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First published online 30 March 2004
doi: 10.1242/jcs.01049

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Polarity and lipid raft association of the components of the ciliary neurotrophic factor receptor complex in Madin-Darby canine kidney cells

¹ Institut für Biologische Chemie und Ernährungswissenschaft, Universität Hohenheim, Garbenstrasse 30, 70599 Stuttgart, Germany
² Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstr. 30, 52057 Aachen, Germany

View larger version (60K): [in a new window]   Fig. 1. Localisation of the human CNTF-R and endogenous caveolin-1 in stably transfected MDCK cells. (A,B) MDCK cells stably transfected with the human CNTF-R were grown on collagen-coated coverslips for 5 days. Indirect immunofluorescence staining was performed using specific antibodies against the CNTF-R and caveolin-1 followed by Cy3-conjugated (CNTF-R) and FITC-conjugated (caveolin-1) secondary antibodies. Microscopy was performed using a Zeiss Axiovert 100 M confocal laser scanning microscope equipped with LSM 5 Pascal (Jena, Germany). (A) The xy-scan shows the localisation of the human CNTF-R (red) and caveolin-1 (green) in serial sections of 0.4 µm from the basal to the apical pole. (B) The xz-scan for CNTF-R and caveolin-1. (C) Parental MDCK (lanes 1 and 4) and stably transfected MDCK-CNTF-R cells (lanes 2+3 and 5+6) were grown on Transwell filters for 5 days. Sulfo-NHS-biotin was employed to selectively label the apical or the basolateral surface. The cells were extracted with lysis buffer and the supernatants were immunoprecipitated with a monoclonal CNTF-R-specific antibody. Immunoprecipitates were analysed by SDS-PAGE and western blot. A molecular mass marker containing albumin (66 kDa) was used for comparison. The biotinylated proteins were detected using HRP-conjugated streptavidin and visualised by ECL+plus. For MDCK-CNTF-R cells two independent determinations are shown.

View larger version (51K): [in a new window]   Fig. 2. (A) Non-polarised expression of LIF-R in MDCK cells. HeLa, HepG2, Ba/F3-gp130AA, and Ba/F3-LIF-R cells or MDCK cells were extracted with lysis buffer and cell lysates immunoprecipitated with a monoclonal LIF-R specific antibody (7G7) or a LIF-R-specific rabbit antiserum, respectively. (B) MDCK and HeLa cells were grown on tissue culture dishes, lysed and incubated either with a LIF-R-specific rabbit antiserum [-LIF-R (rb) +] or non-immune rabbit serum (rabbit serum +). (C) MDCK cells were grown on Transwell filters for 5 days. Sulfo-NHS-biotin was employed to selectively label the apical (ap) or the basolateral (bl) surfaces or cells were left untreated (ctr). The lysates were incubated with a LIF-R-specific rabbit antiserum. Immunoprecipitates (A-C) were analysed by SDS-PAGE and western blotting using either a polyclonal LIF-R-specific antibody followed by an appropriate HRP-conjugated secondary antibody (A,B) or HRP-conjugated streptavidin (C). Blots were developed using ECL+plus.

View larger version (32K): [in a new window]   Fig. 3. Functionality of the CNTF-R and the LIF-R in MDCK cells. Parental MDCK (upper panel), stably transfected MDCK-CNTF-R (middle panel) or MDCK-gp130 cells (lower panel) were grown on Transwell filters for 5 days. The cells were apically stimulated with CNTF (25 ng/ml) or LIF (50 ng/ml) for 15-30 minutes at 37°C. Nuclear extracts were prepared and equal protein aliquots analysed by SDS-PAGE and western blotting. Phosphorylated STAT3 proteins were detected with an activation-specific STAT3 antibody (pY-STAT3), an HRP-conjugated secondary antibody and visualised using the ECL+plus system. Quantitation over basal levels (fold stimulation) was calculated in each set of experiment as indicated in the figure.

View larger version (54K): [in a new window]   Fig. 4. STAT activation in polarised MDCK cells. Parental MDCK (upper panels) and stably transfected MDCK (MDCK-gp130, middle panels; MDCK-CNTF-R, lower panels) were cultivated on Transwell filters for 5 days. CNTF (25 ng/ml), LIF (50 ng/ml), or IL-6 (20 ng/ml)/soluble IL-6R (500 ng/ml), respectively, were added to the apical (ap) or the basolateral (bl) surfaces for 30 minutes at 37°C. Nuclear extracts were prepared and analysed by SDS-PAGE and western blotting. Phosphorylated STAT3 proteins were detected with an activation-specific STAT3 antibody (pY-STAT3), an HRP-conjugated secondary antibody and visualised using the ECL+plus system. Quantitation over basal levels (fold stimulation) was calculated in each set of experiment as indicated in the figure.

View larger version (50K): [in a new window]   Fig. 5. Association with lipid rafts of human CNTF-R, human gp130, and canine LIF-R in stably transfected and parental MDCK cells. MDCK-CNTF-R (A-C), MDCK-gp130 (D-F), parental MDCK (G-I) and MDCK-CNTF-R (K-M) cells were grown on tissue culture dishes and lysed at 4°C using three different protocols: a detergent-free extraction with sodium carbonate buffer, pH 11 (A,D,G,K), extraction with 1% Triton X-100 (B,E,H,L), and extraction with 1% Brij 58 (C,F,I,M) (see Materials and Methods for details). Lysates were subsequently homogenised and adjusted to 45% (A,D,G,K) or 40% sucrose (B,C,E,F,H,I,L,M) respectively. Subcellular fractions were obtained by ultracentrifugation at 4°C in a sucrose gradient (45/35/5% or 40/30/5% sucrose) for 16-20 hours at 192,000 g in a Beckman SW40 rotor. 1 ml fractions were collected, TCA-precipitated and proteins were analysed by 10% SDS-PAGE. Proteins were transferred to a PVDF membrane and the CNTF-R (A-C), gp130 (D-F), or the LIF-R (G-I) and also the endogenous caveolin-1 (A-I) were sequentially detected with specific antibodies and HRP-conjugated secondary antibodies. In the control panels (K-M) blots were incubated with specific antibodies to Rab5 or caveolin-1 and respective HRP-conjugated secondary antibodies. The proteins were visualised using the ECL+plus system.

View larger version (50K): [in a new window]   Fig. 6. Effect of receptor activation on lipid raft association. MDCK-CNTF-R cells (A), MDCK-gp130 (B,C), and parental MDCK cells (D) were stimulated with CNTF (A), IL-6/sIL-6R (B) or LIF (C+D) for 30 minutes at 37°C and lysed at 4°C using the Triton X-100 (A) or the Brij 58 (B-D) protocol (see Materials and Methods). After ultracentrifugation in a 40/30/5% sucrose gradient at 4°C for 16-20 hours at 192,000 g in a SW40 rotor 1 ml fractions were collected, trichloracetic acid-precipitated, and proteins analysed by 10% SDS-PAGE and western blotting. Human CNTF-R (A), human gp130 (B,C), and the endogenous LIF-R (D), respectively, as well as endogenous caveolin-1 (A-D) were sequentially detected with specific antibodies and HRP-conjugated secondary antibodies. The proteins were visualised using the ECL+plus system.

View larger version (39K): [in a new window]   Fig. 7. Cholesterol depletion by methyl-ß-cyclodextrin (MCD) and its effect on CNTF-, IL-6- and LIF-signalling. (A,B) MDCK cells, cultured for 72 hours, were plated on 60 mm tissue culture dishes and incubated with mevalonate (0.25 mM) and lovastatin (4 µM) for 48 hours prior to the experiment. Control cells (0 minutes MCD) were not pretreated. Cholesterol was depleted using 10 mM (A) or 20 mM MCD (B) in DMEM containing 4 µM lovastatin and 0.2% BSA at 37°C for different times as indicated. Total cellular lipids were extracted and cholesterol measured enzymatically at 500 nm. The data represent the mean±s.d. of three independent experiments. The percentage decrease in cellular cholesterol levels is also given above each bar. (C) MDCK-gp130 cells were pretreated (+) with lovastatin (Lov) and mevalonate (Mev) as described above or left untreated (—). The cells were stimulated with IL-6 (20 ng/ml)/sIL-6R (500 ng/ml) in DMEM/0.2% BSA for 30 minutes at 37°C (+) or left untreated (—). Nuclear extracts were prepared and equal protein aliquots analysed by SDS-PAGE and western blotting. The membranes were probed with an activation-specific STAT3 antibody and an HRP-conjugated secondary antibody. Proteins were visualised using the ECL+plus system. (D) MDCK-CNTF-R (upper panel), MDCK-gp130 (middle panel) and parental MDCK cells (lower panel) were grown on tissue culture dishes and incubated with mevalonate and lovastatin as described above. Control cells (first two lanes) were left untreated. Lovastatin (4 µM) was also included in all depletion or stimulation solutions except for controls. The cells were cholesterol-depleted using 10-20 mM MCD at 37°C for different times as indicated and afterwards stimulated (+) with CNTF (25 ng/ml), IL-6 (20 ng/ml)/sIL-6R (500 ng/ml), or LIF (50 ng/ml), respectively. Nuclear extracts were prepared and analysed as outlined above.

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