Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the Notch1 regulatory switch results in sensitivity to metalloprotease cleavage, and bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction.
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We thank Benedikt Bauer and Anthony Nguyen for help in optimizing single molecule experiments and Adam17 expression, Scott Ficarro for mass spectrometry, and Kelly Arnett and Brian McMillan for helpful discussions. W.R.G. was supported by an American Heart Association (AHA) SDG grant. L.H. was supported by a Damon Runyon Postdoctoral Fellowship. This work was supported by NIH grants P01 CA119070 (J.J.L., J.C.A., and S.C.B.) and R01 CA092433 (J.C.A. and S.C.B.), the Howard Hughes Medical Institute (HHMI), and the NIH (N.P.).
© 2015 Elsevier Inc.