N-Methyl-D-aspartate-induced -Amino-3-hydroxy-5-methyl-4- isoxazoleproprionic Acid (AMPA) Receptor Down-regulation Involves Interaction of the Carboxyl Terminus of GluR2/3 with Pick1 LIGAND-BINDING STUDIES USING Sindbis VECTORS CARRYING AMPA RECEPTOR DECOYS*

The dynamics of -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors, as represented by their exocytosis, endocytosis and cytoskeletal linkage, has often been implicated in N-methyl-D-aspartate (NMDA)-dependent synaptic plasticity. To explore the molecular mechanisms underlying the AMPA receptor dynamics, cultured hippocampal neurons were stimulated with 100 M NMDA, and the biochemical and pharmacological changes in the ligand binding activity of AMPA receptor complexes and its subunits, GluR1 and GluR2/3, were investigated. The NMDA treatment reduced the total amount of bound [H]AMPA on the surface of the neurons but not in their total membrane fraction. This process was mimicked by a protein kinase C activator, phorbol ester, but blocked by an inhibitor of the same kinase, calphostin C. The NMDA-induced down-regulation of the ligand binding activity was also reflected by the decreased AMPA-triggered channel activity as well as by the cells’ reduced immunoreactivity for GluR1. In parallel, the NMDA treatment markedly altered the interaction between the AMPA receptor subunits and their associating molecule(s); the association of PDZ molecules, including Pick1, with GluR2/3 was enhanced in a protein-kinaseC-dependent manner. Viral expression vectors carrying GluR1 and GluR2 C-terminal decoys, both fused to enhanced green fluorescent protein, were transfected into hippocampal neurons to disrupt their interactions. The overexpression of the C-terminal decoy for GluR2 specifically and significantly blocked the NMDA-triggered reduction in [H]AMPA binding, whereas that for GluR1 had no effects. Co-immunoprecipitation using anti-Pick1 antibodies revealed that the overexpressed GluR2 C-terminal decoy indeed prevented Pick1 from interacting with the endogenous GluR2/3. Therefore, these observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors involves the interaction between GluR2/3 subunits and Pick1. Synaptic processes in hippocampal plasticity, such as long term potentiation (LTP) and long term depression (LTD), are thought to involve the subcellular dynamics of the -amino-3hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptor channels. The most recent physiological studies have suggested that an exocytotic process of these receptor channels to the postsynaptic sites may form the basis of the NMDA-dependent hippocampal LTP (1). Conversely, the elimination of the receptors from the synaptic surface has been suggested to associate with hippocampal LTD (2). The former synaptic plasticity is known to require the activation of calmodulin-dependent protein kinase (3, 4), whereas the latter involves protein kinase C (PKC) as well as various protein phosphatases (5, 6). Through the NMDA receptor channels, the influx of calcium ions can lead to the activation of these two protein kinases, each of which results in opposing synaptic changes. Although it remains to be established how both types of synaptic plasticity are switched on and off following the postsynaptic influx of calcium ions, postsynaptic density (PSD) proteins should contribute to such synaptic processes. There are a large variety of PSD proteins and other molecules that can interact with AMPA receptor subunits. The AMPA receptor subunit GluR1 is known to interact with SAP97, Narp, and Lyn through various subdomains of this subunit (7–9). Other subunits (e.g. GluR2 and GluR3) can associate with GRIP, ABP, Pick1, and NSF at its carboxylterminal regions (4, 10–12). These adaptor molecules appear to regulate the subcellular distributions of AMPA receptor subunits, altering their cytoskeletal associations and/or metabolic stability (13, 14). Although individual subunits of the AMPA receptor channel complex have been shown to interact with different PSD proteins in parallel with AMPA receptor internalization (5, 6, 15–20), the real dynamics of the functional AMPA receptor complexes and the underlying molecular mechanism(s) have not been fully characterized. For instance, which AMPA receptor subunit governs the dynamics of the whole receptor complex? Which adaptor molecule accordingly regulates the pharmacological change of the AMPA receptors? In cultured hippocampal neurons, we attempted to address these questions by monitoring their ligand binding activity and mo* This work was supported by Japanese Society for the Promotion of Science Grant RFTF-96L00203 and a Grant-in-Aid for Creative Scientific Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address: Tokyo Medical and Dental University, School of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan. ¶ To whom correspondence should be addressed. E-mail: hnawa@bri. niigata-u.ac.jp. 1 The abbreviations used are: LTP, long term potentiation; LTD, long term depression; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid; EGFP, enhanced green fluorescent protein; NMDA, Nmethyl-D-aspartate; PSD, postsynaptic density; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; ANOVA, analysis of variance. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 43, Issue of October 26, pp. 40025–40032, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.