Hrs mediates downregulation of multiple signalling receptors in Drosophila - PubMed (original) (raw)

Hrs mediates downregulation of multiple signalling receptors in Drosophila

Gáspár Jékely et al. EMBO Rep. 2003 Dec.

Abstract

Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-beta signalling. We find that Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp (TGF-beta related). Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal.

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Figures

Figure 1

Figure 1

Accumulation of ubiquitinated proteins in hrs mutant cells. Ubiquitinated proteins accumulate intracellularly in hrs mutant cells. Mosaic egg chambers and wing discs were stained with an antibody against ubiquitinated proteins (red). (A) Schematic diagram of a transverse section through the oocyte and the follicle cells, as shown in (B). (C) Optical section in the plane of the follicular epithelium.hrs mutant cells are marked by the absence of GFP (green) in both ovarian follicle cells (B, C) and wing discs (D). Some ubiquitinated proteins appear to be at the cell cortex (arrows in (C)). Arrowheads in (B) indicate the boundary between mutant and wild-type cells.

Figure 2

Figure 2

Ubiquitinated proteins accumulate in enlarged endosomes in_hrs_ mutant cells. Egg chambers expressing GFP-2xFYVE (A, C) or GFP-Rab5 (B) (green) and carrying patches of hrs mutant follicle cells were stained with an antibody against ubiquitinated proteins (red). In (A) and (B) all cells shown are hrs mutant, and in (C) the boundary between mutant and wild-type cells is indicated with a dashed line. hrs mutant cells can be detected by distinctive staining with the ubiquitinated protein antibody. Note the increased staining with the endosomal marker (FYVE) in mutant cells relative to neighbouring cells. Phalloidin-stained F-actin (blue) outlines cells in the overlay to the right.

Figure 3

Figure 3

Colocalization of signalling receptors and ubiquitinated proteins in_hrs_ mutant cells. Egg chambers with patches of hrs mutant follicle cells were stained with an antibody against ubiquitinated protein (green) and antibodies against the following specific proteins (red): PVR (A), EGFR (B), Ptc (C), Smo (D), Tkv (E), Notch (F) and DE-cadherin (G). Notch could not be analysed for colocalization with the ubiquitinated protein due to antibody incompatibility. Instead, labelled phalloidin (green) is used to mark cell outlines in (F) and (G). The overlap between the signals is yellow in the merged images (right). The boundary between hrs mutant and wild-type cells is indicated with arrowheads (A) or with dashed lines (B–G). Mutant cells are marked by the absence of GFP (blue) in the merged images. (A, D, F, G) Similar transverse sections through the egg chamber, with the apical side of the follicle cells towards the oocyte (bottom of image). (B, C,E) More oblique sections through the follicular epithelium.

Figure 4

Figure 4

Role of Hrs in Dpp signalling and Tkv trafficking in follicle cells. (A) Schematic cross-section of stage 10 egg chamber. A small and a large stippled box indicate areas shown in (B, C) and (D,G, H), respectively. (D, H) Surface views of the epithelium, as are (E) and (F). Anterior follicle cells (red in (A)) express Dpp and Dpp-lacZ (see also (B)). P-MAD staining visualizes Dpp signalling activity in the cells expressing Dpp and in adjacent cells (C). The Dpp receptor Tkv is detected in all follicle cells**(D). Wild-type (C, D) and hrs mosaic (E–H**) stage 10 egg chambers stained with antibodies (red in double-staining) against β-galactosidase (B), P-MAD (C,E, F) or Tkv (D, G, H). (B) and (C) are also stained with phalloidin (green). Mutant cells are marked by the absence of GFP (blue) in (E–H). The boundary between_hrs_ mutant and wild-type cells is also indicated with an arrowhead (G) or a dashed line (H). In (E) no follicle cells are mutant, and in (F) all follicle cells are mutant for hrs. The double-headed arrow (E, F) indicates the P-MAD positive domain, which is expanded when cells are mutant for hrs.

Figure 5

Figure 5

Dpp target gene expression is increased in hrs mutant wing disc cells. Wild-type (A) and hrs mosaic (B, C) wing discs were stained with an antibody against Spalt (red in the merged images). hrs mutant cells are marked by the absence of GFP (green). Dpp is expressed in the middle of the disc and induces Spalt expression in a broad domain. Note the slight increase in Spalt expression in hrs mutant cells within the normal expression domain (B) and the expansion of the Spalt expression domain when hrs mutant cells are at the edge of this domain ((C), most clear in the middle of the disc, to the right).

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References

    1. Bilodeau P.S., Urbanowski J.L., Winistorfer S.C. & Piper R.C. ( 2002) The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting. Nat. Cell Biol., 4, 534–539. - PubMed
    1. Capdevila J., Estrada M.P., Sanchez-Herrero E. & Guerrero I. ( 1994) The Drosophila segment polarity gene patched interacts with decapentaplegic in wing development. EMBO J., 13, 71–82. - PMC - PubMed
    1. Christoforidis S., Miaczynska M., Ashman K., Wilm M., Zhao L., Yip S.C., Waterfield M.D., Backer J.M. & Zerial M. ( 1999) Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat. Cell Biol., 1, 249–252. - PubMed
    1. Denef N., Neubuser D., Perez L. & Cohen S.M. ( 2000) Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell, 102, 521–531. - PubMed
    1. Duchek P., Somogyi K., Jékely G., Beccari S. & Rørth P. ( 2001) Guidance of cell migration by the Drosophila PDGF/VEGF receptor. Cell, 107, 17–26. - PubMed

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