Live cell imaging shows hepatocyte growth factor-induced Met dimerization.
The canonical model of receptor tyrosine kinase (RTK) activation assumes that ligand-induced dimerization of inactive receptor monomers is a prerequisite for autophosphorylation. For several RTK families, recent results of fluorescence microscopy provided evidence for preformed receptor dimers that may or may not require ligand binding for kinase activity. Here we report, for the first time, the application of advanced quantitative fluorescence microscopy techniques to study changes in the oligomerization state of the RTK Met in response to stimulation by its endogenous ligand hepatocyte growth factor (HGF). We used inducible C-terminal fusions between Met and enhanced green fluorescent protein (EGFP) or red fluorescent protein (RFP) in combination with fluorescence resonance energy transfer (FRET)-based fluorescence-lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS). A small fraction of HGF-independent Met dimers appeared to be present in cells even at low receptor density. At high receptor density, both the fraction of Met dimers and the level of Met autophosphorylation increased in the absence of HGF. Stimulation with HGF at low receptor density significantly increased the fraction of Met dimers on live cells. We found no indications of Met oligomers larger than dimers. Our findings thus confirm a model of Met activation through HGF-induced dimerization and at the same time they support previous reports of Met dimers in unstimulated cells. The tools established in this work will be useful to further characterize the mechanism of Met activation and to define the contribution of co-receptors.
Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Postfach 3640, 76021 Karlsruhe, Germany.
David
D
Koschut
UMR 7213 CNRS, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie 74 route du Rhin, 67401 Illkirch, France.
Ludovic
L
Richert
Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Postfach 3640, 76021 Karlsruhe, Germany.
Giuseppina
G
Pace
Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
Hartmut H
HH
Niemann
UMR 7213 CNRS, Laboratoire de Biophotonique et Pharmacologie, Faculté de Pharmacie 74 route du Rhin, 67401 Illkirch, France.
Yves
Y
Mély
Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics (ITG), Postfach 3640, 76021 Karlsruhe, Germany. Electronic address: veronique.orian-rousseau@kit.edu.
Véronique
V
Orian-Rousseau
D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D036641:Microscopy, Fluorescence, Multiphoton; D011485:Protein Binding; D055503:Protein Multimerization; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D017228:Hepatocyte Growth Factor; D008024:Ligands; D008164:Luminescent Proteins; D019859:Proto-Oncogene Proteins c-met; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C066855:HGF protein, human; C485184:enhanced green fluorescent protein; C491743:MET protein, human
FRET; Fluorescence correlation spectroscopy; RTK activation; Receptor dimerization; Transmembrane signaling