Characterization of the Rat GRIK5 Kainate Receptor Subunit Gene Promoter and Its Intragenic Regions Involved in Neural Cell Specificity

Abstract

The GRIK5 (glutamate receptor ionotropic kainate-5) gene encodes the kainate-preferring glutamate receptor subunit KA2. The GRIK5 promoter is TATA-less and GCrich, with multiple consensus initiator sequences. Transgenic mouse lines carrying 4 kilobases of the GRIK5 5-flanking sequence showed lacZ reporter expression predominantly in the nervous system. Reporter assays in central glial (CG-4) and non-neural cells indicated that a 1200-base pair (bp) 5-flanking region could sustain neural cell-specific promoter activity. Transcriptional activity was associated with the formation of a transcription factor IID-containing complex on an initiator sequence located 1100 bp upstream of the first intron. In transfection studies, deletion of exonic sequences downstream of the promoter resulted in reporter gene activity that was no longer neural cell-specific. When placed downstream of the GRIK5 promoter, a 77-bp sequence from the deleted fragment completely silenced reporter expression in NIH3T3 fibroblasts while attenuating activity in CG-4 cells. Analysis of the 77-bp sequence revealed a functional SP1-binding site and a sequence resembling a neuron-restrictive silencer element. The latter sequence, however, did not display cell-specific binding of REST-like proteins. Our studies thus provide evidence for intragenic control of GRIK5 promoter activity and suggest that elements contributing to tissue-specific expression are contained within the first exon.Three structurally related mammalian glutamate receptor (GluR) 1 gene families encode N-methyl-D-aspartate, ␣-amino-3-hydroxymethylisoxazole-4-propionic acid, and kainate receptor subunits (1-3). Two kainate receptor gene subfamilies encode the subunits GluR5-7 and KA1/2, which bind kainate and domoate with high affinity (2-4). When expressed in Xenopus oocytes or heterologous mammalian cells, only GluR5-7 form functional homomeric channels (5-7). KA1 and KA2 are inactive as homomeric ion channels, but associate with members of the GluR5-7 family to form functional heteromeric kainatepreferring receptors with biophysical properties and agonist selectivity distinct from those of homomeric GluR5-7 channels (8 -10).In situ hybridization and immunohistochemical studies have demonstrated that, in different brain regions, specific combinations of members of the KA1/2 and GluR5-7 gene subfamilies are coexpressed (11-13). This indicates that, similar to ␣-amino-3-hydroxymethylisoxazole-4-propionic acid and Nmethyl-D-aspartate receptors, kainate receptor subunits assemble to form diverse subtypes of heteromeric channels. For example, co-immunoprecipitation experiments with antiGluR6 and anti-KA2 antibodies have shown that these subunit proteins associate to form receptor channel complexes in brain cell membranes (14).These molecular studies are consistent with the finding that functional homomeric and hetero-oligomeric kainate-preferring receptors are expressed in cultured neurons and glia (15)(16)(17). Despite the widespread expression of kainate receptor subunits in the mammalia...

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene

“…Independent studies have shown that Sp1 has functional implication in expression of some of NRSF target genes including N -methyl- d -aspartate receptors, dynamin I, synaptophysin and glutamate receptor ionotropic kainate-5 genes (22–24). For MOR gene regulation, Sp1 factor acts as a major positive regulator by binding two cis -elements (distal and proximal promoters) located at −340 through −300 residues from the translation start site while Sp3 has a minor effect by binding to one of these elements (25).…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…Furthermore, a 77-bp sequence termed the kainate cell-specific enhancer region, involved in cell-specific expression, includes a functional Sp1 site that when placed downstream of the Grik5 promoter, silenced reporter expression in NIH3T3 fibroblasts and attenuated activity in CG-4 cells. These studies show that elements contributing to tissue-specific expression are contained within the first exon (Chew et al, 2001).…”

“…Transgenic mouse lines carrying 4 kb of the 5Ј-flanking sequence showed lacZ reporter expression predominantly in the nervous system. Reporter assays in central glial (CG-4) and non-neural cells indicated that a 1200-bp 5Ј-flanking region could sustain neural cell-specific promoter activity (Chew et al, 2001). Sp1 binding suggests a functional role for Sp1 in initiatormediated activation of Grik5 transcription that involves transcription factor II D-mediated basal activity (Chew et al, 2001).…”

“…Reporter assays in central glial (CG-4) and non-neural cells indicated that a 1200-bp 5Ј-flanking region could sustain neural cell-specific promoter activity (Chew et al, 2001). Sp1 binding suggests a functional role for Sp1 in initiatormediated activation of Grik5 transcription that involves transcription factor II D-mediated basal activity (Chew et al, 2001). Removal of two putative AP2 sequences reduced promoter activity in both neural and non-neural cells, suggesting that these sites are also important for basal transcription.…”

Transcriptional Regulation of the Mouse Gene Encoding the α-4 Subunit of the GABAA Receptor

“…Gel Shift Assay-Mouse brain nuclear extract was prepared as described previously with modifications (42). Briefly, the brain tissue was frozen in liquid nitrogen and shattered into tissue powder.…”