Subtype-specific kinetics of inhibitory adenosine receptor internalization are determined by sensitivity to phosphorylation by G protein-coupled receptor kinases.

Despite coupling to the same class of inhibitory G proteins and binding the same physiological ligand, the human A 1 and rat A 3 adenosine receptors (ARs) desensitize at different rates in response to sustained agonist exposure. This is due to the ability of the A 3 AR, but not the A 1 AR, 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 A 3 ARs internalize profoundly in response to short-term exposure to agonist but not activators of second messenger-regulated kinases. The A 3 AR-selective antagonist MRS1523 blocked both A 3 AR phosphorylation and internalization. Moreover, in contrast to the A 1 AR, which internalized quite slowly ( t 1/2 = 90 min), A 3 ARs internalized rapidly ( t 1/2 = 10 min) over a time frame that followed the onset of receptor phosphorylation. A nonphosphorylated A 3 AR mutant failed to internalize over a 60-min time course, suggesting that receptor phosphorylation was essential for rapid A 3 AR internalization to occur. In addition, fusion onto the A 1 AR of the A 3 AR 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 A 3 AR is obligatory to observe rapid agonist-mediated internalization of the receptor.