By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5′-flanking region and a 0.5-kb 5′-untranslated region of the rat A2A adenosine receptor (A2A-R) gene. Various lengths of the 5′-flanking region of the A2A-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A2A-R/NF1), located between bases -2846 and -2827 of the A2A-R gene, functions as a repressor for A2A-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A2A-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A2A-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A2A-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A2A-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A2A-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A2A-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A2A-R. Moreover, addition of the A2A-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A2A-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A2A-R/NF1) that plays a functional role in modulating the expression of A2A-R.
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