N-methyl-D-aspartate-induced alpha-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 alpha -amino-3-hydroxy-5-methyl-4-isox-azoleproprionic acid (AMPA)-type glutamate receptors, as represented by their exocytosis, endoc ytosis and cytoskeletal linkage, has often been implicated in N-methyl-D-as partate (NMDA)-dependent synaptic plasticity. To explore the molecular mech anisms underlying the AMPA receptor dynamics, cultured hippocampal neurons were stimulated with 100 mum NMDA, and the biochemical and pharmacological changes in the ligand binding activity of AMPA receptor complexes and its s ubunits, GluR1 and GluR2/3, were investigated. The NMDA treatment reduced t he total amount of bound [H-3]AMPA on the surface of the neurons but not in their total membrane fraction. This process was mimicked by a protein kina se C activator, phorbol ester, but blocked by an inhibitor of the same kina se, calphostin C. The NMDA-induced down-regulation of the ligand binding ac tivity was also reflected by the decreased AMPA-triggered channel activity as well as by the cells' reduced immunoreactivity for GluR1. In parallel, t he NMDA treatment markedly altered the interaction between the AMPA recepto r subunits and their associating molecule(s); the association of PDZ molecu les, including Pick1, with GluR2/3 was enhanced in a protein-kinase-C-depen dent manner. Viral expression vectors carrying GluR1 and GluR2 C-terminal d ecoys, both fused to enhanced green fluorescent protein, were transfected i nto hippocampal neurons to disrupt their interactions. The overexpression o f the C-terminal decoy for GluR2 specifically and significantly blocked the NMDA-triggered reduction in [H-3]AMPA binding, whereas that for GluR1 had no effects. Co-immunoprecipitation using anti-Pick1 antibodies revealed tha t the overexpressed GluR2 C-terminal decoy indeed prevented Pick1 from inte racting with the endogenous GluR2/3. Therefore, these observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors invo lves the interaction between GluR2/3 subunits and Pick1.