Analyse der Wechselwirkungen von Proteinen des Interleukin-6-Signalwegs in einzelnen Zellen
type: CreativeWork
description: 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 betwe…
abstract: 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 betwe…
author: Giese, Bernd Moritz
language: ger
keywords: info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Interleukin-6, Jak1, GFP, FRAP, FRET
date published: 2005
date modified: 2026-07-30T19:43:19Z
provider: RWTH Aachen University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Publikationsserver der RWTH Aachen University