Dimerization of the cytokine receptors gp130 and LIFR analysed in single cells.
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.
Giese
Bernd
B
Institut für Biochemie, Universitätsklinikum RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany.
Roderburg
Christoph
C
Sommerauer
Michael
M
Wortmann
Saskia B
SB
Metz
Silke
S
Heinrich
Peter C
PC
Müller-Newen
Gerhard
G
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D050822:Cytokine Receptor gp130 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010777:Photochemistry; D021381:Protein Transport / Q000235:genetics; D018121:Receptors, Cytokine / Q000737:chemistry* / Q000378:metabolism; D053606:Receptors, OSM-LIF; D017384:Sequence Deletion; D014162:Transfection
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D015850:Interleukin-6; C507840:LIFR protein, human; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008164:Luminescent Proteins; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; C054562:yellow fluorescent protein, Bacteria; D050822:Cytokine Receptor gp130; D049452:Green Fluorescent Proteins