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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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Hrs regulates EGFR sorting, but not bulk transport to late endosomes. (A and B) As in Fig. 3 A and B, control cells (ctrl) or cells expressing either GFP-2xFYVE (FYVE) or myc-Hrs were labeled with EGF-biotin/streptavidin-phycoerythrin or dextran, except that the marker was endocytosed for 45 min followed by a 60-min chase, and analyzed by triple channel fluorescence microscopy (B). In A, the colocalization of dextran or EGF with the early endosomal marker (EE, GFP-2xFYVE or myc-Hrs) or the late endosomal marker (LE, LBPA) was quantified as in Fig. 3 C. (C) ECV formation in vitro was measured with HRP as a marker, as in Fig. 4, using cytosol depleted of Hrs by immunoprecipitation (Hrs IP), or mock-treated with a control anti-Rab7 antibody (ctrl IP). (D) These cytosols (after Hrs immunoprecipitation or control immunoprecipitation, as in C) were analyzed by SDS electrophoresis and Western blotting with anti-Hrs (αHrs) antibodies. (E) The incorporation of GFP-EGFR into ECVs was followed as in Fig. 5, using Hrs-depleted or mock-treated (Rab7) cytosol (as in C). GFP-EGFR was analyzed in donor membranes (EE; 50% of the donor membranes) and vesicles (whole fraction) formed in vitro (vesicles) by Western blotting with anti-GFP antibodies. The lanes were scanned, and the quantification is shown in C, as a percentage of the total amounts of GFP-EGFR in the vesicle fraction of controls. Bar, 2.5 μm.
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fig6: Hrs regulates EGFR sorting, but not bulk transport to late endosomes. (A and B) As in Fig. 3 A and B, control cells (ctrl) or cells expressing either GFP-2xFYVE (FYVE) or myc-Hrs were labeled with EGF-biotin/streptavidin-phycoerythrin or dextran, except that the marker was endocytosed for 45 min followed by a 60-min chase, and analyzed by triple channel fluorescence microscopy (B). In A, the colocalization of dextran or EGF with the early endosomal marker (EE, GFP-2xFYVE or myc-Hrs) or the late endosomal marker (LE, LBPA) was quantified as in Fig. 3 C. (C) ECV formation in vitro was measured with HRP as a marker, as in Fig. 4, using cytosol depleted of Hrs by immunoprecipitation (Hrs IP), or mock-treated with a control anti-Rab7 antibody (ctrl IP). (D) These cytosols (after Hrs immunoprecipitation or control immunoprecipitation, as in C) were analyzed by SDS electrophoresis and Western blotting with anti-Hrs (αHrs) antibodies. (E) The incorporation of GFP-EGFR into ECVs was followed as in Fig. 5, using Hrs-depleted or mock-treated (Rab7) cytosol (as in C). GFP-EGFR was analyzed in donor membranes (EE; 50% of the donor membranes) and vesicles (whole fraction) formed in vitro (vesicles) by Western blotting with anti-GFP antibodies. The lanes were scanned, and the quantification is shown in C, as a percentage of the total amounts of GFP-EGFR in the vesicle fraction of controls. Bar, 2.5 μm.

Mentions: Hrs is likely to mediate the PI3P-dependent sorting of EGFR into ECVs. Indeed, Hrs contains a FYVE domain, is wortmannin sensitive and is involved in EGF down-regulation, presumably by linking ubiquitinated EGFR with endosomal clathrin (Bishop et al., 2002; Lloyd et al., 2002; Raiborg et al., 2002). Hrs overexpression was previously found either to inhibit (Bean et al., 2000) or not inhibit (Raiborg et al., 2001) the recycling pathway, and to interfere with EGFR and dextran transport from early to late endosomes (Raiborg et al., 2001). After low Hrs-myc overexpression to limit problems associated with high overexpression, EGFR remained trapped in early endosomes containing Hrs-myc, and failed to reach late endosomes (Fig. 6 B, and quantification in A), in agreement with Raiborg et al. (2001). Dextran, however, did not accumulate in Hrs-myc–labeled early endosomes and reached late endosomes containing LBPA. This apparent discrepancy with Raiborg et al. (2001), like perhaps the differential effects of Hrs on the recycling pathway (Bean et al., 2000; Raiborg et al., 2001), may well reflect differences in the levels of Hrs overexpression.


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

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

Hrs regulates EGFR sorting, but not bulk transport to late endosomes. (A and B) As in Fig. 3 A and B, control cells (ctrl) or cells expressing either GFP-2xFYVE (FYVE) or myc-Hrs were labeled with EGF-biotin/streptavidin-phycoerythrin or dextran, except that the marker was endocytosed for 45 min followed by a 60-min chase, and analyzed by triple channel fluorescence microscopy (B). In A, the colocalization of dextran or EGF with the early endosomal marker (EE, GFP-2xFYVE or myc-Hrs) or the late endosomal marker (LE, LBPA) was quantified as in Fig. 3 C. (C) ECV formation in vitro was measured with HRP as a marker, as in Fig. 4, using cytosol depleted of Hrs by immunoprecipitation (Hrs IP), or mock-treated with a control anti-Rab7 antibody (ctrl IP). (D) These cytosols (after Hrs immunoprecipitation or control immunoprecipitation, as in C) were analyzed by SDS electrophoresis and Western blotting with anti-Hrs (αHrs) antibodies. (E) The incorporation of GFP-EGFR into ECVs was followed as in Fig. 5, using Hrs-depleted or mock-treated (Rab7) cytosol (as in C). GFP-EGFR was analyzed in donor membranes (EE; 50% of the donor membranes) and vesicles (whole fraction) formed in vitro (vesicles) by Western blotting with anti-GFP antibodies. The lanes were scanned, and the quantification is shown in C, as a percentage of the total amounts of GFP-EGFR in the vesicle fraction of controls. Bar, 2.5 μm.
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fig6: Hrs regulates EGFR sorting, but not bulk transport to late endosomes. (A and B) As in Fig. 3 A and B, control cells (ctrl) or cells expressing either GFP-2xFYVE (FYVE) or myc-Hrs were labeled with EGF-biotin/streptavidin-phycoerythrin or dextran, except that the marker was endocytosed for 45 min followed by a 60-min chase, and analyzed by triple channel fluorescence microscopy (B). In A, the colocalization of dextran or EGF with the early endosomal marker (EE, GFP-2xFYVE or myc-Hrs) or the late endosomal marker (LE, LBPA) was quantified as in Fig. 3 C. (C) ECV formation in vitro was measured with HRP as a marker, as in Fig. 4, using cytosol depleted of Hrs by immunoprecipitation (Hrs IP), or mock-treated with a control anti-Rab7 antibody (ctrl IP). (D) These cytosols (after Hrs immunoprecipitation or control immunoprecipitation, as in C) were analyzed by SDS electrophoresis and Western blotting with anti-Hrs (αHrs) antibodies. (E) The incorporation of GFP-EGFR into ECVs was followed as in Fig. 5, using Hrs-depleted or mock-treated (Rab7) cytosol (as in C). GFP-EGFR was analyzed in donor membranes (EE; 50% of the donor membranes) and vesicles (whole fraction) formed in vitro (vesicles) by Western blotting with anti-GFP antibodies. The lanes were scanned, and the quantification is shown in C, as a percentage of the total amounts of GFP-EGFR in the vesicle fraction of controls. Bar, 2.5 μm.
Mentions: Hrs is likely to mediate the PI3P-dependent sorting of EGFR into ECVs. Indeed, Hrs contains a FYVE domain, is wortmannin sensitive and is involved in EGF down-regulation, presumably by linking ubiquitinated EGFR with endosomal clathrin (Bishop et al., 2002; Lloyd et al., 2002; Raiborg et al., 2002). Hrs overexpression was previously found either to inhibit (Bean et al., 2000) or not inhibit (Raiborg et al., 2001) the recycling pathway, and to interfere with EGFR and dextran transport from early to late endosomes (Raiborg et al., 2001). After low Hrs-myc overexpression to limit problems associated with high overexpression, EGFR remained trapped in early endosomes containing Hrs-myc, and failed to reach late endosomes (Fig. 6 B, and quantification in A), in agreement with Raiborg et al. (2001). Dextran, however, did not accumulate in Hrs-myc–labeled early endosomes and reached late endosomes containing LBPA. This apparent discrepancy with Raiborg et al. (2001), like perhaps the differential effects of Hrs on the recycling pathway (Bean et al., 2000; Raiborg et al., 2001), may well reflect differences in the levels of Hrs overexpression.

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