Arrestin isoforms dictate differential kinetics of A(2B) adenosine receptor trafficking

Adenosine mediates the activation of adenylyl cyclase via its interaction w ith specific A(2A) and Ate adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of t he A(2B) adenosine receptor (A(2B)AR) in HEK293 cells. In the present study , we investigate the role of arrestins in A(2B)AR trafficking. Initial stud ies demonstrated that HEK293 cells stably expressing arrestin antisense con structs, which reduce endogenous arrestin levels, effectively reduced A(2B) AR internalization. A(2B)AR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interes tingly, while overexpression of arrestin-2 or arrestin-3 rescued A(2B)AR in ternalization and recycling, arrestin-3 promoted a significantly faster rat e of recycling as compared to arrestin-2. The specificity of arrestin inter action with A(2B)ARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwe nt rapid translocation (<1 min) from the cytosol to the plasma membrane fol lowing A(2B)AR activation. However, longer incubations with agonist (>10 mi n) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A(2B)AR i n rab-5 and transferrin receptor containing early endosomes. At later times , the A(2B)AR but not arr-2-GFP was observed in an apparent endocytic recyc ling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-ind uced A(2B)AR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A(2B)AR recycling and resensi tization.