Publikationsserver der RWTH Aachen University
Untersuchung und Spezifizierung der molekularen Interaktion von Ubiquitin und Sumo mit dem rekombinanten a1-Glyzinrezeptor
Abstract
dc:descriptionThe specification of covalent posttranslational modification by sumoylation and ubiquitination of membrane proteins – in this case of the GlyR alpha 1-subunit and its ubiquitin mediated internalisation – is described. The clinical relevance of those mechanisms is wide spread and would in this case produce an inadequate receptor regulation with the expected consequences. In order to show ubiquitination/sumoylation lysates of rat brain and spinal cord were incubated with ubiquitin/sumo as well as different mutants of the glycine subunit. The proteins were isolated as GST-fusion-protein from E. coli or were expressed as radioactive His-tagged protein in Xenopus oocytes. After incubation it was expected that adducts of ubiquitin/sumo with the receptor could be isolated by using GST-pullover or Ni2+-NTA-Chromatographie. However, there were no adducts found. In further experiments the ubiquitination/sumoylation of the receptor was demonstrated in Xenopus oocytes, in which cRNA and Proteins of the different components were injected before incubation with radioactive [S]-methionine. The isolation of adducts was done by GST-pullover or Ni2+-NTA-Chromatographie; the adducts were demonstrated on SDS-PAGES. It was therefore possible to show monoubiquitination of the GlyR alpha 1-subunit. A special mutation without cytoplasmatic lysines was not ubiquitinated, however was degraded into the same proteolytic pieces specific for ubiquitin mediated endocytosis as the wildtype. This implies that there is an alternate way to mediate endocytosis rather than ubiquitination. Further more a non covalent binding of ubiquitin to the wildtype and to the No-K-mutant was observed implicating a non covalent binding before covalent modification. Interestingly, a special mutant I387A,D388A GlyR alpha 1-subunit was ubiquitinated without being expressed at the cell membrane. In this case it might be ubiquitination serves in this case to address the mutant, which was never transported to the golgi, for proteasomal degradation. The endodomain of the receptor could be excluded as substrate of ubiquitination. We supposed that the ubiquitination enzymes cannot recognize the soluble endodomain, they may need it fixed at the membrane. A sumoylation of the GlyR alpha 1-subunit could not be detected even in ubc-09-ligase over expressed cells. But in the sumo expressing oocytes was an increased rate of receptor endocytosis. This effect was independent of the lysins in the Protein, so we suppose an indirect effect or a non-covalent binding.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2008
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
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- Rohde, Uta Julia Maximiliane
- Contributors dc:contributor
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- Schmalzing, Günther
Subjects
dc:subject × 12Rights
dc:rights- Statement dc:rights
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- info:eu-repo/semantics/openAccess
- Language dc:language
- ger