An in vivo screen identifies diverse domains that can act as force-dependent proteolytic switches for Notch activation.

Notch proteins are single-pass transmembrane receptors activated by sequential extracellular and intramembrane cleavages to release the cytosolic domains that function as transcription factors. Transmembrane ligands of the Delta/Serrate/LAG-2 (DSL) family activate Notch on neighboring cells by exerting a pulling force across the intercellular ligand-receptor bridge. This force is generated by Epsin-mediated endocytosis of the ligand into the signal-sending cell and results in the extracellular cleavage of the force-sensing negative regulatory region (NRR) of the receptor by an ADAM10 protease on the signal-receiving cell. Here, we used chimeric Notch and DSL proteins to screen for other domains that could function as ligand-dependent proteolytic switches in place of the NRR in the developing Drosophila melanogaster wing. The domains that could functionally substitute for the NRR in vivo derived from diverse source proteins, varied in sequence, and had different predicted structures, yet all depended on cleavage that was catalyzed by the Drosophila ADAM10 homolog Kuzbanian (Kuz) and stimulated by Epsin-mediated ligand endocytosis. The large sequence space of protein domains that can serve as force-sensing proteolytic switches suggests a widespread potential role for force-dependent, ADAM10-mediated proteolysis in other cell contact-dependent signaling mechanisms.