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First published online 2 September 2003
doi: 10.1242/jcs.00723

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The UIM domain of Hrs couples receptor sorting to vesicle formation

¹ Physiological Laboratory, University of Liverpool, Crown St., Liverpool L69 3BX, UK
² Department of Cell Biology, University Medical Center Utrecht and Institute of Biomembranes, 3584 CX Utrecht, The Netherlands
³ Center for Biomedical Genetics, PO Box 80042, 3508 TA Utrecht, The Netherlands
⁴ Department of Cell Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz, 18a, Martinsried, 82152 Germany

View larger version (37K): [in a new window]   Fig. 1. Tyrosine phosphorylation of Hrs in response to EGF. (A) An antibody directed against an Hrs peptide containing phospho-Y334 recognizes Hrs specifically in response to EGF stimulation. HeLa cells were starved for 16 hours in serum-free medium and either not stimulated or stimulated for 8 minutes with EGF (100 ng/ml). Lysates were analysed by immunoblotting with anti-PY334-Hrs antibody. Molecular mass markers are indicated. (B,C) EGF-dependent tyrosine phosphorylation of Hrs is dependent on the presence of either Y329 or Y334 as well as on an intact UIM domain. (B) HeLa cells were transfected with GFP-Hrs, GFP Y329F, GFP-Y334F, GFP-Y329/334F, GFP-UIM, GFP-LSAA or mock-transfected, starved 16 hours in serum-free medium and then stimulated 22 hours post-transfection with EGF (100 ng/ml). Lysates were prepared and subjected for immunoprecipitation with anti-GFP antibody. Phosphorylation was assessed by immunoblotting with PY20 antibody. (C) Lysates were prepared as described in b and analysed by immunoblotting with anti-GFP and anti-PY334-Hrs antibodies respectively. The arrowhead indicates GFP-PY334-Hrs, the arrow indicates endogenous PY334-Hrs. (D) PY334-Hrs is enriched in the cytosol. HeLa cells were starved for 16 hours in serum-free medium and stimulated for 8 minutes with EGF (100 ng/ml) or left unstimulated. Membrane and cytosol fractions were prepared as described in the Materials and Methods. The relative distribution of tyrosine phosphorylated Hrs was assessed by analysing equal proportions of membrane and cytosolic fractions by immunoblotting with anti-Hrs and anti-PY334-Hrs antibodies. (E) Membrane and cytosol fractions were prepared from cells stimulated for 8 minutes with EGF, and the specific enrichment of Hrs phosphorylated at Y334 was analysed by loading four times more membrane fraction than cytosol fraction on SDS-PAGE followed by immunoblotting with anti-Hrs and anti-PY334-Hrs antibodies as in D.

View larger version (61K): [in a new window]   Fig. 2. Ubiquitination of overexpressed Hrs is constitutive and independent of tyrosine phosphorylation. (A) HeLa cells were transfected with GFP-Hrs, GFP-Y329F, GFP-Y334F, GFPY329/334F or GFP-UIM, starved for 16 hours in serum-free medium and then stimulated 22 hours post-transfection for 8 minutes with EGF (100 ng/ml) or left unstimulated. Lysates were prepared in the presence or absence of 10 mM NEM and subjected to immunoprecipitation with anti-GFP antibody. Ubiquitination was assessed by immunoblotting with anti-ubiquitin antibody. Molecular mass markers are indicated on the left. (B) HeLa cells were transfected with GFP-Hrs, GFP-Y329F, GFP-Y334F, GFPY329/334F, GFP-LSAA, GFP-UIM or mock transfected, and treated as in A. Lysates were prepared according to the `boiling SDS-lysis' method (see Materials and Methods) and subjected to immunoprecipitation with anti-GFP antibody.

View larger version (97K): [in a new window]   Fig. 3. Immunolocalisation of GFP-Hrs, GFP-Y329/334F and GFP-UIM. HeLa cells were transfected with GFP-Hrs (A,D), GFPY329/334F (B,E) or GFP-UIM (C,F) and fixed 22 hrs post-transfection with 3% PFA, permeabilised with Triton X-100 and stained with either anti-transferrin receptor (TrfR, A-C, shown in red) or anti-mannose-6-phosphate receptor (Man6PR, D-F, shown in red) antibodies followed by secondary antibodies coupled to Alexa Fuor 594. All panels show a single confocal section of a group of cells presenting the most typical staining pattern for each construct. Insets show a threefold enlargement of selected areas. Scale bars: 20 µm.

View larger version (108K): [in a new window]   Fig. 4. Colocalisation of Hrs with activated EGFR does not depend on tyrosine phosphorylation of Hrs or on an intact UIM domain. HeLa cells were transfected with GFP-Hrs (A-C), GFP-Y329/334F (D-F) or GFP-UIM (G-I), starved 16 hours in serum-free medium and stimulated 22 hours post-transfection for 8 minutes with EGF (100 ng/ml). The cells were then fixed with 3% PFA, permeabilised with Triton X-100 and stained with anti-EGFR (EGFR shown in red) followed by secondary antibody coupled to Alexa Fluor 594. All panels show a single confocal section of low-expressing cells. Insets show a threefold enlargement of selected areas. Scale bars: 10 µm.

View larger version (92K): [in a new window]   Fig. 5. EGFR downregulation is inhibited by over-expression of Hrs independently of tyrosine phosphorylation. HeLa cells were transfected with GFP-Hrs (A-C), GFP-Y329/334F (D-F) or GFP-UIM (G-I), starved for 16 hours in serum-free medium and treated 22 hrs post-transfection for 4 hours with EGF (100 ng/ml). The cells were then fixed with 3% PFA, permeabilised with Triton X-100 and stained with anti-EGFR (EGFR shown in red) followed by secondary antibody coupled to Alexa Fuor 594. All panels show a single confocal section of cells presenting the most typical staining pattern for each construct. Scale bars: 20 µm.

View larger version (65K): [in a new window]   Fig. 6. Hrs-Y329/334F and Hrs-UIM are present in clathrin coats on endosomes. HeLa cells were transfected with GFP-Hrs, GFPY329/334F or GFP-UIM, processed for immunogold-labelling on cryosections as described in Materials and Methods. Cryosections were double labeled for GFP (10 nm gold) and clathrin (15 nm gold). (A-C) Overexpression of GFP-Hrs, GFP-Y329/334F and GFP-UIM leads to an increased recruitment of clathrin to endosomal vacuoles (E). All three constructs colocalise with clathrin on the peripheral endosomal membrane but label is absent from tubular extensions that emerge from the vacuole (arrows in B and not shown). (C) In contrast to A and B, cells transfected with GFP-UIM show GFP labelling at the limiting membrane as well as on internal vesicles of endosomal vacuoles. PM, plasma membrane. Scale bars: 200 nm.

View larger version (176K): [in a new window]   Fig. 7. The UIM domain of Hrs is required for efficient retention of the EGFR at the limiting membrane of the early endosome. HeLa cells were transfected with GFP-Hrs (B), GFP-Y329/334F (C) or GFP-UIM (D), or mock-transfected (A), starved 16 hours in serum-free medium and stimulated 22 hrs post-transfection for 30 minutes with EGF. Cells were processed for immunogold-labelling on cryosections as described in Materials and methods and cryosections were double-labeled for GFP (10 nm gold) and EGFR (15 nm gold). (A) In control cells, EGFR is mainly localized on internal vesicles of endosomal vacuoles. (B) In cells overexpressing GFP-Hrs, EGFR is retained in the limiting membrane of endosomal vacuoles (arrows). (C) In HeLa cells expressing GFP-Hrs Y329/334F, EGFR is retained at the limiting membrane of endosomal vacuoles (arrows). (D) By contrast, in cells transfected with GFP-Hrs-UIM, EGFR is localized at the limiting membrane (arrows) as well as on internal vesicles (arrowheads). E, endosomal vacuole; N, nucleus; PM, plasma membrane. Scale bars: 200 nm.

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