,

2002; Ling et al., 2002). In addition, b2 adaptin binds to clathrin

through its ear and hinge domains (Thieman et al., 2009) and

to hippocalcin, a protein that translocates to the plasma

membrane in the presence of Ca2+ during hippocampal LTD

(Palmer et al., 2005). Nevertheless, none of these interactions

are reported to be affected by the dephosphorylation of the

C-terminal domain of PIP5Kg661. To examine the specificity of

the dephosphorylation-dependent binding of PIP5Kg661 to b2

adaptin, we performed GST pull-down assays. Although b2

adaptin bound to GST-CT-WT and GST-CT-S645A with sig-

nificantly higher affinity than to GST-CT-S645E, m2 adaptin

equally interacted with all GST-fused C-terminal fragments of

PIP5Kg661 (Figure S5A). In addition, although PIP5Kg661


binding to b2 adaptin was specifically inhibited by the depho-

phomimetic C-terminal peptide of PIP5Kg661 (pep-S645A),

the clathrin binding to b2 adaptin was not affected by either pho-

phomimetic (pep-S645E) or dephosphomimetic (pep-S645A)

peptide (Figure S5B). Furthermore, binding of CT-WT to talin

was inhibited by pep-S645A and pep-S645E to the same degree

(Figure S5C). The interaction between b2 adaptin and hippocal-

cin in the presence of Ca2+ was also similarly inhibited by de-

phosphomimetic or phosphomimetic PIP5Kg661 C-terminal

peptides and fragments (Figures S5D). These results indicate

that the dephosphorylated form-specific effect of PIP5Kg661

(Figure 4) is likely mediated by its specific interaction with b2

adaptin and that this specific interaction is required for NMDA-

induced AMPA receptor endocytosis.

PIP5Kg661 Activity Is Required for NMDA-InducedAMPA Receptor EndocytosisTo examine the requirement of the kinase activity of PIP5Kg661

for NMDA-induced AMPA receptor endocytosis, we coex-

pressed HA-GluA2 and GFP-tagged PIP5Kg661 (PIP5K-WT) or

its kinase-dead mutant (PIP5K-D316A), in which Ala replaced

Glu at 316 (Rao et al., 1998), in cultured hippocampal neurons.

We found that the NMDA-induced reduction in surface HA-

GluA2 was completely blocked by expression of PIP5K-D316A

(Figures 5B and 5C), but not by expression of PIP5K-WT (Figures

5A and 5C). There was no significant difference in surface HA-

GluA2 levels between neurons expressing PIP5K-WT and

Figure 5. PIP5Kg661 Activity Is Required for the

NMDA-Induced AMPA Receptor Endocytosis

(A andB) Effect of a kinase-dead PIP5Kg661mutant on the

NMDA-induced GluA2 endocytosis. Cultured hippo-

campal neurons coexpressing GFP-tagged wild-type

PIP5Kg661 (PIP5K-WT) (A) or the kinase-dead mutant

PIP5K-D316A (B) with HA-GluA2 were treated with 50 mM

NMDA for 10 min and were stained for the surface HA-

GluA2 and total GluA2 as in Figure 4. The dendritic regions

marked by white squares are magnified in the bottom

panels. Scale bars represent 10 mm.

(C) The ratio of surface HA-GluA2/total HA-GluA2 staining

intensity was quantified as in Figure 4. Student’s t test,

*p < 0.005 compared with the control for each condition;

n = 8–15 cells. Error bars indicate SEM.

(D) Cultured hippocampal neurons were treated with

(NMDA) or without (control) 50 mM NMDA for 10 min. After

immunoprecipitation of PIP5Kg from the neurons, the

PIP5K activity was determined. Student’s t test, *p < 0.05;

n = 4. Error bars indicate SEM.

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PIP5K-D316A in the resting state. Next, we performed an in vitro

kinase assay of PIP5Kg661 after its immunorecipitation from

hippocampal neurons treated or untreated with NMDA. The

kinase activity of PIP5Kg661 from neurons treated with NMDA

was significantly increased (Figure 5D). These results suggest

that an NMDA-induced increase in PIP5Kg661’s kinase activity

is necessary for evoking AMPA receptor endocytosis.

To further confirm the role of PIP5Kg661 in NMDA-induced

AMPA receptor endocytosis, we employed a loss-of-function

approach using vectors for shRNA and GFP. Immunoblot anal-

ysis of the cell lysates with an anti-FLAG antibody revealed

that two shRNAs directed against PIP5Kg (shRNA 1 and 2)

specifically inhibited expression of FLAG-PIP5Kg661, but not

FLAG-PIP5Ka or FLAG-PIP5Kb, in HEK293T cells (Figures

S6A and S6B). Similarly, in GFP-positive neurons, endogenous

PIP5Kg661 immunoreactivity was markedly reduced by these

shRNAs, but not by a scrambled shRNA, whereas tubulin

immunoreactivity was not affected by either construct (Figures

S6C–S6E). NMDA-induced reduction in surface HA-GluA2 was

significantly inhibited by these PIP5Kg-specific shRNAs (Fig-

ures 6B, 6C, and 6E), but not by a scrambled shRNA (Figures

6A and 6E). Furthermore, the inhibitory effect of shRNA 2 on

NMDA-induced reduction in surface HA-GluA2 was rescued

by coexpression of the shRNA-resistant GFP-PIP5Kg661

(PIP5Kg661res) in hippocampal neurons (Figures 6D and 6E).

These results indicate that PIP5Kg661 plays a crucial role in

NMDA-induced AMPA receptor endocytosis in hippocampal

neurons.

Neuron 73, 13

LFS-Induced LTD Requiresthe Interaction of PIP5Kg661with AP-2 and Its Enzymatic ActivityFinally, to examine whether LTD is regulated by

similar mechanisms, we introduced the dephos-

phomimetic pep-S645A, which specifically in-

hibited the interaction between PIP5Kg661 and

AP-2 (Figures S5B–S5D), into theCA1 pyramidal

neurons via a patch pipette during LFS-induced

LTD in hippocampal slice preparations (Figure 7A). There was no

difference in the excitatory postsynaptic current (EPSC) ampli-

tude between neurons treated with decoy peptide and control

phosphomimetic peptide pep-S645E. To examine the effect of

peptides on the basal EPSC amplitude in the same neurons,

we measured the EPSC amplitudes just after breaking into

whole-cell mode and 9–10 min later, because it generally takes

at least several minutes for peptides to diffuse from patch

pipettes to synapses. We did not detect differences in EPSC

amplitudes between 0–1 min and 9–10 min after establishing

the whole-cell mode (Figures S7A–S7C; pep-S645A, 170 ±

14 pA at 0–1 min and 187 ± 18 pA at 9–10 min, n = 24, p =

0.152 by a paired t test; pep-S645E, 174 ± 11 pA at 0–1 min

and 195 ± 13 pA at 9–10 min, n = 17, p = 0.186 by a paired t

test; pep-S645A versus pep-S645E, p = 0.672 by a Mann-Whit-

ney U test). In contrast, after LFS was applied to the Schaffer

collateral, LTDwas only observed in neurons treated with control

peptides (the EPSC amplitude at 25–30min after LFSwas 69%±

5% of baseline; Figures 7C and 7D), but not in neurons treated

with a dephosphomimetic peptide pep-S645A (92% ± 7% of

baseline; Figures 7B and 7D). These results indicate that the

interaction of PIP5Kg661 with AP-2 is required for LFS-induced

LTD, but not for basal neurotransmission.

To examine requirement of the kinase activity of PIP5Kg661

for LFS-induced LTD, we infected hippocampal CA1 neurons

with Sindbis virus encoding GFP and wild-type (Sin-GFP-

PIP5K-WT) or kinase-dead PIP5Kg661 (Sin-GFP-PIP5K-

D316A) (Figure 7E). LTD was significantly inhibited in neurons

5–148, January 12, 2012 ª2012 Elsevier Inc. 141

Figure 6. Suppression of NMDA-Induced AMPA Receptor Endocytosis by Knockdown of PIP5Kg661

(A–D) Effect of PIP5Kg661 knockdown on NMDA-induced GluA2 endocytosis. Cultured hippocampal neurons coexpressing GFP, HA-GluA2, and scrambled

shRNA (A), shRNA 1 (B), shRNA 2 (C), or GFP-fused shRNA-resistant form (GFP-PIP5Kres) and shRNA #2 (D) were treated with 50 mM NMDA for 10 min and

were immunostained for the surface HA-GluA2 and total GluA2 as in Figure 4. The dendritic regions enclosed by white squares are enlarged in the bottom panels.

Scale bars represent 10 mm.

(E) Quantification of NMDA-induced endocytosis of surface GluA2 in shRNA-treated neurons. The ratio of surface HA-GluA2/total HA-GluA2 staining

intensity was quantified as in Figure 4. Error bars indicate SEM. Student’s t test, *p < 0.001 compared with the control for each condition; n = 15 cells for A–C,

n = 20 cells for D.

Neuron


expressing Sin-GFP-PIP5K-D316A (the EPSC amplitude at

20–25 min after LFS was 89% ± 6% of baseline) than those ex-

pressing Sin-GFP-PIP5K-WT (64% ± 4% of baseline; p = 0.028,

Figure 7F). These results indicate that the activation of

PIP5Kg661 following its interaction with b2 adaptin is required

for LFS-induced LTD.

DISCUSSION

PIP5Kg661 and the Regulated Endocytosis of AMPAReceptors at PostsynapsesIn the present study, we demonstrated that NMDA receptor acti-

vation induced the dephosphorylation of PIP5Kg661 (Figure 2)

and its association with AP-2 at postsynapses in hippocampal

neurons (Figure 3). NMDA-induced AMPA receptor endocytosis

was blocked by inhibiting the interaction of PIP5Kg661with AP-2

(Figure 4), by inhibiting the PIP5Kg661 activity (Figure 5), or by

PIP5Kg661 knockdown (Figure 6). Furthermore, LFS-induced

LTD was also blocked by inhibiting the interaction of PIP5Kg661

with AP-2 or by inhibiting the kinase activity of PIP5Kg661 in the

CA1 pyramidal neurons (Figure 7). Binding to AP-2 activates

PIP5Kg661 to produce PI(4,5)P2 (Nakano-Kobayashi et al.,


2007), which plays a key role in recruiting AP-2 to the plasma

membrane (Gaidarov and Keen, 1999). Indeed, NMDA treatment

increased the PIP5Kg661 activity in hippocampal neurons (Fig-

ure 5D). Based on these findings, we propose the following

model in which AMPA receptor endocytosis is upregulated at

postsynapses during NMDA receptor-dependent LTD (Figure 8):

(1) activity-induced Ca2+ influx through NMDA receptors

dephosphorylates PIP5Kg661 by activating PP1 and calcineurin;

(2) the dephosphorylated PIP5Kg661 is recruited to the postsyn-

aptic endocytic site by binding to preexisting AP-2 and (3) is

activated to produce PI(4,5)P2; and (4) produced PI(4,5)P2 further

recruits AP-2 and other components of the early endocytic

machinery (Ford et al., 2001; Gaidarov and Keen, 1999; Itoh

et al., 2001; Rohde et al., 2002) and stimulates the clathrin-

dependent AMPA receptor endocytosis.

Several other mechanisms have been proposed for the AMPA

receptor endocytosis during NMDA receptor-dependent LTD in

the hippocampus. For example, hippocalcin, a protein that

possesses a Ca2+/myristoyl switch, binds AP-2 and translocates

to the plasmamembrane (Palmer et al., 2005). However, how the

hippocalcin-AP2 complex is recruited to the endocytic zone

remains unclear. Activation of the small GTPase Rab5 has also

Figure 7. Suppression of Hippocampal CA1-LTD by Inhibiting the Interaction of PIP5Kg661 with AP-2 and by Expression of Kinase-Dead

PIP5Kg661 Mutant

(A) Schematic diagram of the electrophysiological experimental procedure for (B–D). CA1 pyramidal neurons were perfused with the phosphomimetic (pep-

S645E) or dephosphomimetic (pep-S645A) peptide of PIP5Kg661 C-terminus via a recording patch pipette (Rec.). To evoke EPSCs, we stimulated Schaffer

collaterals by a glass stimulating electrode (Stim.) placed on the stratum radiatum on the CA1 region.

(B and C) Representative CA1-LTD data recorded from CA1 pyramidal neurons perfused with pep-S645A (300 mM; B) or pep-S645E (300 mM; C). Inset traces

represent EPSCs recorded just before (a) or 30 min after (b) LFS and their superimposition (a + b).

(D) Averaged CA1-LTD data. EPSC amplitudes were normalized by the amplitudes immediately before LFS and averaged (n = 14 for pep-S645A, n = 12 for pep-

S645E). Error bars indicate SEM.

(E) A representative image showing GFP expression by infection of recombinant Sindbis virus at hippocampal CA1 region in wild-type mice (36 hr after virus

injection). Scale bar represents 100 mm.

(F) Averaged CA1-LTD data recorded from CA1 pyramidal neurons that express a GFP plus wild-type PIP5Kg661 (Sin-GFP-PIP5K-WT; open circles; n = 5) or the

kinase-dead PIP5Kg661mutant (Sin-GFP-PIP5K-D316A; filled circles; n = 7). EPSC amplitudes were normalized by the amplitudes 1min immediately before LFS

(1 Hz, 300 stimuli at –40mV) and averaged. Insets show EPSC traces recorded just before (black traces) and 25 min after (gray traces) LFS in each condition. The

error bars indicate the SEM. The p value was obtained by the Mann-Whitney U test.

Neuron


been shown to be essential for AMPA receptor endocytosis

during hippocampal LTD (Brown et al., 2005). Rab5 regulates

multiple steps on the endocytic pathway, such as invagination

of the clathrin-coated pit, endosomal fusion, and downstream

signaling (Zerial and McBride, 2001). It also mediates the

uncoating of AP-2 from clathrin-coated vesicles by decreasing

the PI(4,5)P2 levels (Semerdjieva et al., 2008). Furthermore,

cpg2, a protein expressed in the hippocampus after increased


Figure 8. ProposedMolecularMechanism for NMDA-InducedAMPA

Receptor Endocytosis and LTD Induction

Upon NMDA receptor activation, PIP5Kg661 is dephosphorylated by calci-

neurin and PP1 (1) and interacts with AP-2 at the restricted area of the

postsynaptic plasma membrane (2). The interaction with AP-2 induces

the activation of PIP5Kg661, resulting in the local production of PI(4,5)P2 (3).

The produced PI(4,5)P2 recruits endocytic components and induces AMPA

receptor endocytosis, thereby inducing LTD (4).

Neuron


neuronal activity, is localized to the postsynaptic endocytic zone

and regulates AMPA receptor endocytosis during LTD (Cottrell

et al., 2004). Similarly, Arc, an immediate early gene transcribed

after increased neuronal activity, enhances AMPA receptor

endocytosis by binding to components of the later endocytic

machinery, such as dynamin and endophilin (Chowdhury et al.,

2006). These findings support the hypothesis that activity-

induced AMPA receptor endocytosis is regulated at the level of

the endocytic process itself. Although all these processes are

not mutually exclusive and are likely to work in concert, our

model proposes an important form of regulation at the initial

step of endocytosis: the recruitment of PIP5Kg661 to the plasma

membrane to induce the clathrin-dependent AMPA receptor

endocytosis.

An alternative scenario is that AMPA receptor endocytosis is

regulated by the lateral movement of AMPA receptors to the

constitutively active endocytic zone, but not by endocytic steps:

the application of NMDA does not cause an appreciable loss of

clathrin from the dendritic spines of hippocampal neurons (Blan-

pied et al., 2002). Similarly, the activity-dependent endocytosis

of epidermal growth factor receptor occurs via its lateral move-

ment to preformed clathrin-coated pits in HeLa cells (Rappoport

and Simon, 2009). PI(4,5)P2 is rapidly dephosphorylated by syn-

aptojanin 1 to uncoat AP-2 and other phosphoinositide-based

membrane associations from clathrin-coated membranes (Cre-

mona et al., 1999; Zoncu et al., 2007). Indeed, NMDA-induced

AMPA receptor endocytosis is severely impaired in synaptojanin

1 null hippocampal neurons, indicating that PI(4,5)P2metabolism

plays an essential role in the regulation of postsynaptic AMPA

receptor trafficking (Gong and De Camilli, 2008). Therefore, ac-

cording to this scenario, the NMDA-dependent activation of


PIP5Kg661 by interaction with AP-2 may serve as a mechanism

for maintaining constant PI(4,5)P2 and AP-2 levels during

enhanced AMPA receptor endocytosis.

Comparison with Regulated Endocytosisat PresynapsesElevated neuronal activities increase SV endocytosis at presy-

napses. PI(4,5)P2 plays a major role in clathrin coat dynamics.

For example, activity-induced SV endocytosis is severely

retarded in Pip5kg null neurons, in which the PI(4,5)P2 levels

are decreased both in the basal state and after KCl stimulation

(Di Paolo et al., 2004). Thus, our findings demonstrate that in

addition to its role at the presynapse, PIP5Kg also has an impor-

tant postsynaptic function.

Several differences exist in the regulation of PIP5Kg between

the pre- and postsynaptic sides. The enhanced SV endocytosis

induced by high KCl or direct stimulation of nerve terminals is

largely mediated by Ca2+ influx through VDCC (Cousin and Rob-

inson, 2001). Indeed, high-KCl-induced dephosphorylation of

PIP5Kg is blocked by VDCC blockers (Figures 2E and S2). In

contrast, NMDA-induced Ca2+ influx (Dayanithi et al., 1995)

and AMPA receptor endocytosis (Beattie et al., 2000) are insen-

sitive to VDCC blockers. Similarly, NMDA-induced dephosphor-

ylation of PIP5Kg (in the presence of TTX) was dependent on

Ca2+ influx through NMDA receptors, but not VDCC (Figures

2E and S2). Furthermore, LFS-induced LTD in CA hippocampal

neurons was largely insensitive to a VDCC blocker nimodipine

under our experimental conditions (Figure S8), as reported

earlier (Oliet et al., 1997; Selig et al., 1995). Such dependency

on NMDA receptors as a predominant Ca2+ source may reflect

the geometrical arrangement of NMDA receptors, which are

highly expressed in spines (Corlew et al., 2008), together with

PSD-95 and A-kinase-anchoring proteins (AKAPs). Indeed,

AKAPs play a crucial role in NMDA-induced AMPA receptor

endocytosis by scaffolding specific protein kinases and calci-

neurin at postsynapses in hippocampal neurons (Bhattacharyya

et al., 2009). Although certain VDCCs are expressed in dendrites,

they may not be fully activated by the depolarization caused by

NMDA receptor activation, which may inhibit VDCC activities

(Chernevskaya et al., 1991).

The second major difference is that, whereas KCl-induced

SV endocytosis and the dephosphorylation of PIP5Kg are largely

blocked by calcineurin inhibitors (Cousin and Robinson, 2001;

Lee et al., 2005; Nakano-Kobayashi et al., 2007), the NMDA-

induced dephosphorylation of PIP5Kg was more potently in-

hibited by PP1 blockers (Figure 2D). Similarly, NMDA receptor-

dependent LTD requires the activation of PP1 and calcineurin

(Mulkey et al., 1993, 1994). PP1 activity is regulated by the calci-

neurin-dependent dephosphorylation of Inhibitor 1 during LTD

induction (Munton et al., 2004). In addition, the activity of PP1

is regulated by its rapid recruitment from the dendrites to

synapses during LTD stimulus (Morishita et al., 2001). Such

multiple regulatory pathways for PP1 activation may explain

why calcineurin only partially inhibited the NMDA-dependent

dephosphorylation of PIP5Kg661. PP1 can dephosphorylate

PIP5Kg661 in vitro (Nakano-Kobayashi et al., 2007). Thus,

although serine 845 of the GluA1 AMPA receptor is one of the

critical substrates of calcineurin and PP1, we propose that

Neuron


PIP5Kg661 is another substrate that may regulate the endocytic

process during LTD.

Various Endocytic Machineries at the Postsynaptic SiteIn addition to NMDA receptor-activated AMPA receptor endocy-

tosis, there are other forms of activity-dependent AMPA receptor

endocytosis in hippocampal neurons. The application of AMPA

or (s)-3,5-dihydroxyphenylglycine (DHPG), a metabotropic

glutamate receptor (mGluR) agonist, induces AMPA receptor

endocytosis (Beattie et al., 2000; Snyder et al., 2001). Similarly,

mGluR-dependent LTD could be induced in hippocampal slices

using paired-pulse LFS. Unlike NMDA receptor-dependent LTD,

mGluR-dependent LTD is independent of an increase in cyto-

solic Ca2+ or the activation of serine/threonine phosphatases,

such as PP1 and calcineurin (Harris et al., 2004; Schnabel

et al., 2001). Similarly, AMPA-induced AMPA receptor endocy-

tosis is relatively insensitive to Ca2+ influx or calcineurin (Beattie

et al., 2000; Lin et al., 2000). Thus, in these forms of activity-

induced AMPA receptor endocytosis, the postsynaptic PI(4,5)P2

level may be regulated by a mechanism distinct from the PP1-

and calcineurin-dependent pathway. Indeed, during the prepa-

ration of this manuscript, it was reported that mGluR-dependent

AMPA receptor endocytosis is regulated by ARF6 activation

through the ARF6-specific guanine-nucleotide exchange factor

BRAG2 (Scholz et al., 2010). Interestingly, in contrast to DHPG,

NMDA did not activate endogenous ARF6 in hippocampal

neurons (Figure S9), indicating the existence of multiple path-

ways leading to PIP5Kg activation.

Other ARF6 guanine nucleotide exchange factors, such as

EFA6 and ARNO, are expressed at postsynapses (Krauss

et al., 2003; Vitale et al., 2002). Because inactive ARF6 also binds

to PIP5Kg (Nakano-Kobayashi et al., 2007), it may also serve to

concentrate PIP5Kg near the postsynaptic endocytic zone and

to work with the PP1- and calcineurin-dependent PIP5Kg activa-

tion pathway during NMDA receptor-mediated AMPA receptor

endocytosis. Some forms of activity-induced AMPA receptor

endocytosis may also be mediated by clathrin-independent

mechanisms, such as caveolar and lipid raft pathways (Frances-

coni et al., 2009; Glodowski et al., 2007). Further study on the

regulation of AMPA receptor endocytosis is warranted to better

understand the various forms of synaptic plasticity underlying

learning and memory.

EXPERIMENTAL PROCEDURES

Plasmid Construction

cDNAs for PIP5Kg splicing variants, the C-terminal fragment of PIP5Kg661

corresponding to amino acids 448–661, and their mutants were prepared as

previously reported (Nakano-Kobayashi et al., 2007). cDNA for HA-GluA2

was prepared by PCR and Pyrobest (Takara). These cDNAs were cloned

into the pCAGGS expression vector. For the BiFC assay, cDNAs for the

Myc-tagged ear domain of b2 adaptin corresponding to amino acids

714–951 and HA-PIP5Kg661 or its phosphomimetic mutant PIP5Kg661

S645E were inserted into the pVenus (1–173) N-1 and pVenus (155–238) C-1

vector, respectively. For experiments with shRNAs, we developed an expres-

sion vector containing dual promoters, CAG and H1. cDNAs encoding GFP

and shRNA were inserted downstream of CAG and H1 promoters, respec-

tively. PIP5Kg targeting sequences employed in this study are 50-GGACCTG

GACTTCATGCAG-30 (Ling et al., 2007; Sun et al., 2007) and 50-CATCAAGACTGTCATGCAC-30 (a mouse version of Mao et al., 2009) for shRNA

1 and 2, respectively. The sequence for a scrambled shRNA was 50-ATTGAC

CATCACTCACTGA-30. For rescue experiments, four nucleotides were

mutated in the region targeted by shRNA 2 without changing the amino acid

sequence to generate PIP5Kres. The cDNAs encoding GFP-PIP5Kres and

shRNA 2 were inserted downstream of CAG and H1 promoters, respectively.

Culture of Hippocampal Neurons and Transfection

Hippocampi dissected from E16/17 ICR mice were treated with 10 U ml�1

papain and 100 U ml�1 DNase in Dulbecco’s modified Eagle’s medium at

37�C for 20 min. The dissociated hippocampal neurons were plated on polye-

thyleneimine (PEI)-coated plates or glass coverslips and cultured in Neuro-

basal medium (Invitrogen) with B-27 supplement (Invitrogen) and 0.5 mM

L-glutamine. After 14–20 DIV culture, the neurons were transiently transfected

with plasmids using Lipofectamine 2,000 and used for assays of AMPA

receptor endocytosis after 24–48 hr or for assays of BiFC after 19–26 hr. For

shRNA experiments, neurons were transfected with shRNA expression

vectors with or without the HA-GluA2 expression vector at 7 DIV and used

for immunostaining or assay of AMPA receptor endocytosis at 14 DIV.

Assay for AMPA Receptor Endocytosis

Hippocampal neurons transfected with plasmids for HA-GluA2 and GFP

proteins or shRNA were stimulated with 50 mM NMDA for 10 min and fixed

in 4% paraformaldehyde without permeabilization for 10 min at room temper-

ature. After neurons were washed with PBS and incubated with a blocking

solution (2% bovine serum albumin and 2% normal goat serum in PBS), the

surface HA-GluA2 was visualized with the anti-HA antibody (1:1,000) and

Alexa 546 secondary antibody (1:1,000; Invitrogen). To label the total HA-

GluA2, we then permeabilized the neurons, blocked them with a blocking

solution containing 0.4% Triton X-100, and incubated them with the anti-HA

antibody (1:1,000) and Alexa 350 secondary antibodies (1:1,000). The expres-

sion of GFP proteins was also detected by anti-GFP (1:3,000) and Alexa 488

secondary antibody (1:1,000). Fluorescence images were captured by a fluo-

rescence microscope (BX60, Olympus) equipped with a charge-coupled

device camera (DP 70, Olympus) and analyzed using IPLab software (Scana-

lytics). Only the transfected neurons with well-developed spines were

analyzed. For statistical analysis of the surface expression level of HA-

GluA2, we measured the intensity of Alexa 546 for the surface HA-GluA2

and normalized it to the intensity of Alexa 350 for the total HA-tagged GluA2.

The fluorescence intensity on dendrites between 20 mm and 100 mm from

the soma was measured.

Immunoprecipitation

To investigate the NMDA-dependent interaction of endogenous PIP5Kg661

with AP-2, we stimulated cultured neurons with 50 mM NMDA for 10 min and

solubilized them in a lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA,

0.1 mM EGTA, 5 mM MgCl2, 10 mM KCl, 5 mM NaF, 2 mM Na3VO4, 4 mM

Na4P2O7, 1 mM PMSF, and 1% Triton X-100) supplemented with a protease

inhibitor cocktail (Nacalai Tesque). PIP5Kg661 was immunoprecipitated with

an anti-PIP5Kg661 antibody conjugated with protein A sepharose (GE Health-

care). Proteins in the immunoprecipitate were blotted with anti-a adaptin,

anti-b adaptin, and anti-PIP5Kg antibodies.

BiFC Assay

For time-lapse imaging, hippocampal neurons were plated on 35 mm

PEI-coated glass-bottom dishes (thickness = 0.12–0.19 mm; Mattek or Asahi

glass) and cultured in Neurobasal mediumwithout phenol red (Invitrogen), with

B-27 supplement (Invitrogen) and 0.5 mM L-glutamineas as described above

for 16–20 DIV. The neurons were transiently transfected with plasmids for

VN-b2 ear and VC-PIP5K-WT or VC-PIP5K-S645E and cultured for another

19–26 hr. During imaging, the glass-bottom dish was kept at 32�C by a micro-

scope incubation system (Tokai Hit). Time-lapse epifluorescent images were

acquired with a Nikon Eclipse Ti inverted microscope equipped with an

Apochromatic 603 oil immersion objective (NA 1.49) and an ORCA-II-ER

camera (Hamamatsu Photonics). Image capture and data acquisition were

performed using NIS-Elements BR3.0 software (Nikon). Image sequences

were subsequently processed with NIS-Elements and ImageJ software

(1.42q, National Institutes of Health).


Neuron


For immunocytochemical analysis, hippocampal neurons cultured on PEI-

coated glass coverslips were transfected as described above. After treatment

with 50 mM NMDA for 5 min, the neurons were fixed, permeabilized, and

blocked with a blocking solution containing 0.4% Triton X-100. PSD-95 and

MAP2 were labeled with anti-PSD-95 (1:1,000) and anti-MAP2 antibodies

(1:1,000), respectively, and visualized with Alexa 546 secondary antibodies

(1:1,000). F-actin was labeled with rhodamine phalloidin. The numbers of the

Venus punctate signals in the dendrites and the total length of the dendrites

between 20 and 100 mm from the soma were measured.

Assay of PIP5Kg661 Activity

PIP5Kg661 activity was determined as previously reported (Honda et al.,

1999). For more detail, see Supplemental Experimental Procedures.

Recombinant Sindbis Virus and In Vivo Injection

The recombinant Sindbis virus for the expression of GFP and wild-type or

kinase-dead PIP5Kg661 was constructed as described (Matsuda et al.,

2003). Under the deep anesthesia with an intraperitoneal injection of ket-

amine/xylazine (80/20mg/kg;Sigma), the recombinantSindbis virus (2.5ml; titer,

1.0 3 108�1.0 3 109 TU/ml) was stereotactically injected into the CA1 region

of dorsal hippocampus of P14–21 ICR mice (2.0–2.3 mm posterior to the

Bregma, 1.5–2.0 mm lateral to the midline, and 1.5–2.0 mm ventral from the

pial surface). After 24–36 hr, infected cells were identified by the GFP expres-

sion, and hippocampal slices were used for electrophysiological analyses.

Electrophysiology

Transverse hippocampal slices (300 mm thickness) were prepared from

P14–21 ICR mice or virus-infected mice according to the institutional guide-

lines. Whole-cell patch-clamp recordings were made from visually identified

CA1 pyramidal neurons using a 603 water-immersion objective attached to

an upright microscope (BX51WI, Olympus) at room temperature. The resis-

tance of the patch pipettes was 4–6 MU when filled with an intracellular solu-

tion consisting of 150mMCs-gluconate, 10 mMHEPES (pH 7.3), 8 mMMgCl2,

2 mM Na2ATP, 0.5 mM Na2GTP, 0.2 mM EGTA, and 5 mM N-ethyl bromide

quaternary salt (QX-314) (290 mOsm/kg). For the experiments shown in

Figures 7B and 7C, synthetic peptide, pep-S645A, or pep-S645E (300 mM

each) was added to the patch pipette solution to be perfused postsynaptically.

The solution used for slice storage and recording consisted of 125 mM NaCl,

2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3,

and 10 mM D-glucose, which was bubbled continuously with a mixture of 95%

O2 and 5% CO2. Picrotoxin (100 mM; Sigma) was always included in the assay

to block inhibitory synaptic transmission. To evoke EPSCs, we stimulated the

Schaffer collaterals with a glass stimulating electrode placed on the stratum

radiatum of the CA1 region (200–300 mm from the recorded neurons). The

stimulus intensity was subsequently adjusted to 50% of the maximal EPSC

amplitude. For LTD experiments, EPSCs were recorded successively from

CA1 neurons voltage clamped at –80mV at a frequency of 0.1 Hz. After stable

EPSCs were observed for least 10 min, LFS (1 Hz, 300 stimuli at –40mV) was

applied. Access resistances were monitored every 10 s by applying hyperpo-

larizing steps (2mV, 50 ms) throughout the experiments; the measurements

were discarded if the resistance changed by more than 20% of its original

value. The current responseswere recorded using an Axopatch 200B amplifier

(Molecular Devices), and the pCLAMP system (version 9.2; Molecular Devices)

was used for data acquisition and analysis. The signals were filtered at 1 kHz

and digitized at 4 kHz.

SUPPLEMENTAL INFORMATION

Supplemental Information includes nine figures, Supplemental Experimental

Procedures, and one movie and can be found with this article online at

doi:10.1016/j.neuron.2011.09.034.

ACKNOWLEDGMENTS

We thank J.Miyazaki (Osaka University), K. Nakayama (Kyoto University), M.A.

Frohman (Stony Brook University), and J.G. Donaldson (National Institutes of


Health) for providing the pCAGGS expression vector, human b2 adaptin

cDNA, pVenus (1–173) N-1 and pVenus (155–238) C-1 vectors, and the anti-

ARF6 antibody, respectively, and S. Narumi for technical assistance. This

work was supported by Ministry of Education, Culture, Sports, Science, and

Technology (MEXT) and/or Japan Society for the Promotion of Science, Grant

in Aid for Scientific Research to Y.K. (20247010), S.M. (22700343), W.K.

(23240053), and M.Y. (23689012), by Core Research for Evolutional Science

and Technology to M.Y., and by Special Coordination Funds for Promoting

Science and Technology to H.H. from MEXT and the Mitsubishi Research

Foundation.

Accepted: September 20, 2011

Published: January 11, 2012

REFERENCES

Akiba, Y., Suzuki, R., Saito-Saino, S., Owada, Y., Sakagami, H.,Watanabe, M.,

and Kondo, H. (2002). Localization of mRNAs for phosphatidylinositol phos-

phate kinases in the mouse brain during development. Brain Res. Gene

Expr. Patterns 1, 123–133.

Bairstow, S.F., Ling, K., Su, X., Firestone, A.J., Carbonara, C., and Anderson,

R.A. (2006). Type Igamma661 phosphatidylinositol phosphate kinase directly

interacts with AP2 and regulates endocytosis. J. Biol. Chem. 281, 20632–

20642.

Beattie, E.C., Carroll, R.C., Yu, X., Morishita, W., Yasuda, H., von Zastrow, M.,

and Malenka, R.C. (2000). Regulation of AMPA receptor endocytosis by

a signaling mechanism shared with LTD. Nat. Neurosci. 3, 1291–1300.

Bhattacharyya, S., Biou, V., Xu, W., Schluter, O., and Malenka, R.C. (2009).

A critical role for PSD-95/AKAP interactions in endocytosis of synaptic

AMPA receptors. Nat. Neurosci. 12, 172–181.

Bialojan, C., and Takai, A. (1988). Inhibitory effect of a marine-sponge toxin,

okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem. J.

256, 283–290.

Blanpied, T.A., Scott, D.B., and Ehlers, M.D. (2002). Dynamics and regulation

of clathrin coats at specialized endocytic zones of dendrites and spines.

Neuron 36, 435–449.

Boritzki, T.J., Wolfard, T.S., Besserer, J.A., Jackson, R.C., and Fry, D.W.

(1988). Inhibition of type II topoisomerase by fostriecin. Biochem.

Pharmacol. 37, 4063–4068.

Bredt, D.S., and Nicoll, R.A. (2003). AMPA receptor trafficking at excitatory

synapses. Neuron 40, 361–379.

Brown, T.C., Tran, I.C., Backos, D.S., and Esteban, J.A. (2005). NMDA

receptor-dependent activation of the small GTPase Rab5 drives the removal

of synaptic AMPA receptors during hippocampal LTD. Neuron 45, 81–94.

Carroll, R.C., Beattie, E.C., Xia, H., Luscher, C., Altschuler, Y., Nicoll, R.A.,

Malenka, R.C., and von Zastrow, M. (1999). Dynamin-dependent endocytosis

of ionotropic glutamate receptors. Proc. Natl. Acad. Sci. USA 96, 14112–

14117.

Chernevskaya, N.I., Obukhov, A.G., and Krishtal, O.A. (1991). NMDA receptor

agonists selectively block N-type calcium channels in hippocampal neurons.

Nature 349, 418–420.

Chowdhury, S., Shepherd, J.D., Okuno, H., Lyford, G., Petralia, R.S., Plath, N.,

Kuhl, D., Huganir, R.L., and Worley, P.F. (2006). Arc/Arg3.1 interacts with the

endocytic machinery to regulate AMPA receptor trafficking. Neuron 52,

445–459.

Cognet, L., Groc, L., Lounis, B., and Choquet, D. (2006). Multiple routes

for glutamate receptor trafficking: surface diffusion and membrane traffic

cooperate to bring receptors to synapses. Sci. STKE 2006, pe13.

Corlew, R., Brasier, D.J., Feldman, D.E., and Philpot, B.D. (2008). Presynaptic

NMDA receptors: newly appreciated roles in cortical synaptic function and

plasticity. Neuroscientist 14, 609–625.

Cottrell, J.R., Borok, E., Horvath, T.L., and Nedivi, E. (2004). CPG2: a brain-

and synapse-specific protein that regulates the endocytosis of glutamate

receptors. Neuron 44, 677–690.

http://dx.doi.org/doi:10.1016/j.neuron.2011.09.034

Neuron


Cousin, M.A., andRobinson, P.J. (2001). The dephosphins: dephosphorylation

by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci. 24,

659–665.

Cremona, O., Di Paolo, G., Wenk, M.R., Luthi, A., Kim, W.T., Takei, K., Daniell,

L., Nemoto, Y., Shears, S.B., Flavell, R.A., et al. (1999). Essential role of

phosphoinositide metabolism in synaptic vesicle recycling. Cell 99, 179–188.

Dayanithi, G., Rage, F., Richard, P., and Tapia-Arancibia, L. (1995).

Characterization of spontaneous and N-methyl-D-aspartate-induced calcium

rise in rat cultured hypothalamic neurons. Neuroendocrinology 61, 243–255.

Di Paolo, G., Pellegrini, L., Letinic, K., Cestra, G., Zoncu, R., Voronov, S.,

Chang, S., Guo, J., Wenk, M.R., and De Camilli, P. (2002). Recruitment and

regulation of phosphatidylinositol phosphate kinase type 1 gamma by the

FERM domain of talin. Nature 420, 85–89.

Di Paolo, G., Moskowitz, H.S., Gipson, K., Wenk, M.R., Voronov, S., Obayashi,

M., Flavell, R., Fitzsimonds, R.M., Ryan, T.A., and De Camilli, P. (2004).

Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in

synaptic vesicle trafficking. Nature 431, 415–422.

Ehlers, M.D. (2000). Reinsertion or degradation of AMPA receptors determined

by activity-dependent endocytic sorting. Neuron 28, 511–525.

Ford, M.G., Pearse, B.M., Higgins, M.K., Vallis, Y., Owen, D.J., Gibson, A.,

Hopkins, C.R., Evans, P.R., and McMahon, H.T. (2001). Simultaneous binding

of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on

membranes. Science 291, 1051–1055.

Francesconi, A., Kumari, R., and Zukin, R.S. (2009). Regulation of group I me-

tabotropic glutamate receptor trafficking and signaling by the caveolar/lipid

raft pathway. J. Neurosci. 29, 3590–3602.

Gaidarov, I., and Keen, J.H. (1999). Phosphoinositide-AP-2 interactions

required for targeting to plasma membrane clathrin-coated pits. J. Cell Biol.

146, 755–764.

Giudici, M.L., Emson, P.C., and Irvine, R.F. (2004). A novel neuronal-specific

splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform

gamma. Biochem. J. 379, 489–496.

Glodowski, D.R., Chen, C.C., Schaefer, H., Grant, B.D., and Rongo, C. (2007).

RAB-10 regulates glutamate receptor recycling in a cholesterol-dependent

endocytosis pathway. Mol. Biol. Cell 18, 4387–4396.

Gong, L.W., and De Camilli, P. (2008). Regulation of postsynaptic AMPA

responses by synaptojanin 1. Proc. Natl. Acad. Sci. USA 105, 17561–17566.

Guo, Y., Rebecchi, M., and Scarlata, S. (2005). Phospholipase Cbeta2 binds to

and inhibits phospholipase Cdelta1. J. Biol. Chem. 280, 1438–1447.

Harris, S.L., Gallyas, F., Jr., and Molnar, E. (2004). Activation of metabotropic

glutamate receptors does not alter the phosphorylation state of GluR1 AMPA

receptor subunit at serine 845 in perirhinal cortical neurons. Neurosci. Lett.

372, 132–136.

Honda, A., Nogami, M., Yokozeki, T., Yamazaki, M., Nakamura, H., Watanabe,

H., Kawamoto, K., Nakayama, K., Morris, A.J., Frohman, M.A., and Kanaho, Y.

(1999). Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream

effector of the small G protein ARF6 in membrane ruffle formation. Cell 99,

521–532.

Ishihara, H., Shibasaki, Y., Kizuki, N., Katagiri, H., Yazaki, Y., Asano, T., and

Oka, Y. (1996). Cloning of cDNAs encoding two isoforms of 68-kDa type I

phosphatidylinositol-4-phosphate 5-kinase. J. Biol. Chem. 271, 23611–23614.

Ishihara, H., Shibasaki, Y., Kizuki, N., Wada, T., Yazaki, Y., Asano, T., and Oka,

Y. (1998). Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the

third isoform and deletion/substitution analysis of members of this novel lipid

kinase family. J. Biol. Chem. 273, 8741–8748.

Itoh, T., Koshiba, S., Kigawa, T., Kikuchi, A., Yokoyama, S., and Takenawa, T.

(2001). Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate

binding and endocytosis. Science 291, 1047–1051.

Kerppola, T.K. (2006). Design and implementation of bimolecular fluorescence

complementation (BiFC) assays for the visualization of protein interactions in

living cells. Nat. Protoc. 1, 1278–1286.

Kerppola, T.K. (2009). Visualization of molecular interactions using bimolecular

fluorescence complementation analysis: characteristics of protein fragment

complementation. Chem. Soc. Rev. 38, 2876–2886.

Krauss, M., Kinuta, M., Wenk, M.R., De Camilli, P., Takei, K., and Haucke, V.

(2003). ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes

by activating phosphatidylinositol phosphate kinase type Igamma. J. Cell

Biol. 162, 113–124.

Lee, S.H., Liu, L., Wang, Y.T., and Sheng, M. (2002). Clathrin adaptor AP2 and

NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA

receptor trafficking and hippocampal LTD. Neuron 36, 661–674.

Lee, S.Y., Voronov, S., Letinic, K., Nairn, A.C., Di Paolo, G., and De Camilli, P.

(2005). Regulation of the interaction between PIPKI gamma and talin by

proline-directed protein kinases. J. Cell Biol. 168, 789–799.

Lin, J.W., Ju, W., Foster, K., Lee, S.H., Ahmadian, G., Wyszynski, M., Wang,

Y.T., and Sheng, M. (2000). Distinct molecular mechanisms and divergent

endocytotic pathways of AMPA receptor internalization. Nat. Neurosci. 3,

1282–1290.

Ling, K., Doughman, R.L., Firestone, A.J., Bunce, M.W., and Anderson, R.A.

(2002). Type I gamma phosphatidylinositol phosphate kinase targets and

regulates focal adhesions. Nature 420, 89–93.

Ling, K., Bairstow, S.F., Carbonara, C., Turbin, D.A., Huntsman, D.G., and

Anderson, R.A. (2007). Type I gamma phosphatidylinositol phosphate kinase

modulates adherens junction and E-cadherin trafficking via a direct interaction

with mu 1B adaptin. J. Cell Biol. 176, 343–353.

Loijens, J.C., and Anderson, R.A. (1996). Type I phosphatidylinositol-4-phos-

phate 5-kinases are distinct members of this novel lipid kinase family. J. Biol.

Chem. 271, 32937–32943.

MacDonald, M.L., Lamerdin, J., Owens, S., Keon, B.H., Bilter, G.K., Shang, Z.,

Huang, Z., Yu, H., Dias, J., Minami, T., et al. (2006). Identifying off-target effects

and hidden phenotypes of drugs in human cells. Nat. Chem. Biol. 2, 329–337.

Man, H.Y., Lin, J.W., Ju, W.H., Ahmadian, G., Liu, L., Becker, L.E., Sheng, M.,

andWang, Y.T. (2000). Regulation of AMPA receptor-mediated synaptic trans-

mission by clathrin-dependent receptor internalization. Neuron 25, 649–662.

Mao, Y.S., Yamaga, M., Zhu, X.H., Wei, Y.J., Sun, H.Q., Wang, J., Yun, M.,

Wang, Y.F., Di Paolo, G., Bennett, M., et al. (2009). Essential and unique roles

of PIP5K-gamma and -alpha in Fcgamma receptor-mediated phagocytosis.

J. Cell Biol. 184, 281–296.

Matsuda, K., Fletcher, M., Kamiya, Y., and Yuzaki, M. (2003). Specific

assembly with the NMDA receptor 3B subunit controls surface expression

and calcium permeability of NMDA receptors. J. Neurosci. 23, 10064–10073.

Matsuda, S., Miura, E., Matsuda, K., Kakegawa, W., Kohda, K., Watanabe, M.,

and Yuzaki, M. (2008). Accumulation of AMPA receptors in autophagosomes

in neuronal axons lacking adaptor protein AP-4. Neuron 57, 730–745.

Morgan, J.R., Di Paolo, G., Werner, H., Shchedrina, V.A., Pypaert, M.,

Pieribone, V.A., and De Camilli, P. (2004). A role for talin in presynaptic func-

tion. J. Cell Biol. 167, 43–50.

Morishita, W., Connor, J.H., Xia, H., Quinlan, E.M., Shenolikar, S., and

Malenka, R.C. (2001). Regulation of synaptic strength by protein phosphatase

1. Neuron 32, 1133–1148.

Mulkey, R.M., Herron, C.E., and Malenka, R.C. (1993). An essential role for

protein phosphatases in hippocampal long-term depression. Science 261,

1051–1055.

Mulkey, R.M., Endo, S., Shenolikar, S., and Malenka, R.C. (1994). Involvement

of a calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term

depression. Nature 369, 486–488.

Munton, R.P., Vizi, S., and Mansuy, I.M. (2004). The role of protein phospha-

tase-1 in the modulation of synaptic and structural plasticity. FEBS Lett.

567, 121–128.

Nakano-Kobayashi, A., Yamazaki, M., Unoki, T., Hongu, T., Murata, C.,

Taguchi, R., Katada, T., Frohman, M.A., Yokozeki, T., and Kanaho, Y. (2007).

Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle

endocytosis. EMBO J. 26, 1105–1116.


Neuron


Oliet, S.H., Malenka, R.C., and Nicoll, R.A. (1997). Two distinct forms of long-

term depression coexist in CA1 hippocampal pyramidal cells. Neuron 18,

969–982.

Palmer, C.L., Lim, W., Hastie, P.G., Toward, M., Korolchuk, V.I., Burbidge,

S.A., Banting, G., Collingridge, G.L., Isaac, J.T., and Henley, J.M. (2005).

Hippocalcin functions as a calcium sensor in hippocampal LTD. Neuron 47,

487–494.

Rao, V.D., Misra, S., Boronenkov, I.V., Anderson, R.A., and Hurley, J.H. (1998).

Structure of type IIbeta phosphatidylinositol phosphate kinase: a protein

kinase fold flattened for interfacial phosphorylation. Cell 94, 829–839.

Rappoport, J.Z., and Simon, S.M. (2009). Endocytic trafficking of activated

EGFR is AP-2 dependent and occurs through preformed clathrin spots.

J. Cell Sci. 122, 1301–1305.

Rohde, G., Wenzel, D., and Haucke, V. (2002). A phosphatidylinositol (4,5)-bi-

sphosphate binding site within mu2-adaptin regulates clathrin-mediated

endocytosis. J. Cell Biol. 158, 209–214.

Schmidt, C., Peng, B., Li, Z., Sclabas, G.M., Fujioka, S., Niu, J., Schmidt-

Supprian, M., Evans, D.B., Abbruzzese, J.L., and Chiao, P.J. (2003).

Mechanisms of proinflammatory cytokine-induced biphasic NF-kappaB

activation. Mol. Cell 12, 1287–1300.

Schnabel, R., Kilpatrick, I.C., and Collingridge, G.L. (2001). Protein phospha-

tase inhibitors facilitate DHPG-induced LTD in the CA1 region of the hippo-

campus. Br. J. Pharmacol. 132, 1095–1101.

Scholz, R., Berberich, S., Rathgeber, L., Kolleker, A., Kohr, G., and Kornau,

H.C. (2010). AMPA receptor signaling through BRAG2 and Arf6 critical for

long-term synaptic depression. Neuron 66, 768–780.

Selig, D.K., Lee, H.K., Bear, M.F., and Malenka, R.C. (1995). Reexamination of

the effects of MCPG on hippocampal LTP, LTD, and depotentiation.

J. Neurophysiol. 74, 1075–1082.


Semerdjieva, S., Shortt, B., Maxwell, E., Singh, S., Fonarev, P., Hansen, J.,

Schiavo, G., Grant, B.D., and Smythe, E. (2008). Coordinated regulation of

AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6. J. Cell

Biol. 183, 499–511.

Snyder, E.M., Philpot, B.D., Huber, K.M., Dong, X., Fallon, J.R., and Bear, M.F.

(2001). Internalization of ionotropic glutamate receptors in response to mGluR

activation. Nat. Neurosci. 4, 1079–1085.

Sun, Y., Ling, K., Wagoner, M.P., and Anderson, R.A. (2007). Type I gamma

phosphatidylinositol phosphate kinase is required for EGF-stimulated direc-

tional cell migration. J. Cell Biol. 178, 297–308.

Thieman, J.R., Mishra, S.K., Ling, K., Doray, B., Anderson, R.A., and Traub,

L.M. (2009). Clathrin regulates the association of PIPKIgamma661 with the

AP-2 adaptor beta2 appendage. J. Biol. Chem. 284, 13924–13939.

Vitale, N., Chasserot-Golaz, S., Bailly, Y., Morinaga, N., Frohman, M.A., and

Bader, M.F. (2002). Calcium-regulated exocytosis of dense-core vesicles

requires the activation of ADP-ribosylation factor (ARF)6 by ARF nucleotide

binding site opener at the plasma membrane. J. Cell Biol. 159, 79–89.

Wang, Y.T., and Linden, D.J. (2000). Expression of cerebellar long-term

depression requires postsynaptic clathrin-mediated endocytosis. Neuron 25,

635–647.

Wenk, M.R., Pellegrini, L., Klenchin, V.A., Di Paolo, G., Chang, S., Daniell, L.,

Arioka, M., Martin, T.F., and De Camilli, P. (2001). PIP kinase Igamma is the

major PI(4,5)P(2) synthesizing enzyme at the synapse. Neuron 32, 79–88.

Zerial, M., andMcBride, H. (2001). Rab proteins asmembrane organizers. Nat.

Rev. Mol. Cell Biol. 2, 107–117.

Zoncu, R., Perera, R.M., Sebastian, R., Nakatsu, F., Chen, H., Balla, T., Ayala,

G., Toomre, D., and De Camilli, P.V. (2007). Loss of endocytic clathrin-coated

pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc.

Natl. Acad. Sci. USA 104, 3793–3798.

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