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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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Transport from early to late endosomes after PI3 kinase inhibition. (A) BHK cells expressing EGFR-GFP were preincubated with EGF for 1 h at 4°C and washed; this pretreatment was always used for EGF or EGFR endocytosis. Cells were incubated for 10 min at 37°C with rhodamine-dextran (pulse), labeled with antibodies against the indicated antigens, and analyzed by triple channel fluorescence microscopy. (B) BHK cells overexpressing EGFR-GFP were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C, followed by a 90-min chase in the presence of 0.5 mg/ml leupeptin and analyzed as in described for A; high magnification views of the indicated areas are shown below the micrographs. (C) A pulse of rhodamine dextran was endocytosed as described for A and then chased for 45 min; cells were analyzed as described for A. (D) A pulse of rhodamine dextran was endocytosed as described for A into BHK cells with or without chase (as in C); 100 nM wortmannin (Wort) was present during both the pulse and the chase. Cells were analyzed as described for A. Bars: (A–C) 5 μm; (D) 2.5 μm.
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fig1: Transport from early to late endosomes after PI3 kinase inhibition. (A) BHK cells expressing EGFR-GFP were preincubated with EGF for 1 h at 4°C and washed; this pretreatment was always used for EGF or EGFR endocytosis. Cells were incubated for 10 min at 37°C with rhodamine-dextran (pulse), labeled with antibodies against the indicated antigens, and analyzed by triple channel fluorescence microscopy. (B) BHK cells overexpressing EGFR-GFP were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C, followed by a 90-min chase in the presence of 0.5 mg/ml leupeptin and analyzed as in described for A; high magnification views of the indicated areas are shown below the micrographs. (C) A pulse of rhodamine dextran was endocytosed as described for A and then chased for 45 min; cells were analyzed as described for A. (D) A pulse of rhodamine dextran was endocytosed as described for A into BHK cells with or without chase (as in C); 100 nM wortmannin (Wort) was present during both the pulse and the chase. Cells were analyzed as described for A. Bars: (A–C) 5 μm; (D) 2.5 μm.

Mentions: Bulk transport from early to late endosomes was followed with soluble markers that are incorporated nonselectively within the endosomal content, e.g., fluorescent dextran or HRP, whereas the EGFR was used as a reporter for the trafficking of down-regulated receptors. Since solutes may follow more than one endocytic pathways (Nichols and Lippincott-Schwartz, 2001), the fate of endocytosed dextran was first compared with that of down-regulated EGFR after EGF treatment. After 10 min, both markers colocalized in early endosomes (Fig. 1 A), containing the Rab5 effector early endosomal antigen 1 (EEA1) (Simonsen et al., 1998). After subsequent incubation for 45 min (not shown) or 90 min at 37°C, both EGFR and its ligand EGF reached vesicles that contained the late endosomal-specific lipid, lysobisphosphatidic acid (LBPA) (Kobayashi et al., 1998) (not shown) or Lamp1 (Fig. 1 B), a very abundant protein of late endosomes and lysosomes (Aniento et al., 1993a; Griffiths et al., 1988). Similarly, under the same conditions, dextran reached vesicles that contained Lamp1 (not shown) or LBPA (Fig. 1 C). Hence, both solutes and EGFR follow the same pathway to early endosomes and then to late endocytic compartments.


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

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

Transport from early to late endosomes after PI3 kinase inhibition. (A) BHK cells expressing EGFR-GFP were preincubated with EGF for 1 h at 4°C and washed; this pretreatment was always used for EGF or EGFR endocytosis. Cells were incubated for 10 min at 37°C with rhodamine-dextran (pulse), labeled with antibodies against the indicated antigens, and analyzed by triple channel fluorescence microscopy. (B) BHK cells overexpressing EGFR-GFP were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C, followed by a 90-min chase in the presence of 0.5 mg/ml leupeptin and analyzed as in described for A; high magnification views of the indicated areas are shown below the micrographs. (C) A pulse of rhodamine dextran was endocytosed as described for A and then chased for 45 min; cells were analyzed as described for A. (D) A pulse of rhodamine dextran was endocytosed as described for A into BHK cells with or without chase (as in C); 100 nM wortmannin (Wort) was present during both the pulse and the chase. Cells were analyzed as described for A. Bars: (A–C) 5 μm; (D) 2.5 μm.
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Related In: Results  -  Collection

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fig1: Transport from early to late endosomes after PI3 kinase inhibition. (A) BHK cells expressing EGFR-GFP were preincubated with EGF for 1 h at 4°C and washed; this pretreatment was always used for EGF or EGFR endocytosis. Cells were incubated for 10 min at 37°C with rhodamine-dextran (pulse), labeled with antibodies against the indicated antigens, and analyzed by triple channel fluorescence microscopy. (B) BHK cells overexpressing EGFR-GFP were incubated with EGF-biotin and streptavidin-phycoerythrin for 1 h at 4°C and then for 10 min at 37°C, followed by a 90-min chase in the presence of 0.5 mg/ml leupeptin and analyzed as in described for A; high magnification views of the indicated areas are shown below the micrographs. (C) A pulse of rhodamine dextran was endocytosed as described for A and then chased for 45 min; cells were analyzed as described for A. (D) A pulse of rhodamine dextran was endocytosed as described for A into BHK cells with or without chase (as in C); 100 nM wortmannin (Wort) was present during both the pulse and the chase. Cells were analyzed as described for A. Bars: (A–C) 5 μm; (D) 2.5 μm.
Mentions: Bulk transport from early to late endosomes was followed with soluble markers that are incorporated nonselectively within the endosomal content, e.g., fluorescent dextran or HRP, whereas the EGFR was used as a reporter for the trafficking of down-regulated receptors. Since solutes may follow more than one endocytic pathways (Nichols and Lippincott-Schwartz, 2001), the fate of endocytosed dextran was first compared with that of down-regulated EGFR after EGF treatment. After 10 min, both markers colocalized in early endosomes (Fig. 1 A), containing the Rab5 effector early endosomal antigen 1 (EEA1) (Simonsen et al., 1998). After subsequent incubation for 45 min (not shown) or 90 min at 37°C, both EGFR and its ligand EGF reached vesicles that contained the late endosomal-specific lipid, lysobisphosphatidic acid (LBPA) (Kobayashi et al., 1998) (not shown) or Lamp1 (Fig. 1 B), a very abundant protein of late endosomes and lysosomes (Aniento et al., 1993a; Griffiths et al., 1988). Similarly, under the same conditions, dextran reached vesicles that contained Lamp1 (not shown) or LBPA (Fig. 1 C). Hence, both solutes and EGFR follow the same pathway to early endosomes and then to late endocytic compartments.

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