MET receptor tyrosine kinase activation studied at the single-molecule level
The receptor tyrosine kinase MET is a central player in vertebrate development, wound healing, and tissue regeneration. MET mediates these responses by activating different signaling pathways including signal transduction via the mitogen-activated protein kinase (MAPK) and the phosphoinositide 3-kinase (PI3K) pathways. Next to its important physiological function, MET is involved in numerous diseases. In cancer, MET was reported to be aberrantly expressed leading to constitutively active receptors inducing tumor growth and metastasis. MET is also exploited by pathogenic bacteria during infection, e.g. Listeria monocytogenes, the causative agent of listeriosis. L. monocytogenes induces its uptake via proteins of the internalin family and MET is one of their targets.
We use single-molecule techniques to study the initial processes occurring directly on the membrane upon activation of MET by its bacterial ligand internalin B (InlB). Single-molecule photobleaching microscopy reveals MET receptor dimerization upon activation with InlB as well as pre-formed dimers in the absence of activating ligands (Figure 1). We established a method to measure ligand affinities directly on cells, and determined the binding constant of InlB321 to MET (Figure 2). With single-molecule tracking in live cells, we studied MET mobility and its changes following ligand binding as well as its link to different endocytosis pathways (Figure 3).
Harwardt, M.-L. I. E., Young, P., Bleymüller, W. M., Meyer, T., Karathanasis, C., Niemann, H. H., Heilemann, M. & Dietz, M. S. (2017) Membrane dynamics of resting and InlB-bound MET receptor tyrosine kinase studied by single-molecule tracking. FEBS Open Bio. doi: .
Volz, Y., Koschut, D., Matzke-Ogi, A., Dietz, M. S., Karathanasis, C., Richert, L., Wagner, M. G., Mély, Y., Heilemann, M., Niemann, H. H. & Orian-Rousseau, V. (2015) Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6. Bioscience Reports. 35 (4), e00236.
Dietz, M. S., Fricke, F., Krüger, C. L., Niemann, H. H. & Heilemann, M. (2014) Receptor–ligand interactions: Binding affinities studied by single-molecule and super-resolution microscopy on intact cells. ChemPhysChem. 15 (4), 671.
Dietz, M. S., Haße, D., Ferraris, D. M., Göhler, A., Niemann, H. H. & Heilemann, M. (2013) Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells. BMC Biophysics 6, 6.