Untersuchungen zu Mechanismen der Co-Internalisierung von Arrestin-3 mit GPCRs

Arrestine sind eine kleine Familie von vier Proteinen, die eine wichtige Rolle in der Signaltransduktion von G-Protein-gekoppelten Rezeptoren (GPCRs) spielen. Neben ihrer Funktion in der Desensibilisierung des G-Protein-Signalwegs sind sie auch für die Internalisierung aktivierter GPCRs zuständig. J...

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Bibliographic Details
Main Author: Mößlein, Nadja
Contributors: Bünemann, Moritz (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2021
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Arrestins are a small family of four proteins that play an important role in the signal transduction of G protein-coupled receptors (GPCRs). Besides desensitizing the G protein signaling, they also induce the internalization of GPCRs. Based on the apparent stability of arrestin-receptor complexes, GPCRs are classified into two groups. Class A receptors such as the b2 adrenergic receptor (b2AR) interact only transiently with arrestins and arrestin dissociates during the endocytic process, whereas class B receptors like the angiotensin type 1 (AT1R), or parathyroid hormone receptor (PTHR) form very stable complexes with arrestins that co-internalize into endosomes. However, the underlying mechanisms explaining why arrestins only co-internalize with class B receptors are still largely unknown and were therefore examined in more detail in this thesis. The most prominent hypothesis to date is that class B receptors induce sustained ubiquitination of arrestin, which is necessary for the co-internalization of arrestin-receptor complexes. Therefore, in the first part of this thesis, several lysine residues of arrestin-3, which usually serve as acceptors for ubiquitin, were eliminated to prevent the co-internalization with class B receptors. So far it is thought that the ubiquitination of the lysine residues 11 and 12 is necessary to induce the internalization of arrestin-3, as mutation of these lysines to arginines prevents the co-internalization of the arrestin-mutant with the AT1R (Shenoy and Lefkowitz, 2005). In addition, Arr3 KK11RR interacts only transiently with several other class B receptors (Zindel, 2015). However, docking experiments showed that the inserted arginines display stronger intramolecular interactions with the arrestin C-terminus and thereby stabilize its inactive conformation. Therefore, further destabilizing mutations were inserted into Arr3 KK11RR to reverse this effect. On the one hand, the interaction partners of the arginines were mutated to alanines (ED389AA), and on the other hand, the mutation R170E was introduced, leading to a pre- activated conformation of arrestin (Granzin et al., 2015). Indeed, the combination of KK11RR with these rescue mutations led to the formation of stable complexes with class B receptors and their co-internalization. Furthermore, both Arr3 KK11RR R170E and Arr3 KK11RR ED389AA displayed robust and sustained ubiquitination typically observed after stimulation of class B receptors similar to wild-type arrestin-3. Thus, we could confirm that Arr3 KK11RR can internalize with class B receptors after insertion of destabilizing mutations, and the lysine residues 11 and 12 do not have to be available as ubiquitination sites. As the elimination of all potential ubiquitination sites in arrestin-3 by mutation of all lysine residues to arginines resulted in a mutant that showed barely any recruitment to agonist-activated receptors, only proven ubiquitination sites (identified by mass-spectrometry or literature review) were removed. Since eight lysine to arginine single mutants of arrestin-3 still co-internalized with the PTHR, we next combined all of these mutations. The resulting arrestin mutant displayed an inconsistent internalization with the PTHR and colocalized with the receptor only in some cells. As the cause for this variable phenotype could not be identified, the number of mutations in arrestin-3 was next reduced successively to identify those lysine to arginine mutations that influence the co-internalization with class B receptors. This also led to a more consistent co-internalization with these GPCRs. In the end, the relevant mutations could be narrowed down to the lysine residues 108 and 178 as the double mutant Arr3 K108R K178R was no longer co-internalized with several class B receptors. Moreover, FRAP (fluorescence recovery after photobleaching) measurements showed that the complexes between this mutant and an agonist-activated class B receptor were as stable as with wild-type arrestin-3, suggesting that the reduced co-internalization of Arr3 K108R K178R was not based on a less stable receptor binding. However, the question remains whether the impaired co-internalization can indeed be attributed to the reduced ubiquitination of this arrestin mutant. To examine the role of posttranslational modifications in the co-internalization of arrestin-3 with class B receptors, we first prevented the cellular ubiquitination with an inhibitor of the most prominent E1-ligase Uba1, TAK-243. Even though this did not impair the co-internalization of arrestin with class B receptors, it is not completely sure whether TAK-243 indeed prevents the ubiquitination of arrestin-3. As the SUMOylation inhibitor 2-D08 did not affect the internalization of arrestin-3 either, both ubiquitination and SUMOylation do not play a major role in the co-internalization of arrestin with class B receptors according to this thesis. In the last part of the thesis, several agonists for the µ-opioid receptor (µOR) with different dissociation half-lives were employed to examine whether ligands with particularly slow off-rates also induce the formation of more stable receptor-arrestin complexes. However, FRAP experiments demonstrated that the exchange rate of arrestin-3 at the µOR is not affected by the dissociation half-life of the agonist. Furthermore, agonists with very slow off-rates could not induce co-internalization of arrestin-3 with the class A receptors µOR and b2AR. In conclusion, the dissociation half-lives of agonists do not affect class A or class B behavior of GPCRs and therefore do not influence the co-internalization of arrestins.