Characterization of the Rat A2a Adenosine Receptor Gene

Abstract

To understand the molecular basis for the regulation of rat A2a adenosine receptor (A2a-R) expression, we have characterized the rat A2a-R gene and defined its promoter regions. Through a combination of restriction mapping and sequence analysis, we have demonstrated that the rat A2a-R gene is composed of two exons interrupted by a 7.2-kb intron. Primer extension and RNase protection on RNA isolated from PC12 cells suggested that the A2a-R gene encoded two clusters of alternative transcripts. The most upstream transcription start site was designated as +1. The sequence of the proximal 1.5 kb of 5'-flanking region demonstrated no potential TATA box, CCAAT box, or initiator element in the appropriate location. Varying lengths of 5'-flanking regions were inserted into a transient expression vector (pGL2-basic), which contained bacterial luciferase as the reporter gene, to determine its promoter region(s) in PC12 cells, CHOP cells, and C6 cells. Consistent with two clusters of transcription start sites, two independent functional promoter regions (designated P1, -67/-1; and P2, +272/+304) for the rat A2a-R gene were identified. Although both promoters are in use in PC12 cells, only P2 is active in CHOP cells, indicating possible cell line-specific usage of these two promoters.

Cloning and Characterization of the Promoter Region of the Wilson Disease Gene

“…2B). Since the size of the RNA fragment obtained in RPA is generally estimated to be 5-10% smaller than that of the DNA fragment on a polyacrylamide gel (17), the band size agreed well with the result obtained by primer extension experiments. It should be noted that MLS6 and MREd site were located at the downstream region of the transcription start site in the reverse orientation, and further investigation will elucidate whether this site is relevant to the transcriptional or post-transcriptional activity of the WD gene.…”

cAMP-response Element-binding Protein Contributes to Suppression of the A2A Adenosine Receptor Promoter by Mutant Huntingtin with Expanded Polyglutamine Residues

“…To determine whether mutant Htt affects expression of the A 2A -R gene by regulating its promoter, the pGL2-(Ϫ1997/Ϫ145) construct, which contains two promoters of the rat A 2A -R gene (18), and the expression construct encoding mutant Htt with the indicated number of CAG repeats (Q) were cotransfected into PC12 and primary striatal neurons. As shown in Fig.…”

“…Because of the instability of the CAG repeat in bacteria, three pcDNA3.1-Htt-(Q) n -hrGFP constructs containing different numbers of CAG repeats (48,73, and 109) were created while subcloning the original pcDNA3.1-Htt-(Q) 158 -hrGFP construct. The pGL2-luciferase reporter constructs, which contain various lengths of the promoter fragment of the rat A 2A -R gene, and the CREB-VP16 construct were created as described elsewhere (6,18). The pRL-SV40 and pRL-TK constructs were purchased from Promega (Madison, WI).…”

“…The pCMV-␤-galactosidase was from Clontech. The expression constructs, which encode a dominant negative S133A-CREB mutant (CREBm1), and a dominant negative CREBR287L mutant, as well as the A 2A -R promoter constructs are described elsewhere (18,35). Promoter constructs harboring mutations at the CRE site (Ϫ246 to Ϫ241; TCCAGG) were created by PCR using pGL2-A 2A R -250/-145 as the template together with the indicated sense primer (M2, 5Ј-AACATAACCCTCTTGCCCGG-TATC-3Ј; M4, 5Ј-AACATAACCCTCTTGACCTGTATC-3Ј; M6, 5Ј-AA-CATAACCCTCTTGAACTTTATC-3Ј) and the same antisense primer (5Ј-CTTTATGTTTTTGGCGTCTTCCA-3Ј).…”

“…We previously cloned cDNA and the gene of the A 2A -R, which contains seven transmembrane domains and belongs to the G protein-coupled receptor family (18 -22). The A 2A -R gene contains two independent TATA-less promoters (P1 and P2), which produce transcripts whose 5Ј-untranslated regions are of different lengths but that encode the same protein (18,19). Our previous study demonstrated that P2 is the major promoter of the A 2A -R gene, which is active in various tissues including the brain (19,21).…”