An screen for proteolytic switch domains that can mediate Notch activation by force.

Notch proteins are single pass transmembrane receptors activated by sequential extracellular and intramembrane cleavages that release their cytosolic domains to function as transcription factors in the nucleus. Upon binding, Delta/Serrate/LAG-2 (DSL) ligands activate Notch by exerting a "pulling" force across the intercellular ligand/receptor bridge. This pulling force is generated by Epsin-mediated endocytosis of 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 [Kuzbanian (Kuz) in ]. Here, we have used chimeric Notch and DSL proteins to screen for other domains that can function as ligand-dependent proteolytic switches in place of the NRR in the developing wing. While many of the tested domains are either refractory to cleavage or constitutively cleaved, we identify several that mediate Notch activation in response to ligand. These NRR analogues derive from widely divergent source proteins and have strikingly different predicted structures. Yet, almost all depend on force exerted by Epsin-mediated ligand endocytosis and cleavage catalyzed by Kuz. We posit that the sequence space of protein domains that can serve as force-sensing proteolytic switches in Notch activation is unexpectedly large, a conclusion that has implications for the mechanism of target recognition by Kuz/ADAM10 proteases and is consistent with a more general role for force dependent ADAM10 proteolysis in other cell contact-dependent signaling mechanisms. Our results also validate the screen for increasing the repertoire of proteolytic switches available for synthetic Notch (synNotch) therapies and tissue engineering.