TY - JOUR T1 - Engineered Hyperphosphorylation of the <em>β</em><sub>2</sub>-Adrenoceptor Prolongs Arrestin-3 Binding and Induces Arrestin Internalization JF - Molecular Pharmacology JO - Mol Pharmacol SP - 349 LP - 362 DO - 10.1124/mol.114.095422 VL - 87 IS - 2 AU - Diana Zindel AU - Adrian J. Butcher AU - Suleiman Al-Sabah AU - Peter Lanzerstorfer AU - Julian Weghuber AU - Andrew B. Tobin AU - Moritz Bünemann AU - Cornelius Krasel Y1 - 2015/02/01 UR - http://molpharm.aspetjournals.org/content/87/2/349.abstract N2 - G protein–coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C-terminal tail of the β2-adrenoceptor (β2AR) and demonstrate that this mutant, termed β2ARSSS, showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the β2AR, whereas the interaction with β2ARSSS was 2- to 4-fold prolonged. In contrast, arrestin-3 interaction with a β2-adrenoceptor fused to the carboxyl-terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the β2-adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild-type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, β2ARSSS internalized more efficiently than the wild-type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor-arrestin affinity are sufficient to alter arrestin trafficking. ER -