Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A2A adenosine receptor

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'-untransiated region of the rat A(2A) adenosine receptor (A(2A)-R) gene. Various lengths of the 5'-flanking region of the A(2A)-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 A(2A)-R/NF1), located between bases -2846 and -2827. of the A(2A)-R gene, functions as a repressor for A(2A)-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A(2A)-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A(2A)-R/NF1-protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A(2A)-R/NF1-protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A(2A)-R/NF1 element disturbed DNA-protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A(2A)-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A(2A)-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A(2A)-R. Moreover, addition of the A(2A)-R/NF1-element 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 A(2A)-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A(2A)-R/NF1) that plays a functional role in modulating the expression of A(2A)-R. (C) 2003 Elsevier Science B.V. All rights reserved.