Abstract

Despite coupling to the same class of inhibitory G proteins and binding the same physiological ligand, the human A₁ and rat A₃ adenosine receptors (ARs) desensitize at different rates in response to sustained agonist exposure. This is due to the ability of the A₃AR, but not the A₁AR, to serve as a substrate for rapid phosphorylation and desensitization by members of the G protein-coupled receptor kinase (GRK) family. The aim of this study was to investigate whether these differences were also manifested in their abilities to undergo agonist-dependent receptor internalization. For the first time, we report that A₃ARs internalize profoundly in response to short-term exposure to agonist but not activators of second messenger-regulated kinases. The A₃AR-selective antagonist MRS1523 blocked both A₃AR phosphorylation and internalization. Moreover, in contrast to the A₁AR, which internalized quite slowly (t _1/2 = 90 min), A₃ARs internalized rapidly (t _1/2 = 10 min) over a time frame that followed the onset of receptor phosphorylation. A nonphosphorylated A₃AR mutant failed to internalize over a 60-min time course, suggesting that receptor phosphorylation was essential for rapid A₃AR internalization to occur. In addition, fusion onto the A₁AR of the A₃AR C-terminal domain containing the sites for phosphorylation by GRKs conferred rapid agonist-induced internalization kinetics (t _1/2 = 10 min) on the resulting chimeric AR. In conclusion, these data suggest that GRK-stimulated phosphorylation of threonine residues within the C-terminal domain of the A₃AR is obligatory to observe rapid agonist-mediated internalization of the receptor.