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• W2050489286 abstract "NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca2+ influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 at postsynapses in situ. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses. NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca2+ influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 at postsynapses in situ. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses. NMDA-evoked Ca2+ influx induces dephosphorylation of PIP5Kγ661 at postsynapses Dephosphorylated PIP5Kγ661 associates with AP-2 at postsynapses NMDA-induced AMPA receptor endocytosis requires the PIP5Kγ661/AP-2 interaction NMDA-dependent LTD requires the PIP5Kγ661/AP-2 interaction in hippocampal slices AMPA receptors mainly mediate fast excitatory neurotransmission 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, 2003Bredt D.S. Nicoll R.A. AMPA receptor trafficking at excitatory synapses.Neuron. 2003; 40: 361-379Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar). 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, 2003Bredt D.S. Nicoll R.A. AMPA receptor trafficking at excitatory synapses.Neuron. 2003; 40: 361-379Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar, Cognet et al., 2006Cognet L. Groc L. Lounis B. Choquet D. Multiple routes for glutamate receptor trafficking: surface diffusion and membrane traffic cooperate to bring receptors to synapses.Sci. STKE. 2006; 2006: pe13PubMed Google Scholar, Wang and Linden, 2000Wang Y.T. Linden D.J. Expression of cerebellar long-term depression requires postsynaptic clathrin-mediated endocytosis.Neuron. 2000; 25: 635-647Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Adaptor protein complex-2 (AP-2) binds to the AMPA receptor and plays a crucial role in recruiting clathrin (Carroll et al., 1999Carroll R.C. Beattie E.C. Xia H. Lüscher C. Altschuler Y. Nicoll R.A. Malenka R.C. von Zastrow M. Dynamin-dependent endocytosis of ionotropic glutamate receptors.Proc. Natl. Acad. Sci. USA. 1999; 96: 14112-14117Crossref PubMed Scopus (347) Google Scholar, Lee et al., 2002Lee S.H. Liu L. Wang Y.T. Sheng M. Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD.Neuron. 2002; 36: 661-674Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Man et al., 2000Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization.Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). 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)-bisphosphate (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., 2001Ford M.G. Pearse B.M. Higgins M.K. Vallis Y. Owen D.J. Gibson A. Hopkins C.R. Evans P.R. McMahon H.T. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes.Science. 2001; 291: 1051-1055Crossref PubMed Scopus (602) Google Scholar, Gaidarov and Keen, 1999Gaidarov I. Keen J.H. Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits.J. Cell Biol. 1999; 146: 755-764Crossref PubMed Scopus (231) Google Scholar, Itoh et al., 2001Itoh T. Koshiba S. Kigawa T. Kikuchi A. Yokoyama S. Takenawa T. Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis.Science. 2001; 291: 1047-1051Crossref PubMed Scopus (386) Google Scholar, Rohde et al., 2002Rohde G. Wenzel D. Haucke V. A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis.J. Cell Biol. 2002; 158: 209-214Crossref PubMed Scopus (134) Google Scholar). PI(4,5)P2 is produced predominantly from phosphatidylinositol 4-phosphate by phosphatidylinositol 4-phosphate 5-kinase (PIP5K) (Loijens and Anderson, 1996Loijens J.C. Anderson R.A. Type I phosphatidylinositol-4-phosphate 5-kinases are distinct members of this novel lipid kinase family.J. Biol. Chem. 1996; 271: 32937-32943Crossref PubMed Scopus (185) Google Scholar). Of three PIP5Kα-γ isozymes, PIP5Kγ is highly and predominantly expressed in the brain (Akiba et al., 2002Akiba Y. Suzuki R. Saito-Saino S. Owada Y. Sakagami H. Watanabe M. Kondo H. Localization of mRNAs for phosphatidylinositol phosphate kinases in the mouse brain during development.Brain Res. Gene Expr. Patterns. 2002; 1: 123-133Crossref PubMed Scopus (18) Google Scholar, Wenk et al., 2001Wenk M.R. Pellegrini L. Klenchin V.A. Di Paolo G. Chang S. Daniell L. Arioka M. Martin T.F. De Camilli P. PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse.Neuron. 2001; 32: 79-88Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and has three splicing variants, PIP5Kγ635, PIP5γ661, and PIP5Kγ687 (Giudici et al., 2004Giudici M.L. Emson P.C. Irvine R.F. A novel neuronal-specific splice variant of Type I phosphatidylinositol 4-phosphate 5-kinase isoform gamma.Biochem. J. 2004; 379: 489-496Crossref PubMed Scopus (50) Google Scholar, Ishihara et al., 1996Ishihara H. Shibasaki Y. Kizuki N. Katagiri H. Yazaki Y. Asano T. Oka Y. Cloning of cDNAs encoding two isoforms of 68-kDa type I phosphatidylinositol-4-phosphate 5-kinase.J. Biol. Chem. 1996; 271: 23611-23614Crossref PubMed Scopus (181) Google Scholar, Ishihara et al., 1998Ishihara H. Shibasaki Y. Kizuki N. Wada T. Yazaki Y. Asano T. Oka Y. 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. 1998; 273: 8741-8748Crossref PubMed Scopus (253) Google Scholar, Loijens and Anderson, 1996Loijens J.C. Anderson R.A. Type I phosphatidylinositol-4-phosphate 5-kinases are distinct members of this novel lipid kinase family.J. Biol. Chem. 1996; 271: 32937-32943Crossref PubMed Scopus (185) Google Scholar). The small GTPase ARF6 activates both PIP5Kγ (Krauss et al., 2003Krauss M. Kinuta M. Wenk M.R. De Camilli P. Takei K. Haucke V. ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma.J. Cell Biol. 2003; 162: 113-124Crossref PubMed Scopus (235) Google Scholar) and PIP5Kα (Honda et al., 1999Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). PIP5Kγ661 is also activated by talin (Morgan et al., 2004Morgan J.R. Di Paolo G. Werner H. Shchedrina V.A. Pypaert M. Pieribone V.A. De Camilli P. A role for talin in presynaptic function.J. Cell Biol. 2004; 167: 43-50Crossref PubMed Scopus (69) Google Scholar). In addition, increased neuronal activity induces dephosphorylation of PIP5Kγ661 (Akiba et al., 2002Akiba Y. Suzuki R. Saito-Saino S. Owada Y. Sakagami H. Watanabe M. Kondo H. Localization of mRNAs for phosphatidylinositol phosphate kinases in the mouse brain during development.Brain Res. Gene Expr. Patterns. 2002; 1: 123-133Crossref PubMed Scopus (18) Google Scholar, Wenk et al., 2001Wenk M.R. Pellegrini L. Klenchin V.A. Di Paolo G. Chang S. Daniell L. Arioka M. Martin T.F. De Camilli P. PIP kinase Igamma is the major PI(4,5)P(2) synthesizing enzyme at the synapse.Neuron. 2001; 32: 79-88Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) by calcineurin, which is activated by Ca2+ influx through voltage-gated Ca2+ channels (VDCCs) (Lee et al., 2005Lee S.Y. Voronov S. Letinic K. Nairn A.C. Di Paolo G. De Camilli P. Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases.J. Cell Biol. 2005; 168: 789-799Crossref PubMed Scopus (97) Google Scholar, Nakano-Kobayashi et al., 2007Nakano-Kobayashi A. Yamazaki M. Unoki T. Hongu T. Murata C. Taguchi R. Katada T. Frohman M.A. Yokozeki T. Kanaho Y. Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.EMBO J. 2007; 26: 1105-1116Crossref PubMed Scopus (65) Google Scholar). The dephosphorylated PIP5Kγ661 becomes enzymatically active by binding AP-2 at presynaptic endocytic spots and produces PI(4,5)P2 to further recruit AP-2 and other components of the early endocytic machinery (Nakano-Kobayashi et al., 2007Nakano-Kobayashi A. Yamazaki M. Unoki T. Hongu T. Murata C. Taguchi R. Katada T. Frohman M.A. Yokozeki T. Kanaho Y. Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.EMBO J. 2007; 26: 1105-1116Crossref PubMed Scopus (65) Google Scholar). 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., 1993Mulkey R.M. Herron C.E. Malenka R.C. An essential role for protein phosphatases in hippocampal long-term depression.Science. 1993; 261: 1051-1055Crossref PubMed Scopus (541) Google Scholar). We found that Ca2+ influx through the NMDA receptor, but not through VDCC, activated protein phosphatase 1 (PP1) and calcineurin and dephosphorylated PIP5Kγ661, which then bound to AP-2 at postsynapses. NMDA-induced AMPA receptor endocytosis and the LFS-induced LTD were completely blocked by inhibiting the association between PIP5Kγ661 and AP-2 and by overexpression of a kinase-dead PIP5Kγ661 mutant in postsynaptic neurons. These results suggest that NMDA receptor activation dynamically controls early steps of the clathrin-mediated endocytosis during hippocampal LTD by regulating the PIP5Kγ661 activity. Of the three splice variants of PIP5Kγ, PIP5Kγ661 was selectively expressed in mouse hippocampus in vivo and in vitro (Figures 1A and 1B ). PIP5Kγ661 expression was observed in mouse hippocampus from approximately 2 weeks after birth in vivo and in vitro, and it increased during late postnatal development (Figures 1A and 1B). To examine whether PIP5Kγ661 is present on postsynapses, we first analyzed cultured hippocampal neurons at 18 days in vitro (DIV) by immunocytochemistry. When green fluorescent protein (GFP)-tagged PIP5Kγ661 (GFP-PIP5Kγ661) was expressed in hippocampal neurons, the GFP signal was observed in dendrites, which were immunopositive for microtubule-associated 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 PIP5Kγ661 was enriched in dendritic spine-like protrusions (see Figures S1A–S1E available online). Endogenous PIP5Kγ661 partially colocalized with PSD-95 and F-actin (Figures 1D and 1E). Furthermore, immunoblot analysis of the subcellular fractions of adult mouse brain showed PIP5Kγ661 not only in the SV fraction, which was immunonegative for PSD-95, but also in the PSD fractions, which were immunonegative for an SV marker synaptophysin (Figure 1F). Together, these results indicate that PIP5Kγ661 localizes at least in part to postsynapses. The dephosphorylation of PIP5Kγ661 by calcineurin plays an essential role in the activity-dependent production of PI(4,5)P2 at presynapses (Lee et al., 2005Lee S.Y. Voronov S. Letinic K. Nairn A.C. Di Paolo G. De Camilli P. Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases.J. Cell Biol. 2005; 168: 789-799Crossref PubMed Scopus (97) Google Scholar, Nakano-Kobayashi et al., 2007Nakano-Kobayashi A. Yamazaki M. Unoki T. Hongu T. Murata C. Taguchi R. Katada T. Frohman M.A. Yokozeki T. Kanaho Y. Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.EMBO J. 2007; 26: 1105-1116Crossref PubMed Scopus (65) Google Scholar). To examine whether PIP5Kγ661 is also dephosphorylated at postsynapses, we treated hippocampal neurons with NMDA, which induces AMPA receptor endocytosis and LTD (Beattie et al., 2000Beattie E.C. Carroll R.C. Yu X. Morishita W. Yasuda H. von Zastrow M. Malenka R.C. Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD.Nat. Neurosci. 2000; 3: 1291-1300Crossref PubMed Scopus (616) Google Scholar, Carroll et al., 1999Carroll R.C. Beattie E.C. Xia H. Lüscher C. Altschuler Y. Nicoll R.A. Malenka R.C. von Zastrow M. Dynamin-dependent endocytosis of ionotropic glutamate receptors.Proc. Natl. Acad. Sci. USA. 1999; 96: 14112-14117Crossref PubMed Scopus (347) Google Scholar, Lee et al., 2002Lee S.H. Liu L. Wang Y.T. Sheng M. Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD.Neuron. 2002; 36: 661-674Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Lin et al., 2000Lin J.W. Ju W. Foster K. Lee S.H. Ahmadian G. Wyszynski M. Wang Y.T. Sheng M. Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization.Nat. Neurosci. 2000; 3: 1282-1290Crossref PubMed Scopus (408) Google Scholar). To block action potential-induced VDCC activation at presynapses, we included tetrodotoxin (TTX) in the culture medium. Immunoblot analysis of the cell lysates with an anti-PIP5Kγ antibody revealed that an additional PIP5Kγ661 band, which migrated faster on electrophoresis gels, appeared after NMDA treatment (Figure 2A ). This band likely corresponds to the dephosphorylated form of PIP5Kγ661, because PIP5Kγ661 migrated to the same position when the lysates were treated with λ-phosphatase before electrophoresis (Figure 2A). NMDA treatment increased the dephosphorylated form of PIP5Kγ661 in a dose-dependent manner, with an EC50 of approximately 30 μM (Figure 2B). The dephosphorylation of PIP5Kγ661 was observed as early as 5 min and was saturated by 20 min after 50 μM NMDA treatment (Figure 2C). These results indicate that PIP5Kγ661 is mostly phosphorylated at the basal level and is rapidly dephosphorylated upon NMDA treatment. The concentration and duration of NMDA treatment were similar to those used previously to induce AMPA receptor endocytosis in cultured neurons (Beattie et al., 2000Beattie E.C. Carroll R.C. Yu X. Morishita W. Yasuda H. von Zastrow M. Malenka R.C. Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD.Nat. Neurosci. 2000; 3: 1291-1300Crossref PubMed Scopus (616) Google Scholar, Carroll et al., 1999Carroll R.C. Beattie E.C. Xia H. Lüscher C. Altschuler Y. Nicoll R.A. Malenka R.C. von Zastrow M. Dynamin-dependent endocytosis of ionotropic glutamate receptors.Proc. Natl. Acad. Sci. USA. 1999; 96: 14112-14117Crossref PubMed Scopus (347) Google Scholar, Lee et al., 2002Lee S.H. Liu L. Wang Y.T. Sheng M. Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD.Neuron. 2002; 36: 661-674Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, Lin et al., 2000Lin J.W. Ju W. Foster K. Lee S.H. Ahmadian G. Wyszynski M. Wang Y.T. Sheng M. Distinct molecular mechanisms and divergent endocytotic pathways of AMPA receptor internalization.Nat. Neurosci. 2000; 3: 1282-1290Crossref PubMed Scopus (408) Google Scholar). To examine the molecular mechanism responsible for the NMDA-induced dephosphorylation of PIP5Kγ661, 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 PIP5Kγ661 (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 PIP5Kγ661 (Figure 2D). In contrast, the dephosphorylation of PIP5Kγ661 was almost completely blocked by a high concentration (1 μM) of okadaic acid (Figure 2D), which inhibits both PP1 and protein phosphatase 2A (PP2A). Because a low concentration (10 nM) of okadaic acid and fostriecin, which specifically inhibit PP2A (Bialojan and Takai, 1988Bialojan C. Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics.Biochem. J. 1988; 256: 283-290Crossref PubMed Scopus (1513) Google Scholar, Boritzki et al., 1988Boritzki T.J. Wolfard T.S. Besserer J.A. Jackson R.C. Fry D.W. Inhibition of type II topoisomerase by fostriecin.Biochem. Pharmacol. 1988; 37: 4063-4068Crossref PubMed Scopus (130) Google Scholar), were ineffective (Figure 2D), the NMDA-induced dephosphorylation of PIP5Kγ661 was probably mediated by PP1 and partially by calcineurin. These results were in contrast to the dominant role of calcineurin in the high-KCl-induced dephosphorylation of PIP5Kγ661 at presynapses (Nakano-Kobayashi et al., 2007Nakano-Kobayashi A. Yamazaki M. Unoki T. Hongu T. Murata C. Taguchi R. Katada T. Frohman M.A. Yokozeki T. Kanaho Y. Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.EMBO J. 2007; 26: 1105-1116Crossref PubMed Scopus (65) Google Scholar). Indeed, the NMDA-induced dephosphorylation of PIP5Kγ661 was not inhibited by a cocktail of Ca2+ channel blockers (Figures 2E and S2). Therefore, the direct calcium influx through NMDA receptors likely activates a specific pathway that involves PP1 and calcineurin and dephosphorylates PIP5Kγ661 at postsynapses. The AP-2 subunit β2 adaptin was previously shown to interact directly with the dephosphorylated form of PIP5Kγ661 in vitro (Nakano-Kobayashi et al., 2007Nakano-Kobayashi A. Yamazaki M. Unoki T. Hongu T. Murata C. Taguchi R. Katada T. Frohman M.A. Yokozeki T. Kanaho Y. Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.EMBO J. 2007; 26: 1105-1116Crossref PubMed Scopus (65) Google Scholar). To examine whether NMDA treatment induces the interaction of PIP5Kγ661 with AP-2 in neurons, we performed a coimmunoprecipitation assay using cultured hippocampal neurons. AP-2 subunits (α and β adaptins) were coimmunoprecipitated with PIP5Kγ661 upon NMDA treatment (Figure 3A ). Furthermore, immunocytochemical analysis of hippocampal neurons expressing hemagglutinin (HA)-tagged PIP5Kγ661 and FLAG-tagged β2 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 β2 adaptin (VN-β2 ear) and wild-type PIP5Kγ661 (VC-PIP5K-WT), respectively. In this assay, interaction of fused proteins mediates the reconstitution of Venus, resulting in efficient fluorescence emission (Kerppola, 2006Kerppola T.K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.Nat. Protoc. 2006; 1: 1278-1286Crossref PubMed Scopus (380) Google Scholar). Time-lapse imaging of hippocampal neurons expressing these fusion proteins showed Venus fluorescent puncta appearing 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 chromophore maturation in BiFC studies (Kerppola, 2006Kerppola T.K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.Nat. Protoc. 2006; 1: 1278-1286Crossref PubMed Scopus (380) Google Scholar), but several studies of interaction between various proteins have reported rapid changes in fluorescence intensity in response to stimuli (Guo et al., 2005Guo Y. Rebecchi M. Scarlata S. Phospholipase Cbeta2 binds to and inhibits phospholipase Cdelta1.J. Biol. Chem. 2005; 280: 1438-1447Crossref PubMed Scopus (53) Google Scholar, MacDonald et al., 2006MacDonald M.L. Lamerdin J. Owens S. Keon B.H. Bilter G.K. Shang Z. Huang Z. Yu H. Dias J. Minami T. et al.Identifying off-target effects and hidden phenotypes of drugs in human cells.Nat. Chem. Biol. 2006; 2: 329-337Crossref PubMed Scopus (260) Google Scholar, Schmidt et al., 2003Schmidt C. Peng B. Li Z. Sclabas G.M. Fujioka S. Niu J. Schmidt-Supprian M. Evans D.B. Abbruzzese J.L. Chiao P.J. Mechanisms of proinflammatory cytokine-induced biphasic NF-kappaB activation.Mol. Cell. 2003; 12: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Furthermore, immunocytochemical analysis confirmed 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-transferase (GST) and VC, VN-β2 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 activation triggers the interaction of PIP5Kγ661 with AP-2 at postsynapses in hippocampal neurons. The phosphorylation of Ser at position 645 of PIP5Kγ661 blocks its interaction with β2 adaptin (Nakano-Kobayashi et al., 2007Nakano-Kobayashi A. Yamazaki M. Unoki T. Hongu T. Murata C. Taguchi R. Katada T. Frohman M.A. Yokozeki T. Kanaho Y. Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.EMBO J. 2007; 26: 1105-1116Crossref PubMed Scopus (65) Google Scholar). To examine whether such a dephosphorylation-dependent interaction occurs at postsynapses, we used the phosphomimetic mutant of PIP5Kγ661, VC-PIP5K-S645E, in which Glu replaced Ser at 645. The BiFC assay using VN-β2 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 μM) or okadaic acid (1 μM), the NMDA-induced formation of Venus fluorescent puncta was significantly reduced in neurons expressing VN-β2 ear and VC-PIP5K-WT (Figures 3F and 3G). Because Ser645 of PIP5Kγ661 is phosphorylated by Cdk5 (Lee et al., 2005Lee S.Y. Voronov S. Letinic K. Nairn A.C. Di Paolo G. De Camilli P. Regulation of the interaction between PIPKI gamma and talin by proline-directed protein kinases.J. Cell Biol. 2005; 168: 789-799Crossref PubMed Scopus (97) Google Scholar), we performed the BiFC assay in the presence of a Cdk5 inhibitor olomoucine. The NMDA-induced formation of Venus fluorescent puncta in neurons expressing VN-β2 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 reflects specific interaction between β2 adaptin and PIP5Kγ661. They also indicate that the NMDA-induced dephosphorylation of PIP5Kγ661 at Ser 645 plays an essential role in its association 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 PI5Kγ661 was dephosphorylated 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, 2009Kerppola T.K. Visualization of molecular interactions using bimolecular fluorescence complementation analysis: characteristics of protein fragment complementation.Chem. Soc. Rev. 2009; 38: 2876-2886Crossref PubMed Scopus (155) Google Scholar) in spines, whereas the immunoblot assay reflects total endogenous PIP5Kγ661. The dephosphorylated form of PIP5Kγ661 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. Association with AP-2 activates PIP5Kγ661, leading to the production of PI(4,5)P2 in vitro (Nakano-Kobayashi et al., 2007Nakano-Kobayashi A. Yamazaki M. Unoki T. Hongu T. Murata C. Taguchi R. Katada T. Frohman M.A. Yokozeki T. Kanaho Y. Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.EMBO J. 2007; 26: 1105-1116Crossref PubMed Scopus (65) Google Scholar); PI(4,5)P2 triggers further accumulation of AP-2 and other endocytic components at presynapses. Thus, we hypothesized that the NMDA-induced association of PIP5Kγ661 with AP-2 (Figure 3) plays an essential role in NMDA-induced AMPA receptor endocytosis at postsynapses. To test this hypothesis, we expressed the GluA2 subunit tagged with HA at its extracellular N terminus in cultured hippocampal neurons. As reported previously (Beattie et al., 2000Beattie E.C. Carroll R.C. Yu X. Morishita W. Yasuda H. von Zastrow M. Malenka R.C. Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD.Nat. Neurosci. 2000; 3: 1291-1300Crossref PubMed Scopus (616) Google Scholar, Carroll et al., 1999Carroll R.C. Beattie E.C. Xia H. Lüscher C. Altschuler Y. Nicoll R.A. Malenka R.C. von Zastrow M. Dynamin-dependent endocytosis of ionotropic glutamate receptors.Proc. Natl. Acad. Sci. USA. 1999; 96: 14112-14117Crossref PubMed Scopus (347) Google Scholar, Ehlers, 2000Ehlers M.D. Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting.Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar), the surface HA-GluA2, which is mainly localized to the somatodendritic domain (Matsuda et al., 2008Matsuda S. Miura E. Matsuda K. Kakegawa W. Kohda K. Watanabe M. Yuzaki M. Accumulation of AMPA receptors in autophagosomes in neuronal axons lacking adaptor protein AP-4.Neuron. 2008; 57: 730-745Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), rapidly decreased following NMDA stimulation (Figures 4A and 4E ). When the GFP-tagged C-terminal fragment of PIP5Kγ661 (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 of the PIP5Kγ661 C-terminal fragment (PIP5K-CT-S645A), in which Ala replaced Ser 645, blocked NMDA-induced AMPA receptor endocytosis (Figures 4C and 4E). In contrast, expression of GFP-tagged phosphomimetic mutant of the PIP5Kγ661 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 β2 adaptin (Figure S5A). These results suggest that overexpression of the wild-type and dephosphomimetic C-terminal fragment of PIP5Kγ661 interferes with the interaction between endogenous PIP5Kγ661 and AP-2, thereby inhibiting the NMDA-induced AMPA receptor endocytosis. The C terminus of PIP5Kγ661 is also reported to bind to the μ2 adapti" @default.
• W2050489286 created "2016-06-24" @default.
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• W2050489286 date "2012-01-01" @default.
• W2050489286 modified "2023-10-12" @default.
• W2050489286 title "NMDA Receptor-Mediated PIP5K Activation to Produce PI(4,5)P2 Is Essential for AMPA Receptor Endocytosis during LTD" @default.
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• W2050489286 doi "https://doi.org/10.1016/j.neuron.2011.09.034" @default.
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