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First published online 7 April 2009
doi: 10.1242/jcs.040030

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Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots

Joshua Z. Rappoport^* and Sanford M. Simon

The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA

View larger version (54K): [in this window] [in a new window]   Fig. 1. EGFR does not reside in caveolae prior to EGF stimulation. (A) TIR-FM and (B) quantification demonstrates that prior to addition of EGF, EGFR does not colocalize with caveolin 1. Fluorescence of EGFR at sites of caveolae is nearly identical to the intensity outside caveolin 1 spots.

View larger version (73K): [in this window] [in a new window]   Fig. 2. Activated EGFR colocalizes with clathrin, and not caveolin 1. (A) EGFR-EGFP and clathrin-dsRed, or caveolin 1-mRFP, were imaged by simultaneous two-color TIR-FM 10 minutes after addition of EGF. Colocalization of EGFR and clathrin appears yellow in overlay. (B,C) Quantification demonstrates colocalization of clathrin at the sites of EGFR clusters (B) but not caveolin 1 (C).

View larger version (42K): [in this window] [in a new window]   Fig. 3. Activated EGFR predominantly clusters at sites of preformed clathrin. (A) EGFR-EGFP and clathrin-dsRed were imaged by simultaneous two-color TIR-FM. Colocalization between clathrin imaged prior to addition of EGF (red), and EGFR observed at 10 minutes after stimulation (green) appears yellow in the overlay. (B) The number of clathrin spots on the cell surface counted before and 10 minutes after EGF addition is almost the same. (C) The percentage of EGFR clusters that formed at sites where clathrin was present prior to EGF addition was compared with the percentage that appeared where clathrin was not identified at time 0. (D) The clathrin and EGFR fluorescence intensities were logged every 2 minutes following EGF addition at the sites of EGFR clusters that colocalized with preformed clathrin.

View larger version (91K): [in this window] [in a new window]   Fig. 4. The AP-2 complex is involved in the trafficking of activated EGFR. (A) TIR-FM comparing cells transfected with siRNA against -adaptin demonstrates reduction of transferrin (Tf) uptake as well as apparent effects on EGFR distribution. Scale bars shown in image in upper left of (A) can be applied to all images in figure. (B) Western blot probed with anti--adaptin, as well as control anti-GAPDH, reveals potent and specific silencing. (C) Cells transfected with siRNA, demonstrating reductions in transferrin uptake, do not show alterations in the number of EGFR spots but do show (D) a 40% reduction in EGF spot intensity relative to local background.

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