Arrestin Isoforms Dictate Differential Kinetics of A2B Adenosine Receptor Trafficking

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Abstract
Adenosine mediates the activation of adenylyl cyclase via its interaction with specific A2A and A2B adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of the A2B adenosine receptor (A2BAR) in HEK293 cells. In the present study, we investigate the role of arrestins in A2BAR trafficking. Initial studies demonstrated that HEK293 cells stably expressing arrestin antisense constructs, which reduce endogenous arrestin levels, effectively reduced A2BAR internalization. A2BAR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interestingly, while overexpression of arrestin-2 or arrestin-3 rescued A2BAR internalization and recycling, arrestin-3 promoted a significantly faster rate of recycling as compared to arrestin-2. The specificity of arrestin interaction with A2BARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwent rapid translocation (10 min) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A2BAR in rab-5 and transferrin receptor containing early endosomes. At later times, the A2BAR but not arr-2-GFP was observed in an apparent endocytic recycling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-induced A2BAR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A2BAR recycling and resensitization.

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