(A) Schematic of Synthetic Notch Assay for Proteolytic Switches (SNAPS). Cells co-expressing Flag-Notch-X-Gal4 chimeras, where X is a putative proteolysis region of another receptor, and luciferase reporter constructs are co-cultured with DLL4 ligand-expressing cells to induce Notch activation and expression of luciferase. (B) Schematic of chimeric constructs utilized in the signaling assay. Protein domains are color coded and labeled below. Amino acid ranges used for each construct are in parentheses under the names. Note that Notch's SEA-like domain is also referred to as the Heterodimerization Domain (HD) in the literature. Abbreviations used: Cad: cadherin. EGF: Epidermal growth factor. LBD: Ligand binding domain. LNR: Lin-12 Notch-like repeats. ND: N-terminal domain. PKD: polycystic kidney disease domain. S/T rich: serine-threonine rich. TFP: Teal fluorescent protein. TM: transmembrane domain. TY: thyroglobulin type-1A domain. (C) Luciferase reporter gene activity profile of Notch and Notch chimera constructs (1 ng transfected in 96wp) co-cultured with MS5 cells or MS5 cells stably expressing DLL4. BB-94 = Batimastat (pan-metalloproteinase inhibitor) GSI = Compound E (γ-secretase inhibitor). Data shown are triplicate measurements from a representative experiment. Error bars represent the SEM of triplicate measurements. (D) Cell surface ELISA of Notch and Notch chimera constructs. Anti-Flag primary and goat anti-mouse HRP secondary antibodies were used to detect cell surface expression levels of each chimera. The horizontal dotted line corresponds to Notch expression levels. Error bars represent the SEM of triplicate measurements. (E) Structures and PDB IDs of SEA-like domains (gray) with applicable adjacent domains (purple). The Notch adjacent domain is comprised of three cysteine-rich, calcium binding Lin12 Notch repeats. Protocad15 (De-la-Torre et al., 2019; Dionne et al., 2018; Ge et al., 2018) has an Ig-like adjacent domain and EpCAM (Pavšič et al., 2014) has a cysteine-rich thyroglobulin adjacent domain. The buried surface area of the adjacent domains are 3800, 1300, and 2800 square Angstroms for Notch, Protocad15, and EpCAM, respectively. SEA-like domains were structurally aligned to the Notch SEA-like domain.