Cousins, Sarah Louise; (2007) A study of NMDA receptor / PSD-95 membrane associated guanylate kinase interactions. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

N-Methyl-D-aspartate (NMDA) receptors are a subclass of excitatory ionotropic glutamate neurotransmitter receptors. There are four major subclasses of NMDA receptors formed by the co-assembly of different NMDA receptor subunits, i.e. NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D. These subclasses have distinct physiological properties and distinct temporal and spatial patterns of distribution in neurones. NMDA receptors are trafficked to dendritic spines in neurons where they are thought to be anchored in the post- synaptic membrane by their association with the postsynaptic density-95 (PSD-95) membrane associated guanylate kinase (MAGUK) family of scaffolding proteins. PSD-95 is the prototypic member of this family which also includes channel associated protein of synapse-110 (Chapsyn-110), synapse associated protein97 (SAP97) and SAP 102. The association between PSD-95 and the NR1/NR2A and NR1/NR2B receptor subtypes has been well characterized and shown to be important for the regulation of receptor cell surface expression. However there is a paucity of knowledge with regard to the association between NR1/NR2C and NR1/NR2D subtypes and PSD-95 and also all four major NMDA receptor subclasses and the other members of the PSD-95 MAGUK family. The aim of this thesis therefore was to investigate the association between the PSD-95 MAGUK proteins and the four major NMDA receptor subclasses and the effects of this association on NMDA receptor expression and cell surface trafficking. Each major NMDA receptor subclass was co-expressed with PSD-95 MAGUK family members in human embryonic kidney (HEK) 293 cells and the expressed proteins analysed by immunoprecipitation assays, quantitative immunoblotting and cell surface ELISA assays. In addition, the association between NR1/NR2A NMDA receptors and PSD-95 was studied in more detail which included the mapping of a new PSD-95 binding motif in the NR2A C-terminal domain and a study of the role of protein kinase A phosphorylation in receptor/scaffold association and cell surface trafficking. Finally, a yeast two-hybrid screen was conducted to identify novel NR1/NR2D NMDA receptor associated proteins. Differential association with the PSD-95 MAGUK family and NMDA receptors was discovered. This may be important for the regulation of cell surface receptor number, the stabilisation, clustering, turnover and compartmentalisation of NMDA receptor subtypes in neurons during development and in the mature brain.

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