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Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

Futter CE, Collinson LM, Backer JM, Hopkins CR - J. Cell Biol. (2001)

Bottom Line: In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation.Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs.Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

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The effects of wortmannin on EGF and EGFR degradation. (a) Cells were incubated with 125I-EGF for 1 h at 20°C, surface stripped, and chased at 37°C in the absence or presence of wortmannin (wo) for up to 2 h. Media samples were TCA precipitated to determine the percentage of degradation. (b) Cells were incubated with EGF for 1 h at 20°C and then chased at 37°C for up to 2 h in the absence or presence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with an antibody against the cytoplasmic domain of the EGFR. Percentage degradation is calculated by comparison with the amount of EGFRs in cells not treated with EGF.
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fig2: The effects of wortmannin on EGF and EGFR degradation. (a) Cells were incubated with 125I-EGF for 1 h at 20°C, surface stripped, and chased at 37°C in the absence or presence of wortmannin (wo) for up to 2 h. Media samples were TCA precipitated to determine the percentage of degradation. (b) Cells were incubated with EGF for 1 h at 20°C and then chased at 37°C for up to 2 h in the absence or presence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with an antibody against the cytoplasmic domain of the EGFR. Percentage degradation is calculated by comparison with the amount of EGFRs in cells not treated with EGF.

Mentions: To further investigate the efficiency of delivery of EGF and EGFRs to the lysosome in the presence of wortmannin, the effects of wortmannin on 125I-EGF degradation and EGFR degradation were determined. In HEp-2 cells, ∼70% of endocytosed 125I-EGF is delivered to the lysosome and degraded to TCA soluble radioactivity, and 20% is released into the extracellular medium intact (Fig. 2 a). Wortmannin treatment had very little effect on the magnitude or kinetics of EGF degradation, although EGF recycling was inhibited. We have shown previously that MVB–lysosome fusion is required for EGF degradation to TCA-soluble products (Futter et al., 1996), and so these data together with the EM data described above indicate that wortmannin treatment does not inhibit MVB–lysosome fusion. EGFRs are normally delivered to the lysosome primarily on internal vesicles after MVB–lysosome fusion. In the presence of wortmannin, a considerable proportion of EGFRs are delivered to the lysosome on the perimeter membrane, and so EGF and the lumenal domain of the EGFR are exposed to a degradative environment, whereas the COOH terminus of the receptor remains exposed to the cytoplasm. Therefore, we determined the rate of degradation of the EGFR in the presence and absence of wortmannin by Western blotting with an antibody against the cytoplasmic domain of the EGFR. Although the rate of EGFR degradation was reduced in wortmannin-treated cells (Fig. 2 b), no fragments of the EGFR indicative of partial degradation were observed.


Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

Futter CE, Collinson LM, Backer JM, Hopkins CR - J. Cell Biol. (2001)

The effects of wortmannin on EGF and EGFR degradation. (a) Cells were incubated with 125I-EGF for 1 h at 20°C, surface stripped, and chased at 37°C in the absence or presence of wortmannin (wo) for up to 2 h. Media samples were TCA precipitated to determine the percentage of degradation. (b) Cells were incubated with EGF for 1 h at 20°C and then chased at 37°C for up to 2 h in the absence or presence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with an antibody against the cytoplasmic domain of the EGFR. Percentage degradation is calculated by comparison with the amount of EGFRs in cells not treated with EGF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199316&req=5

fig2: The effects of wortmannin on EGF and EGFR degradation. (a) Cells were incubated with 125I-EGF for 1 h at 20°C, surface stripped, and chased at 37°C in the absence or presence of wortmannin (wo) for up to 2 h. Media samples were TCA precipitated to determine the percentage of degradation. (b) Cells were incubated with EGF for 1 h at 20°C and then chased at 37°C for up to 2 h in the absence or presence of wortmannin (wo). Cell lysates were analyzed by SDS-PAGE followed by Western blotting with an antibody against the cytoplasmic domain of the EGFR. Percentage degradation is calculated by comparison with the amount of EGFRs in cells not treated with EGF.
Mentions: To further investigate the efficiency of delivery of EGF and EGFRs to the lysosome in the presence of wortmannin, the effects of wortmannin on 125I-EGF degradation and EGFR degradation were determined. In HEp-2 cells, ∼70% of endocytosed 125I-EGF is delivered to the lysosome and degraded to TCA soluble radioactivity, and 20% is released into the extracellular medium intact (Fig. 2 a). Wortmannin treatment had very little effect on the magnitude or kinetics of EGF degradation, although EGF recycling was inhibited. We have shown previously that MVB–lysosome fusion is required for EGF degradation to TCA-soluble products (Futter et al., 1996), and so these data together with the EM data described above indicate that wortmannin treatment does not inhibit MVB–lysosome fusion. EGFRs are normally delivered to the lysosome primarily on internal vesicles after MVB–lysosome fusion. In the presence of wortmannin, a considerable proportion of EGFRs are delivered to the lysosome on the perimeter membrane, and so EGF and the lumenal domain of the EGFR are exposed to a degradative environment, whereas the COOH terminus of the receptor remains exposed to the cytoplasm. Therefore, we determined the rate of degradation of the EGFR in the presence and absence of wortmannin by Western blotting with an antibody against the cytoplasmic domain of the EGFR. Although the rate of EGFR degradation was reduced in wortmannin-treated cells (Fig. 2 b), no fragments of the EGFR indicative of partial degradation were observed.

Bottom Line: In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation.Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs.Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Show MeSH
Related in: MedlinePlus