Characterization of genomic organization of the adenosine A_2A receptor gene by molecular and bioinformatics analyses

Abstract

The adenosine A_2A receptor (A_2AR) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A_2AR gene expression, we have characterized the genomic organization of the mouse and human A_2AR genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5′ untranslated regions (5′-UTRs) of mouse A_2AR mRNA were identified by rapid amplification of 5′ cDNA end (5′ RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding “exon 1” (h1A, h1B, h1C, h1D, h1E and h1F) in the human A_2AR gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5′ oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A_2AR transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A_2AR gene by 5′ oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A_2AR gene expression. Finally, our analysis indicates that A_2AR mRNA and a novel transcript partially overlapping with the 3′ exon h3, but in opposite orientation to the A_2AR gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A_2AR genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5′-UTR. This raises the possibility of generating multiple tissue-specific A_2AR mRNA species by alternative promoters with varying regulatory susceptibility.

Introduction

Adenosine, a naturally occurring nucleoside, is distributed ubiquitously throughout the body as a metabolic intermediary. Adenosine also acts through multiple G-protein coupled receptors to exert a variety of physiological effects on many mammalian systems [17], [23], [24], [70]. The biological functions of adenosine are mediated through four adenosine receptor subtypes: A₁, A_2A, A_2B and A₃. Adenosine A_2A receptors (A_2ARs) are expressed at high levels in the striatum and at moderate levels in thymus, polymorphonuclear leukocytes, platelets and other tissues [23], [24]. Pharmacologically, activation of the A_2AR has been associated with the following adenosine effects: vasodilatation, immunosuppression, tissue protection, sleep promotion and psychomotor depression [18], [24], [40], [47], [61], [70]. Mice lacking A_2ARs exhibited high blood pressure, aggression, anxious behavior [38] and extensive inflammation [47]. Genetic inactivation A_2A receptors also reduces withdrawal symptoms after chronic alcohol consumption [38] and spinal delta opioid receptor-associated pain responses [3], and attenuates brain injury in response to both ischemia [8] and MPTP-induced neurotoxicity [9].

From these pharmacological and genetic studies, the A_2A receptor has recently emerged as a promising drug target for treating many neurological and psychiatric disorders such as Parkinson's disease [9], [20], [58], [64], schizophrenia and affective disorders [18], [19], [59]. Based on profound antagonistic interaction between A_2A receptor and dopaminergic systems in brain, A_2A antagonists and A_2A agonists have been shown to have anti-parkinsonian [9], [20], [58], [64] and anti-psychotic properties [18], [59], respectively. A_2A antagonists also enhance motor activity and protect against neurodegeneration of dopaminergic neurons in several animal models of Parkinson's disease, and are now entering clinical phase II trials for the treatment of Parkinson's disease. To fully realize the therapeutic potential of the A_2AR requires a refined understanding of the A_2AR genomic structure and corresponding transcripts.

A_2ARs are regulated at the transcriptional level by physiological and pathophysiological signals as well as pharmacological manipulations. For example, in cultured PC12 cells, treatment with nerve growth factor or exposure to hypoxic conditions decreases and increases A_2AR mRNA levels, respectively [31], [65]. Treatment with tetradecanoyl phorbol acetate (TPA) increases A_2AR mRNA levels in human SHSY5Y neuroblastoma cells [54]. In intact animals, lesioning the dopaminergic system with 6-hydroxydoapmine in rats or chronic l-dopa treatment in parkinsonian monkeys has been shown to up-regulate striatal A_2AR mRNA [55], [79]. In an animal model of Huntington's disease, transgenic mice expressing mutant huntington protein display a profound decrease in A_2AR protein in striatum [5], [6] accompanied by a decrease in A_2AR mRNA, suggesting transcriptional down-regulation of the A_2AR gene [5], [6]. A better understanding of the mechanism of A_2AR gene regulation by such physiological and pathophysiological signals may provide important insights into A_2AR function in vivo. However, a significant impediment to the study of A_2AR transcriptional regulation is the fact that the promoter region of A_2AR remains largely undetermined. Thus, characterization of A_2AR genomic structure, particularly the accurate determination of the transcription start site for the human A_2AR gene, is an important step toward understanding regulation of the A_2AR gene.

The cDNA structure for the A_2AR has been cloned from various species including mouse [42], rat [10], [21], [62], guinea pig [44] and human [26]. The human A_2AR gene has been localized to chromosome 22 [37], [41]. However, the genomic structure and the promoter region of the A_2AR gene have not been fully characterized. The A_2AR gene for human and rat consists of the two coding exons separated by a single intron [11], [53]. Chu et al. [11] provide evidence for two independent promoters in the rat A_2AR gene. This genomic organization, however, excludes the possibility of generating multiple A_2AR mRNA species with varying regulatory susceptibility.

During the construction of an A_2AR gene-targeting vector, our preliminary characterization of the genomic structure of the mouse A_2AR gene revealed a non-coding exon 5′-upstream of the previously identified exons [7]. Recent publications of drafts of complete human [36], [75] and mouse [77] genomes provide an opportunity to further characterize the A_2AR gene in these two species. In this study, we have used molecular and bioinformatics analyses to identify multiple, tissue-specific exons encoding 5′-untranslated regions (5′-UTRs) of mouse and human A_2AR mRNAs. Such analyses have indicated the presence of a newly identified genomic structure with the potential to generate tissue-specific A_2AR mRNA variants by alternative promoter usage.