Abstract
Abstract: :
Purpose:Glutamate is the main excitatory neurotransmitter in the CNS, acting through distinct receptor populations (GluRs). The NMDA subtype of GluR is assembled as a tetramer including NR1, NR2, and possibly NR3 subunits. The alternative splicing of exons 5, 21 and 22 in NR1 pre–mRNA generates eight isoforms which contribute to the functional diversity of NMDARs in the CNS. Biochemical and pharmacological differences in chick retinal NMDARs compared to the brain have been shown, which could relate to the NR1 splice variant included in the heteromer. The aim of this work was to investigate differences in NR1 splice variants of the chick retina compared to the brain, and the cloning of chick NR1 gene, in order explore its relationship with the tissue–specific regulation of NMDARs in the retina. Methods: The expression of NR1 isoforms was analyzed using semiquantitative RT–PCR. The NR1 chick gene was cloned using a chick genomic library, from which four partially overlapping clones containing the structural and regulatory regions of NR1 were obtained. Results:The postnatal chick retina expresses primarily NR1 isoforms containing exon 5, whereas the cerebral ones mainly lack this exon. The differential expression between the retina and the brain, was also observed in the rat, however, it was more pronounced in the chick. Retinal NR1 also differs form the brain in its C–terminal splice variants, since it expresses in addition to a splice variant containing C1 and C2 cassettes, a novel isoform containing an additional putative alternative splice exon. The 41bp insertion encodes a 6 amino acid sequence (C3 cassette) containing a predicted casein kinase II phosphorylation site. Molecular cloning of chick NR1 gene confirmed the existence of this novel cassette. Noticeably, the chick gene 3’ region differs significantly from the mammalian gene, besides the existence of C3 cassette, the C2 cassette was shown to contain a myristoylation site; in contrast, no PDZ binding domains were identified in C2’–containing isoforms. The promoter region despite its low homology with previously described genes contains multiple regulatory sites that may contribute to the particular modulation of NR1 expression in avian neurons. Conclusions:Expression of exon 5 containing isoforms might explain some of the biochemical characteristics of retinal NMDA receptor, which differ from those observed in the CNS. Differences in the C–terminal splice variants suggest distinct targeting and/or anchoring mechanisms for NMDARs in avian retina.
Keywords: excitatory amino acid receptors • ion channels • gene/expression