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-aspartat e receptors. We have previously characterized 3 kilobases (kb) of 5'-f lanking sequence of the NMDAR1 gene and now report on the ability of t his region to direct transcription of a reporter gene and on its inter action with nuclear proteins, The sequence 356 base pairs (bp) 5' of t he 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-f old. A labeled 112-bp fragment (position -356 to -245) formed six comp lexes (C1A and -B, C2A and -B, and C3A and -B), grouped as three doubl e bands, with nuclear extracts from PC12 cells, Competition with Sp1 o ligonucleotides abolished formation of C2A and -B and C3A and -B compl exes. Sp1 antibody recognized the C3A complex in supershift experiment s, Prior immunoprecipitation of nuclear extracts with Spl antibody abo lished 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 fragm ent was protected from DNase I digestion by nuclear extract, These res ults suggest that a 356-bp sequence comprises the NMDAR1 basal promote r, and that NMDAR1 gene expression may be regulated by Sp1-like nuclea r factors.