Structure

The 50 kb ADAM17 gene, which is located at chromosome 2p25, consists of 19 exons, and encodes an 824 amino acid protein. ADAM17 is synthesized as an inactive precursor protein consisting of five domains: the pro-, metalloprotease, cysteine-rich, transmembrane, and cytoplasmic domains. Prior to ADAM17 maturation, a conserved cysteine residue within the pro-domain interacts with the active site zinc atom maintaining the enzyme biologically inert. The active site of the metalloprotease domain contains a histidine consensus sequence (HExxHxxGxxH) that coordinates zinc atoms and water required for the enzymatic processing of ADAM17 substrates. Removal of the pro-domain occurs through a  furin cleavage site (RVKR), by an unidentified furin or proprotein convertase, enabling the active site zinc to interact with the required histidine residues and to generate the active protease.

While the structural and functional aspects of the pro- and metalloprotease domains have been studied extensively and are well defined, the precise functions of the remaining ADAM17 domains are still somewhat obscure. The cysteine-rich domain consists of two subdomains: the disintegrin and EGF-like domains. A role in cellular  adhesion has been proposed for the disintegrin domain. In support of this hypothesis, ADAM17 has been shown to interact with at least one integrin (α5β1) and modulate cell migration as a result of this interaction. It has also been demonstrated that the cysteine-rich domain is indispensable for the ectodomain shedding of select ADAM17 substrates and, thus, might function in substrate recognition through the recruitment of accessory proteins or direct contact with the substrates themselves. The transmembrane domain tethers mature ADAM17 in the cell membrane where it exerts most of its physiological functions. Finally, the cytoplasmic domain comprises several Src homology 2 (SH2) and 3 (SH3) domain binding sites as well as phosphorylation sites, and is likely involved in regulatory signal transduction pathways.

Expression and Regulation

ADAM17 mRNA is ubiquitously expressed in most adult tissues, albeit at lower levels than those observed in fetal tissues at various stages of development. The ADAM17 zymogen is synthesized in the rough endoplasmic reticulum and is processed in the late Golgi compartment to produce the mature protease lacking the inhibitory pro-domain. This maturation step seems to entail a constitutive process as the majority of cellular ADAM17 exists in its mature form. The greater part of ADAM17 protein is localized in the perinuclear area while the remaining fraction resides at the cell surface, as expected. Notably, it appears that the membrane-bound ADAM17 population is exclusively in the processed form. This surface pool of ADAM17 is relatively stable with a half-life of ~8 h.

The mechanism by which ADAM17 function is regulated is not entirely clear; however, two methods by which the protease can be activated have been described. The first method involves the activation of ADAM17 by growth factors, such as the  fibroblast growth factor (FGF) and the  platelet-derived growth factor (PDGF). ADAM17-mediated ligand shedding can also be induced by non-physiological stimuli such as phorbol esters (phorbol myristate acetate). Treatment of cells with phorbol esters, such as PMA, results in increased ligand shedding without affecting the quantity or localization of endogenous ADAM17 in the cell. There is conflicting evidence with respect to the mechanism by which this stimulation occurs. One study demonstrated that PMA exerts its effects by activating the extracellular signal-regulated kinase (ERK) signaling pathway, which results in the phosphorylation of ADAM17 at Thr735 in its cytoplasmic tail, while another group showed that the cytoplasmic tail of ADAM17 is not required for PMA-induced ligand shedding. Although there is no evidence that phorbol esters regulate ADAM17 activity in vivo, the ERK signaling pathway has also been implicated in growth factor stimulated ADAM17 activation. For this reason, the ERK signaling pathway will likely be the focus of future studies aimed at delineating the mechanisms involved in the positive regulation of ADAM17 activity.

In addition to stimulating ADAM17-mediated ligand cleavage, the treatment of cells with PMA also triggers the establishment of a negative feedback mechanism. Following an increase in ADAM17 activity and ligand shedding, the protease itself is internalized and degraded in response to prolonged treatment with PMA. This negative regulatory mechanism is probably in place to prevent over-stimulation of ligand-activated signaling pathways. In attempt to identify potential regulators of ADAM17 activity, two ADAM17 binding partners were uncovered by at least two-hybrid screens: synapse associated protein 97 (SAP97) and protein tyrosine phosphatase PTPH1. Overexpression of either molecule results in decreased ligand shedding implicating them in the negative regulation of ADAM17 activity. Whether either of these two proteins regulates ADAM17 activity in vivo remains to be seen. The only known endogenous inhibitor of ADAM17 is the tissue metalloprotease inhibitor, TIMP3. The mechanism by which TIMP3 expression results in reduced ADAM17 activity is unknown.

Biological Function

ADAM17 was initially identified as the secretase responsible for the cleavage of tumor necrosis factor-alpha (TNFα), a pro-inflammatory cytokine. The generation of transgenic mice expressing ADAM17 lacking the zinc-binding sequence in its metalloprotease domain (ADAM17^ΔZn/ΔZn) allowed for the identification of a multitude of additional ADAM17 substrates. The vast majority of the ADAM17^ΔZn/ΔZn mice die at birth as a result of severe deficiencies in skin, muscle, lung, and neuronal system development that cannot be entirely attributed to loss of TNFα shedding. This indicates the existence of other biologically relevant ADAM17 substrates. Interestingly, the few animals that do survive display a phenotype that is comparable to that of transforming growth factor alpha (TGFα) or  epidermal growth factor receptor (EGFR) knockout mice. This includes the failure of eyelids to fuse as well as defects in skin and hair follicle development. Upon further investigation it was confirmed that TGFα, an EGFR ligand, is in fact an ADAM17 substrate. Moreover, ADAM17 appears to be the major convertase of several EGFR ligands which are involved in a variety of cellular processes including cellular proliferation, survival, migration, and differentiation. The bulk of ADAM17 substrates, including the EGFR ligands, are involved in cell development and differentiation. Other examples include the neurogenic signaling molecule Notch, the neurotrophin receptor TrkA, and the EGFR-family receptor HER4. The remaining substrates can be classified as those involved in cellular immunity and regulation of immunogenic responses, like TNFα. These substrates include the TNF receptors (TNF-RI and TNF-RII), the chemokine fractalkine, and the leukocyte adhesion molecule L-selectin to name a few. While many ADAM17 substrates have been identified to date, there is no obvious sequence or structural homology between their cleavage sites. How ADAM17 achieves substrate specificity is a key question that remains to be answered. Nonetheless, it is evident that ADAM17 substrates play an important role in a broad range of fundamental cellular processes.

Clinical Relevance

Due to its involvement in TNFα processing, ADAM17 is considered to be a central mediator in human inflammatory diseases such as rheumatoid arthritis. Direct inhibition of TNFα or ADAM17 in arthritis-affected cartilage has been shown to reduce inflammation. For these reasons ADAM17-based therapies, such as zinc-chelating sulfonamide hydroxamates, are in use for the treatment of such diseases.

In addition to its role in inflammatory diseases, ADAM17 is becoming increasingly implicated in the development and progression of cancer as a result of its role in the processing of EGFR ligands. The upregulation of EGFR expression and signaling is a common feature in human cancer. Unfortunately, EGFR inhibitors have rendered disappointing results in  clinical trials and there is an apparent resistance of several cancer cell lines to these agents. Importantly, ADAM17 is also overexpressed in several neoplastic tissues including breast carcinomas, colon carcinomas, pancreatic ductal adenocarcinomas, and ovarian carcinomas. There is also a positive correlation between ADAM17 expression and the aggressiveness of the malignancy. Thus ADAM17 is most highly expressed in advanced tumors, suggesting that ADAM17 and its substrates play a role in tumor progression.

In accordance with these observations, there is a growing amount of evidence supporting the use of anti-ADAM17 drugs in the treatment of cancer. Several studies have shown that inhibition of ADAM17 activity using a variety of approaches is sufficient to inhibit EGFR ligand release and to prevent the proliferation, migration, and survival of squamous cell, kidney cancer,  bladder cancer, and  breast cancer cell lines in vitro. It was demonstrated that  siRNA-mediated silencing of ADAM17 inhibits the release of soluble TGFα in highly malignant renal carcinoma cells, thereby abolishing their ability to form tumors in nude mice. This was the first in vivo evidence that ADAM17-mediated ligand cleavage is a pivotal step in the establishment of the TGFα/EGFR autocrine ( autocrine signaling) growth stimulatory loop and thus in tumorigenesis. Another study revealed that targeting ADAM17, using a small molecule inhibitor, prevents heregulin cleavage and hence HER3 activation in non–small cell lung cancer cells. Not only did this inhibition abolish tumor growth in vivo but it also enhanced the sensitivity of the cancer cells to gefitinib, an anti-EGFR based therapy. This result suggests that the concomitant inhibition of ADAM17 and EGFR should improve patient responsiveness to such agents and increase survival. Thus targeting ADAM17 is a promising new alternative to traditional EGFR-based therapies in the treatment of human cancer.

Summary

ADAM17 was originally characterized for its role in TNFα processing and the regulation of inflammatory responses. It has since been demonstrated that ADAM17 is also a physiological convertase of a wide variety of signaling molecules implicated in the development and progression of cancer. The importance of ADAM17 in these oncogenic pathways is highlighted by the finding that silencing of ADAM17 is sufficient to abolish tumor formation in vivo. These results validate ADAM17 as a rational therapeutic target and endorse the use of ADAM17 inhibitors in the treatment of human cancer.