A simple cell-based assay for the detection of surface protein shedding by rhomboid proteases

Abstract

Rhomboids are intramembrane serine proteases that cleave their substrates within or immediately adjacent to their transmembrane domains, a process known as regulated intramembrane proteolysis. In eukaryotes, two main types of rhomboid proteases can be distinguished based on their subcellular localization: mitochondrial rhomboids and secretase-type rhomboids that target the secretory pathway. The latter class can cleave and release the extracellular domain of all epidermal growth factor-like proteins in Drosophila and can liberate epidermal growth factor (EGF) in mammals, in a process known as ectodomain shedding. These released EGFs can then activate the EGF receptor (EGFR). EGFR signaling is crucial for mammalian development and is often deregulated in human cancer. Here we describe a cell-based protocol for detecting the ability of rhomboid proteases to release EGFR ligands into the medium. First, cells are transfected with the corresponding protease- and substrate-expressing vectors; second, cells condition the medium and accumulate shed protein. After this, protein lysates from cells and media are prepared and Western blotting is performed to detect the EGFR ligands that have been released into the medium.