PMU-Autor/inn/en
Wortmann SaskiaAbstract
The cytokine receptor gp130 is the shared signalling subunit of the IL-6-type cytokines. Interleukin-6 (IL-6) signals through gp130 homodimers whereas leukaemia inhibitory factor (LIF) exerts its action through a heterodimer of gp130 and the LIF receptor (LIFR). Related haematopoietic receptors such as the erythropoietin receptor have been described as preformed dimers in the plasma membrane. Here we investigated gp130 homodimerization and heterodimerization with the LIFR by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC). We detected a FRET signal between YFP- and CFP-tagged gp130 at the plasma membrane of unstimulated cells that does not increase upon IL-6 stimulation. However, FRET between YFP-tagged gp130 and CFP-tagged LIFR considerably increased upon LIF stimulation. Using a BiFC approach that detects stable interactions we show that fluorescence complementation of gp130 constructs tagged with matching "halves" of fluorescent proteins increases upon IL-6 stimulation. Taken together, these findings suggest that transient 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.
Useful keywords (using NLM MeSH Indexing)
Animals
Bacterial Proteins/metabolism
COS Cells
Cell Line
Cell Membrane/metabolism
Cercopithecus aethiops
Cytokine Receptor gp130/chemistry*
Cytokine Receptor gp130/genetics
Cytokine Receptor gp130/metabolism
Dimerization
Fluorescence Resonance Energy Transfer
Green Fluorescent Proteins/genetics
Green Fluorescent Proteins/metabolism
Humans
Interleukin-6/genetics
Interleukin-6/metabolism
Leukemia Inhibitory Factor Receptor alpha Subunit
Luminescent Proteins/metabolism
Microscopy, Confocal
Microscopy, Fluorescence
Photochemistry
Protein Transport/genetics
Receptors, Cytokine/chemistry*
Receptors, Cytokine/metabolism
Receptors, OSM-LIF
Sequence Deletion
Transfection
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cytokine receptor