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Functional analysis of the cytoplasmic domain of complement receptor type II (CR2/CD21)

Functional analysis of the cytoplasmic domain of complement receptor type II (CR2/CD21)

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HOEFER, Melanie Merja, 2006. Functional analysis of the cytoplasmic domain of complement receptor type II (CR2/CD21) [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Hoefer2006Funct-7004, title={Functional analysis of the cytoplasmic domain of complement receptor type II (CR2/CD21)}, year={2006}, author={Hoefer, Melanie Merja}, address={Konstanz}, school={Universität Konstanz} }

eng 2011-03-24T17:30:47Z deposit-license The complement receptor type 2 (CR2/CD21) is functionally regulated by a proteolytic cleavage event termed ectodomain shedding which results in the release of the extracellular domain as functional active, soluble protein. Deletion of the extracellular membrane-adjacent short consensus repeat 16 (SCR16) abolished shedding.<br />The aim of this work was to investigate the role of the cytoplasmic domain in CD21-shedding. Shedding was induced either by the thiol antioxidants glutathione and N-acetylcysteine or by activation of the cells through the phosphotyrosine-specific phosphatase inhibitor pervanadate. A negative regulatory role for the CD21 cytoplasmic domain could be established, as murine B-cells expressing CD21 mutants lacking this domain revealed higher basal and induced shedding rates. Interestingly, CD21 cell surface expression was up-regulated upon stimulation with low (20 uM) pervanadate concentrations, while higher (200 uM) concentrations lead to CD21-shedding. This prompted us to suggest CD21-shedding as mechanism to fine-tune B-cell activation.<br />It was of further interest, whether CD21 would undergo regulated intramembrane proteolysis (RIP), a process often taking place after initial ectodomain shedding. In fact, in human B-lymphocytes and several B-cell lines, two constitutively present, membrane-tethered carboxy-terminal fragments were identified, p8CD21-CTF and p16CD21-CTF. Human T-lymphocytes, suspected not to shed CD21, featured neither CTF, although the full length protein could be detected. In murine splenocytes, only the smaller p8CD21-CTF was seen. In the SCR16-lacking mutant the p8CD21-CTF was still present, suggesting that CD21 could be cleaved within the membrane, independent of ectodomain shedding. In B-cells, the p8CD21-CTF was degraded by the proteasomal pathway, independent of additional shedding induction by thiol antioxidants. Moreover, when CD21 was overexpressed in HEK293 epithelial cells, the two CD21-CTFs were degraded both by the proteasomal and the lysosomal pathways.<br />To identify potential intracellular binding partners, the CD21 cytoplasmic domain was used in a Yeast Two-Hybrid screening. One candidate, the proteasome maturation protein POMP, was further investigated. Although the interaction with CD21 only occurred in yeast and could not be confirmed in mammalian cells, the protein revealed interesting physicochemical properties and a peculiar behavior towards standard staining techniques. Localization studies revealed that the protein is expressed in the cytosol as well as in the nucleus. Comparison of POMP-expression levels in transformed and non-transformed cells showed that malignancy often led to enhanced POMP-expression. Finally, POMP could possibly form tetramers to perform its chaperoning function in 20S proteasome assembly. 2006 Hoefer, Melanie Merja Hoefer, Melanie Merja 2011-03-24T17:30:47Z Functional analysis of the cytoplasmic domain of complement receptor type II (CR2/CD21) Funktionelle Analyse der zytoplasmatischen Domäne des Komplement Rezeptors Typ II (CR2/CD21) application/pdf

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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