Knock-in of phosphorylation sites at the beta-2-adrenoceptor C-terminal tail results in changes in arrestin affinity and trafficking pattern

D. Zindel1, A.J. Butcher2, S.A. Sabah3, P. Lanzerstorfer4, J. Weghuber4, A.B. Tobin2, M. Bünemann1, C. Krasel1. 1Philipps-Universität, Marburg, Germany, 2University of Leicester, Leicester, UK, 3University of Kuwait, Kuwait, Kuwait, 4Fachhochschule Wels, Wels, Austria

G-Protein coupled receptor (GPCR) phosphorylation plays a major role in receptor desensitization and arrestin binding. It is as yet unclear, however, how distinct receptor phosphorylation patterns may influence arrestin binding and subsequently downstream signalling. Here we demonstrate that a novel β2AR mutant rationally designed on serine cluster analysis, in which G361, E362 and Q363 were changed to serine (β2AR (G361S, E362S, Q363S) or β2ARSSS) showed increased isoprenaline-stimulated phosphorylation compared to β2AR wild-type (β2AR WT) and also showed differences in arrestin3 affinity, trafficking and ERK activation. We measured arrestin3 recruitment and stability of arrestin3-receptor complexes in real time using fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP). We found that arrestin3 dissociates quickly and almost completely from β2AR WT, (t0.5 of 29.4 s) however, the interaction with β2ARSSS was significantly more prolonged (t0.5 of 61.5 s, p<0.001). In contrast, arrestin3 interaction with a β2AR fused to the carboxyl-terminal tail of the vasopressin type 2 receptor (β2V2R)(1) was nearly irreversible. The coupling efficiency of agonist-induced receptor-Gs protein interaction was very similar for both β2AR WT and β2ARSSS, as determined by FRET based single cell assays. Further analysis of arrestin3 localization revealed that β2ARSSS was able to change the trafficking pattern from class A in which arrestin3 interacts only transiently with the β2AR at the membrane to class B in which arrestin3 cointernalizes with receptors on endosomes. These findings imply that β2ARSSS stimulation promotes arrestin3 recruitment potentially resulting in increased signalling through arrestin3. In addition, we have examined the time course of ligand-induced phosphorylation of extracellular signal-regulated kinases (pERK) to gain further insight into a possible regulation of downstream signalling. Interestingly, we found that β2ARSSS was able to provoke sustained ERK phosphorylation compared to β2AR WT. These results suggest that the stability of the receptor-arrestin interaction is a sensitive parameter that determines signalling and trafficking properties of arrestin.

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