Size-dependent protein segregation creates a spatial switch for Notch and APP signaling

Aberrant cleavage of Notch and amyloid precursor protein (APP) by γ-secretase is implicated in developmental disorders, cancer, and dementia. How cleavage is regulated in space and time is unclear. Here, we report that size-dependent protein segregation acts as a spatial switch regulating the dynamic distribution of these receptors relative to their enzymatic partner, γ-secretase. Because the size of the extracellular domain (ECD) greatly exceeds the width of the junctional cleft, Notch is excluded from cadherin-based adherens junctions (cadAJs) where γ-secretase localizes, prohibiting the enzyme-substrate interaction. ECD elimination by ADAM 10/17 drives a spatial rearrangement of Notch into cadAJs where γ-secretase cleavage occurs. Spatial mutation of Notch by decreasing (or increasing) the ECD size promotes (or inhibits) downstream signaling, respectively, regardless of ligand-presentation or S2 cleavage. Elimination of cadAJs abolishes Notch activation, confirming the indispensable role of cadAJs in regulating Notch-γ-secretase interactions. We further show that this spatial switch regulates APP cleavage and amyloid beta production. Therefore, size-dependent protein segregation creates membrane domains with unique proteolytic activities, choreographing the proteolytic sequence while suppressing nonspecific signal activation.