Monitoring protein-protein interactions in live mammalian cells by beta-galactosidase complementation.

Publisher Summary Conditional protein–protein interactions are critical to a vast number of intracellular regulatory mechanisms. A case in point is the activation and autophosphorylation of growth factor receptors, which are often determined by the ligand-dependent induction of receptor dimerization. Docking sites on the activated receptor become accessible to downstream components of the relevant signaling pathways, thereby leading to further protein–protein interactions. Although efficient genetic methods to identify interacting partners exist and have been successfully applied by a number of laboratories, a technique that allows the monitoring of interactions in real time in the cellular compartment in which they normally take place would extend the types of interactions that could be studied. The β -galactosidase ( β -Gal)-based intracistronic complementation methodology described in the chapter is the first technology that fulfills these requirements and can be applied to live mammalian cells. The novel β -Gal complementation assay for monitoring protein–protein interactions in intact mammalian cells has many advantages. Unlike the yeast two-hybrid assay, this assay is independent of transcription. Instead, protein interactions are monitored directly in situ . Because it is based on the detection of an enzymatic activity, it provides amplification of the signal.