Analyse von ligandenunabhängiger Assoziation der IL-6-Typ-Zytokinrezeptoren mit Schwerpunkt auf der gp130-gp80-Interaktion

Cytokines are essential messengers of the human organism. The IL-6-type cytokines, namely IL-6, IL-11, IL-27, LIF, CT-1, CNTF, CLC, NP and OSM, play an essential role for the acute-phase-reaction, the hematopoesis, the embryonic development as well as for inflammation and distinct tumors. The signal mechanism of this family functions via receptor-complexes consistent of multiple type-I transmembrane-proteins. As gp130 is involved in all receptor-complexes, it is the essential receptor of this family. Within this thesis, the IL-6 receptor-complex consisting of gp130 and gp80 as well as the LIF receptor complex of gp130 and LIFR have been investigated for possible ligand-independent associations. In the first part experiments with lysis of the cells were applied. A co-immuno precipitation of gp80 on gp130 was only possible after stimulation with IL-6. Also, a higher molecular complex of receptors became evident only after ligand-binding in a native gel-electrophoresis assay. A similar result was obtained for the LIF-receptor-complex in native gels. When the crosslinker BS3 was added to the cells before lysis, a complex formation of gp130 at the plasma membrane could be observed with and without stimulation. These results point to an already existing weak ligand-independent interaction of the receptors before stimulation. The second part of the study comprises methods without lysis of the cells in order to elicit weaker interactions. BiFC experiments showed the same results for the receptor combination gp130-gp80 with and without IL-6 stimulation. In contrast, an increase of the fluorescence after stimulation with LIF was noticed for the pair of gp130-LIFR. A homodimerisation of gp130 was seen without stimulation; still it was markedly pronounced afterwards. These results can be interpreted as follows: gp130 tends to ligand-independent homodimerisation, there exists a preassociation with gp80 but no stimulation independent interaction with the LIFR. Based on these results, further investigations of the gp130-gp80 interaction have been accomplished. A truncation of gp130 up to the box1 region could not diminish the coimmobilisation of gp80 by linking gp130 at the plasmamembran with antibodies. The exchange of the transmembrane region of gp80 with the one of the LIFR did not show any effect either. Furthermore, the extracellular deletion of the domains D2 and D3 of gp80 did not result in any change of mobility of the receptor. In addition to that, the preassociation of the two receptors was confirmed by FRET-experiments: no significant rising of the FRET-efficiency was obtained by stimulation with IL-6. Neither the deletion of the whole cytoplasmatic part of gp80, nor the exchange of its transmembrane region by the one of the EpoR resulted in a significant change of the FRET efficiency. Mutated receptors, that lack the whole extracellular part of gp80 or where an exchange of the whole extracellular region from gp130 to the EpoR was done, were not translocated to the plasmamembrane. Due to this, further investigations of this region were not possible. The last part ist concerned with investigations about the stoichiometry of the IL-6 receptor complex. An exchange of the fluorophores between gp130 and gp80 did not result in any change of the FRET efficiency, not without stimulation nor with a stimulation of 5, 15 or 25ng/ml IL-6. Thus both receptors in the complex must be equimolar with and without addition of IL-6, otherwise the FRET efficiency would have changed due to a transformed YFP-CFP relation during fluorophor exchange