Identification of cell signaling molecules by expression cloning

Using expression cloning one can isolate proteins with specific biological functions. This methodology can be adapted for the identification of novel players in the regulation of cell signaling. Here, we describe an expression cloning strategy to identify suppressors of Ras signaling. This screen is based on the observation that the activation of the small guanosine triphosphate (GTP)-binding protein H-Ras initiates a mitogen-activated protein kinase (MAPK)-dependent signaling pathway that inactivates integrin ligand binding. Our strategy depends on flow cytometry and a monoclonal antibody that recognizes integrin activation states. Flow cytometry enhances the screen's sensitivity thereby allowing us to examine function quantitatively at the level of a single cell millions of times in one screen. The following protocol provides a detailed method for the isolation of proteins that regulate cell signaling.