N-methyl-D-aspartate (NMDA) receptors have a high affinity for glutamate and are important for control of dendritic growth, gene transcription, and synaptic plasticity. However, high levels of NMDA receptor stimulation cause excitotoxicity. In the present work I have explored NMDA receptor-mediated regulation of two independent pathways: p38 mitogen-activated protein kinase (MAPK) activation and excitatory amino acid carrier 1 (EAAC1) cell surface expression. In the current research, using NMDA as a specific agonist, the pharmacological requirements for NMDA receptor-mediated p38 MAPK phosphorylation and dephosphorylation were examined. Low concentrations of NMDA resulted in robust, sustained phosphorylation, and high concentrations of NMDA produced only transient phosphorylation of p38 MAPK. Antagonists of NR2B-containing NMDA receptors and calcineurin each prevented NMDA-induced p38 MAPK phosphorylation. Alternatively, antagonists of NR1/2B subtype NMDA receptors and phosphoinositide-3 kinase (PI3K) inhibitors prevented the rapid loss of p38 MAPK phosphorylation with high concentrations of NMDA. The p85 (regulatory) subunit of PI3K was coimmunoprecipitated with the NR2B, but not the NR2A subunit, and increased phosphorylation of NR2B-Y1336 was observed after treatment with high concentrations of NMDA. These data suggest that p38 MAPK phosphorylation (and thereby activation) occurs through NR1/2A/2B subtype NMDA receptors through calcineurin, and dephosphorylation occurs through phosphorylation of NR2B-Y1336, resulting in PI3K activation, in conjunction with NR1/2B subtype NMDA receptors. Independently, an association and regulation of EAAC1 by the NMDA receptor was identified. Antibodies that pull-down EAAC1 coimmunoprecipitated NMDA receptor subunits NR1, NR2A, and NR2B in hippocampal cultures and in C6 glioma cotransfected with myc-EAAC1, NR1, NR2A, and NR2B. C6 glioma, transfected with both NR1 and NR2 subunits, increased levels of cell surface EAAC1. Furthermore, high concentrations of NMDA produced a substantial loss of cell surface EAAC1 in hippocampal cultures. These effects of NMDA were inhibited by NMDA receptor antagonists, Ca2+ chelators, and hypertonic sucrose. These data suggest that NMDA receptors interact with EAAC1, facilitating its cell surface expression, and that NMDA receptor activation induces internalization of EAAC1 through overall increases in intracellular calcium and clathrin-mediated endocytosis. This work has identified novel mechanisms by which NMDA receptors may affect synaptic plasticity and/or excitotoxicity
