A toolkit for studying cell surface shedding of diverse transmembrane receptors.

Amanda N Hayward,Eric J Aird,Wendy R Gordon

doi:10.7554/elife.46983

Abstract

Proteolysis of transmembrane receptors is a critical cellular communication mechanism dysregulated in disease, yet decoding proteolytic regulation mechanisms of hundreds of shed receptors is hindered by difficulties controlling stimuli and unknown fates of cleavage products. Notch proteolytic regulation is a notable exception, where intercellular forces drive exposure of a cryptic protease site within a juxtamembrane proteolytic switch domain to activate transcriptional programs. We created a Synthetic Notch Assay for Proteolytic Switches (SNAPS) that exploits the modularity and unequivocal input/response of Notch proteolysis to screen surface receptors for other putative proteolytic switches. We identify several new proteolytic switches among receptors with structural homology to Notch. We demonstrate SNAPS can detect shedding in chimeras of diverse cell surface receptors, leading to new, testable hypotheses. Finally, we establish the assay can be used to measure modulation of proteolysis by potential therapeutics and offer new mechanistic insights into how DECMA-1 disrupts cell adhesion.

Highlights

Proteolysis of cell surface transmembrane proteins is a tightly regulated cellular mechanism that controls communication of cells with their extracellular environment
To determine if receptors bearing juxtamembrane domains predicted to be structurally homologous to Notch could function as proteolytic switches, we created chimeric receptors in which we replaced the Notch Negative Regulatory Region (NRR) proteolytic switch domain with SEA and SEA-like domains from other cell surface receptors and included any previously characterized tandem N-terminal domains (Fig. 1B, Supplementary Table 1)
We hypothesized that other putative proteolytic switches could functionally substitute for the Notch NRR and initiate a transcriptional response in response to contact with a cell expressing DLL4

Summary

Introduction

Proteolysis of cell surface transmembrane proteins is a tightly regulated cellular mechanism that controls communication of cells with their extracellular environment Diverse adhesion receptors, such as cadherins, as well as receptors that respond to soluble factors, such as receptor tyrosine kinases (RTKs), have been shown to be cleaved at sites close to the extracellular side of the membrane by metalloproteinases such as ‘A Disintegrin And Metalloproteinases’ (ADAMs) and matrix metalloproteinases (MMPs) (Miller, Sullivan, and Lauffenburger 2017; Kessenbrock, Plaks, and Werb 2010; McCawley and Matrisian 2001; Seals and Courtneidge 2003; White 2003), resulting in ectodomain shedding. Cancer cells evade the immune response by shedding MICA receptors (Groh et al 2002; Kaiser et al 2007; Boutet et al 2009; Waldhauer et al 2008), which are normally deployed to the cell surface in response to cellular damage

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