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First published online October 27, 2005
doi: 10.1242/10.1242/jcs.02627

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Assigning functions to distinct regions of the N-terminus of the prion protein that are involved in its copper-stimulated, clathrin-dependent endocytosis

Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, UK

View larger version (40K): [in a new window]   Fig. 1. The copper-stimulated endocytosis of PrPC is blocked by tyrphostin A23. (A) SH-SY5Y cells expressing PrPC were surface biotinylated and then either untreated or treated with tyrphostin A23 in the presence or absence of 100 µM Cu2+. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrPC. Cells were then lysed, and total PrPC was immunoprecipitated from the sample using antibody 3F4 and then subjected to western blot analysis. The biotin-labelled PrPC fraction was detected with peroxidase-conjugated streptavidin. (B) The same samples from (A) were also immunoprecipitated using an anti-transferrin receptor antibody. (C) Cells were treated with tyrphostin A63 and processed as in (A). (D) Biotin-labelled N2a cells were processed as described in (A), but PrPC was immunoprecipitated using the antibody SAF-32. Densitometric analysis (mean±s.e.m.) of multiple blots from three separate experiments is shown.

View larger version (56K): [in a new window]   Fig. 2. Tyrphostin A23 blocks PrPC and transferrin endocytosis but has no effect on ganglioside GM1 endocytosis. (A) SH-SY5Y cells stably expressing PrPC were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C, washed three times in PBS and then incubated for 20 minutes at 37°C in OptiMEM in the absence of Cu2+, in the presence of 100 µM Cu2+ or in the presence of Cu2+ and 500 µM tyrphostin A23. Cells were also incubated with Texas-Red-conjugated transferrin at a dilution of 1:1000. (B) Cells were incubated with BODIPY FL C5-ganglioside GM1 for 20 minutes in the presence of either 500 µM tyrphostin A23 or 1 mM MßCD. Cells were fixed, incubated with Alexa488-conjugated rabbit anti-mouse antibody and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Bars, 10 µm.

View larger version (29K): [in a new window]   Fig. 3. Overexpression of AP180-C inhibits the Cu2+-stimulated endocytosis of PrPC. SH-SY5Y cells stably expressing PrPC were seeded onto coverslips and transiently transfected with either an expression plasmid encoding Myc-tagged AP180-C or an expression plasmid encoding AP180-N. After 24 hours, cells were processed as described in Fig. 2, with AP180-C detected using a polyclonal anti-Myc primary antibody and Marina Blue goat anti-rabbit IgG secondary antibody, and AP180-N detected using a polyclonal anti-AP180 primary antibody and Alexa488-conjugated donkey anti-goat IgG secondary antibody. In AP180-N-transfected cells, 3F4-labelled PrP was detected using an Alexa594-conjugated rabbit anti-mouse IgG secondary antibody. (A) Cells expressing AP180-C are unable to endocytose either transferrin or PrPC, whereas endocytosis is unaffected in AP180-N-expressing cells. (B) Quantification of (A). Results are the mean±s.e.m. of four individual experiments, in each of which 30 cells were visualised to determine their ability to endocytose transferrin and PrPC. The number of AP180-C- and AP180-N-transfected cells showing endocytosis is shown as a percentage of the number of untransfected cells showing endocytosis. (C) AP180-C cells in which clathrin-mediated endocytosis is blocked are not senescent, as shown by the uptake of dextran. Bars, 10 µm.

View larger version (37K): [in a new window]   Fig. 4. Effect of Cu2+ on the cell-surface distribution of PrPC. SH-SY5Y cells stably expressing PrPC were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C, washed three times in PBS and then incubated for 20 minutes at 37°C in OptiMEM in the absence or presence of 100 µM Cu2+ along with 500 µM tyrphostin A23. Where indicated, cells were incubated at 4°C for 10 minutes with PBS containing 1% Triton X-100 prior to fixation. Cells were fixed, incubated with Alexa488-conjugated secondary antibody and viewed using a DeltaVision Optical Restoration Microscopy System. (A) Images were taken of ten Z-slices from the top of the cell, and of an individual Z-slice from the middle of the cell. Bars, 10 µm. (B) Cell-surface distribution of PrP was measured using ImageJ as described in the Materials and Methods. Average fluorescent intensities per unit length of membrane were as follows: –Cu2+, 15.14 units; –Cu2+ + Triton X-100, 13.46 units; +Cu2+, 16.22 units; +Cu2+ + Triton X-100, 8.58 units. (C) The percentage of the cell surface stained for PrPC was determined from an individual Z-slice image in panel (A). Results are the mean±s.e.m. of three individual experiments, in each of which 30 cells were measured. Statistical differences, using Student's t-test (n=90), with probability values of P<0.05 were taken as significant.

View larger version (21K): [in a new window]   Fig. 5. Effect of Cu2+ on the distribution of PrP in detergent-insoluble rafts. SH-SY5Y cells expressing PrPC, PrP-N or PrP-oct were surface biotinylated and incubated in the absence or presence of 100 µM Cu2+ along with 500 µM tyrphostin A23. Cells were homogenised in the presence of 1% (v/v) Triton X-100 and subjected to buoyant sucrose density gradient centrifugation. PrP constructs were immunoprecipitated from equal volumes of each gradient fraction using 3F4 and subjected to western blotting. The biotin-labelled PrPC fraction was detected with peroxidase-conjugated streptavidin. Flotillin-1 and transferrin receptor (TfR) were detected using anti-flotillin-1 and H68.4 antibodies, respectively.

View larger version (23K): [in a new window]   Fig. 6. Effect of Cu2+ on the cell-surface distribution of PrP-N and PrP-oct. (A) Schematic of the PrP constructs used in this study. PrPC is shown as the mature, full-length protein (residues 23-231) with its C-terminal GPI anchor, the N-terminal polybasic region (KKRP, residues 23-26, chequered box) and the octapeptide repeats (residues 51-91, shaded). PrP-oct lacks the entire octapeptide repeat region and PrP-N lacks the N-terminal polybasic region. In PrP-CTM, the GPI anchor attachment signal is replaced with the transmembrane (filled box) and cytoplasmic domains (cross-hatched) from angiotensin-converting enzyme (Walmsley et al., 2001). (B) SH-SY5Y cells expressing PrP-N were surface biotinylated and then incubated in the absence or presence of 100 µM Cu2+. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrP. Cells were then lysed, and total PrP was immunoprecipitated from the sample using antibody 3F4 and then subjected to western blot analysis. The biotin-labelled PrP fraction was detected with peroxidase-conjugated streptavidin. SH-SY5Y cells stably expressing (C) PrP-N or (D) PrP-oct were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C, washed three times in PBS and then incubated for 20 minutes at 37°C in OptiMEM in the absence or presence of 100 µM Cu2+ along with 500 µM tyrphostin A23. Where indicated, cells were incubated at 4°C for 10 minutes with PBS containing 1% Triton X-100 prior to fixation. Cells were fixed, incubated with Alexa488-conjugated secondary antibody and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Bar, 10 µm. Average fluorescent intensity per unit length of membrane for PrP-N and PrP-oct were as follows: –Cu2+, 11.58 and 10.54 units; +Cu2+, 12.61 and 10.10 units; +Cu2+ + Triton X-100, 9.09 and 8.81 units, respectively. (E) The percentage of the cell surface stained for PrP-N and PrP-oct was determined from the images in (C) and (D), respectively, as described in Fig. 4. Results are the mean±s.e.m. of three individual experiments, in each of which 30 cells were measured. Statistical differences (n=90), using Student's t-test, with probability values of P<0.05 were taken as significant.

View larger version (35K): [in a new window]   Fig. 7. Disruption of rafts or displacement of PrP from rafts promotes the endocytosis of PrP in the absence of Cu2+. (A) SH-SY5Y cells expressing PrPC were surface biotinylated and either untreated or treated with 1 mM MßCD in the absence or presence of 500 µM tyrphostin A23. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrPC. Cells were then lysed, and PrPC was immunoprecipitated from the sample using antibody 3F4 and then subjected to western blot analysis. The biotin-labelled PrPC fraction was detected with peroxidase-conjugated streptavidin. The same samples were also immunoprecipitated using an anti-transferrin receptor antibody. (B) SH-SY5Y cells stably expressing PrPC or PrP-CTM were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C and then washed three times in PBS. Where indicated, cells were incubated either at 4°C for 10 minutes with PBS containing 1% Triton X-100, or with 1 mM MßCD at 37°C for 45 minutes, prior to fixation. Bar, 10 µm. (C) SH-SY5Y cells expressing PrP-CTM were surface biotinylated and either untreated or treated with 500 µM tyrphostin A23. Prior to lysis, the cells were incubated with trypsin to digest cell-surface PrP. Cells were lysed, and PrP-CTM immunoprecipitated and analysed as described in (A). Results are the mean±s.e.m. of three separate experiments done in duplicate each time (n=6), with values of P<0.05 taken as statistically significant where indicated.

View larger version (36K): [in a new window]   Fig. 8. Schematic showing the mechanism of internalisation of PrPC. PrPC is attached to the exoplasmic leaflet of the plasma membrane by its GPI anchor and localises within detergent-insoluble rafts through interactions between its N-terminal region [residues 23-90, red (Walmsley et al., 2003)] and a raft-resident protein or lipid. Upon Cu2+ binding to the octapeptide repeats (blue), the protein undergoes a conformational change that dissociates it from the raft-resident partner and PrPC then moves laterally out of rafts into detergent-soluble regions of the plasma membrane. The polybasic N-terminal region then interacts with the ectodomain of a transmembrane protein that engages, via its cytoplasmic domain, with the adaptor protein AP-2 and the endocytic machinery of clathrin-coated pits.

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