AbstractThe conserved transmembrane receptor Notch mediates cell fate decisions in all animals. In the absence of ligand, a Negative Regulatory Region (NRR) in the Notch ectodomain adopts an autoinhibited confirmation, masking an ADAM protease cleavage site [1, 2]; ligand binding makes the cleavage site accessible, leading to shedding of the Notch ectodomain as the first step of signal transduction [3, 4]. In Drosophila and vertebrates, the ligands are all single-pass transmembrane Delta/Serrate/LAG-2 (DSL) proteins; the endocytic adaptor Epsin binds to the ubiquitinated intracellular domain, and the resulting Clathrin-mediated endocytosis exerts a “pulling force” that exposes the cleavage site in the NRR [4–6]. However, in C. elegans, the presence of natural secreted DSL proteins [7] and other observations suggested that Epsin-mediated endocytosis may not be required to activate the Notch proteins LIN-12 and GLP-1. Here, we confirm that neither Epsin nor the cytosolic domains of DSL proteins are required for Notch signaling in C. elegans. Furthermore, we provide evidence that the NRRs of LIN-12 and GLP-1 are tuned to a lower force level than the NRR of Drosophila Notch. Finally, we show that adding a Leucine “plug” that occludes the cleavage site in vertebrate and Drosophila Notch proteins but is absent in the C. elegans Notch proteins [1, 2] renders the LIN-12 and GLP-1 NRRs dependent on Epsin-mediated ligand endocytosis, indicating that greater force is now required to expose the cleavage site. Thus, the NRRs of LIN-12 and GLP-1 appear to be tuned to a lower force threshold, accounting for the different requirements for signaling in C. elegans.