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Cholesterol is important in control of EGF receptor kinase activity but EGF receptors are not concentrated in caveolae

¹ Institute of Pathology, University of Oslo, Rikshospitalet University Hospital, N-0027 Oslo, Norway
² MSD Cardiovascular Research Center, Rikshospitalet University Hospital and Department of Pharmacology, University of Oslo, N-0316 Oslo, Norway

View larger version (92K): [in a new window]   Fig. 1. Immuno-EM shows that EGFR is localized mainly outside caveolae. Ultrathin cryosections of nonstimulated cells were double labeled to localize EGFR (large gold particles) and caveolin (small gold particles). A431 cells (A,B) showed strong labeling for EGFR but very limited colocalization of EGFR (large arrowheads) with caveolin in caveolae. In HEp-2 cells (C,D) EGFR labeling was less intense but, as in A431 cells, EGFR labeling was mainly at smooth noninvaginated areas of the plasma membrane (small arrowheads) with only small amounts colocalizing (large arrowheads) with caveolin in caveolae. Bars, 100 nm.

View larger version (73K): [in a new window]   Fig. 2. Immuno-EM shows that binding of EGF mainly occurs outside caveolae. Ultrathin cryosections of cells incubated with EGF (10 nM) for 15 minutes on ice were double-labeled to localize bound EGF (large gold particles) and caveolin (small gold particles). In both A431 cells (A,B) and HEp-2 cells (C,D) bound EGF was found at smooth noninvaginated areas of the plasma membrane (small arrowheads), and only small amounts colocalized with caveolin in caveolae (large arrowheads). Bars, 100 nm.

View larger version (96K): [in a new window]   Fig. 3. Immuno-EM shows that whereas EGFR in nonstimulated cells localizes both within and outside rafts, TfR localizes outside rafts. To patch PLAP, HEp-2 cells were incubated with mouse anti-PLAP antibodies, followed by rabbit anti-mouse IgG and finally 5 nm protein A-gold. Each incubation was for 30 minutes on ice. Ultrathin cryosections of HEp-2 cells with patched PLAP (small gold particles) were labeled (large gold particles) to detect localization of EGFR (A-C) or TfR (D). Labeling for EGFR was found both outside (small arrowheads in A) and within (large arrowheads in B,C) PLAP patches. Labeling for TfR was mainly found outside PLAP patches. Bars, 100 nm.

View larger version (109K): [in a new window]   Fig. 4. Immuno-EM shows that binding of EGF occurs to EGFR both inside and outside rafts. PLAP was patched as described in Fig. 3, except that EGF (10 nM) was added together with 5 nm protein A-gold. Labeling for EGFR (A) showed that incubation with EGF on ice did not change the localization of the EGFR. Labeling for EGFR was found both outside (small arrowheads) and within (large arrowheads) rafts. Labeling for EGF (B) showed that EGF bound to EGFR both outside (small arrowheads) and within (large arrowheads) rafts. Bars, 100 nm.

View larger version (70K): [in a new window]   Fig. 5. Incubation with U18666A causes intracellular accumulation of cholesterol. A431 cells (A,B) and HEp-2 cells (C,D) were incubated with or without 1 µg/ml U18666A for 48 hours prior to fixation. Cholesterol was labeled using filipin, as described in Materials and Methods. In untreated cells (A,C) filipin labeled mainly the plasma membrane. Upon treatment with U18666A (B,D) filipin labeling revealed accumulation of cholesterol in intracellular vesicles.

View larger version (24K): [in a new window]   Fig. 6. Cholesterol depletion induces increased tyrosine phosphorylation of the EGFR. (A) A431 cells and HEp-2 cells were pre-incubated with (+) or without (-) 10 mM MßCD for 15 minutes at 37°C, cooled with ice-cold PBS and further incubated with or without EGF in MEM with or without 10 mM MßCD for 15 minutes on ice. Whereas A431 cells were incubated with 0.1 nM EGF, HEp-2 cells were incubated with 1 nM EGF to achieve detectable ligand-induced phosphorylation. The cells were lysed and subjected to SDS-PAGE and western blotting, using antibodies to EGFR and PY1173. (B) A431 cells were pre-incubated with (+) or without (-) 1 µg/ml U18666A for 48 hours prior to incubation with or without EGF (0.1 or 1.0 nM) for 15 minutes on ice. To demonstrate differences in EGFR tyrosine phosphorylation (PY1173) upon incubation with 1 nM EGF, two different exposures of the blot are shown.

View larger version (17K): [in a new window]   Fig. 7. Incubation with water-soluble cholesterol decreases tyrosine phosphorylation of the EGFR. A431 cells and HEp-2 cells were pre-incubated with (+) or without (-) 0.4 mg/ml water-soluble cholesterol for 30 minutes at 37°C, cooled with ice-cold PBS, and further incubated with 10 nM EGF in the absence or presence of cholesterol for 15 minutes on ice. Blotting with antibody to PY1173 revealed a large decrease in EGFR phosphorylation in cells incubated with water-soluble cholesterol. However, blotting with antibody to EGFR showed equal amounts of EGFR in cell lysates irrespective of addition of cholesterol.

View larger version (12K): [in a new window]   Fig. 8. Cholesterol depletion and incubation with water-soluble cholesterol has opposite effects on EGF binding. Untreated HEp-2 cells (control) and HEp-2 cells preincubated with either 10 mM MßCD for 15 minutes (MßCD), 1 µg/ml U18666A for 48 hours (U18666A), or 0.4 mg/ml water-soluble cholesterol for 30 minutes (cholesterol) were incubated with 125I-EGF for 15 minutes on ice. Bound 125I-EGF was measured as described in Materials and Methods. EGF bound to control cells was set to be 1 (approx. 1x105 binding sites/cell). The figure shows one representative experiment of three measured in triplicate (means±s.e.m.).

View larger version (14K): [in a new window]   Fig. 9. Incubation with MßCD or water-soluble cholesterol has opposite effects on EGFR dimerization. HEp-2 cells were preincubated either with (+) or without (-) 10 mM MßCD for 15 minutes, or with 0.4 mg/ml water-soluble cholesterol for 30 minutes at 37°C. The cells were subsequently incubated with or without 10 nM EGF for 15 minutes on ice. The EGFR was crosslinked by the crosslinker BS3, before cell lysis, SDS-PAGE and western blotting, using an antibody to EGFR, as described in Materials and Methods. Bands at 170 kDa represent EGFR monomers, whereas bands at 340 kDa represent EGFR dimers.

View larger version (14K): [in a new window]   Fig. 10. Cholesterol depletion affects the cellular distribution of EGFR. Untreated HEp-2 cells (control) or HEp-2 cells preincubated with 10 mM MßCD for 15 minutes (MßCD), 1 ug/ml U18666A for 48 hours (U18666A), or 0.4 mg/ml water-soluble cholesterol for 30 minutes (cholesterol), were biotinylated as described in Materials and Methods. The fraction of biotinylated EGFR (plasma-membrane-localized EGFR) was calculated. Biotinylated EGFR in control cells was set to be 1. The figure shows pooled data from three independent experiments (means±s.e.m.).

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