Abstract
The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. 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 induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.3,4 In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a "pulling force" on Notch that is essential to expose the cleavage site in the NRR.4-6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,6 we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a "leucine plug" that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.1,2 Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.