ADAM17 stabilizes its interacting partner inactive Rhomboid 2 (iRhom2) but not inactive Rhomboid 1 (iRhom1)

Gisela Weskamp,Johanna Tüshaus,Daniel Li,Regina Feederle,Thorsten Maretzky,Steven Swendemann,Erik Falck-Pedersen,David R Mcilwain,Tak W Mak,Jane E Salmon,Stefan F Lichtenthaler,Carl P Blobel

doi:10.1074/jbc.ra119.011136

Abstract

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) is a key regulator of tumor necrosis factor α (TNFα), interleukin 6 receptor (IL-6R), and epidermal growth factor receptor (EGFR) signaling. ADAM17 maturation and function depend on the seven-membrane-spanning inactive rhomboid-like proteins 1 and 2 (iRhom1/2 or Rhbdf1/2). Most studies to date have focused on overexpressed iRhom1 and -2, so only little is known about the properties of the endogenous proteins. Here, we show that endogenous iRhom1 and -2 can be cell surface-biotinylated on mouse embryonic fibroblasts (mEFs), revealing that endogenous iRhom1 and -2 proteins are present on the cell surface and that iRhom2 also is present on the surface of lipopolysaccharide-stimulated primary bone marrow-derived macrophages. Interestingly, very little, if any, iRhom2 was detectable in mEFs or bone marrow-derived macrophages lacking ADAM17, suggesting that iRhom2 is stabilized by ADAM17. By contrast, the levels of iRhom1 were slightly increased in the absence of ADAM17 in mEFs, indicating that its stability does not depend on ADAM17. These findings support a model in which iRhom2 and ADAM17 are obligate binding partners and indicate that iRhom2 stability requires the presence of ADAM17, whereas iRhom1 is stable in the absence of ADAM17.

Highlights

ADAM17 is a cell surface metalloprotease that is required for the proteolytic processing of tumor necrosis factor a (TNFa) and is referred to as TACE (TNFa convertase)
Since mouse embryonic fibroblasts (mEFs) lacking both iRhom1 and iRhom2 only have pro-ADAM17, but no detectable mature ADAM17, we were interested in exploring whether endogenous iRhom2 and the related iRhom1 can be detected on the cell surface of wild type mEFs, as would be predicted if they can function as regulators of endogenous mature ADAM17
If any iRhom2 is detectable by Western blot in myeloid cells or in embryos lacking ADAM17 under conditions where it can readily be detected in wild-type controls, provides the first evidence that ADAM17 is required for the stabilization of endogenous iRhom2

Summary

Introduction

ADAM17 is a cell surface metalloprotease that is required for the proteolytic processing of tumor necrosis factor a (TNFa) and is referred to as TACE (TNFa convertase). Additional insight into the relationship of ADAM17 and iRhom was provided by a point mutation in the first transmembrane domain (TMD) of iRhom, termed sinecure, which results in a strong reduction of ADAM17dependent TNFa release from BMDM [26]. Dependent shedding [28] suggested that iRhom and ADAM17 form a heteromeric complex This complex associates in the endoplasmic reticulum (ER) and remains together to regulate iRhom2/ADAM17-dependent shedding on the cell surface or in the late secretory pathway. This model is further supported by recent studies demonstrating that mutations in cytoplasmic phosphorylation sites of iRhom affect the activation of ADAM17 [30,31]. Since iRhom has been shown to interact with the multimembrane spanning protein stimulator of interferon genes (STING) [35], this raised questions about the role of STING in the stability and maturation of iRhom and ADAM17

Results

Discussion

A17 iR2 iR1