Die zeitliche Stabilität und zelluläre Lokalisation von Arrestin-Rezeptorkomplexen wird durch die Rezeptorphosphorylierung determiniert.
Die Phosphorylierung G-Protein-gekoppelter Rezeptoren spielt eine bedeutende Rolle für die Interaktion mit Arrestinen und die Rezeptordesensibilisierung. Es ist jedoch unklar, wie unterschiedliche Phosphorylierungsmuster die Interaktion von Arrestin mit Rezeptoren und den nachfolgende Transportmuste...
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Agonist-induced phosphorylation of GPCRs is a significant prerequisite for the interaction with arrestins and receptor desensitization. Yet, it is unclear how different phosphorylation patterns impact on the interaction of arrestins with receptors and determine subsequent trafficking patterns of arrestin receptor complexes. In the present thesis this question was investigated at the β2-adrenoceptor and the parathyroid hormone receptor. First, an additional serine cluster was inserted into the proximal part of the β2-adrenoceptor´s C terminus. This receptor mutant, termed β2AR SSS, showed enhanced agonist dependent phosphorylation and an increase in the affinity for arrestin 3. In addition, the intracellular trafficking of arrestin 3 with this receptor was altered. The arrestin recruitment and the stability of arrestin receptor complexes were determined in real time by FRET and FRAP. It could be demonstrated that arrestin 3 dissociated quickly from the β2AR whereas the interaction with the β2AR SSS was two-to fourfold prolonged. The interaction of arrestin with a β2V2 chimera was poorly reversible during the timescale of the experiment. Further experiments revealed that the additional phosphorylation sites in the β2AR SSS led to colocalization of arrestin receptor complexes on early endosomes in response to agonist. In contrast, the wild type receptor interacted only transiently with arrestin at the plasma membrane. Furthermore the β2AR SSS internalized more efficiently than the β2AR whereas the recycling kinetics were very similar for both receptors. It was thus possible to show that the affinity between arrestins and receptors could be increased with minimal receptor modification. This was sufficient to alter the trafficking pattern of arrestin. In the second part of this thesis the ligand-dependent phosphorylation sites of the human PTHR were determined by LC-MS/MS. Ten phosphorylation sites could be identified of which three had not been described so far: T503, S519 and T547. The quantification of the ligand-dependent phosphorylation in PTHR mutants revealed that the first large serine cluster between S489-S495 in the C-terminal tail of the PTHR was massively phosphorylated or important for the phosphorylation of subsequent serine and threonine residues. Mutation of Ser and Thr residues between S501 and T506 showed that these residues play a crucial role for the interaction with arrestin 3. However, the extent of ligand dependent phosphorylation within this second cluster was smaller than the phosphorylation within the first cluster. T503 und S504 within the second cluster were essential for the robust interaction with arrestin-3. Although the mutation of phosphorylation sites in the proximal part of the C-terminus of the PTHR to alanine massively reduced the interaction with arrestin 3, in none of the receptor mutants was the interaction with arrestin abolished entirely The stability of arrestin-receptor-complexes can not only be influenced on the level of receptors but can also be modified by modifications in arrestin. Two lysine to arginine mutations within phosphate-sensing elements in the N-terminus of arrestin 3 (K11,12R) were able to reduce the arrestin 3 affinity towards the β2AR, the β2AR SSS and the PTHR. Notably this impacted on the compartmentalization of these receptors with arrestin. Whereas the colocalization of Arrestin K11,12R on endosomes with the β2AR SSS or the β2V2R was reduced, no cotrafficking of ArrestinK11,12R could be observed with the PTHR. This suggests that, depending on the receptor subtype, phosphate-sensing elements within arrestin may be of varying significance. In summary the experiments in this thesis show that arrestin is able to sense differential phosphorylation patterns in the proximal part of the C-terminus of GPCRs. The temporal stability of arrestin-receptor complexes is determined by the number and the localization of phosphorylation sites in the C-terminus of the GPCRs that were examined in this thesis. Importantly, this impacts on the cellular localization of arrestin-receptor complexes. The higher the temporal stability of an arrestin receptor complex, the greater the probability will be that it can be found in endocytic compartments. These findings collectively suggest that arrestin is able to form receptor specific and phosphorylation specific complexes.