Abstract
Here we demonstrate that overexpression of the human A(2A) adenosine receptor (A(2A)AR)in vascular endothelial cells confers an ability of interferon-alpha and a soluble IL-6 receptor/ IL-6 (sIL-6R alpha/IL-6) trans-signaling complex to trigger the downregulation of signal transducer and activator of transcription (STAT) proteins. It is noteworthy that STAT down-regulation could be reversed by coincubation with A 2A AR-selective inverse agonist 4-(2-[7-amino-2-(2-furyl)[1,2,4] triazolo[2,3a][ 1,3,5] triazin-5-ylamino] ethyl) phenol (ZM241385) but not adenosine deaminase, suggesting that constitutive activation of the receptor was responsible for the effect. Moreover, STAT down-regulation was selectively abolished by proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132), whereas lysosome inhibitor chloroquine was without effect. Down-regulation required Janus kinase (JAK) activity and a Tyr7053Phe-mutated STAT3 was resistant to the phenomenon, suggesting that JAK-mediated phosphorylation of this residue is required. Consistent with this hy-pothesis, treatment of A 2A AR-overexpressing cells with (sIL-6R alpha/IL-6) triggered the accumulation of polyubiquitylated wild-type but not Tyr7053Phe-mutated STAT3. Support for a functional role of this process was provided by the observation that A 2A AR overexpression attenuated the JAK/ STATdependent up-regulation of vascular endothelial growth factor receptor-2 by (sIL-6R alpha/IL-6). Consistent with a role for endogenous A 2A ARs in regulating STAT protein levels, prolonged exposure of endogenous A 2A ARs in endothelial cells with ZM241385 in vitro triggered the up-regulation of STAT3, whereas deletion of the A 2A AR in vivo potentiated STAT1 expression and phosphorylation. Together, these experiments support a model whereby the A 2A AR can prime JAKphosphorylated STATs for polyubiquitylation and proteasomal degradation and represents a new mechanism by which an anti-inflammatory seven-transmembrane receptor can negatively regulate JAK/STAT signaling.