PT - JOURNAL ARTICLE AU - Diana Zindel AU - Adrian J Butcher AU - Suleiman Al-Sabah AU - Peter Lanzerstorfer AU - Julian Weghuber AU - Andrew B Tobin AU - Moritz Bunemann AU - Cornelius Krasel TI - Engineered Hyperphosphorylation of the β2-adrenoceptor Prolongs Arrestin-3 Binding and Induces Arrestin Internalization AID - 10.1124/mol.114.095422 DP - 2014 Jan 01 TA - Molecular Pharmacology PG - mol.114.095422 4099 - http://molpharm.aspetjournals.org/content/early/2014/12/10/mol.114.095422.short 4100 - http://molpharm.aspetjournals.org/content/early/2014/12/10/mol.114.095422.full AB - 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 two- to four-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 β2-adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes following 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.