Background:Adora2a encodes A(2A) adenosine receptor (A(2A)R) and a new protein (uORF5) translated from an out-of-frame AUG. Results: uORF5 exists in tissues where the Adora2a transcript is detected and is up-regulated by A(2A)R activation. uORF5 suppresses AP1-mediated transcription. Conclusion:Adora2a encodes two distinct proteins (A(2A)R and uORF5) in an A(2A)R-dependent manner. Significance: uORF5 may participate in the functions of A(2A)R during pathophysiological conditions. The A(2A) adenosine receptor (A(2A)R) is a G protein-coupled receptor and a major target of caffeine. The A(2A)R gene encodes alternative transcripts that are initiated from at least two independent promoters. The different transcripts of the A(2A)R gene contain the same coding region and 3-untranslated region and different 5-untranslated regions that are highly conserved among species. We report here that in addition to the production of the A(2A)R protein, translation from an upstream, out-of-frame AUG of the rat A(2A)R gene produces a 134-amino acid protein (designated uORF5). An anti-uORF5 antibody recognized a protein of the predicted size of uORF5 in PC12 cells and rat brains. Up-regulation of A(2A)R transcripts by hypoxia led to increased levels of both the A(2A)R and uORF5 proteins. Moreover, stimulation of A(2A)R increased the level of the uORF5 protein via post-transcriptional regulation. Expression of the uORF5 protein suppressed the AP1-mediated transcription promoted by nerve growth factor and modulated the expression of several proteins that were implicated in the MAPK pathway. Taken together, our results show that the rat A(2A)R gene encodes two distinct proteins (A(2A)R and uORF5) in an A(2A)R-dependent manner. Our study reveals a new example of the complexity of the mammalian genome and provides novel insights into the function of A(2A)R.

Adenosine is a constituent of many molecules of life; increased free extracellular adenosine indicates cell damage or metabolic stress. The importance of adenosine signaling in basal physiology, as opposed to adaptive responses to danger/damage situations, is unclear. We generated mice lacking all four adenosine receptors (ARs), Adora1(-/-);Adora2a(-/-);Adora2b(-/-);Adora3(-/-) (quad knockout [QKO]), to enable investigation of the AR dependence of physiologic processes, focusing on body temperature. The QKO mice demonstrate that ARs are not required for growth, metabolism, breeding, and body temperature regulation (diurnal variation, response to stress, and torpor). However, the mice showed decreased survival starting at about 15 weeks of age. While adenosine agonists cause profound hypothermia via each AR, adenosine did not cause hypothermia (or bradycardia or hypotension) in QKO mice, indicating that AR-independent signals do not contribute to adenosine-induced hypothermia. The hypothermia elicited by adenosine kinase inhibition (with A134974), inosine, or uridine also required ARs, as each was abolished in the QKO mice. The proposed mechanism for uridine-induced hypothermia is inhibition of adenosine transport by uridine, increasing local extracellular adenosine levels. In contrast, adenosine 5-monophosphate (AMP)-induced hypothermia was attenuated in QKO mice, demonstrating roles for both AR-dependent and AR-independent mechanisms in this process. The physiology of the QKO mice appears to be the sum of the individual knockout mice, without clear evidence for synergy, indicating that the actions of the four ARs are generally complementary. The phenotype of the QKO mice suggests that, while extracellular adenosine is a signal of stress, damage, and/or danger, it is less important for baseline regulation of body temperature. Author summary Elevated extracellular adenosine generally indicates metabolic stress or cell damage and regulates many aspects of physiology. We studied QKO mice lacking all four adenosine receptors. Young QKO mice do not appear obviously ill, but do show decreased survival later in life. QKO mice demonstrate that adenosine receptors are not required for growth, metabolism, breeding, and body temperature regulation. QKO mice are missing the pharmacologic effects of adenosine on body temperature, heart rate, and blood pressure. Therefore, all of these effects are mediated by the four adenosine receptors. We also determined that the hypothermic effects of a pharmacologic adenosine kinase inhibitor (A134974), uridine, or inosine each requires adenosine receptors. The uridine-induced hypothermia is likely due to its inhibition of adenosine uptake into cells. QKO mouse physiology appears to be the sum of the individual knockout mice, without evidence for synergy, indicating that the actions of the four adenosine receptors are generally complementary.