Document Detail

Dynamic conformational transitions of the EGF receptor in living mammalian cells determined by FRET and fluorescence lifetime imaging microscopy.
MedLine Citation:
PMID:  23839800     Owner:  NLM     Status:  Publisher    
We have revealed a reorientation of ectodomain I of the epidermal growth factor receptor (EGFR; ErbB1; Her1) in living CHO cells expressing the receptor, upon binding of the native ligand EGF. The state of the unliganded, nonactivated EGFR was compared to that exhibited after ligand addition in the presence of a kinase inhibitor that prevents endocytosis but does not interfere with binding or the ensuing conformational rearrangements. To perform these experiments, we constructed a transgene EGFR with an acyl carrier protein sequence between the signal peptide and the EGFR mature protein sequence. This protein, which behaves similarly to wild-type EGFR with respect to EGF binding, activation, and internalization, can be labeled at a specific serine in the acyl carrier tag with a fluorophore incorporated into a 4'-phosphopantetheine (P-pant) conjugate transferred enzymatically from the corresponding CoA derivative. By measuring Förster resonance energy transfer between a molecule of Atto390 covalently attached to EGFR in this manner and a novel lipid probe NR12S distributed exclusively in the outer leaflet of the plasma membrane, we determined the apparent relative separation of ectodomain I from the membrane under nonactivating and activating conditions. The data indicate that the unliganded domain I of the EGFR receptor is situated much closer to the membrane before EGF addition, supporting the model of a self-inhibited configuration of the inactive receptor in quiescent cells. © 2013 International Society for Advancement of Cytometry.
Iwona Ziomkiewicz; Anastasia Loman; Reinhard Klement; Cornelia Fritsch; Andrey S Klymchenko; Gertrude Bunt; Thomas M Jovin; Donna J Arndt-Jovin
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-7-9
Journal Detail:
Title:  Cytometry. Part A : the journal of the International Society for Analytical Cytology     Volume:  -     ISSN:  1552-4930     ISO Abbreviation:  Cytometry A     Publication Date:  2013 Jul 
Date Detail:
Created Date:  2013-7-10     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101235694     Medline TA:  Cytometry A     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
© 2013 International Society for Advancement of Cytometry.
Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
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