Distribution of an NMDA receptor:GFP fusion protein in sensory neurons is altered by a C-terminal construct.
The NMDA receptor plays an important role in mediating sensory input to the spinal cord. Domains within the C-terminus of the NMDA receptor bind to cytoskeletal proteins and facilitate membrane targeting and synaptic clustering, and may participate in regulation of receptor function. One strategy to manipulate NMDA receptor function is to express C-terminal constructs in neurons to disrupt synaptic clustering via competition for binding motifs in cytoskeletal proteins and postsynaptic densities. Biolistic particle-mediated gene transfer was used to deliver plasmid DNA into organotypic cultures of dorsal root ganglia (DRG). Fusion proteins consisting of recombinant (r)NMDA receptor subunit 1-1 (rNR1-1) deletion constructs and enhanced green fluorescent protein (GFP) were expressed in sensory neurons and demonstrated unique distribution patterns within the cell. Expression of the full length rNR1-1:GFP construct was cytosolic and localized to membranous patches similar to endogenous NR1-1 protein expression in sensory neurons. Expression of a construct containing only the C-terminus, GFP:C0C1C2, demonstrated nuclear and membranous localization. When the GFP:C0C1C2 construct was co-expressed with rNR1-1 in sensory neurons, membranous localization of rNR1-1 was disrupted. In contrast, co-expression of a C-terminal cassette lacking the C1 exon cassette, GFP:C0C2, with rNR1-1 did not alter the membranous distribution of rNR1-1. This observation verifies the utility of a gene transfer strategy to diminish membranous NR1-1 content by expressing a construct containing the C1 exon cassette.
Neurodegeneration Research Group, The John P. Robarts Research Institute, London, Ontario, Canada.
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Marsh
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Holmes
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Dekaban
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Weaver
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000818:Animals; D019470:Biolistics; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009475:Neurons, Afferent / Q000166:cytology / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000096:biosynthesis; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism
D008164:Luminescent Proteins; C409466:NR1 NMDA receptor; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C104722:glutamate receptor ionotropic, AMPA 2