Abstract
Rhomboids are intramembrane serine proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of rhomboid protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloproteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.
Highlights
Proteins of the rhomboid family are the most widely occurring intramembrane proteases and are spread throughout the tree of life
Considering the described functions of the identified substrates basal cell adhesion molecule (BCAM), Spint-1, Discoidin domain receptor 1 (DDR1), Cadm[1], KIRREL, CLCP1 and interleukin-6 receptor (IL6R), which involve mainly cell adhesion and migration (Fig. 5B), we propose that RHBDL2 functions in epithelial homeostasis
In this study we developed and applied a quantitative proteomics technique to identify the substrate repertoire of the mammalian rhomboid protease RHBDL2
Summary
Proteins of the rhomboid family are the most widely occurring intramembrane proteases and are spread throughout the tree of life. The best understood mammalian ‘secretase’ rhomboid far is RHBDL2, which localizes to the plasma membrane[2] where it can catalyse proteolysis of cell surface transmembrane proteins. Candidate screens have identified several substrates of RHBDL2, such as B-type ephrins[7], which act as ligands for Eph tyrosine kinase receptors, thrombomodulin[2], a cell-surface membrane protein involved in the regulation of blood coagulation, epidermal growth factor (EGF)[8], and C-type lectin CLEC14A9. Ectodomain-shedding enzymes on the surface of mammalian cells, suggesting redundancy between rhomboids and ADAMs. Overall, the lack of systematic and objective approaches to identify rhomboid substrates severely limits our ability to understand the physiological roles of rhomboid proteases. We find that a range of type I membrane proteins are cleaved and shed into the media upon co-expression with RHBDL2. Our data indicate that mammalian rhomboids have a specific substrate repertoire and can function autonomously from metalloproteases, in distinct signalling pathways
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