Abstract
The dynamics of alpha-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 microm 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 [(3)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-kinase-C-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 [(3)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.
Highlights
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
Down-regulation of the Ligand Binding Activities of AMPA Receptors Expressed on Neuronal Surfaces—The radiolabeled receptor assay method allows measurement of the amounts of radiolabeled ligands bound to cell membranes and enables characterization of the pharmacological properties of its receptors, such as the number of functional receptors (Bmax) and the dissociation constant for their ligands (Kd) [25, 26]. This assay was used with 3H-labeled AMPA to assess the effects of NMDA treatment on the ligand binding activity of functional AMPA receptors expressed on the surface of cultured hippocampal neurons
By monitoring [3H]AMPA binding to the receptors, we were able to examine the subcellular dynamics of functional AMPAtype glutamate receptors in young hippocampal neurons
Summary
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. Co-immunoprecipitation using anti-Pick antibodies revealed that the overexpressed GluR2 C-terminal decoy prevented Pick from interacting with the endogenous GluR2/3 These observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors involves the interaction between GluR2/3 subunits and Pick. 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. Other subunits (e.g. GluR2 and GluR3) can associate with GRIP, ABP, Pick, 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]. Using the Sindbis viral expression vector carrying AMPA receptor decoys, here we investigated the AMPA receptor subunits and domains responsible for the NMDA-induced pharmacological changes of AMPA receptors
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