Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera

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.