Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy.
Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 x 10(-9) cm(2) s(-1) independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.
Neugart
Felix
F
Institut für Biologische Chemie und Ernährungswissenschaft, Universität Hohenheim, Emil-Wolff-Str. 14, D-70599 Stuttgart, Germany.
Zappe
Andrea
A
Buk
Deborah M
DM
Ziegler
Inna
I
Steinert
Steffen
S
Schumacher
Monika
M
Schopf
Eva
E
Bessey
Ralph
R
Wurster
Kathrin
K
Tietz
Carsten
C
Börsch
Michael
M
Wrachtrup
Jörg
J
Graeve
Lutz
L
D000818:Animals; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000378:metabolism; D050822:Cytokine Receptor gp130 / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D018613:Microscopy, Confocal; D008956:Models, Chemical; D020801:Receptor, Ciliary Neurotrophic Factor / Q000737:chemistry* / Q000378:metabolism; D053606:Receptors, OSM-LIF / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D020934:Ciliary Neurotrophic Factor; D020801:Receptor, Ciliary Neurotrophic Factor; D053606:Receptors, OSM-LIF; D050822:Cytokine Receptor gp130