Endocytosis of Functional Epidermal Growth Factor Receptor-Green Fluorescent Protein Chimera*
- From the Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262
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.
Footnotes
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↵* This work was supported by National Institutes of Health Grant DK 46817 and University of Colorado Health Sciences/Howard Hughes Medical Institute Grant (to A. S.) and Cancer League of Colorado Fellowship (to R. E. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262. Tel.: 303-315-7252; Fax: 303-315-7097; E-mail:alexander.sorkin{at}uchsc.edu.
- Abbreviations:
- EGFR
-
EGF receptor
- GFP
-
green fluorescent protein
- wt
-
wild-type
- EGF
-
epidermal growth factor
- EGF-TR
-
Texas Red-conjugated EGF
- AP-2
-
clathrin adaptor protein complex
- MVE
-
multi-vesicular endosome
- DMEM
-
Dulbecco’s modified Eagle’s media
- TGH
-
Triton X-100-glycerol-Hepes buffer
- PAE
-
porcine aortic endothelial
- Ab
-
antibody
- PAGE
-
polyacrylamide gel electrophoresis
- MAP
-
mitogen-activated protein.
-
- Received August 17, 1998.
- Revision received October 7, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
