FRET basierte Analyse der Oligomerisierung von NMDA Rezeptoruntereinheiten

Der N-Methyl-D-Aspartat Rezeptor (NMDAR), ein Mitglied der Familie ionotroper Glutamatrezeptoren, ist maßgeblich an synaptischer Plastizität und neurodegenerativen Prozessen im zentralen Nervensystem beteiligt. Der NMDAR ist ein differenziell regulierter heterooligomerer Proteinkomplex aus 7 möglich...

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Bibliographic Details
Main Author: Schüler, Thomas
Contributors: McGregor, Gerald P. (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2005
Online Access:PDF Full Text
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The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors (iGluR) is a ligand-gated cation channel, which mediates excitatory neurotransmission in the central nervous system and plays a key role in brain development, synaptic plasticity and memory formation. NMDA receptors are obligatory heteromeric membrane proteins composed of the homologous NR1, NR2 and/or NR3 subunits. The combination of eight splice variants of the essential NR1 subunit with the differentially expressed NR2 isoforms (A, B, C, or D) and/or NR3 (A, B) isoforms gives rise to a multiplicity of temporarily and spatially regulated subtypes of NMDA receptors with distinct pharmacological properties and channel characteristics. Using fluorescently tagged NR1, NR2A, NR2B and NR3A subunits and the biophysical approach fluorescence resonance energy transfer (FRET) putative protein-protein interactions of different subunit combinations were analyzed after heterologous expression in HEK cells. When expressed alone, only the NR1 subunits do self-aggregate. Neither NR2 nor NR3 subunits could form homo-oligomeric complexes. Whereas the presence of NR1 resulted in an interaction between NR2 subunits, the additional presence of NR2 did not affect the strong interaction between NR1 subunits significantly. The combination of NR1 and NR2 as well as the combination of NR1 and NR3 resulted in the expression of functional channels and the detection of strong interactions between the different subunits. The NR1/NR3 NMDA receptor was activated exclusively by glycine and was not affected by glutamate or glutamatergic antagonists. No indications for a hetero-oligomerization of NR2 and NR3 subunits were found. The presence of NR1 resulted in an interaction between NR2 and NR3 subunits. The data show a differential homo-oligomerization behavior of the NR1 versus the NR2 and NR3 subunits. The results indicate that homo-oligomerization of the NR1 subunit might be an initial step in receptor assembly and mandatory for the stable aggregation of each of the other subunits in the NMDA complex. Thus the NR1 might control the stoichiometry and the subunit arrangement of the NMDA receptor. The data are indirect evidence for the presence of two NR1 and two NR2 subunits in a tetrameric NMDA receptor. Based on additional analyses of the assembly of ionotropic glutamate receptors the data suggest a tetrameric NMDA receptor with an alternating arrangement of the subunits. The results give rise to a better understanding of the subunit assembly and the biogenesis of a mature of NMDA receptor complex. In addition, a model of the receptor stoichiometry provides insight into the structure-function relation of NMDA receptors.