Assembly and trafficking of nmda receptors: a biochemical approach

The N-methyl-D-aspartate (NMDA) receptor is a subtype of the excitatory, ionotropic L- glutamate neurotransmitter receptor family. Functional NMDA receptors are composed of the obligatory NR1 subunits together with NR2 subunits. The NMDA receptor subunits are encoded by seven genes: NR1, NR2A-NR2D, NR3A and NR3B. The NR1 subunit gene undergoes alternative splicing to yield eight splice variants: NR1-1a, NR1-1b- NR1-4a., NR1-4b. NMDA receptors are targeted to dendritic spines of neurones where they associate with proteins of the post-synaptic density such as the scaffolding protein, post-synaptic density 95 (PSD-95). The aim of this thesis was to study the assembly and trafficking of NMDA receptors. This investigation was initiated by observing that insertion of the c-Myc epitope at position 81 of the NR1-2a subunit (NRl-2ac-Myc) did not result in NMDA receptor-mediated cell cytotoxicity following co-expression with NR2A in human embryonic kidney (HEK) 293 cells. NR1-2a/NR2 and NR1-2ac-Myc/NR2 were transiently expressed in HEK 293 cells and analysed by one and two dimensional immunoblotting, cell surface ELISAs, [3H]MDL105,519 and [3H]MK801 radioligand binding activities and co-immunoprecipitation. NR1-2ac-Myc/NR2 behaved as wild-type except that mutant receptors were not expressed on the cell surface and m immunoblots only an NR1 Mr 116 kDa species was detected compared to Mr 115 and a 226 kDa species for wild-type. Two-dimensional electrophoresis of the NR1 Mr 226 kDa species revealed that it represented disulphide-1 inked NR1 subunits, suggesting that insertion of the c-Myc tag may interfere with NR1-NR1 disulphide-linked dimer formation. To investigate this further, cysteines 22, 79 and 308 of the NRl-2a subunit were mutated to alanine, co-expressed with NR2 m HEK 293 cells and analysed as above. Receptors containing the C79A and C308A mutations in the NR1-2a subunit, showed impaired cell surface expression and NR1-2ac79A/NR2A lacked the NR1 Mr 226 kDa species. These results reveal that NR1-NR1 inter-subunit disulphide bond formation involving cysteine 79 is requisite for the efficient trafficking of NR1/NR2 NMDA receptors to the cell surface. The synaptic targeting of NMDA receptors is mediated by PSD-95. Recently, an alternative splice form of PSD-95 was identified, termed PSD-95beta. Co-immunoprecipitation experiments demonstrated that PSD-95beta associates with NR1-1a/NR2A receptors in HEK 293 cells. This association resulted in an ˜ 4-fold increase in the expression levels of the NR2A subunit relative to NR1-1a/NR2A receptors expressed alone. Collectively, these results offer new grounds towards understanding the complicated exocytotic machinery involved in the assembly of NMDA receptors.