iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding

Abstract

Protein ectodomain shedding by ADAM17 (a disintegrin and metalloprotease 17), a principal regulator of EGF-receptor signaling and TNFα release, is rapidly and posttranslationally activated by a variety of signaling pathways, and yet little is known about the underlying mechanism. Here, we report that inactive rhomboid protein 2 (iRhom2), recently identified as essential for the maturation of ADAM17 in hematopoietic cells, is crucial for the rapid activation of the shedding of some, but not all substrates of ADAM17. Mature ADAM17 is present in mouse embryonic fibroblasts (mEFs) lacking iRhom2, and yet ADAM17 is unable to support stimulated shedding of several of its substrates, including heparin-binding EGF and Kit ligand 2 in this context. Stimulated shedding of other ADAM17 substrates, such as TGFα, is not affected in iRhom2 −/− mEFs but can be strongly reduced by treating iRhom2 −/− mEFs with siRNA against iRhom1. Activation of heparin-binding EGF or Kit ligand 2 shedding by ADAM17 in iRhom2 −/− mEFs can be rescued by wild-type iRhom2 but not by iRhom2 lacking its N-terminal cytoplasmic domain. The requirement for the cytoplasmic domain of iRhom2 for stimulated shedding by ADAM17 may help explain why the cytoplasmic domain of ADAM17 is not required for stimulated shedding. The functional relevance of iRhom2 in regulating shedding of EGF receptor (EGFR) ligands is established by a lack of lysophasphatidic acid/ADAM17/ EGFR-dependent crosstalk with ERK1/2 in iRhom2 −/− mEFs, and a significant reduction of FGF7/ADAM17/EGFR-stimulated migration of iRhom2 −/− keratinocytes. Taken together, these findings uncover functions for iRhom2 in the regulation of EGFR signaling and in controlling the activation and substrate selectivity of ADAM17-dependent shedding events.

iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

“…2H), demonstrating that the functions of ADAM17 in microglia and circulating leukocytes depend on iRhom2 and not iRhom1. The normal appearance and behavior of iR1 −/− mice (this study) or iR2 −/− mice (24,26,28) and the finding that treatment of iR2 −/− mEFs with iRhom1 siRNA reduced the function of ADAM17 (27,29) led us to examine possible compensatory or redundant roles of iRhoms 1 and 2 during mouse development by simultaneously inactivating both iRhoms. Matings of iR1 +/− iR2 −/− mice yielded iR1/2 −/− double knockout offspring at the expected Mendelian ratio (Fig.…”

“…This raises interesting questions about putative interacting regulatory membrane proteins. The discovery of iRhom2 as a crucial regulator of ADAM17 in hematopoietic cells (24,26,29) and in vitro studies in mouse embryonic fibroblasts demonstrating that iRhom2 controls the substrate selectivity of ADAM17-dependent shedding (27) and that iRhoms 1 and 2 are required for the function of ADAM17 in mouse embryonic fibroblasts (27,30) raised the possibility that iRhom2 and the related iRhom1 could be the long-sought-after membrane regulators of ADAM17-dependent EGFR signaling. However, this hypothesis had not been previously corroborated by in vivo genetic studies.…”

“…S6 A-D). However, iR1/2 −/− mEFs resembled Adam17 −/− mEFs (4,27) in that they showed almost no constitutive or PMA-stimulated shedding of amphiregulin, epiregulin, HB-EGF, or TGF-α (Fig. 5 A-D).…”

“…EGFR Ligand Shedding from iR1/2 −/− mEFs and EGFR Phosphorylation in iR1/2 −/− Tissues. Previous experiments had uncovered a role for iRhom2 in regulating the substrate selectivity of stimulated ADAM17-dependent shedding events in mEFs (27). To test how inactivation of iRhom1 or both iRhoms 1 and 2 affects the function of ADAM17 in cell-based assays, we performed shedding experiments with iR1 −/− mEFs, which have marginally reduced levels of mature ADAM17 compared with wild-type controls, or iR1/2 −/− mEFs, which have no mature ADAM17 ( Fig.…”

“…Recent studies have shown that the maturation and function of ADAM17 in myeloid cells depend on inactive Rhomboid 2 (iRhom2), a catalytically inactive member of the Rhomboid family of seven membrane-spanning intramembrane serine proteinases (24)(25)(26)(27)(28). Myeloid cells lacking iRhom2 release very little TNF-α in response to activation of Toll-like receptor 4 by lipopolysaccharide (LPS) (24,26,28).…”