L-Glutamate is the primary excitatory neurotransmitter of the vertebrate central nervous system. Activation of the N-methyl-D-aspartate (NMDA) class of L-glutamate receptors initiates various forms of synaptic plasticity and learning and memory. However, overactivation of these receptors may be responsible for cell death in a wide variety of neurological conditions. To better understand cellular processes regulated by NMDA receptors and cellular signals that regulate NMDA receptors, we sought to identify proteins that associate with a specific NMDA receptor subunit, the NR2D, a subunit recently implicated in Alzheimer's and prion disease. Receptors that contain the NR2D subunit have distinct biochemical and electrophysiological properties and have a distinct cytoplasmic domain that contains a large number of protein-protein interaction motifs. These protein-binding domains are likely to be responsible for mediating a variety of signals to and from the receptor complex.

Using bioinformatics, yeast two-hybrid screening, and mass spectrometry, this study identified a large number of proteins (>100) that can associate with the NR2D COOH-terminal region. This includes various adaptors, calcium-responsive proteins, kinases, phosphatases, and cytoskeletal and scaffold proteins. Of the interactions identified, several were PDZ-containing scaffold proteins that all selectively associated with the region of the NR2D COOH-terminal that contains the PDZ binding motif. Thus, these proteins are likely responsible for localizing NR2D-containing NMDA receptors and linking NR2D to specific signaling pathways. Yet other interactions reveal potential mechanisms by which NR2D-containing receptors may distinctly signal to pathways underlying the initiation of long-term depression (e.g. the calcium signaling linker protein Homer, the JNK pathway adapter protein JIP1b, and PLC-γ). Select protein-protein interactions were further evaluated in a variety of systems to confirm the nature of their interaction. The specific sites of molecular interaction between NR2D and JIP1b, and Nedd4 were identified. In the case of Nedd4, the association with NR2D was found to lead to ubiquitination of the receptor and thus may be responsible for downregulating NR2D-containing receptors. Taken together, these studies provide a large number of leads to determine the specific molecular mechanisms of NR2D-containing NMDA receptor function and regulation.