Proteolysis has emerged as a key posttranslational regulator of ligands of the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor with important roles in development and diseases such as cancer. All EGFR-ligands are made as membrane anchored precursors whose ectodomains frequently require proteolytic release or "shedding" to trigger EGFR-signaling. Metalloproteases of the ADAM (a disintegrin and metalloprotease) protein family have key roles in shedding six EGFR-ligands, and mice lacking ADAM17 resemble animals lacking the EGFR, or animals lacking the ADAM17 substrates TGFa, HB-EGF and amphiregulin. The main goal of the proposed research is to elucidate the mechanism underlying the critical role of ADAMs in shedding and activating ligands of the EGFR. Specifically, we will: 1) Perform a structure/function analysis to understand the substrate selectivity and regulation ofADAMslO and 17. We will generate chimera between ADAMslO and 17 as well as between the ADAM10 substrate EGF and the ADAM17 substrate TGFa to identify which domains of these enzymes and substrates are required for their substrate selectivity. Moreover, we will establish how different activators and inhibitors of intracellular signaling pathways affect the function of ADAMslO and 17. 2) Study the role ofADAM17 in juxtacrine signaling. Signaling via the EGFR is unusual in that it requires two separate ligand binding events before the occupied receptors can dimerize. Uncleaved membrane tethered ligands are predicted to impede receptor dimerization at low ligand concentrations, which might explain why cleavage of these ligands is critical for juxtacrine signaling (cell-cell signaling) under certain conditions. However, clustering or overexpression of ligands might allow receptor dimerization and thus juxtacrine signaling even when they are not cleaved. We will test this hypothesis by assessing how low or high concentrations of uncleavable TGFa, or of TGFa that is clustered by the tetraspanin CD9 or by chemical inducers of dimerization, affect EGFR-signaling. 3) Address the in vivo relevance of the results of aim 2 using conditional knockout mice forADAM17 crossed with transgenic mice expressing different levels of TGFa in the mammary gland. We anticipate that the proposed studies will provide exciting new insights into the upstream regulation of the EGFR pathway by proteolysis of its ligands. Because EGFR-signaling has a crucial role in diseases such as cancer, we hope this work will uncover new targets for the design of drugs that can affect EGFR signaling.