Polarity and lipid raft association of the components of the ciliary neurotrophic factor receptor complex in Madin-Darby canine kidney cells.
Ciliary neurotrophic factor (CNTF) signals via a tripartite receptor complex consisting of the glycosyl-phosphatidylinositol (GPI)-anchored CNTF receptor (CNTF-R), the leukaemia inhibitory factor receptor (LIF-R) and the interleukin-6 (IL-6) signal transducer gp130. We have recently reported that gp130 is endogenously expressed in the polarised epithelial model cell line Madin-Darby canine kidney (MDCK) and we have demonstrated a preferential basolateral localisation of this protein. In the present study we show that MDCK cells also express the LIF-R and respond to stimulation with human LIF by activation of tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT3), both however in an unpolarised fashion. This suggests that MDCK cells may be target cells for LIF. We have furthermore stably expressed the human CNTF-R in MDCK cells and by two different assays we found an apical localisation. Consistent with these findings, stimulation of CNTF-R-positive cells resulted only in an activation of STAT3 when CNTF was added apically. These data demonstrate that each subunit of the CNTF receptor complex has a distinct distribution in polarised cells which may reflect the different roles the respective cytokines play in vivo. Since it is currently believed that lipid rafts are involved in signal transduction as well as protein sorting we studied the association of the three receptor complex components with membrane rafts using different protocols. Whereas the CNTF-R cofractionated quantitatively with lipid rafts independently of the method used, gp130 and the LIF-R were found to associate with lipid rafts only partially when detergents were used for isolation. These findings could indicate that either the three receptor complex subunits are localised to the same kind of raft but with different affinities to the liquid-ordered environment, or that they are localised to different types of rafts. CNTF-, LIF-, and IL-6-dependent STAT3 activation was sensitive to the cholesterol-depleting drug methyl-beta-cyclodextrin (MCD) suggesting that the integrity of lipid rafts is important for IL-6-type cytokine-induced STAT activation.
Buk
Deborah M
DM
Institut für Biologische Chemie und Ernährungswissenschaft, Universität Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany. dbuk@uni-hohenheim.de
Waibel
Michaela
M
Braig
Claudia
C
Martens
Astrid S
AS
Heinrich
Peter C
PC
Graeve
Lutz
L
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D036022:Capsid Proteins; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D020934:Ciliary Neurotrophic Factor / Q000378:metabolism; D058966:Contactins; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D005822:Genetic Vectors / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D015850:Interleukin-6 / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D020801:Receptor, Ciliary Neurotrophic Factor / Q000378:metabolism; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D047392:beta-Cyclodextrins / Q000378:metabolism
D036022:Capsid Proteins; D051242:Caveolin 1; D022461:Caveolins; D020934:Ciliary Neurotrophic Factor; D058966:Contactins; D016207:Cytokines; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D015850:Interleukin-6; D008055:Lipids; D019006:Neural Cell Adhesion Molecules; D020801:Receptor, Ciliary Neurotrophic Factor; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C079841:portal protein, bacteriophage P22; D002784:Cholesterol