Members of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases are crucial to the proper regulation of a number of important cellular processes, including cell proliferation, differentiation, and migration. Abnormal behavior of these receptors is associated with a range of diseases, including cancer. Significant advances towards understanding EGFR-mediated signal transduction have relied on crystallographic methods. However, many questions about this fundamental mechanism still remain, largely due to the paucity of existing methods available to study the dynamic processes of EGFR dimerization in vivo. As a complement to existing biochemical methods, we will apply the novel bipartite tetracysteine display technique to monitor the dimerization and activation of epidermal growth factor receptor (EGFR) in live cells. Biarsenical labeling of linear tetracysteine motifs is a well established method for the site-specific labeling of proteins. Recent work from the Schepartz laboratory at Yale has shown that efficient labeling can also be achieved when the tetracysteine motif is displayed in a bipartite fashion, where the cysteine pairs are far away from each other in primary sequence, but closely associated when the target protein is in a folded or assembled state. This proposal outlines the utilization of bipartite tetracysteine display in the context of monitoring EGFR dimerization and activation in live cells through three specific aims. Initially (Aim 1), we conduct a series of experiments to validate the extension of the bipartite display methodology to the EGFR. Next (Aim 2), we ask whether this novel system for monitoring protein dimerization in vivo can be used to answer longstanding questions about the mechanism of EGFR signal transduction. Finally (Aim 3), we extend this platform to be the basis of a sensitive, in-cell, high-throughput screen for agonist and antagonists of the EGFR family. Public Health Relevance: The proposed research will contribute to our knowledge about the fundamental basis of EGFR activity. This will lead to an improved understanding of how aberrant EGFR behavior can lead to cancer and could have implications for new strategies to target EGFR for the treatment or detection of cancer. The proposed research also involves the development of a high-throughput screen for inhibitors of EGFR that may lead to the discovery of new, potent EGFR inhibitors and/or to the innovative use of known inhibitors as highly effective and selective combination therapies for the treatment of cancer.