N-methyl-D-aspartate receptors (NMDARs) belong to a subclass of excitatory ionotropic glutamate receptors that play important roles in neuronal differentiation, synaptic transmission, synaptic integration and plasticity. NMDARs are widely distributed in the mammalian central nervous system (CNS). Their heteromultimeric structures are composed of at least two essential NR1 subunits, one or more regulatory NR2A-D subunits and/or the additional NR3A-B subunits. Overstimulation of NMDARs contributes to the neurodegeneration observed in acute and progressive degenerative neurological disorders. It was reported previously that the association of the serine/threonine protein phosphatase 2A (PP2A) with the NR3A subunit is involved in the regulation of NMDAR function through modulation of the ph...[ Read more ]

N-methyl-D-aspartate receptors (NMDARs) belong to a subclass of excitatory ionotropic glutamate receptors that play important roles in neuronal differentiation, synaptic transmission, synaptic integration and plasticity. NMDARs are widely distributed in the mammalian central nervous system (CNS). Their heteromultimeric structures are composed of at least two essential NR1 subunits, one or more regulatory NR2A-D subunits and/or the additional NR3A-B subunits. Overstimulation of NMDARs contributes to the neurodegeneration observed in acute and progressive degenerative neurological disorders. It was reported previously that the association of the serine/threonine protein phosphatase 2A (PP2A) with the NR3A subunit is involved in the regulation of NMDAR function through modulation of the phosphorylation state of NR1. Therefore it is important to gain insight into the interaction between the NR3A subunit and PP2A. In this study, data from co-immunoprecipitation experiments using rat brain synaptic plasma membranes (SPM) revealed that NR3A was associated with the PP2A holoenzyme but not the core enzyme. In addition, the molecular determinants required for the formation of the NR3A-PP2A complex were characterized by site-directed mutagenesis. In particular, alanine-scanning mutagenesis was performed in the previously identified 37-amino acid PP2A-binding domain of the intracellular carboxyl terminal of NR3A subunit (NR3Ac). A yeast two-hybrid system was then used to investigate the effects of the mutations on the interaction of NR3Ac and the catalytic subunit of PP2A (PP2A-C). The data suggest that mutation of either one of three critical amino acids reduced the interaction between the NR3Ac and the PP2A-C by more than 80%. These amino acids are conserved in both human and rat NR3Ac. Co-immunoprecipitation studies indicated that at least one of these critical amino acids is essential for the interaction of NR3A with PP2A in mammalian cells. This project delineates molecular determinants of the association of NR3A with PP2A.