Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Han K, Kim M-H, Seeburg D, Seo J, Verpelli C, et al. (2009) Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD. PLoS Biol 7(9): e1000187. doi:10.1371/journal.pbio.1000187 As Published http://dx.doi.org/10.1371/journal.pbio.1000187 Publisher Public Library of Science Version Final published version Citable link http://hdl.handle.net/1721.1/52609 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/2.5/ Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD Kihoon Han1., Myoung-Hwan Kim1., Daniel Seeburg2,3, Jinsoo Seo4, Chiara Verpelli5, Seungnam Han1, Hye Sun Chung6, Jaewon Ko1, Hyun Woo Lee1, Karam Kim1, Won Do Heo1, Tobias Meyer7, Hyun Kim6, Carlo Sala5, Se-Young Choi4, Morgan Sheng2,3, Eunjoon Kim1¤* 1 National Creative Research Initiative Center for Synaptogenesis and Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea, 2 The Picower Institute for Learning and Memory, RIKEN-MIT Neuroscience Research Center, 3 Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 4 Department of Physiology and Dental Research Institute, Seoul National University School of Dentistry, Seoul, Korea, 5 CNR Institute of Neuroscience and Department of Neurological Sciences, University of Milan, Milan, Italy, 6 Department of Anatomy and Division of Brain Korea 21 Biomedical Science, College of Medicine, Korea University, Seoul, Korea, 7 Department of Chemical and Systems Biology, Stanford University, Stanford, California, United States of America Abstract Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)– dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD- 95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner. Citation: Han K, Kim M-H, Seeburg D, Seo J, Verpelli C, et al. (2009) Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD. PLoS Biol 7(9): e1000187. doi:10.1371/journal.pbio.1000187 Academic Editor: Michael D. Ehlers, Duke University Medical Center, United States of America Received February 12, 2009; Accepted July 30, 2009; Published September 8, 2009 Copyright: ß 2009 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Creative Research Initiatives Program of the Korean Ministry of Science and Technology (to EK); the Korea Science and Engineering Foundation M10500000049-05J0000-04900 (to HK); Telethon-Italy GGP06208, Fondazione Cariplo 2006-0779, and Compagnia di San Paolo 2005-1964 (to CS); and a National Institute of Health grant R01MH064801 (to TM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: AMPAR, AMPA receptor; CA1, CA1 pyramidal cell; EPSC, excitatory postsynaptic current; GVDN, G23VD49N; LFS, low-frequency electrical stimulation; LTD, long-term depression; mEPSCs, miniature EPSCs; mGluR, metabotropic glutamate receptor; NMDAR, NMDA receptor; PKA, protein kinase A; POB1 CC, CC domain of POB1; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PPF, paired-pulse facilitation; PP-LFS, paired-pulse LFS; RalBD, Ral binding domain of RalBP1; SC, Schaffer collateral; shRNA, short hairpin RNA; TBS, theta-burst stimulation; WT, wild-type. * E-mail: [email protected] ¤ Current address: Department of Biological Sciences, Korea Advances Institute of Science and Technology (KAIST), Daejeon, Korea . These authors contributed equally to this work. Introduction NMDAR-dependent LTD and AMPAR endocytosis, including the GluR1 subunit of AMPARs [11–13], stargazin [14], and PSD- Long-term depression (LTD), a long-lasting activity-dependent 95 [15]. However, their dephosphorylation has not been clearly decrease in synaptic strength, has been implicated in brain linked to the regulation of AMPAR endocytosis. In addition, the development, learning and memory, drug addiction, and mental clathrin adaptor complex AP2, which directly binds to AMPARs retardation [1]. NMDA receptor (NMDAR)–dependent LTD, an and is required for NMDAR-dependent AMPAR endocytosis and extensively studied form of LTD, involves a rise in postsynaptic LTD [5,16], should be brought close to AMPARs in a tightly Ca2+ concentration, activation of a serine-threonine protein regulated manner, but little is known about the underlying phosphatase cascade, and clathrin-dependent rapid endocytosis mechanism. of AMPA receptors (AMPARs) [1–10]. However, little is known AMPA receptors have two distinct auxiliary subunits, TARP/ about the identity of key phosphatase substrates and, more stargazin and cornichons, that regulate AMPAR trafficking and importantly, how their dephosphorylation promotes AMPAR gating [17,18]. Of these, TARP/stargazin is known to anchor endocytosis during NMDAR-dependent LTD. Previous studies AMPARs to synapses by directly interacting with PSD-95, an have identified several phosphatase substrates associated with abundant postsynaptic scaffolding protein implicated in the PLoS Biology | www.plosbiology.org 1 September 2009 | Volume 7 | Issue 9 | e1000187 RalBP1, RalA, and PSD-95 in Synaptic Depression Author Summary Results Neurons adapt over time in order to dampen their PSD-95 Interacts with RalBP1 response to prolonged or particularly strong stimuli. This Yeast two-hybrid screens with PSD-95 identified RalBP1 as a process, termed long-term depression (LTD), results in a novel PSD-95-interacting protein, which is coupled to other long-lasting decrease in the efficiency of synaptic trans- endocytic proteins including POB1, RalA, AP2, epsin, and Eps15 mission. One extensively studied form of LTD requires the (Figure 1A) [34,36]. The PDZ-binding motif at the RalBP1 C- activation of a specific class of receptors known as NMDA terminus interacted with the PDZ domains of PSD-95 (Figure 1B). glutamate receptors (NMDARs). A key molecular event RalBP1 binding to PSD-95 was further confirmed by in vitro and initiated by NMDA receptor activation is the stimulation of in vivo pull down and coimmunoprecipitation assays (Figure 1C– protein phosphatases. Another key event is internalization 1H). In addition to interacting with PSD-95, RalBP1 formed an in via endocytosis of synaptic AMPA glutamate receptors vivo complex with PSD-93/chapsyn-110 (a PSD-95 relative), but (AMPARs). However, the mechanism by which protein dephosphorylation is coupled to AMPAR endocytosis has no associations of other PSD-95 family proteins (SAP97 and remained unclear. Here, we help to define this mechanism. SAP102) with RalBP1 were detected. RalBP1 also formed a We show that endocytic proteins, including RalBP1, are complex with POB1 and a-adaptin (a subunit of the AP2 complex) widely distributed in neurons under normal conditions. in the brain (Figure 1I and 1J). Notably, RalBP1 tightly associated Upon NMDAR activation, the small GTPase RalA becomes with POB1, but relatively weakly with a-adaptin and PSD-95. activated and binds to RalBP1, resulting in the transloca- tion of RalBP1 and RalBP1-associated endocytic proteins to Expression Patterns of RalBP1, RalA, and POB1 in the synapses. At the same time, RalBP1 becomes dephosphor- Brain ylated, which promotes its binding to the postsynaptic In situ hybridization revealed that mRNAs of RalBP1, RalA, scaffold protein PSD-95, a protein that itself associates and POB1 are widely expressed in various brain regions (Figure with AMPARs. This concerted recruitment of endocytic proteins to the vicinity of AMPARs results in AMPAR S1). The three different mRNAs showed overlapping as well as endocytosis. On the basis of our data, we propose a model distinct distribution patterns. in which dual binding of RalBP1 to both RalA and PSD-95 In the brain, RalBP1 and POB1 antibodies recognized a single following RalBP1 dephosphorylation is essential