| dc.contributor.advisor | Oppermann, Martin Prof. Dr. | |
| dc.contributor.author | Liebick, Marcel | |
| dc.date.accessioned | 2015-02-26T09:57:46Z | |
| dc.date.available | 2015-02-26T09:57:46Z | |
| dc.date.issued | 2015-02-26 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0022-5DD3-C | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-4939 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | |
| dc.subject.ddc | 610 | |
| dc.title | Chemokine receptors CXCR4 and CCR5: Cell surface expression, signaling and modulation by β-arrestin 2 | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Lutz, Susanne Prof. Dr. | |
| dc.date.examination | 2014-10-23 | |
| dc.description.abstracteng | G protein-coupled receptors which mediate a large variety of different cellular effects have always been a field of significant interest for pharmaceutical research. Since it was shown that the chemokine receptors CXCR4 and CCR5 act as essential co-receptors for HIV-1 entry the interest in understanding the regulation of cell surface expression of these specific GPCR increased even more. The receptor expression at the cell surface is regulated by different mechanisms such as agonist induced receptor endocytosis and subsequent receptor recycling, whereas boths effects are more or less distinct for different receptors. During recent years it was also shown that constitutive receptor endocytosis plays a prominent role in the circulation of receptors. Previous methods for the analysis of receptor cycling which used receptor-specific antibodies were not sensitive enough and lacked the potential to monitor constitutive receptor endocytosis in quantitative terms.
In this work an antibody-independent method based on specific biotinylation of an acceptor peptide (AP) by the enzyme biotin ligase A was established. Exemplified by the quantification of the ligand induced internalization and recycling of CXCR4 and CCR5 the robustness and high sensivity of the assay was demonstrated. Furthermore, the assay was not influenced by receptors which were freshly translocated to the cell surface without prior ligand binding. As an additional feature the assay provided the possibility for a detailed quantification of constitutive receptor endocytosis. In order to detect all receptors within a cell regardless of whether they had previously been expressed at the cell surface AP-specific monoclonal antibodies were generated which can be used for double immunofluorescence microscopy. These antibodies allow discriminations of biotinylated and non biotinylated receptors or detection of transmembrane proteins lacking high specific antibodies.
β-Arrestin 2 is a multivalent adaptor protein involved in receptor signaling as well as endocytosis which binds to various intracellular proteins. Recent reports challenged the classical concept of GPCR signaling via heterotrimeric G proteins and postulated a higher relevance of receptor homodimerization or binding of β-arrestins to the receptor. To circumvent G protein activation after ligand binding a chemical-induced dimerization system consisting of three components was used. Either a FKBP12 (DmrA) or FRB (DmrC) domain was fused to the C-terminus of CXCR4/CCR5 and β arrestin 2. Treatment of Rec DmrA/βArr DmrC cell lines with AP21967 led to dose- and time dependent recruitment of β arrestin 2 to the receptor in the absence of ligand stimulation. AP21967-induced translocation of β arrestin 2 to the receptor significantly decreased ligand-induced G protein mediated calcium release. In cell lines without βArr-DmrC expression no alterations were obtained. AP21967-binding also provoked a ligand-independent internalization of CXCR4/CCR5 which was on a comparable level as ligand-induced internalization. Interestingly, the AP21967 induced recruitment of β-arrestin 2 to the receptor was sufficient to mimic the specific, ligand-induced intracellular receptor distribution of either CXCR4 or CCR5. Whereas AP21967 treatment led to a β arrestin 2 receptor desensitization and internalization it was not sufficient to mediate receptor signaling via the MAP kinases ERK 1/2. AP20187-induced receptor homodimerization had no detectable effect on either receptor desensitization or the phsophorylation level of ERK 1/2. However AP20187 pretreament led to an enhanced ligand-induced internalization in Rec-DmrA cell lines. In summary, the results obtained within this work contribute to a more detailed understanding of β arrestin-mediated functions during chemokine receptor trafficking and demonstrated the applicability of a highly sensitive, biotin-based detection system for the analysis of trafficking of transmembrane proteins. | de |
| dc.contributor.coReferee | Kube, Dieter Prof. Dr. | |
| dc.subject.eng | CXCR4 | de |
| dc.subject.eng | CCR5 | de |
| dc.subject.eng | GPCR | de |
| dc.subject.eng | Heterodimerization | de |
| dc.subject.eng | Homodimerization | de |
| dc.subject.eng | Chemokine | de |
| dc.subject.eng | Chemokine receptor | de |
| dc.subject.eng | G protein | de |
| dc.subject.eng | Arrestin | de |
| dc.subject.eng | G protein coupled receptor | de |
| dc.subject.eng | Internalization | de |
| dc.subject.eng | AP TAG | de |
| dc.subject.eng | AP21967 | de |
| dc.subject.eng | AP20187 | de |
| dc.subject.eng | Biotion ligase A | de |
| dc.subject.eng | BirA | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0022-5DD3-C-7 | |
| dc.affiliation.institute | Medizinische Fakultät | |
| dc.subject.gokfull | Medizin (PPN619874732) | de |
| dc.identifier.ppn | 81903780X | |