INTERACTION OF THE N-METHYL-D-ASPARTATE RECEPTOR COMPLEX WITH A NOVELSYNAPSE-ASSOCIATED PROTEIN, SAP102

Ionotropic glutamate receptors are known to cluster at high concentrat ion on the postsynaptic membrane of excitatory synapses, but the mecha nism by which this occurs is poorly understood. Studies on the neuromu scular junction and central inhibitory synapses suggest that clusterin g of neurotransmitter receptors requires its interaction with a cytopl asmic protein. Recently, in vitro studies have shown that members of t he N-methyl-D-aspartate (NMDA) class of glutamate receptors interact w ith a synapse-associated protein, SAP90 (PSD-95). However, evidence fo r the in vivo interaction of NMDA receptors with SAPs is still lacking . In the present study, we demonstrate the specific interaction betwee n SAP102, a novel synapse-associated protein, and the NMDA receptor co mplex from the rat cortical synaptic plasma membranes using co-immunop recipitation techniques. No association was observed between SAP102 an d GluR1, a member of the pha-amino-3-hydroxy-5-methyl-4-isoxazolepropi onate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidi ne fusion proteins hom different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, th e NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino a cids of the NR2 subunit as indicated by the interaction of the synthet ic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex a nd a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.