Dissecting the epidermal growth factor receptor signal transactivation pathway

Interreceptor cross-talk has emerged as a general concept in cellular signaling cascades. Therein epidermal growth factor receptor (EGFR) signal transactivation represents the so far best investigated cross-talk mechanism comprising heterogeneous receptor families. In this signaling process G protein-coupled receptor (GPCR) stimulation induces phosphorylation of the EGFR, combining the broad diversity of GPCRs with the potent signaling capacities of this receptor tyrosine kinase. Early reports attributed this transactivation mechanism to solely intracellular pathways as no EGF-like ligands could be detected in conditioned media of GPCR agonist-stimulated cells. However, Prenzel and colleagues demonstrated the involvement of metalloproteinase-mediated release of EGF-like ligands as the predominant mechanism of EGFR signal transactivation, providing a point of convergence for different intracellular effector proteins. Since this discovery, numerous investigations revealed the broad relevance of metalloproteinase-mediated ligand-dependent EGFR signal transactivation for coupling GPCRs to various cellular signaling responses. Here we describe methods to investigate GPCR-stimulated EGFR signal transactivation allowing the identification of both the EGF-like ligands and the metalloproteinases involved.