Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.487137
Title: The folding and assembly of NMDA receptor subunits
Author: Atlason, Palmi por
ISNI:       0000 0001 3432 3244
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2007
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Abstract:
The NMDA receptor is a member of the ionotropic glutamate receptor family, which includes the AMPA and kainate receptors. Functional NMDA receptors are heteromeric and made up from three receptor subunit families; NR1, NR2 and NR3. Currently, the receptor is thought to form in a dimer-of-dimer assembly with two NRI subunits and two NR2 subunits. The NR3 can associate with both NRI and NR2 to form a unique receptor or NRI alone to form an excitatory glycine receptor. The exact assembly pathway and the nature of assembly intermediates are currently unknown. The folding status of NR1, NR2A and NR3A was examined in heterologous systems, both individually expressed and co-expressed. It was found that while NRI appeared to fold well when expressed on its own and contain relatively few free cysteine residues, the NR3A subunit appeared to form aggregates when analysed under non-reducing conditions. BothNR2A and NR3A aggregated when treated with the sulfhydryl crosslinker BMH, indicating mUltiple free cysteine residues and a degree of misfolding. NRI migrated as a dimer when treated with BMH. A small pool of fast degrading NRI was detected but the majority of the protein was very stable in cells. NR2A and NR3A were turned over rapidly when expressed on their own with NR2A showing stabilisation in the presence of NRI. FRAP analysis indicated a degree of misfolding of NR2A and NR3A. NRI readily associated with both NR2A and NR3A but no association of NR2A and NR3A was detected in the absence ofNRl. The analysis of receptor assembly using BiFC confirmed the homodimerization of NRI but failed to give any evidence for homodimerization of either NR2A or NR3A. The BiFC analysis further indicates that the homodimer of NRI is readily disassociable. The robust complementation seen between NRI and NR2A, coupled with the absence of complementation ofNR2A and NR2A in the presence ofNRl suggest the preferential assembly of heterodimers. Possibly, steric constraints prevent complementation in the tetramer, suggesting a 1-2-1-2 symmetry around the pore. A similar constraint due to a coiled-coil domain in NR3A might explain the absence of complementation between NRI and NR3A. . Taken together, the results suggest the central role of NRI in the folding and assembly of the other subunit families. The probable assembly pathway suggests recruitment of NRI from an intracellular pool to associate with newly synthesised NR2 or NR3 to form heterodimers which then assemble to form the functional tetrameric receptor.
Supervisor: Not available Sponsor: Not available
Qualification Name: University of Oxford, 2007 Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.487137  DOI: Not available
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