Re. Carter et A. Sorkin, Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera, J BIOL CHEM, 273(52), 1998, pp. 35000-35007
A chimera of the epidermal growth factor receptor (EGFR) and green fluoresc
ent protein (GFP) has been engineered by fusing GFP to the carboxyl terminu
s of EGFR. Data are provided to demonstrate that the GFP moiety does not af
fect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at t
yrosine residues in response to EGF and is capable of phosphorylating endog
enous substrates and initiating signaling cascades. EGF-dependent associati
on of the chimeric receptor with the clathrin adaptor protein AP-2, involve
d in endocytosis, and with Shc adaptor protein, which binds in close proxim
ity to the fusion point, is not affected by the GFP moiety. Receptor down-r
egulation and internalization occur at rates similar to those in cells expr
essing wild-type EGFR. Western blot analysis reveals that lysosomal degrada
tion of EGFR-GFP proceeds from the extracellular domain and that GFP is not
preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Tex
as Red-conjugated EGF in living cells using digital deconvolution microscop
y demonstrates the trafficking of ligand-receptor complexes through the ear
ly and multivesicular endosomes followed by segregation of the ligand and r
eceptor at the late stages of endocytosis. Time-lapse optical analysis of t
he early stages of endocytosis reveals localization of EGFR-GFP in the tubu
lar-vesicular endosomal compartments. Rapid dynamics of membrane movement a
nd fusion within these compartments were observed. This approach and the fi
delity of the biochemical properties of the EGFR-GFP demonstrate that real-
time visualization of trafficking and protein interactions of tyrosine kina
se receptors in the presence or absence of the ligand are feasible.