FUNCTIONAL-ANALYSIS OF THE PROXIMAL 5'-FLANKING REGION OF THE N-METHYL-D-ASPARTATE RECEPTOR SUBUNIT GENE, NMDAR1

The NMDAR1 receptor subunit is a common subunit of N-methyl-D-aspartate receptors. We have previously characterized 3 kilobases (kb) of 5'-flanking sequence of the NMDAR1 gene and now report on the ability of this region to direct transcription of a reporter gene and on its interaction with nuclear proteins, The sequence 356 base pairs (bp) 5' of the first nucleotide of codon 1 was sufficient to express a luciferase reporter gene in rat PC12 pheochromocytoma cells, Additional sequences upstream of nucleotide -356 influenced the activity approximately 2-fold. A labeled 112-bp fragment (position -356 to -245) formed six complexes (C1A and -B, C2A and -B, and C3A and -B), grouped as three double bands, with nuclear extracts from PC12 cells, Competition with Sp1 oligonucleotides abolished formation of C2A and -B and C3A and -B complexes. Sp1 antibody recognized the C3A complex in supershift experiments, Prior immunoprecipitation of nuclear extracts with Spl antibody abolished formation of C2A and -B and C3A and -B complexes. Purified Sp1 protein alone did not form a C3A complex but potentiated its formation when PC12 nuclear extract was added, A CC-rich sequence in this fragment was protected from DNase I digestion by nuclear extract, These results suggest that a 356-bp sequence comprises the NMDAR1 basal promoter, and that NMDAR1 gene expression may be regulated by Sp1-like nuclear factors.