[en] Receptor tyrosine kinases (RTKs) are transmembrane receptors activated by a wide diversity of growth factors, cytokines or hormones. They ensure multiple roles in cellular processes, including proliferation, differentiation and survival. They are also crucial drivers of development and progression of multiple cancer types, and represent important drug targets. Generally, ligand binding induces dimerization of RTK monomers, which induces auto-/transphosphorylation of tyrosine residues on the intracellular tails leading to the recruitment of adaptor proteins and modifying enzymes to promote and modulate various downstream signaling pathways. This chapter details easy, rapid, sensitive and versatile methods based on split Nanoluciferase complementation technology (NanoBiT) to monitor activation and modulation of two models of RTKs (EGFR and AXL) through the measurement of their dimerization and the recruitment of the adaptor protein Grb2 (SH2 domain-containing growth factor receptor-bound protein 2) and the receptor-modifying enzyme, the ubiquitin ligase Cbl.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Dosquet, Hugo; Department of Cancer Research, NORLUX Neuro-Oncology Laboratory, Luxembourg Institute of Health (LIH), Luxembourg City, Luxembourg
Neirinckx, Virginie ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques
Meyrath, Max; Department of Infection and Immunity, Immunopharmacology and Interactomics, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg
Wantz, May; Department of Infection and Immunity, Immunopharmacology and Interactomics, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg
Haan, Serge; Faculty of Science, Technology and Medicine, Department of Life Sciences and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg
Niclou, Simone P; Department of Cancer Research, NORLUX Neuro-Oncology Laboratory, Luxembourg Institute of Health (LIH), Luxembourg City, Luxembourg, Department of Biomedicine, University of Bergen, Bergen, Norway
Szpakowska, Martyna; Department of Infection and Immunity, Immunopharmacology and Interactomics, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg
Chevigné, Andy; Department of Infection and Immunity, Immunopharmacology and Interactomics, Luxembourg Institute of Health (LIH), Esch-sur-Alzette, Luxembourg. Electronic address: andy.chevigne@lih.lu
Language :
English
Title :
Nanoluciferase-based complementation assays to monitor activation, modulation and signaling of receptor tyrosine kinases (RTKs).
This study was supported by the Luxembourg Institute of Health (LIH), Luxembourg National Research Fund (PRIDE 15/10675146/ “CANBIO”; PRIDE-16749720 “NextImmune2”, Pathfinder “Interceptor” 19/14260467, INTER/FWO “Nanokine” grant 15/10358798, INTER/FNRS grants 20/15084569, and PoC “Megakine” 19/14209621), F.R.S.-FNRS-Télévie (grants 7.4529.19, 7.8504.20, 7.4502.21 and 7.8508.22) and Foundation Cancer Luxembourg (PAN-RTK). AC and MS are part of the Marie Skłodowska-Curie Innovative Training Network ONCORNET2.0 “ONCOgenic Receptor Network of Excellence and Training” (MSCA-ITN-2020-ETN). The authors wish to thank Manuel Counson, Nadia Beaupain and Jean-Marc Plesseria for technical help and support. Figures were created with BioRender.com.
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