A proximity ligation assay using transiently transfected, epitope-tagged proteins: application for in situ detection of dimerized receptor tyrosine kinases.
The development of small molecule and antibody inhibitors targeting the interaction of receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), is of high pharmacological and biological interest. Unfortunately, conventional biochemical techniques using cell or tissue lysates and co-immunoprecipitation experiments to investigate EGFR dimerization are not always conclusive. Here we describe a series of technical and biological validation experiments demonstrating the utility of a proximity ligation assay (PLA)-based methodology for in situ visualization and quantification of ligand-dependent EGFR receptor dimerization in intact cells. Using the PLA approach combined with a universally applicable epitope tagging strategy, we detected EGFR dimers in cells transiently co-expressing FLAG-tagged and MYC-tagged human EGFRs. Our data strongly suggest that PLA can be used to detect ligand-dependent EGFR dimerization and this signal is generated in a protein interaction-based manner, rather than solely due to proximity of target proteins. This application represents a generalized RTK expression strategy for protein-interaction analysis in a transient expression system where antibody epitopes are not known or not unique enough to discriminate between interaction partners. This assay also holds promise as a general RTK dimerization screening tool in tissue specimens to identify potential dimerization inhibitors with clinical relevance.
Gajadhar
Aaron
A
Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital for Sick Children, University of Toronto, Canada.
Guha
Abhijit
A
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D014162:Transfection
D000939:Epitopes; D066246:ErbB Receptors