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, 2 (1), 68-74

Regulation of Intracellular Trafficking of the EGF Receptor by Rab5 in the Absence of Phosphatidylinositol 3-kinase Activity

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Regulation of Intracellular Trafficking of the EGF Receptor by Rab5 in the Absence of Phosphatidylinositol 3-kinase Activity

X Chen et al. EMBO Rep.

Abstract

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110delta kin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.

Figures

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Fig. 1. EGFR endocytosis after inhibition of EGF-stimulated PI3K activity. (A) SKBR-3 cells were transfected with myc-tagged mutant p110Δkin or p110* for 48 h and then stimulated with EGF for 2 h or not stimulated. Indirect immunofluorescence was performed as described in Methods. Photographs are triple exposures with EGFR localization in the green channel (arrows), Lamp1 localization in the red channel (arrowheads) and transfected p110 in the blue channel. Size bar = 15 µm. (B) 293T cells were transfected with human EGFR, wild-type PI3K p110, mutant p110Δkin and/or p110* for 48 h and then stimulated with EGF for the indicated time. Cell lysates were subjected to immunoblot analysis with anti-EGFR, anti-myc or anti-phospho-Akt (p-Akt) antibodies.
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Fig. 2. Effects of wortmannin on the intracellular trafficking of EGFR in SKBR-3 and MDCK cells. (A) SKBR-3 cells. Left panel, control. Right panel, cells treated with wortmannin (100 nM). Cells were either kept in serum-deprived media or stimulated with EGF for 15 min or 2 h. Photographs are double exposures with EGFR localization in the green channel (arrows), Lamp1 localization in the red channel (arrowheads). (B) MDCK cells. Left most panels: control. Second panels: cells treated with wortmannin (100 nM). Third panels: cells treated with LY294002 (20 µM). Right most panels: cells treated with a high concentration of wortmannin (10 µM). Cells were stimulated with EGF for 30 min or 2 h. EGFR localization (arrows) was revealed by indirect immunofluorescence. Size bar = 20 µm.
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Fig. 3. Effects of wortmannin on the lysosomal degradation of EGFR in MDCK, SKBR-3 and BT20 cells. (A) Cells were treated with wortmannin and then stimulated with EGF for the indicated times. Cell lysates were subjected to immunoblot analysis with anti-EGFR or anti-phospho-Akt (p-Akt) antibodies. (B) Quantification of the results from (A). Quantification was performed with a digital imaging system from Alpha Innotech Corporation (San Leandro, CA).
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Fig. 4. Regulation of the intracellular trafficking of EGFR by Rab5. SKBR-3 cells were transfected with wild-type Rab5, mutant Rab5 S34N or Q79L for 48 h and then stimulated with EGF for 30 min (right panels) or kept in serum-deprived media (left panels). Photographs are double exposures with EGFR localization in the green channel (arrows) and Rab5 localization in the red channel (arrowheads). Size bar = 20 µm.
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Fig. 5. Effects of Rab5 effectors on the intracellular trafficking of EGFR. (A) Dissociation of EEA1 from membrane fractions by wortmannin treatment of MDCK, SKBR-3 and BT20 cells. Cells were subjected to subcellular fractionation into a soluble fraction (S100) and a particulate fraction (P100) after treatment with wortmannin (100 nM) and EGF (30 min), as indicated. Subcellular fractions were subjected to immunoblot analysis with anti-EEA1 antibodies. (B) Effects of microinjection with anti-Rabaptin5 antibodies on EGFR endocytosis in MDCK cells. Cells were stimulated with EGF for 15 min or 2 h after injection. Photographs are double exposures, with EGFR localization in the red channel (arrows) and microinjected antibodies in the green channel. Size bar = 20 µm.

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