Figure 2.

Grb2 with mutations in both SH3 domains (d.n. Grb2) inhibits endocytosis of EGF, but not Tf. (A) Hep2 cells transiently transfected with d.n. Grb2 were incubated with EGF (60 ng/ml) for 60 min on ice and prepared for immuno-EM. Thawed cryosections were double-labeled for EGFR (15-nm gold particles) and d.n. Grb2 (rabbit anti-Myc; 10-nm gold particles). Labeling for EGFR (large arrows) was restricted to smooth plasma membrane areas, no labeling was found in flat or invaginated coated plasma membrane areas (coat indicated by small arrows). (B–H) HeLa cells transiently transfected with d.n. Grb2 were incubated with EGF (60 ng/ml) for 10 min at 37°C before preparation for immuno-EM. Thawed cryosections were double-labeled for EGF (B–E) or TfR (F–H; 15-nm gold particles) and d.n. Grb2 (mouse anti-Myc; 5-nm gold particles, arrowheads). In nontransfected cells (B) the EGF-EGFR complex was efficiently endocytosed, and labeling for EGF was found in multivesicular endosomes. In transfected cells (C–H) labeling for EGF (large arrows) was restricted to smooth areas of the plasma membrane (C–E). No labeling was found in coated pits (D) or in endosomes (E). Endocytosis of TfR (F–H) was not inhibited by overexpression of d.n. Grb2, and labeling for TfR was found both in shallow (F) and fully invaginated coated pits (G) as well as in coated vesicles (H). Bars, 100 nm.