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.

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Science Flemingovo n 2 Prague 166 10 Czech Republic

Instituto Gulbenkian de Ciência Lisbon Portugal

MRC Laboratory of Molecular Biology Cambridge CB2 2QH United Kingdom

Sir William Dunn School of Pathology Oxford OX1 3RE United Kingdom

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Urban S, Lee JR, Freeman M. A family of Rhomboid intramembrane proteases activates all Drosophila membrane-tethered EGF ligands. EMBO J. 2002;21:4277–4286. doi: 10.1093/emboj/cdf434. PubMed DOI PMC

Lohi O, Urban S, Freeman M. Diverse substrate recognition mechanisms for rhomboids; thrombomodulin is cleaved by Mammalian rhomboids. Curr. Biol. 2004;14:236–241. PubMed

Fleig L, et al. Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD of membrane proteins. Molecular cell. 2012;47:558–569. doi: 10.1016/j.molcel.2012.06.008. PubMed DOI

Song W, et al. Rhomboid domain containing 1 promotes colorectal cancer growth through activation of the EGFR signalling pathway. Nat. Commun. 2015;6:8022. doi: 10.1038/ncomms9022. PubMed DOI PMC

Wunderle L, et al. Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFalpha. Sci. Rep. 2016;6:27342. doi: 10.1038/srep27342. PubMed DOI PMC

Lemberg MK, Freeman M. Functional and evolutionary implications of enhanced genomic analysis of rhomboid intramembrane proteases. Genome Res. 2007;17:1634–1646. doi: 10.1101/gr.6425307. PubMed DOI PMC

Pascall JC, Brown KD. Intramembrane cleavage of ephrinB3 by the human rhomboid family protease, RHBDL2. Biochem. Biophys. Res. Commun. 2004;317:244–252. doi: 10.1016/j.bbrc.2004.03.039. PubMed DOI

Adrain C, et al. Mammalian EGF receptor activation by the rhomboid protease RHBDL2. EMBO Rep. 2011;12:421–427. doi: 10.1038/embor.2011.50. PubMed DOI PMC

Noy PJ, Swain RK, Khan K, Lodhia P, Bicknell R. Sprouting angiogenesis is regulated by shedding of the C-type lectin family 14, member A (CLEC14A) ectodomain, catalyzed by rhomboid-like 2 protein (RHBDL2) FASEB J. 2016;30:2311–2323. doi: 10.1096/fj.201500122R. PubMed DOI

Menschikowski M, Hagelgans A, Eisenhofer G, Tiebel O, Siegert G. Reducing agents induce thrombomodulin shedding in human endothelial cells. Thromb. Res. 2010;126:e88–93. doi: 10.1016/j.thromres.2010.05.006. PubMed DOI

Atapattu L, Lackmann M, Janes PW. The role of proteases in regulating Eph/ephrin signaling. Cell Adhesion & Migration. 2014;8:294–307. doi: 10.4161/19336918.2014.970026. PubMed DOI PMC

Sahin U, et al. Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands. J Cell Biol. 2004;164:769–779. doi: 10.1083/jcb.200307137. PubMed DOI PMC

Ong SE, et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics. 2002;1:376–386. doi: 10.1074/mcp.M200025-MCP200. PubMed DOI

Higy M, Junne T, Spiess M. Topogenesis of membrane proteins at the endoplasmic reticulum. Biochemistry. 2004;43:12716–12722. doi: 10.1021/bi048368m. PubMed DOI

Kall L, Krogh A, Sonnhammer EL. A combined transmembrane topology and signal peptide prediction method. J Mol Biol. 2004;338:1027–1036. doi: 10.1016/j.jmb.2004.03.016. PubMed DOI

Junttila MR, Saarinen S, Schmidt T, Kast J, Westermarck J. Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells. Proteomics. 2005;5:1199–1203. doi: 10.1002/pmic.200400991. PubMed DOI

Strisovsky K, Sharpe HJ, Freeman M. Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates. Molecular cell. 2009;36:1048–1059. doi: 10.1016/j.molcel.2009.11.006. PubMed DOI PMC

Zoll S, et al. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures. EMBO J. 2014;33:2408–2421. doi: 10.15252/embj.201489367. PubMed DOI PMC

Horiuchi K, et al. Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx. Mol Biol Cell. 2007;18:176–188. doi: 10.1091/mbc.E06-01-0014. PubMed DOI PMC

Hruz T, et al. Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinformatics. 2008;2008:420747. doi: 10.1155/2008/420747. PubMed DOI PMC

Cheng TL, et al. Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing. J Invest Dermatol. 2011;131:2486–2494. doi: 10.1038/jid.2011.230. PubMed DOI

Conway K, Ruge F, Price P, Harding KG, Jiang WG. Hepatocyte growth factor regulation: an integral part of why wounds become chronic. Wound Repair Regen. 2007;15:683–692. doi: 10.1111/j.1524-475X.2007.00296.x. PubMed DOI

Hou G, Vogel W, Bendeck MP. The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair. Journal of Clinical Investigation. 2001;107:727–735. doi: 10.1172/JCI10720. PubMed DOI PMC

Lin ZQ, Kondo T, Ishida Y, Takayasu T, Mukaida N. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. J. Leukoc. Biol. 2003;73:713–721. doi: 10.1189/jlb.0802397. PubMed DOI

Cheng TL, et al. RHBDL2 is a critical membrane protease for anoikis resistance in human malignant epithelial cells. ScientificWorldJournal. 2014;2014:902987. PubMed PMC

Logue SE, Cleary P, Saveljeva S, Samali A. New directions in ER stress-induced cell death. Apoptosis. 2013;18:537–546. doi: 10.1007/s10495-013-0818-6. PubMed DOI

Ribatti D, Crivellato E. “Sprouting angiogenesis”, a reappraisal. Dev. Biol. 2012;372:157–165. doi: 10.1016/j.ydbio.2012.09.018. PubMed DOI

Zhong X, Rescorla FJ. Cell surface adhesion molecules and adhesion-initiated signaling: understanding of anoikis resistance mechanisms and therapeutic opportunities. Cell. Signal. 2012;24:393–401. doi: 10.1016/j.cellsig.2011.10.005. PubMed DOI

Oberst MD, et al. HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease. Am J Physiol Cell Physiol. 2005;289:C462–470. doi: 10.1152/ajpcell.00076.2005. PubMed DOI

Huang HP, et al. Persistent elevation of hepatocyte growth factor activator inhibitors in cholangiopathies affects liver fibrosis and differentiation. Hepatology. 2012;55:161–172. doi: 10.1002/hep.24657. PubMed DOI

Marchand-Adam S, et al. Defect of pro-hepatocyte growth factor activation by fibroblasts in idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 2006;174:58–66. doi: 10.1164/rccm.200507-1074OC. PubMed DOI

Phin S, et al. Imbalance in the pro-hepatocyte growth factor activation system in bleomycin-induced lung fibrosis in mice. Am J Respir Cell Mol Biol. 2010;42:286–293. doi: 10.1165/rcmb.2008-0305OC. PubMed DOI

Leitinger B. Transmembrane collagen receptors. Annu. Rev. Cell Dev. Biol. 2011;27:265–290. doi: 10.1146/annurev-cellbio-092910-154013. PubMed DOI

Carafoli F, Hohenester E. Collagen recognition and transmembrane signalling by discoidin domain receptors. Biochim. Biophys. Acta. 2013;1834:2187–2194. doi: 10.1016/j.bbapap.2012.10.014. PubMed DOI PMC

Hayashida K, Bartlett AH, Chen Y, Park PW. Molecular and cellular mechanisms of ectodomain shedding. Anat Rec (Hoboken) 2010;293:925–937. doi: 10.1002/ar.20757. PubMed DOI PMC

Brambrink T, et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell. 2008;2:151–159. doi: 10.1016/j.stem.2008.01.004. PubMed DOI PMC

Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat. Methods. 2009;6:359–362. doi: 10.1038/nmeth.1322. PubMed DOI

Nilse L, et al. Yeast membrane proteomics using leucine metabolic labelling: Bioinformatic data processing and exemplary application to the ER-intramembrane protease Ypf1. Biochim. Biophys. Acta. 2016;1864:1363–1371. doi: 10.1016/j.bbapap.2016.07.002. PubMed DOI

Vizcaino JA, et al. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 2013;41:D1063–1069. doi: 10.1093/nar/gks1262. PubMed DOI PMC

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