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PI3P signaling regulates receptor sorting but not transport in the endosomal pathway.

Petiot A, Faure J, Stenmark H, Gruenberg J - J. Cell Biol. (2003)

Bottom Line: We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes.Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting.They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211-Geneva-4, Switzerland.

ABSTRACT
While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

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PI3P signaling regulates EGFR sorting. (A) EGF was pulsed for 10 min at 37°C in cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A). Early endosome (EE), late endosome (LE), and heavy membrane (HM) fractions (Aniento et al., 1993a) were then analyzed by Western blotting using anti-GFP antibodies. (B–D) The in vitro formation of ECVs was measured (as in Fig. 4 C) using donor early endosomes from cells expressing EGFR-GFP (as in A) in the presence or absence of ATP, wortmannin (Wort), GST-2xFYVE, or C215S mutant. Fractions containing donor early endosomes (EE) and vesicles formed in vitro were analyzed by Western blotting using antibodies against the indicated antigens. In B and D, 50 and 10% of the donor membranes (EE), respectively, were analyzed. Scanning of the lanes showed that, within the 30-min incubation of the assay, ∼10% of the EGFR present in untreated donor membranes was incorporated into newly formed ECVs. This value is somewhat lower than EGF transport to late endosomes in vivo (∼60% after 45 min; Fig. 3 C), in part because one round of vesicle formation only is reconstituted in vitro (Aniento et al., 1993a).
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fig5: PI3P signaling regulates EGFR sorting. (A) EGF was pulsed for 10 min at 37°C in cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A). Early endosome (EE), late endosome (LE), and heavy membrane (HM) fractions (Aniento et al., 1993a) were then analyzed by Western blotting using anti-GFP antibodies. (B–D) The in vitro formation of ECVs was measured (as in Fig. 4 C) using donor early endosomes from cells expressing EGFR-GFP (as in A) in the presence or absence of ATP, wortmannin (Wort), GST-2xFYVE, or C215S mutant. Fractions containing donor early endosomes (EE) and vesicles formed in vitro were analyzed by Western blotting using antibodies against the indicated antigens. In B and D, 50 and 10% of the donor membranes (EE), respectively, were analyzed. Scanning of the lanes showed that, within the 30-min incubation of the assay, ∼10% of the EGFR present in untreated donor membranes was incorporated into newly formed ECVs. This value is somewhat lower than EGF transport to late endosomes in vivo (∼60% after 45 min; Fig. 3 C), in part because one round of vesicle formation only is reconstituted in vitro (Aniento et al., 1993a).

Mentions: We then used the same assay, but with the EGFR as a marker instead of HRP. EGF-induced endocytosis of the receptor was allowed to proceed for a few minutes to accumulate EGFR in early endosomes. These were then purified (Fig. 5 A) and used as donor membranes. In a highly selective manner, EGFR was incorporated into newly formed ECVs (Fig. 5 C), in contrast to TfR, which remained in donor membranes (Fig. 5 B). Interestingly, incorporation of EGFR into newly formed ECVs was significantly reduced when GST-2xFYVE, but not the inactive C215S mutant, was added to the assay (Fig. 5 C). GST-2xFYVE was recruited by donor membranes (Fig. 5 D) and to a lesser extent by ECVs depleted in EGFR (Fig. 5 C), consistently with the distribution of PI3P on early endosomes and ECVs (Gillooly et al., 2000). Similarly, EGFR incorporation into newly formed ECVs was also inhibited by wortmannin (Fig. 5 C). The selective inhibition of EGFR, but not HRP, incorporation into ECVs by GST-2xFYVE, but not by the C215S mutant, could be reproduced in vitro using donor membranes containing both endocytosed HRP and EGFR (not shown).


PI3P signaling regulates receptor sorting but not transport in the endosomal pathway.

Petiot A, Faure J, Stenmark H, Gruenberg J - J. Cell Biol. (2003)

PI3P signaling regulates EGFR sorting. (A) EGF was pulsed for 10 min at 37°C in cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A). Early endosome (EE), late endosome (LE), and heavy membrane (HM) fractions (Aniento et al., 1993a) were then analyzed by Western blotting using anti-GFP antibodies. (B–D) The in vitro formation of ECVs was measured (as in Fig. 4 C) using donor early endosomes from cells expressing EGFR-GFP (as in A) in the presence or absence of ATP, wortmannin (Wort), GST-2xFYVE, or C215S mutant. Fractions containing donor early endosomes (EE) and vesicles formed in vitro were analyzed by Western blotting using antibodies against the indicated antigens. In B and D, 50 and 10% of the donor membranes (EE), respectively, were analyzed. Scanning of the lanes showed that, within the 30-min incubation of the assay, ∼10% of the EGFR present in untreated donor membranes was incorporated into newly formed ECVs. This value is somewhat lower than EGF transport to late endosomes in vivo (∼60% after 45 min; Fig. 3 C), in part because one round of vesicle formation only is reconstituted in vitro (Aniento et al., 1993a).
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fig5: PI3P signaling regulates EGFR sorting. (A) EGF was pulsed for 10 min at 37°C in cells expressing EGFR-GFP (pretreated with EGF, as in Fig. 1 A). Early endosome (EE), late endosome (LE), and heavy membrane (HM) fractions (Aniento et al., 1993a) were then analyzed by Western blotting using anti-GFP antibodies. (B–D) The in vitro formation of ECVs was measured (as in Fig. 4 C) using donor early endosomes from cells expressing EGFR-GFP (as in A) in the presence or absence of ATP, wortmannin (Wort), GST-2xFYVE, or C215S mutant. Fractions containing donor early endosomes (EE) and vesicles formed in vitro were analyzed by Western blotting using antibodies against the indicated antigens. In B and D, 50 and 10% of the donor membranes (EE), respectively, were analyzed. Scanning of the lanes showed that, within the 30-min incubation of the assay, ∼10% of the EGFR present in untreated donor membranes was incorporated into newly formed ECVs. This value is somewhat lower than EGF transport to late endosomes in vivo (∼60% after 45 min; Fig. 3 C), in part because one round of vesicle formation only is reconstituted in vitro (Aniento et al., 1993a).
Mentions: We then used the same assay, but with the EGFR as a marker instead of HRP. EGF-induced endocytosis of the receptor was allowed to proceed for a few minutes to accumulate EGFR in early endosomes. These were then purified (Fig. 5 A) and used as donor membranes. In a highly selective manner, EGFR was incorporated into newly formed ECVs (Fig. 5 C), in contrast to TfR, which remained in donor membranes (Fig. 5 B). Interestingly, incorporation of EGFR into newly formed ECVs was significantly reduced when GST-2xFYVE, but not the inactive C215S mutant, was added to the assay (Fig. 5 C). GST-2xFYVE was recruited by donor membranes (Fig. 5 D) and to a lesser extent by ECVs depleted in EGFR (Fig. 5 C), consistently with the distribution of PI3P on early endosomes and ECVs (Gillooly et al., 2000). Similarly, EGFR incorporation into newly formed ECVs was also inhibited by wortmannin (Fig. 5 C). The selective inhibition of EGFR, but not HRP, incorporation into ECVs by GST-2xFYVE, but not by the C215S mutant, could be reproduced in vitro using donor membranes containing both endocytosed HRP and EGFR (not shown).

Bottom Line: We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes.Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting.They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, 1211-Geneva-4, Switzerland.

ABSTRACT
While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.

Show MeSH
Related in: MedlinePlus