Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity.

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration.

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. Here, we reveal an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. We show that the glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but that in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, we demonstrate the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, we have discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina.

Downregulation of the metalloproteinases ADAM10 or ADAM17 promotes osteoclast differentiation.

Bone resorption is driven through osteoclast differentiation by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL). We noted that a disintegrin and metalloproteinase (ADAM) 10 and ADAM17 are downregulated at the expression level during osteoclast differentiation of the murine monocytic cell line RAW264.7 in response to RANKL. Both proteinases are well known to shed a variety of single-pass transmembrane molecules from the cell surface. We further showed that inhibitors of ADAM10 or ADAM17 promote osteoclastic differentiation and furthermore enhance the surface expression of receptors for RANKL and M-CSF on RAW264.7 cells. Using murine bone marrow-derived monocytic cells (BMDMCs), we demonstrated that a genetic deficiency of ADAM17 or its required regulator iRhom2 leads to increased osteoclast development in response to M-CSF and RANKL stimulation. Moreover, ADAM17-deficient osteoclast precursor cells express increased levels of the receptors for RANKL and M-CSF. Thus, ADAM17 negatively regulates osteoclast differentiation, most likely through shedding of these receptors. To assess the time-dependent contribution of ADAM10, we blocked this proteinase by adding a specific inhibitor on day 0 of BMDMC stimulation with M-CSF or on day 7 of subsequent stimulation with RANKL. Only ADAM10 inhibition beginning on day 7 increased the size of developing osteoclasts indicating that ADAM10 suppresses osteoclast differentiation at a later stage. Finally, we could confirm our findings in human peripheral blood mononuclear cells (PBMCs). Thus, downregulation of either ADAM10 or ADAM17 during osteoclast differentiation may represent a novel regulatory mechanism to enhance their differentiation process. Enhanced bone resorption is a critical issue in osteoporosis and is driven through osteoclast differentiation by specific osteogenic mediators. The present study demonstrated that the metalloproteinases ADAM17 and ADAM10 critically suppress osteoclast development. This was observed for a murine cell line, for isolated murine bone marrow cells and for human blood cells by either preferential inhibition of the proteinases or by gene knockout. As a possible mechanism, we studied the surface expression of critical receptors for osteogenic mediators on developing osteoclasts. Our findings revealed that the suppressive effects of ADAM17 and ADAM10 on osteoclastogenesis can be explained in part by the proteolytic cleavage of surface receptors by ADAM10 and ADAM17, which reduces the sensitivity of these cells to osteogenic mediators. We also observed that osteoclast differentiation was associated with the downregulation of ADAM10 and ADAM17, which reduced their suppressive effects. We therefore propose that this downregulation serves as a feedback loop for enhancing osteoclast development.

A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity.

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure-function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

The collectrin-like part of the SARS-CoV-1 and -2 receptor ACE2 is shed by the metalloproteinases ADAM10 and ADAM17.

The transmembrane protease angiotensin converting enzyme 2 (ACE2) is a protective regulator within the renin angiotensin system and additionally represents the cellular receptor for SARS-CoV. The release of soluble ACE2 (sACE2) from the cell surface is hence believed to be a crucial part of its (patho)physiological functions, as both, ACE2 protease activity and SARS-CoV binding ability, are transferred from the cell membrane to body fluids. Yet, the molecular sources of sACE2 are still not completely investigated. In this study, we show different sources and prerequisites for the release of sACE2 from the cell membrane. By using inhibitors as well as CRISPR/Cas9-derived cells, we demonstrated that, in addition to the metalloprotease ADAM17, also ADAM10 is an important novel shedding protease of ACE2. Moreover, we observed that ACE2 can also be released in extracellular vesicles. The degree of either ADAM10- or ADAM17-mediated ACE2 shedding is dependent on stimulatory conditions and on the expression level of the pro-inflammatory ADAM17 regulator iRhom2. Finally, by using structural analysis and in vitro verification, we determined for the first time that the susceptibility to ADAM10- and ADAM17-mediated shedding is mediated by the collectrin-like part of ACE2. Overall, our findings give novel insights into sACE2 release by several independent molecular mechanisms.

Interleukin-6 controls recycling and degradation, but not internalization of its receptors.

Interleukin-6 (IL-6) is a cytokine implicated in proinflammatory as well as regenerative processes and acts via receptor complexes consisting of the ubiquitously expressed, signal-transducing receptor gp130 and the IL-6 receptor (IL-6R). The IL-6R is expressed only on hepatocytes and subsets of leukocytes, where it mediates specificity of the receptor complex to IL-6 as the subunit gp130 is shared with all other members of the IL-6 cytokine family such as IL-11 or IL-27. The amount of IL-6R at the cell surface thus determines the responsiveness of the cell to the cytokine and might therefore be decisive in the development of inflammatory disorders. However, how the expression levels of IL-6R and gp130 at the cell surface are controlled is largely unknown. Here, we show that IL-6R and gp130 are constitutively internalized independent of IL-6. This process depends on dynamin and clathrin and is temporally controlled by motifs within the intracellular region of gp130 and IL-6R. IL-6 binding and internalization of the receptors is a prerequisite for activation of the Jak/STAT signaling cascade. Targeting of gp130, but not of the IL-6R, to the lysosome for degradation depends on stimulation with IL-6. Furthermore, we show that after internalization and activation of signaling, both the IL-6R and gp130 are recycled back to the cell surface, a process that is enhanced by IL-6. These data reveal an important function of IL-6 beyond the pure activation of signaling.

Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling.

Interleukin-11 (IL-11) is a pleiotropic cytokine with both pro- and anti-inflammatory properties. It activates its target cells via binding to the membrane-bound IL-11 receptor (IL-11R), which then recruits a homodimer of the ubiquitously expressed, signal-transducing receptor gp130. Besides this classic signaling pathway, IL-11 can also bind to soluble forms of the IL-11R (sIL-11R), and IL-11/sIL-11R complexes activate cells via the induction of gp130 homodimerization (trans-signaling). We have previously reported that the metalloprotease ADAM10 cleaves the membrane-bound IL-11R and thereby generates sIL-11R. In this study, we identify the rhomboid intramembrane protease RHBDL2 as a so far unrecognized alternative sheddase that can efficiently trigger IL-11R secretion. We determine the cleavage site used by RHBDL2, which is located in the extracellular part of the receptor in close proximity to the plasma membrane, between Ala-370 and Ser-371. Furthermore, we identify critical amino acid residues within the transmembrane helix that are required for IL-11R proteolysis. We also show that ectopically expressed RHBDL2 is able to cleave the IL-11R within the early secretory pathway and not only at the plasma membrane, indicating that its subcellular localization plays a central role in controlling its activity. Moreover, RHBDL2-derived sIL-11R is biologically active and able to perform IL-11 trans-signaling. Finally, we show that the human mutation IL-11R-A370V does not impede IL-11 classic signaling, but prevents RHBDL2-mediated IL-11R cleavage.

The metalloproteinase ADAM10 requires its activity to sustain surface expression.

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss.

The iRhom homology domain is indispensable for ADAM17-mediated TNFα and EGF receptor ligand release.

Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation, migration and malignant signalling events in hepatoma cells.

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of ß1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.

Distance dependent shedding of IL-6R.

Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process. This finding contributes to the understanding of the complex and multilayered regulation of ADAM17 at the cell surface.

Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.

The iRhom2/ADAM17 Axis Attenuates Bacterial Uptake by Phagocytes in a Cell Autonomous Manner.

Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity led to increased phagocytosis of pHrodo labelled Gram-negative and -positive bacteria (E. coli and S. aureus, respectively) by human and murine monocytic cell lines or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics.

The SNP rs4252548 (R112H) which is associated with reduced human height compromises the stability of IL-11.

Height is a complex human phenotype that is influenced by variations in a high number of genes. Recently, a single nucleotide polymorphism (SNP) within IL11 (rs4252548) has been described to be associated with height in adults of European ancestry. This coding SNP leads to the exchange of Arg-112 to His-112 within the cytokine Interleukin-11 (IL-11), which has a well-established role in osteoclast development and bone turnover. The functional consequences of the R112H mutation are unknown so far. In this study, we show by molecular replacement that Arg-112 does not participate in binding of IL-11 to its receptors IL-11R and glycoprotein 130 (gp130). Recombinant IL-11 R112H expressed in E. coli displays a correct four-helix-bundle folding topology, and binds with similar affinity to IL-11R and the IL-11/IL-11R/gp130 complex. IL-11 R112H induces cell proliferation and phosphorylation of the downstream transcription factor STAT3 indistinguishable from IL-11. However, IL-11 R112H fails to support the survival of osteoclast progenitor cells and is less thermally stable, which is caused by the loss of the positive charge on the protein surface since protonation of the histidine side chain recovers stability.

Amphiregulin Regulates Phagocytosis-Induced Cell Death in Monocytes via EGFR and the Bcl-2 Protein Family.

Neonates are extremely susceptible to bacterial infections, and evidences suggest that phagocytosis-induced cell death (PICD) is less frequently triggered in neonatal monocytes than in monocytes from adult donors. An insufficient termination of the inflammatory response, leading to a prolonged survival of neonatal monocytes with ongoing proinflammatory cytokine release, could be associated with the progression of various inflammatory diseases in neonates. Our previous data indicate that amphiregulin (AREG) is increasingly expressed on the cell surface of neonatal monocytes, resulting in remarkably higher soluble AREG levels after proteolytic shedding. In this study, we found that E. coli-infected neonatal monocytes show an increased phosphorylation of ERK, increased expression of Bcl-2 and Bcl-XL, and reduced levels of cleaved caspase-3 and caspase-9 compared to adult monocytes. In both cell types, additional stimulation with soluble AREG further increased ERK activation and expression of Bcl-2 and Bcl-XL and reduced levels of cleaved caspase-3 and caspase-9 in an EGFR-dependent manner. These data suggest that reduced PICD of neonatal monocytes could be due to reduced intrinsic apoptosis and that AREG can promote protection against PICD. This reduction of the intrinsic apoptosis pathway in neonatal monocytes could be relevant for severely prolonged inflammatory responses of neonates.

Rhomboid proteases in human disease: Mechanisms and future prospects.

Rhomboids are intramembrane serine proteases that cleave the transmembrane helices of substrate proteins, typically releasing luminal/extracellular domains from the membrane. They are conserved in all branches of life and there is a growing recognition of their association with a wide range of human diseases. Human rhomboids, for example, have been implicated in cancer, metabolic disease and neurodegeneration, while rhomboids in apicomplexan parasites appear to contribute to their invasion of host cells. Recent advances in our knowledge of the structure and the enzyme function of rhomboids, and increasing efforts to identify specific inhibitors, are beginning to provide important insight into the prospect of rhomboids becoming future therapeutic targets. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Molecular insights into the multilayered regulation of ADAM17: The role of the extracellular region.

In contrast to many other signalling mechanisms shedding of membrane-anchored proteins is an irreversible process. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. Due to its central role in signalling the shedding activity of ADAM17 is tightly regulated, especially on the cell surface, where shedding events take place. The activity of ADAM17 can be subdivided into a catalytic activity and the actual shedding activity. Whereas the catalytic activity is constitutively present, the shedding activity has to be induced and is tightly controlled to prevent pathological situations induced by the release of its substrates. The regulation of the shedding activity of ADAM17 is multilayered and different regions of the protease are involved. Intriguingly, its extracellular domains play crucial roles in different regulatory mechanisms. We will discuss the role of these domains in the control of ADAM17 activity. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Secreted Frizzled-related protein 3 (sFRP3)-mediated suppression of interleukin-6 receptor release by A disintegrin and metalloprotease 17 (ADAM17) is abrogated in the osteoarthritis-associated rare double variant of sFRP3.

To avoid malformation and disease, tissue development and homoeostasis are co-ordinated precisely in time and space. Secreted Frizzled-related protein 3 (sFRP3), encoded by the Frizzled-related protein gene (FRZB), acts as an antagonist of Wnt signalling in bone development by delaying the maturation of proliferative chondrocytes into hypertrophic chondrocytes. A disintegrin and metalloprotease 17 (ADAM17) is a transmembrane protease that is essential for developmental processes and promotes cartilage maturation into bone. sFRP3 is chondroprotective and is expressed in chondrocytes of healthy articular cartilage. Upon damage to cartilage, sFRP3 is down-regulated. Rare variants of sFRP3 are associated with osteoarthritis. The present study demonstrates a novel function of sFRP3 in suppression of the enzymatic activity of ADAM17 which results in the inhibition of ADAM17-meditated interleukin-6 receptor (IL-6R) shedding. By contrast, the rare double variant of sFRP3 failed to suppress ADAM17. The shed soluble IL-6R (sIL-6R) is linked to inflammation, cartilage degeneration and osteolysis. Accordingly, enhanced activity of ADAM17 in cartilage, caused by the expression of the rare double sFRP3 variant, provides an explanation for the genetic effect of sFRP3 variants in joint disease. The finding that sFRP3 interacts with the ADAM17 substrate IL-6R also suggests a new regulatory mechanism by which the substrate is protected against shedding.

Extracellular Juxtamembrane Segment of ADAM17 Interacts with Membranes and Is Essential for Its Shedding Activity.

A wide variety of biological processes including differentiation, regeneration, and cancer progression are regulated by shedding of membrane-anchored proteins. One of the major sheddases is A Disintegrin And Metalloprotease-17 (ADAM17) whose extracellular region consists of a pro-, a catalytic, a disintegrin-, and a membrane-proximal domain (MPD) as well as a short juxtamembrane segment of 17 amino acid residues that has been named "Conserved ADAM-seventeeN Dynamic Interaction Sequence" (CANDIS). This segment is involved in substrate recognition. Key mediators of inflammation including interleukin-6 receptor (IL-6R) and tumor necrosis factor (TNF-α) are substrates of ADAM17. The shedding activity of ADAM17 is regulated by the conformation of the membrane-proximal domain preceding the CANDIS segment. Here, we show that CANDIS, besides being involved in substrate recognition, is able to interact with lipid bilayers in vitro and that this property could be involved in regulating ADAM17 shedding activity.

A disintegrin and metalloprotease 17 dynamic interaction sequence, the sweet tooth for the human interleukin 6 receptor.

A disintegrin and metalloprotease 17 (ADAM17) is a major sheddase involved in the regulation of a wide range of biological processes. Key substrates of ADAM17 are the IL-6 receptor (IL-6R) and TNF-α. The extracellular region of ADAM17 consists of a prodomain, a catalytic domain, a disintegrin domain, and a membrane-proximal domain as well as a small stalk region. This study demonstrates that this juxtamembrane segment is highly conserved, α-helical, and involved in IL-6R binding. This process is regulated by the structure of the preceding membrane-proximal domain, which acts as molecular switch of ADAM17 activity operated by a protein-disulfide isomerase. Hence, we have termed the conserved stalk region "Conserved ADAM seventeen dynamic interaction sequence" (CANDIS). Finally, we identified the region in IL-6R that binds to CANDIS. In contrast to the type I transmembrane proteins, the IL-6R, and IL-1RII, CANDIS does not bind the type II transmembrane protein TNF-α, demonstrating fundamental differences in the respective shedding by ADAM17.