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Figure 7


Fig. 7. Clathrin binding by Hrs is important for EGF degradation. (A,B) For EGF degradation assay, HEp-2 cells were treated with Hrs-specific siRNA oligonucleotides and at the same time transiently transfected with (A) siRNA-resistant myc-Hrswt or (B) myc-Hrs{Delta}CB. The cells were stimulated with 100 ng/ml Rhodamine-labelled EGF for 15 minutes, washed and incubated for 3 hours to allow degradation of internalised EGF. The cells were then prepared for confocal immunofluorescence microscopy and labelled with anti-Hrs and anti-myc (not shown) antibodies. (A) Hrs-depleted cells transfected with myc-Hrswt showed normal degradation of EGF (arrowheads) compared with Hrs-depleted neighbouring cells, where the degradation was inhibited. (B) Hrs-depleted cells transfected with myc-Hrs{Delta}CB accumulated EGF (arrowheads) similar to Hrs-depleted neighbouring cells. White lines indicate the cell borders. Bar, 10 µm. Arrows point at endosomes where myc-Hrs{Delta}CB and EGF colocalise. (C) The amount of EGF remaining after 3 hours of chase was quantified as described in Materials and Methods and represented as percentage of total amount of internalised EGF after 15 minutes. Control cells were treated with scrambled RNA. Error bars give the mean + s.e.m. from four independent experiments. Control, n=33 cells; siRNA, n=96; siRNA + myc-Hrswt, n=73; siRNA + myc-Hrs{Delta}CB, n=89.





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