TheN-Methyl-d-aspartate Receptor Splice Variant NR1–4 C-terminal Domain

Abstract

The intracellular C-terminal domain of the N-methyl-D-aspartate receptor (NMDAR) subunits 1 (NR1) and 2 (NR2) are important, if not essential, to the process of NMDAR clustering and anchoring at the plasma membrane and the synapse. Eight NR1 splice variants exist, four of which arise from alternative splicing of the Cterminal exon cassettes. Alternative splice variants may display a differential ability to interact with synaptic anchoring proteins, and splicing of C-terminal exon cassettes may alter the mechanism(s) of subcellular localization and targeting. The NR1-4 isoform has a significantly different C-terminal composition than the prototypic NR1-1 isoform. Whereas the NR1-1 C terminus is composed of C0, C1, and C2 exon cassettes, the NR1-4 C terminus is composed of the C0 and C2 cassettes. In the present study, we address the importance of the NR1-4 C-terminal exon cassettes (C0C2) in subcellular localization in differentiated pheochromocytoma (PC12) cells, in organotypic cultures of dorsal root ganglia, and also in heterologous cells. NR1-4-green fluorescent protein chimeras were created with deletion of either C0, C2, or both cassettes to address their importance in subcellular distribution and cell surface expression of the NR1-4 subunit. These experiments demonstrate that the NR1-4 splice variant found predominantly in the spinal cord uses the C0 cassette, to a large degree, to organize the subcellular distribution of this receptor subunit. Although the role of the C2 subunit is less clear, it may be involved in subunit clustering. However, this clustering is not always as efficient as that attributed to C0 alone or to the natural combination of C0C2. Finally, although an intact C-terminal domain is neither necessary for interaction with the NR2A subunit nor surface expression of the NR1-4 subunit, the C-terminal domain fragment alone blocks surface expression of native NR1-4, in a dominant negative fashion, when the two are coexpressed.Glutamate, the major excitatory neurotransmitter of the central nervous system, acts upon the N-methyl-D-aspartate receptor (NMDAR), 1 a member of the ionotropic class of glutamate receptors (1). Functional NMDARs are heteromers composed of the two subunits NR1 and NR2 (2, 3). A third NMDAR subunit, NR3, has been described, but its biological significance is only just beginning to be investigated (4, 5). Although much is known about the physiological role that NMDARs play in long term potentiation, learning, and memory (6, 7), much remains to be learned about the mechanisms by which these receptors are sorted, targeted, and anchored, both at the synapse and at extra-synaptic sites. To date, considerable research has focused on NMDAR C-terminal domain interactions, specifically those interactions between the NR2 subunit C-terminal threonine/ serine, any amino acid, and valine ((T/S)XV) motif and PDZ domain-containing proteins located at the synapse (8 -10). PDZ domains contain an 80 -90-amino acid sequence that forms the binding site for proteins containing the C-termina...

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