Endocytosis of Functional Epidermal Growth Factor Receptor-Green Fluorescent Protein Chimera

Abstract

A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible.

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

“…To this end, a fusion protein of mRFP and EGFR was prepared and transiently expressed in COS-1 cells. The pattern of subcellular distribution of EGFR-mRFP was similar to that previously observed in cells transiently expressing EGFR-GFP 27 . Western-blot analysis demonstrated correct size and EGF-dependent tyrosine phosphorylation of EGFR-mRFP ( Supplementary Fig.…”

Adaptor protein Shc undergoes translocation and mediates up‐regulation of the tyrosine kinase c‐Src in EGF‐stimulated A431 cells

“…4B,C). It is known that activated EGFR undergoes endocytosis involving dynamin-regulated and clathrincoated endosomes (Vieira et al 1996;Carter & Sorkin 1998;Wilde et al 1999). Thus, we conclude that the cytoplasmic vesicle structures containing both GFP-P52 and EGFR are endosomes.…”

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

“…Firstly, we reduced the cross-correlation amount by coexpressing wild-type EGFR alongside the labeled receptor. Secondly, we performed measurements in HEK293 and COS-7 cell lines, which express low to intermediate levels of endogenous EGFR (HEK293~20,000 EGFR/cell (36) and COS-7 100,000 EGFR/cell (35)). The cross-correlation amounts were low to negligible in cells expressing <200 labeled receptors per mm 2 .…”

“…To investigate the influence of endogenous receptors, we used three different cell lines. Experiments were performed in CHO-K1 cells, a cell line selected for its lack of endogenous EGFR, and on two lines of fibroblasts that express endogenously intermediate and low levels of EGFR (COS-7~100,000 EGFR/cell (35) and HEK-293~20,000 EGFR/cell (36)). To modulate EGFR dimerization, we performed experiments with highand low-level stimulation by EGF, with the dimer-deficient EGFR mutant (37), and with transiently coexpressed unlabeled receptors.…”