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µ1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate

¹ Zentrum für Biochemie und Molekulare Zellbiologie, Biochemie II, Universität Göttingen, Heinrich-Düker-Weg 12, D-37073 Göttingen, Germany
² School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK

View larger version (92K): [in a new window]   Fig. 1. Surface expression of MPR300 in control (ct) and µ1A-deficient cells (µ1A–/–). Cells were rapidly cooled to 0°C and biotinylated. Biotinylated proteins were precipitated with streptavidin-agarose from 300-500 µg of cell extract proteins. Precipitates of biotinylated proteins and 100 µg of total protein extracts were analyzed in western blots by anti-MPR300 antibodies. Signals were quantified by densitometry and normalized for the protein content (see Materials and Methods for experimental details).

View larger version (65K): [in a new window]   Fig. 2. Internalization rate of MPR300 in control (ct) and µ1A-deficient cells (µ1A–/–). Cell-surface receptors were biotinylated on ice (t), warmed to 37°C for 1 minute and rapidly cooled on ice. Biotin was removed from proteins exposed at the cell surface. MPR300 was immunoprecipitated from cell extracts and precipitates were analyzed by western blots and streptavidin-HRP conjugates. Removal of biotin was quantitative, as indicated by the complete loss of biotinylated MPR300 in cells that were stripped without warming the cultures to 37°C (c).

View larger version (28K): [in a new window]   Fig. 3. Exocytosis of internalized MPR300 in control (ct) and µ1A-deficient cells (µ1A–/–). (A) Cells were biotinylated on ice, warmed to 37°C for 2 minutes, cooled on ice and subjected to biotin stripping. The fraction of the internal biotinylated receptors is set to 100% (t). Parallel cultures were subjected to up to three additional cycles of warming to 37°C for two minutes, cooling on ice and biotin stripping. Numbers below give the signal intensities for biotinylated MPR300 as a percentage of the total. (B) Kinetics of exocytosis for biotinylated MPR300, see (A). Numbers correspond to the decrease in intracellular biotinylated receptors. Closed circles and solid line; control cells, open circles; and dashed line, µ1A-deficient cells. Data are from three independent experiments.

View larger version (76K): [in a new window]   Fig. 4. MPR300-containing endosomes in control (ct) and µ1A-deficient cells (µ1A–/–). Steady-state labeling for MPR300 is shown in red, and the early endosome antigen 1 EEA1 is shown in green. Quantificaton of the yellow spectra revealed a 2.3 fold more intense staining in µ1A–/– cells compared with ct cells. Labelling of late endosomes with LBPA is shown in red and MPR300 is shown in green. Steady-state labelling for MPR300 (green) and Cy3-labelled transferrin (red) is shown endocytosed over 15 minutes. The distribution of EEA1 (green) and furin (red) is also shown.

View larger version (43K): [in a new window]   Fig. 5. Immunogold electron microscopy for plasma membrane MPR300. (A) and (B) plasma membrane and coated pit of control cell. (C) and (D) plasma membrane and coated pits of µ1A-deficient cell.