Abstract
Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl--aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from 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, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.
Highlights
These glutamate receptor subunits are differentially expressed in various regions of the brain at both the mRNA and protein level as demonstrated by in situ hybridization (Meguro et al, 1992; Monyer et al, 1992; Monyer et al, 1994) and immunohistochemical studies (Brose et al, 1993), respectively
The present studies show that a novel member of the SAP family, SAP102, interacts with the native NMDA receptor complex from cortical synaptic plasma membranes
SAP90/PSD-95 has been shown to co-localize with the NR2B subunit in cultured hippocampal neurons (Kornau et al, 1995), direct biochemical evidence showing interaction between native proteins has not been developed
Summary
These glutamate receptor subunits are differentially expressed in various regions of the brain at both the mRNA and protein level as demonstrated by in situ hybridization (Meguro et al, 1992; Monyer et al, 1992; Monyer et al, 1994) and immunohistochemical studies (Brose et al, 1993), respectively. Studies from the yeast two-hybrid system (Kornau et al, 1995; Niethammer et al, 1996) have suggested that the NR2 subunits of the NMDA receptor interact with a peripheral membrane protein called PSD-95 (for a 95-kDa protein found in the postsynaptic density)
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