Analyse der Wechselwirkungen von Proteinen des Interleukin-6-Signalwegs in einzelnen Zellen

In the first part of this thesis the long term association of the shared signalling subunit of the IL-6-type cytokines gp130 and the Janus kinase Jak1 was revealed by fluorescence recovery after photobleaching (FRAP) analysis. Signal transduction through cytokine receptors is mediated mainly by non-covalently associated Jak tyrosine kinases. By confocal microscopy, the cytokine receptor gp130 and Jak1, fused with either yellow (YFP) or cyan (CFP) fluorescent protein, were found to be colocalized predominantly at intracellular vesicular structures and at the plasma membrane. Quantitative FRAP analysis at the plasma membrane revealed equal mobilities for gp130-YFP and Jak1-YFP. Thus, Jak1-YFP diffuses like a transmembrane protein indicating that membrane-bound Jak1 does not exchange rapidly with cytosolic Jaks. Applying a dual-color FRAP approach it turned out that immobilization of gp130-CFP by a pair of monoclonal antibodies led to a corresponding immobilization of cotransfected Jak1-YFP. The conclusion from these findings is that Jak1, once bound to a gp130 molecule, does not exchange between different receptors at the plasma membrane neither via the cytoplasmic compartment nor via a membrane-associated state. Thus, cytokine receptor/Jak complexes have closest analogy to receptor tyrosine kinases. The second part of the thesis is focused on the analysis of gp130 homodimerization and gp130/LIFR heterodimerization in single cells. Interleukin-6 (IL-6) signals through gp130 homodimers while leukemia inhibitory factor (LIF) exerts its action through a heterodimer of gp130 and the LIF receptor (LIFR). Related hematopoietic receptors such as the erythropoietin receptor have been described as preformed dimers in the plasma membrane. Here gp130 homodimerization and heterodimerization with the LIFR was investigated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC). It was found that in unstimulated cells a FRET signal between YFP- and CFP-tagged gp130 is clearly detectable at the plasma membrane that does not increase upon IL-6 stimulation. However, FRET between YFP- gp130 and CFP-tagged LIFR considerably increased upon LIF stimulation. Using a BiFC approach that detects stable interactions it is shown that fluorescence complementation of gp130 constructs tagged with matching "halves" of fluorescent proteins increases upon IL-6-stimulation. Taken together, these findings suggest that preformed gp130 homodimers on the plasma membrane are stabilized by IL-6 whereas heterodimerization of gp130 with the LIFR is mainly triggered by the ligand. This view is supported by the observation that the simultaneous action of two IL-6 binding domains on two gp130 molecules is required to efficiently recruit a fluorescent IL-6 (YFP-IL-6) to the plasma membrane.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2005

Author dc:creator

• Giese, Bernd Moritz

Contributors dc:contributor

• Müller-Newen, Gerhard

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Interleukin-6
• Jak1
• GFP
• FRAP
• FRET

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123421%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61797