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Titolo:
OLIGOMERIZATION OF EPIDERMAL GROWTH-FACTOR RECEPTORS ON A431 CELLS STUDIED BY TIME-RESOLVED FLUORESCENCE IMAGING MICROSCOPY - A STEREOCHEMICAL MODEL FOR TYROSINE KINASE RECEPTOR ACTIVATION
Autore:
GADELLA TWJ; JOVIN TM;
Indirizzi:
MAX PLANCK INST BIOPHYS CHEM,DEPT MOLEC BIOL,POSTFACH 2841 D-37018 GOTTINGEN GERMANY MAX PLANCK INST BIOPHYS CHEM,DEPT MOLEC BIOL D-37018 GOTTINGEN GERMANY
Titolo Testata:
The Journal of cell biology
fascicolo: 6, volume: 129, anno: 1995,
pagine: 1543 - 1558
SICI:
0021-9525(1995)129:6<1543:OOEGRO>2.0.ZU;2-1
Fonte:
ISI
Lingua:
ENG
Soggetto:
RESONANCE ENERGY-TRANSFER; HIGH-AFFINITY RECEPTORS; SIGNAL TRANSDUCTION; CROSS-LINKING; DIRECT VISUALIZATION; PLASMA-MEMBRANES; MEDIATED ENDOCYTOSIS; CARCINOMA-CELLS; FACTOR BINDING; LIVING CELLS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
61
Recensione:
Indirizzi per estratti:
Citazione:
T.W.J. Gadella e T.M. Jovin, "OLIGOMERIZATION OF EPIDERMAL GROWTH-FACTOR RECEPTORS ON A431 CELLS STUDIED BY TIME-RESOLVED FLUORESCENCE IMAGING MICROSCOPY - A STEREOCHEMICAL MODEL FOR TYROSINE KINASE RECEPTOR ACTIVATION", The Journal of cell biology, 129(6), 1995, pp. 1543-1558

Abstract

The aggregation states of the epidermal growth factor receptor (EGFR)on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to thecells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). Theaverage FRET efficiency increased dramatically to 28% at 4 degrees C,indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance withprior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431cells are present in a predimerized or oligomerized state. We proposethat the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.

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Documento generato il 28/03/20 alle ore 22:50:19