The ADAM disintegrin metalloproteinases (where ADAM is ‘a disintegrin and metalloproteinase’) are a family of transmembrane cell-surface proteins with essential roles in adhesion and proteolytic processing in all animals. The archetypal family member is ADAM17 {also known as TACE [TNFα (tumour necrosis factor α)-converting enzyme]}, which is involved in processing pro-TNFα and in the activation of ligands for the EGFR [EGF (epidermal growth factor) receptor], as well as cleavage of diverse cell-surface receptors and adhesion molecules. ADAM-mediated shedding is itself influenced via cell signalling pathways. In this issue of the Biochemical Journal, Willems et al. make the observation that phorbol ester activates shedding by ADAM17 by affecting the activity of PDI (protein disulfide isomerase). They propose that PDI maintains ADAM17 in an inactive ‘closed’ state and PMA stimulation generates ROS (reactive oxygen species) and thus an altered redox environment, which in turn inactivates PDI and allows ADAM17 to adopt an ‘open’ active conformation. This activation is accompanied by changes in disulfide bonds in the ADAM17 ectodomain. This is a novel and exciting finding that could help to unlock the actions of ADAM sheddases, as well as a host of other mechanisms that rely upon rapid alterations in protein conformation on the cell surface.

The ADAMs (where ADAM is ‘a disintegrin and metalloproteinase’) are modular cell-surface proteins that perform essential functions in cell adhesion and proteolysis. The biological processes to which ADAMs have been linked include sperm–egg interactions, cell-fate determination in the nervous system, cell migration, axon guidance, epithelial tissue development and immune activation [1]. The typical ADAM is approx. 750 amino acids in length and comprises an N-terminal pro-domain involved in maintenance of enzyme latency, followed successively by discrete metalloproteinase, disintegrin, cysteine-rich and EGF (epidermal growth factor)-like domains, then a transmembrane region and variable length cytoplasmic tail (Figure 1). There are 21 functional members of the human ADAM family, but only 13 of these are active metalloproteinases as the remainder lack the essential zinc-binding catalytic-site motif. Isolated disintegrin or cysteine-rich modules of many ADAMs, as well as the full ectodomains of some family members, have been shown to interact with integrins (reviewed in [1]), and the non-proteolytic members can function as adhesion receptors, as is the case for ADAM22 in the nervous system [2]. The proteolytically active ADAMs act as ‘ectodomain sheddases’, in some cases (typified mostly by ADAM10) carrying out initial cleavages on molecules, such as Notch, that are then subject to ‘regulated intramembrane proteolysis’ generating intracellular signals, whereas ADAM17 acts on a huge variety of cell-surface molecules leading to their activation or release. Thus ADAM17 orchestrates inflammatory responses via activation of pro-TNFα (tumour necrosis factor α) and diverse receptors, but also regulates cell growth via activation of membrane-associated ligands of the epidermal growth factor receptor [3].

C-shaped structure of full-length ADAMs

Figure 1
C-shaped structure of full-length ADAMs

The domain organization of mammalian ADAMs is presented based on crystallographic data from the study of snake venom metalloproteinases [7]. MP, metalloproteinase domain (black); TM, transmembrane domain (black-and-white checkerboard), ds (blue) and da (green) are subdomains of the disintegrin domain (shoulder and arm respectively); cw (white) and ch (grey checkerboard) are subdomains of the cysteine-rich domain (wrist and hard respectively); Cyto, cytoplasmic. Further details are given in the text and in reference [1].

Figure 1
C-shaped structure of full-length ADAMs

The domain organization of mammalian ADAMs is presented based on crystallographic data from the study of snake venom metalloproteinases [7]. MP, metalloproteinase domain (black); TM, transmembrane domain (black-and-white checkerboard), ds (blue) and da (green) are subdomains of the disintegrin domain (shoulder and arm respectively); cw (white) and ch (grey checkerboard) are subdomains of the cysteine-rich domain (wrist and hard respectively); Cyto, cytoplasmic. Further details are given in the text and in reference [1].

One of the striking points about ADAM17 is that its shedding activity can be rapidly induced by treating cells with phorbol esters and many other agents. A logical place to look for the explanation of this phenomenon was the cytoplasmic tail of the protein, which was found to be a target for kinases and therefore potentially able to direct trafficking or protein–protein interactions [1]. However, it was found previously that the intracellular domain of ADAM17 was dispensable for PMA-induced shedding; all that was necessary was insertion into the membrane [4]. It has since become clear that for several ADAMs, the cytoplasmic domain does indeed play a role in regulating ADAM function, as has recently been shown for ADAM10 in ephrin–Eph (ephrin receptor) signalling [5] and ADAM15 in shedding of FGFR2B [FGF (fibroblast growth factor) receptor 2B] [6]. However, to determine the mechanism for PMA-induced ADAM17 shedding, we need to look outside the cell.

A major conceptual advance in ADAM research came with the first detailed crystal structure, which emerged from analysis of related snake venom metalloproteinases that have a similar domain arrangement to the mammalian ADAMs [7]. This revealed a C-shaped structure with the metalloproteinase and cysteine-rich domains as the top and bottom of the C (Figure 1). The disintegrin domain between them was held in a conformation such that the ‘disintegrin loop’, which had been proposed previously as the ligand for integrin interaction, lay buried inside the structure. In contrast the cysteine-rich domain, and in particular a region contained within it that is hypervariable in different ADAMs, was presented as the sideward-facing part of the C in a way that might allow docking with potential ADAM substrates close to the cell membrane.

In a surprisingly simple opening experiment, Willems et al. [8] now show that treatment of HeLa cells with reducing agents inhibits PMA-induced shedding of a HB-EGF (heparin-binding EGF) reporter substrate by ADAM17, arguing that correct disulphide bonding is important for ADAM17 function. As ADAM17 has potentially 16 intrachain disulfide bonds, this raised the notion that perhaps enzymes with thioredoxin-like thiol isomerase activity might modulate the ADAM17 structure. The authors tested this initially by using bacitracin, a rather non-specific inhibitor of PDI (protein disulfide isomerase). This was found to further enhance PMA-induced activity of ADAM17, whereas exogenous recombinant PDI had the opposite effect; reduction and alkylation of PDI wiped out its inhibitory effect. In contrast, knockdown of endogenous PDI expression or use of a function-blocking anti-PDI antibody gave the same stimulatory effects as bacitracin. The authors show that endogenous PDI was on the extracellular side of the cell surface and that ADAM17 and PDI could under certain conditions be co-precipitated.

Some useful new tools brought to bear by the authors are scFv (single chain Fv) antibodies that recognize different domains in ADAM17: the A9 antibody recognizes the metalloproteinase domain, whereas both the D3 and A7 antibodies recognize the combined disintegrin–cysteine-rich domains. Two of these scFvs (A9 and A7) were sensitive to reduction of the disulfide bonds in ADAM17, whereas D3 was not. The authors showed that if they treated the recombinant ADAM17 ectodomain with reduced (and thus active) PDI, they ablated the binding of D3 and A7 to ADAM17, but that this had no effect on A9 binding. This argued strongly that PDI is able to alter the topology of ADAM17 epitopes in the disintegrin–cysteine-rich domains, but not the metalloproteinase domain.

Hence, the notion is that under resting conditions PDI interacts with ADAM17 generating a structure that makes ADAM17 inactive and that PMA treatment alters PDI which in turn allows ADAM17 to adopt an active conformation. How might this be achieved? The authors speculated that ROS (reactive oxygen species) generated by PMA activation of mitochondrial metabolic activity would alter the cellular redox state and this in turn would affect PDI activity. This was supported by the inhibitory effects of ROS scavengers on ADAM17-mediated HB-EGF shedding and subsequent experiments using a thiol-biotinylating reagent showed that PMA stimulation reduced the level of free thiol residues in cell-surface PDI, but not ADAM17.

This paper from Murphy and colleagues [8] is an intriguing and provocative study that throws up a lot of new questions. First, there is the issue of how specific is the effect of PDI. The authors have demonstrated the involvement of PDI with ADAM17 in multiple cell types, suggesting that this is a general mechanism. But what about other types of PDI? The PDI family of disulfide isomerases consists of at least 19 members with thioredoxin-like catalytic domains containing the CxxC active-site motif and C-terminal ER (endoplasmic reticulum)-retention sequences, such as Lys-Asp-Glu-Leu (KDEL) [9]. They are molecular chaperones, mediating correct protein folding and disulfide bond formation in the ER and disulfide bonds are required for stabilization of the structure of all extracellular proteins. Willems et al. [8] show that the link to ADAM17 activity in their model system primarily involves PDI itself as participation of the other PDI family members ERp5 (ER protein 5) and thioredoxin was ruled out. However, this does raise the possibility that interactions between ADAM17 amd other PDI family members might modulate interactions with particular substrates, particularly as the C-shaped ADAM structure positions the cysteine-rich domain to face out towards potential substrates (Figure 1). Recently it was demonstrated that ADAM17 was responsible for PMA-induced shedding of MIC (MHC I-related chain), which acts as an activating ligand for NK (natural killer) cells in pathological conditions [10]. It had been shown previously that MIC associates with the PDI ERp5 and that this association was necessary for shedding to occur [11]. Given the number of players in the ADAM and PDI families, there is thus the potential for building in multiple levels of specificity in terms of substrate preference, cell type and stimulus.

Recent crystallographic data show a radically different I-shaped conformation in one of the snake venom metalloproteinases that is attributed to a distinct disulfide-bonding pattern in its disintegrin domain [12]. In addition, the structure of the non-proteolytic ADAM22 has been revealed as a four-leaf-clover-type arrangement, with the potential for movement between the metalloproteinase domain on top and a three-lobed disintegrin–cysteine-rich–EGF-module upon which it rests [2]. PDIs on the cell surface have been separately suggested to influence the affinity of integrins for their ligands via a structural shift from a bent form to an extended form (reviewed in [13]); it has been demonstrated that this requires disulfide exchange [14] and can be facilitated by PDI family members [15]. Thus the actions of PDIs on ADAM and integrin topology could be very profound indeed and much like the ‘open’ (PDI inactive) and ‘closed’ (PDI active) structures of ADAM17 proposed by Willems et al. [8], PDIs may be major factors in directing ADAM- and integrin-mediated adhesive interactions.

Another big question is how are ADAMs and PDIs brought together? In addition to their well-established role in the ER, there is a growing number of reports of PDI proteins having roles on the cell surface, modulating the conformation and function of cell-surface proteins (reviewed in [16]). However it is intriguing how these KDEL-motif-containing proteins escape the ER and become extracellular. Possibilities include cleavage of this part of the molecule, chemical modification or masking by another protein. Willems et al. [8] show that the levels of PDI do not change on the cell surface with PMA treatment, thus a key aspect may involve the regulated movement of ADAMs, PDIs and substrates within the membrane. It has been shown previously that ADAM17-mediated shedding of MIC depends on recruitment of both proteins to detergent-resistant microdomains [10]. This movement is thus a likely point for control via the cell's signalling machinery.

The study by Willems et al. [8] has revealed a new tier of regulation on ADAM function that will initiate a lot of further investigations. For instance, it would be intriguing to use the new conformation-specific scFvs as tools to study dynamic changes in ADAM conformation in real-time in response to cell stimulation, or perhaps by using FRET (fluorescence resonance energy transfer) to study ADAM–PDI interactions. We also need to know a lot more about the detailed structures of full-length ADAMs, which is quite a challenge for crystallography. This should keep the ADAM field busy for a while!

Abbreviations

     
  • ADAM

    a disintegrin and metalloproteinase

  •  
  • EGF

    epidermal growth factor

  •  
  • ER

    endoplasmic reticulum

  •  
  • ERp5

    ER protein 5

  •  
  • HB-EGF

    heparin-binding EGF

  •  
  • MIC

    MHC I-related chain

  •  
  • PDI

    protein disulfide isomerase

  •  
  • ROS

    reactive oxygen species

  •  
  • scFv

    single chain Fv

  •  
  • TNFα

    tumour necrosis factor α

Our laboratory is supported by Cancer Research UK; the Breast Cancer Campaign; Big C Cancer Charity; the British Heart Foundation; and by the Suzie Wright fund.

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