Etude d ’interactions protéines-protéines in vivo (in cellulo) : Exemple des récepteurs de la famille erbB/HER Pierre Hubert (LISM - IBSM) phubert@ibsm.cnrs-mrs.fr 19/10/04

19/10/04 Douglas Hanahan & Robert A. Weinberg The Hallmarks of Cancer Cell, 100, 57–70, 2000 19/10/04

Récepteurs à activité tyrosine-kinase Human ~ 70 C. elegans ~ 40 Yeast ~ 0 (dual) 19/10/04

Organisation en domaines du récepteur de l ’EGF, et mécanisme d ’activation par trans-autophosphorylation 19/10/04

Dimérisation 19/10/04

> 10 4 Signalisation 19/10/04

Src kinase Interactions Protéines-protéines 19/10/04

PPPVPPRR EYINQ 19/10/04

D Grb2 PH-PTB SH2 EGFR ErbB2 FGFR + egf + anticorps +fgf et héparine Bennasroune et al., Crit. Rev. Oncol. Hematol. 2004, 50, 23-38 19/10/04

dans des myoblastes de souris (C2C12) 1- Dimérisation de récepteurs de l ’EGF par une méthode « double hybride », dans des myoblastes de souris (C2C12) Nature Biotech. 2000, 18, 218 - 222 Epidermal growth factor receptor dimerization monitored in live cells Bruce T. Blakely, Fabio M. V. Rossi, Bonnie Tillotson, Michelle Palmer, Angeles Estelles & Helen M. Blau 19/10/04

Méthode « double-hybride » classique, rappel 19/10/04

Complémentation de mutants de la ß-galactosidase FACS, FDG = fluorescein digalactoside 19/10/04

? Complémentation de mutants de la ß-galactosidase Dimérisation - EGF 19/10/04

Cinétiques, courbes effet-dose 19/10/04

Effets de ≠ anticorps 19/10/04

Rapporteurs fluorescents de phosphorylation 2- FRET I : Rapporteurs fluorescents de phosphorylation (cellules HeLa, NIH 3T3 & B82) PNAS | December 18, 2001 | vol. 98 | no. 26 | 15003-15008 Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells Alice Y. Ting, Kristin H. Kaindagger, Richard L. Klemkedagger, and Roger Y. Tsien 19/10/04

Rappel : Interactions between two proteins can be imaged by detecting fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. FRET is a photophysical phenomenon where energy is transferred non-radiatively from a donor fluorophore to an acceptor fluorophore with an efficiency defined by: where r is the distance between the two fluorophores, and R0 is the distance at which 50% energy transfer takes place (typically 2–6 nm). There are a number of ways to measure FRET in a microscope that can be broadly divided into intensity-based methods and fluorescence decay-kinetics-based methods 19/10/04

Rappel bis : 19/10/04

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Copyright ©2001 by the National Academy of Sciences Fig. 1. General design of ratiometric indicators for monitoring tyrosine kinase activities In vitro 19/10/04

Principe de la microscopie confocale 19/10/04

F. Gaits, K. Hahn, Shedding light on cell signaling: Interpretation of FRET biosensors. Sci. STKE 2003, pe3 (2003) 19/10/04

Localisation IP + WB Réversibilité Fig. 2. B82 cells transfected with the EGFR indicator responded to EGF stimulation with an [approx] 30% increase in yellow to cyan emission ratio (transition from blue to red pseudocolor) Src reporter Localisation IP + WB Réversibilité Egf - + + AG - - + 19/10/04

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Fig. 3. Cellular response of the Crk-based reporter WB Localisation 19/10/04

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Imagerie dynamique de la phosphorylation du récepteur de l ’EGF 3- FRET II (FLIM) : Imagerie dynamique de la phosphorylation du récepteur de l ’EGF (cellules MCF7 et COS7) J. Biol. Chem., 279, 36972-36981, 2004 Imaging Phosphorylation Dynamics of the Epidermal Growth Factor Receptor Martin Offterdinger, Virginie Georget, Andreas Girod, and Philippe I. H. Bastiaens 19/10/04

Overview presenting the described constructs FLAME = Sensors (détecteurs) ratiométriques 19/10/04

Translocation and binding of soluble PTB domain to EGFR-ECFP in MCF7 cells + +pb 19/10/04

4- Fluorescence et transduction du signal : Récepteurs-GFPs et EGF-QD (quantum dots) (cellules CHO et A431) Nature Biotechnology 22, 198 - 203 (2004) Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction Diane S Lidke, Peter Nagy, Rainer Heintzmann, Donna J Arndt-Jovin, Janine N Post, Hernan E Grecco, Elizabeth A Jares-Erijman & Thomas M Jovin 19/10/04

Comparative sizes of common fluorophores and protein carriers used in FRET imaging. Elizabeth A Jares-Erijman1 & Thomas M Jovin Nature Biotech. 2003; 21, 1387 - 1395 19/10/04

Internalisation Phosphorylation = endocytose (transferrine) 200 pM egf-QDs 19/10/04

Cinétiques 19/10/04

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Transport rétrograde des egf-QDs 19/10/04

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Fusion d ’endosomes 19/10/04

Analyse quantitative Binding erbB1 / erbB3 Internalisation masques 19/10/04

Localisation dynamique de complexes protéiques 5- FRET III : Localisation dynamique de complexes protéiques (cellules A431, NIH 3T3, endothéliales B11) Molecular Biology of the Cell, 2002, 13, 1522-1535 Coordinated Traffic of Grb2 and Ras during Epidermal Growth Factor Receptor Endocytosis Visualized in Living Cells Xuejun Jiang, and Alexander Sorkin 19/10/04

Interaction Grb2 / shc Ea = efficacité du transfert d ’énergie 19/10/04

Etc… 19/10/04