FEBS Letters 586 (2012) 1093–1100

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The membrane-proximal domain of A and Metalloprotease 17 (ADAM17) is responsible for recognition of the interleukin-6 receptor and interleukin-1 receptor II

Inken Lorenzen a, Juliane Lokau a, Stefan Düsterhöft a, Ahmad Trad a, Christoph Garbers b, Jürgen Scheller b, ⇑ Stefan Rose-John a, Joachim Grötzinger a, a Institute of Biochemistry, Christian-Albrechts-University, Olshausenstr. 40 24118 Kiel, Germany b Institute of Biochemistry and Molecular Biology II, Heinrich-Heine-University, Universitätsstr. 1, 40225 Düsseldorf, Germany article info abstract

Article history: A great number of physiological processes are regulated by the release of ectodomains of membrane Received 11 October 2011 proteins. A Disintegrin And Metalloprotease 17 (ADAM17) is one of the important , which Revised 5 March 2012 mediate this process called shedding. Today, more than 70 substrates of this transmembrane metal- Accepted 8 March 2012 loprotease are known. This broad spectrum raises the question how ADAM17 recognizes its sub- Available online 21 March 2012 strates specifically. Differently tagged ADAM17 deletion variants were used to demonstrate that Edited by Gianni Cesareni exclusively the extracellular domains of ADAM17 are needed for interaction with two of its sub- strates, the IL-6R and the IL-1RII; whereas the transmembrane- and cytoplasmic-region are dispens- able for this process. In the extracellular part solely the membrane-proximal domain of ADAM17 is Keywords: Metalloprotease mandatory for recognition of the two type-I transmembrane proteins, but not for the interaction ADAM17 with the type-II transmembrane molecule TNF-a. EGF-like domain Cysteine-rich domain Structured summary of protein interactions: Membrane-proximal domain IL-6R physically interacts with ADAM17 by anti bait coimmunoprecipitation (View interaction) ADAM17 Substrate recognition physically interacts with IL-1RII by anti tag coimmunoprecipitation (View interaction)

Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction ADAM17 in mice leads to perinatal lethality, highlighting the impor- tance of ectodomain shedding [6]. ADAM17 (A Disintegrin And Metalloprotease 17) is involved in Members of the ADAM-family are type-I transmembrane multi- many physiological processes, including cell migration and prolifer- domain metalloproteases consisting of an N-terminal signal ation. More than 70 substrates of ADAM17 are described so far [1]. peptide, a pro-, a catalytic-, a disintegrin-, a cysteine-rich- and an These substrates include pro-inflammatory cytokines and cytokine Epidermal Growth Factor- (EGF-) like-domain followed by a single receptors, like Tumor Necrosis Factor-a (TNF-a) [2,3], interleukin-6 transmembrane region and a cytoplasmic tail. It is described that receptor (IL-6R) [4] and IL-1RII [5], which are proteolytically cleaved the cytoplasmic domain of ADAM17 is involved in regulatory events and released from the membrane, a process named ectodomain like protein trafficking [7], but is not needed for phorbol 12-myris- shedding. This does not only provide a possibility for protein tate 13-acetate (PMA) or src induced shedding [5,8]. Furthermore, down-regulation but also for initiating or inhibiting autocrine and the transmembrane region of ADAM17 is necessary for shedding paracrine signaling via soluble proteins. disruption of of Transforming Growth Factor-a and TNF-a, since coupling of the extracellular part of ADAM17 to the plasma membrane by a glyco- Abbreviations: ADAM17, A Disintegrin And Metalloprotease 17; TNF-a, Tumor sylphosphatidylinositol (GPI) anchor leads to a loss of shedding Necrosis Factor-a; IL-6R, interleukin-6 receptor; IL-1RII, interleukin-1 receptor type activity [9]. ADAM10 and ADAM17 are atypical members of the II; EGF, Epidermal Growth Factor; MPD, membrane-proximal domain; PMA, ADAM-family; they contain a pro-, a catalytic-, a disintegrin- and phorbol 12-myristate 13-acetate; GPI, glycosylphosphatidylinositol; AP, alkaline a membrane-proximal domain (MP) with a novel a/b fold. phosphatase ⇑ Corresponding author. Address: Biochemisches Institut, Christian-Albrechts- (Fig. 1A) [10–12]. The extracellular part of mature ADAM17 most Universität Kiel, Olshausenstr. 40, 24118 Kiel, Germany. Fax: +49 431 8805007. likely adopts a C-shaped conformation [11,13], thereby localizing E-mail address: [email protected] (J. Grötzinger). the membrane-proximal domain close to the . This

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.03.012 1094 I. Lorenzen et al. / FEBS Letters 586 (2012) 1093–1100

Fig. 1. Expression of ADAM17 deletion variants. (A) A schematic drawing of ADAM17. (B–E) Schematic pictures of each construct are depicted in the first row. Expression analysis by Western blotting and flow cytometry analysis is shown in the second and third column, respectively. The used antibody for Western blot analysis is indicated. The left lane of the Western blots shows the sample of interest, whereas the right lane shows the corresponding control. Flow cytometry was performed using the antibody A300E (see Materials and methods) and demonstrates that all constructs are expressed on the cell surface (red: non-transfected cells; blue: transfected cells).

supports the idea that the two membrane-proximal domains are human ADAM17 and human N-terminally tagged substrates were involved in substrate recognition and activation [5,11,13– cloned using standard procedures. Detailed information about 17]. Here we show that the membrane-proximal domain of the constructs is provided in Supplementary Fig. 1. Oligonucleo- ADAM17 is the minimal requirement for recognizing two type-I tides used for insert amplification and insertions of the respective transmembrane substrates, IL-1RII and IL-6R, but not for the tags are listed in Supplementary data Table S1. type-II transmembrane protein TNF-a. 2.2. Cells and transfection 2. Materials and methods HEK-293 cells and ADAM17ex/ex mEFs were cultured in DMEM 2.1. Cloning of ADAM17 variants and substrates high-glucose containing 10% fetal calf serum (FCS), penicillin (60 mg/ l) and streptomycin (100 mg/l) (PAA Laboratories, Marburg, Germany). Murine full-length ADAM17, murine full-length chimera of Transfection was performed using TurboFect (Fermentas, St. Leon-Rot, ADAM17 and ADAM22 (ADAM17-EGF22), deletion variants of Germany) according to manufacturer’s instructions. I. Lorenzen et al. / FEBS Letters 586 (2012) 1093–1100 1095

2.3. Co-immunoprecipitation and Western blotting co-immunoprecipitation experiments. Therefore ADAM17 variants were tagged and expressed in HEK-293 cells (Fig. 1 and Supple- Co-immunoprecipitation experiments and Western blotting were mentary Fig. 1A). performed as described earlier [18,19] and in the Supplementary data. The murine full-length ADAM17 was C-terminally tagged either Depending on the constructs, rabbit anti-HA- (C29F4), rabbit anti- with a FLAG- (Fig. 1B), an HA- or PC-tag (data not shown) (Supple- FLAG- (M2), rabbit anti-myc- (71E10), rabbit anti-TNF-a,rabbitanti- mentary Fig. 1A). To analyze the impact of the transmembrane- caveolin-1 (D46G3), murine anti-myc- (New England, Biolabs – Cell and the cytoplasmic region for substrate recognition a construct Signaling, Germany) or murine anti-PC- (HPC4), anti-IL-6R- as well which lacks these parts was used. Thereby the extracellular region asamurineanti-ADAM17antibodydirected against its disintegrin do- of murine ADAM17 (amino-acid residue 1 to 657 of the native main (A300) [20] were used. To avoid the appearance of heavy and ADAM17 sequence) was fused to a GPI-membrane anchor light chain in the blots of immunoprecipitated samples a combination (ADAM17-GPI, Fig. 1C). ADAM17-GPI comprises all domains of its of murine anti-PC- or anti-IL-6R-antibodies and rabbit derived anti- extracellular part and contains a myc- as well as a PC-tag between bodies was used. In case of ADAM17-GPI two antibodies of murine its coding sequence and the GPI attachment sequence (Supplemen- origin were used, since the HA-tagged variant could not be expressed. tary Fig. 1A). To reduce a part of the artificial elongation resulting by When anti-PC-tag antibody was applied, buffers were supple- the insertion of the two tags, amino acid 658–671 of the extracellu- mented with 4 mM CaCl2; lysis buffer contained 20 mM Tris/HCl lar region was deleted. pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and ‘‘complete’’ To examine if the catalytic domain is needed for substrate rec- inhibitor mixture without EDTA (Roche applied science, ognition, a GPI-variant without this domain, DCat-ADAM17-GPI, Mannheim, Germany). was cloned (Fig. 1D and Supplementary Fig. 1A). Therefore human DCat-ADAM17 [21] was used as template to generate a construct 2.4. Shedding assay which starts with the human IL-6 signal peptide followed by the coding sequence of human ADAM17 from amino-acid residue HEK-293 cells were transfected with plasmids coding for the 475 (Supplementary Fig. 1A). respective substrates and ADAM17 variants at a ratio of 1:10. As To distinguish between the roles of the disintegrin- and mem- substrate either human AP-IL-6R or human AP-IL-1RII were used. Next brane-proximal-domain the disintegrin domain of DCat-ADAM17- day, medium was replaced by DMEM high-glucose. Cells were either GPI was deleted resulting in a construct which encodes only a mem- left untreated, stimulated with 100 nM PMA (Merck, Darmstadt, brane bound membrane-proximal domain starting from amino- Germany) or stimulated with 100 nM PMA and treated with 50 lM acid residue 581 of the native ADAM17 sequence (MPD17-GPI, metalloprotease inhibitor GM6001 (Millipore, Schwalbach/Ts., Fig. 1E and Supplementary Fig. 1A). DCat-ADAM17-GPI as well as Germany). After 2 h, supernatant and cells were harvested. For detec- MPD17-GPI was obtained by replacing the murine ectodomain of tion of cell associated AP activity, cells were lysed in lysis buffer as de- ADAM17-GPI with the respective inserts. For co-immunoprecipita- scribed above, and mixed, like the supernatant, with the same volume tion experiments with the IL-6R, where antibodies from murine origin had to be used for precipitating the receptor, the PC-tag of of substrate solution [100 mM glycine, 1 mM MgCl2 and 1 mM ZnCl2 containing 1 mg/ml p-nitrophenylphosphate (Sigma, Taufkirchen, the GPI constructs was replaced by an HA-tag (data not shown), to Germany)] and absorption was measured at 405 nm. The absorption give the possibility of using antibodies from non-murine origin. As of the supernatant was divided by the absorption of cell lysate and depicted in Fig. 1 all variants could be expressed in HEK-293 cells the ratio normalized against the respective sample treated with PMA and were located at the cell surface as demonstrated by Western and metalloprotease inhibitor. Student’s t-test was performed using blotting and flow cytometry, respectively. http://www.physics.csbsju.edu/stats/t-test.html. 3.2. Determination of ADAM17 domains involved in substrate recognition 2.5. ADAM17 activity assay For co-immunoprecipitation of ADAM17 with the human IL-6R ADAM17ex/ex mEFs [23] were transfected either with constructs of as substrate a very sensitive monoclonal antibody against this murine ADAM17, the murine ADAM17-EGF22 chimera or peGFPN1 transmembrane type-I protein was used. As shown in Fig. 2A, this (as mock) and AP-IL-1RII in an 1:5 ratio. One day after transfection antibody was able to pull-down the IL-6R and full-length ADAM17 media was replaced and cells treated like in the shedding assays with was co-precipitated. Since the transmembrane- and the cytoplas- the exception that instead of 50 lM GM6001 10 lM marimastat mic-region are important for the regulation and the function of this (Merck, Darmstadt, Germany) was used to inhibit metalloprotease enzyme [7,9,22] we tested whether these regions are mandatory activity. After 2 h medium and cells were harvested and analyzed for for substrate binding using the GPI anchored variant comprising AP activity as described for the shedding assay. the extracellular part of murine ADAM17 (Fig. 1C). As shown in Fig. 2B this variant could be precipitated by the human IL-6R dem- 2.6. Flow cytometry onstrating that the cytoplasmic- and transmembrane-region are not crucial for substrate recognition. The same result was obtained HEK-293 cells were harvested two days after transfection [20]. with a corresponding variant lacking the catalytic domain, DCat- For detection of ADAM17 variants, 2 lg/ml of A300E antibody, di- ADAM17-GPI. The co-precipitation of DCat-ADAM17-GPI with rected against the membrane-proximal domain of ADAM17, were the IL-6R implicates that the catalytic domain is also dispensable used to stain one million cells [20]. for binding of the IL-6R (Fig. 2C). Previous work suggested that the disintegrin domain has scaf- 3. Results folding functions and places its neighboring domains in a correct orientation relative to each other [13], whereas the membrane- 3.1. Construction and cell surface expression of ADAM17 variants proximal domain is thought to be involved in substrate recogni- tion. Accordingly, the IL-6R precipitated MPD17-GPI (Fig. 2D), the To gain insight into the function of the non-catalytic extracellu- construct containing solely the membrane anchored membrane- lar domains of ADAM17 the full-length molecule as well as proximal domain, suggesting that the membrane-proximal domain deletion variants thereof were analyzed for substrate binding by alone is sufficient for substrate binding. 1096 I. Lorenzen et al. / FEBS Letters 586 (2012) 1093–1100

Fig. 2. The membrane-proximal domain of ADAM17 recognizes the IL-6R. HEK-293 cells were co-transfected with human IL-6R and different ADAM17 variants. Three days later cells were harvested, lysed and subjected to co-immunoprecipitation experiments, thereby the IL-6R was precipitated. (A) Precipitation of the receptor leads to a pull- down of full-length ADAM17, which could be detected by its HA-tag. (B-D) Deletion of the transmembrane domain and the cytoplasmic region, using GPI anchored proteins, does not inhibit these effects; as shown for the ectodomain detected by its PC-tag (B), DCat-ADAM17-GPI (C) as well as for MPD17-GPI (D) both detected by their HA-tag.

To confirm these results, we repeated some of the previous Additional co-immunoprecipitation experiments of MPD17-GPI experiments with a second substrate, the IL-1RII. This time not together with humane TNF-a show the same shift in size, whereby the substrate, but ADAM17 or its variants, were precipitated. In proTNF-a (25 kDa) runs between 26 and 34 kDa. Interestingly, the first immunoprecipitation an HA-tagged full-length ADAM17 pull-down of the PC-tagged variant of ADAM17 leads not to a co- was chosen, because the FLAG-tagged variant could not be precip- precipitation of pro-TNF-a (Fig. 3F), indicating that the co-precipi- itated using the respective antibody. Immunoprecipitation of full- tation experiments are specific and not due to overexpression arti- length ADAM17 leads to co-precipitation of the N-terminally facts. In addition this result shows that the membrane-proximal myc-tagged IL-1RII (Fig. 3A and Supplementary Fig. 1B). Further- domain is most likely not the minimal module for recognizing more a precipitation of DCat-ADAM17-GPI as well as MPD17-GPI, the type-II transmembrane protein TNF-a. via their PC-tag, co-precipitates the IL-1RII (Fig. 3B and C). The GPI constructs were, according to their second tag, the myc-tag, 3.3. Influence of ADAM17 variants on the activity of endogenous detectable by anti-myc antibodies in the respective blots (Fig. 3B ADAM17 and C). To rule out the possibility that endogenous ADAM17 interferes It was described that DCat-ADAM17 acts as an inhibitor of with the co-immunoprecipitation experiments, we used ADA- ADAM17 [25]. If this effect is due to competition of DCat-ADAM17 M17ex/ex mEFs generated from ADAM17ex/ex mice [23] in which with the wild-type molecule for substrates, the same effect should ADAM17 expression is reduced more than 95%. These cells were be true for all variants which contain the membrane-proximal co-transfected with vectors coding for the IL-6R and MPD17-GPI. domain. Hence, we performed shedding assays using AP-tagged Although the expression level was low (the substrate could not variants of IL-6R and IL-1RII as substrates (Supplementary Fig. 1B be detected in the input control) pull-down of the IL-6R lead to and Fig. 4). In addition to DCat-ADAM17-GPI, a construct compris- an accumulation of the protein, which co-precipitated the ing the transmembrane domain but lacking the cytoplasmic por- HA-tagged MPD17-GPI (Fig. 3D) confirming that the membrane- tion (DCat-ADAM17Dcyto, Supplementary Fig. 1A) was tested to proximal domain is the minimal requirement for substrate analyze the influence of the transmembrane region. Both PC- binding. tagged ADAM17 variants, DCat-ADAM17Dcyto and DCat- ADAM17 is located and active within lipid rafts [24]. To exclude ADAM17-GPI, significantly inhibit shedding of the IL-6R and the the possibility that whole lipid rafts were precipitated, we probed IL-1RII in a comparable range (<0.001, 0.003 and P = 0.017, against caveolin-1 as a marker for lipid rafts (Fig. 3E). In this exper- P = 0.003, Fig. 4A). Accordingly the described inhibitory property iment caveolin-1 could only be detected in the input control but of DCat-ADAM17 is most probably located in its extracellular part not in the lanes loaded with the immunoprecipitated sample. For and there is no detectable contribution of the transmembrane this experiments a new lot of marker was used, which runs lower domain. than the lot used before, therefore all bands appear a little too high. The same inhibitory effect is observed if the disintegrin domain For example MPD17-GPI appears in the experiments above just be- is missing and only the membrane-proximal domain is anchored to low 26 kDa and is shifted now towards to 26 kDa; and caveolin-1, the membrane (MPD17-GPI). Co-transfection of MPD17-GPI with which should appear at 21–24 kDa, is detected little above 26 kDa. IL-6R or IL-1RII leads to a significant decrease in their release from I. Lorenzen et al. / FEBS Letters 586 (2012) 1093–1100 1097

Fig. 3. The membrane-proximal domain of ADAM17 is the minimal requirement for substrate recognition. Full-length ADAM17 (A) as well as DCat-ADAM17-GPI (B) and MPD17-GPI (C) were able to precipitate myc-tagged IL-1RII. (B, C) The PC-tagged deletion variants were pulled down using anti-PC-tag antibodies. Due to the presence of the second tag within the GPI-constructs (a myc-tag) both proteins ADAM17 variant and IL-1RII could be detected using rabbit anti-myc antibody as depicted by the arrows. (D) ADAM17ex/ex mEFs were transfected with IL-6R and MPD17-GPI containing an HA-tag. The IL-6R was precipitated and both proteins could be detected. (E) HEK-293 cells were co-transfected with IL-6R and MPD17-GPI. Similar to Fig. 2D pull-down of the substrate leads to a co-precipitation of the HA-tagged membrane anchored membrane- proximal domain. The blot was additionally probed for caveolin-1 as a marker for lipid rafts. (F) Pull-down of MPD17-GPI by its PC-tag from lysate of HEK-293 cells co- transfected with human TNF-a and MPD17-GPI leads to no precipitation of the substrate. the cell surface compared to the control (P = 0.001 and P < 0.001, 3.4. Activity of ADAM17-EGF22 chimeric molecules Fig 4A). Interestingly, also the soluble expressed membrane-prox- imal domain reveals, despite its low expression, a slight but signif- To further validate that the membrane-proximal domain of icant inhibition of IL-1RII shedding (Supplementary Fig. 2A). The ADAM17 is essential for substrate recognition this domain was poor expression of the soluble domain becomes apparent by replaced by a different domain. Hence a chimeric molecule was Western blotting. All membrane bound ADAM17 variants could generated by replacing solely the membrane-proximal domain of be detected by Western blotting against their PC-tags after one murine PC-tagged ADAM17 by the EGF-like domain of murine day of transfection (Fig. 4B and C). This proof could not be achieved ADAM22 (ADAM17-EGF22, Supplementary Fig. 1A). The proteo- in case of the soluble membrane-proximal domain; its expression lytic activity of ADAM17-EGF22 was tested by co-transfecting could only be detected in an independent experiment after three ADAM17ex/ex mEFs with this constructs and AP tagged IL-1RII days of expression. Therefore these results support the importance and compared with wild-type ADAM17. The activity of the wild- of the membrane-proximal domain in the recognition of the two type molecule could be stimulated by the addition of 100 nM type-I transmembrane proteins. PMA (Fig. 5A). In contrast, the measured activity of the chimeric 1098 I. Lorenzen et al. / FEBS Letters 586 (2012) 1093–1100

Fig. 5. The ADAM17-EGF22 chimera is catalytically inactive. ADAM17ex/ex mEFs were co-transfected either with murine wild-type ADAM17, ADAM17-EGF22 or peGFPN1 (mock) and AP-tagged IL-1RII. After 24 h the medium was exchanged and cells were either left untreated, stimulated by the addition of 100 nM PMA or stimulated in the presence of a 10 lM marimastat. After 2 h supernatants and cells were harvested. (A) AP activity in the supernatant and in cell lysates was determined and their ratios were normalized against the sample treated with marimastat. Data are mean values from at least four independent experiments. Fig. 4. Inhibition of ADAM17 mediated shedding is most prominent by the Comparison of the PMA treated samples (ADAM17:ADAM17-EGF22 and ADAM17:- membrane bound membrane-proximal domain. HEK-293 cells were co-transfected mock) are statistically significant (P-value <0.0001). (B) Expression of murine with AP-tagged IL-1RII or IL-6R and ADAM17 variants containing a PC-tag. One day ADAM17 and murine ADAM17-EGF22, both PC-tagged, were tested by Western after transfection, medium was changed and cells were left untreated, stimulated blotting using an a-PC antibodies. with PMA alone or stimulated with PMA in the presence of a metalloprotease inhibitor (GM6001). Two hours later, supernatants and cells were harvested. (A) AP activity was measured in the supernatant as well as in the cell lysate. The ratios of activities (supernatant/lysate) were normalized against the sample treated with for human ADAM17 but also for murine ADAM17, whereas murine PMA and metalloprotease inhibitor. P-values below 0.05 are considered as statistically significant and marked with one asterisk (⁄); values below 0.01 are IL-6R is not. labeled with two and below 0.001 with three asterisks. (B–C) Expression of the Overexpression of full-length ADAM17 in HEK-293 leads to a ADAM17 variants was confirmed by Western blots against their PC-tag for strong appearance of immature ADAM17, but hardly any detect- experiments regarding the IL-1RII (B) and IL-6R (C). able band of the mature enzyme (Fig. 1B). This disappearance was explained by autoproteolysis of mature ADAM17 during cell lysis [27]. But addition of a metalloprotease inhibitor (10 lM ADAM17-EGF22 is decreased down to the level observed with the marimastat) during and after cell-lysis did not change this phe- mock control. Fig. 5B confirms the expression of the wild-type and nomenon. Additionally, the lack of the mature form of ADAM17 ADAM17-EGF22 chimera by Western blotting using their PC-tag. seems to be unique for strong overexpression, since expression of These results clearly demonstrate that the chimeric ADAM17- the same constructs in ADAM17ex/ex mEFs reveals both forms (data EGF22 is inactive and confirmed the importance of the mem- not shown). The absence of the mature form might be due to an brane-proximal domain of ADAM17 in substrate recognition. overload of the ER/Golgi transport, an impaired maturation in the Golgi apparatus, a rapid degradation of the mature form or the 4. Discussion concentration of metalloprotease inhibitor during lysis was too low. Interestingly, co-transfection and/or co-precipitation of full- The aim of this work was to understand the role of the extracel- length ADAM17 with its substrates leads to an accumulation of lular domains of ADAM17 in substrate recognition. Therefore, dif- the mature form implicating its stabilization or an enhanced mat- ferent deletion variants of ADAM17 were constructed and their uration (Fig. 2A and 3A). expression was analyzed by Western blotting and flow cytometry The appearance of both ADAM17 forms in the co-immunopre- analysis (Fig. 1). Comparison of the flow cytometry measurements cipitation experiments is somewhat surprising, since the mature suggests that the construct containing the whole intracellular part form is the catalytically active one. Enzyme and substrate might of ADAM17 was expressed at the cell surface to a lesser degree meet already in the secretory pathway, which is even more likely than the GPI anchored variants, which is in agreement with the in case of overexpression. Under these conditions it is conceivable finding that in unstimulated cells most ADAM17 is localized within that the pro-domain does not interfere with the interaction be- the cell [7]. tween the enzyme and the substrates. First, co-immunoprecipitation experiments of murine full- To analyze the minimal structural requirement for substrate length ADAM17 with human substrates were performed (Fig. 2A binding, additional co-precipitation experiments with deletion and Fig. 3A). Species difference of enzyme and substrate in case mutants were performed. Deletion of the transmembrane- and of the IL-6R is in agreement with the findings of Garbers et al. cytoplasmic-region by anchoring the extracellular part to the [26] who demonstrated that human IL-6R is not only a substrate membrane by GPI (Fig. 1C) does not abolish its interaction with I. Lorenzen et al. / FEBS Letters 586 (2012) 1093–1100 1099 the IL-6R (Fig. 2B), indicating that neither the transmembrane- nor finding that not only constructs lacking the catalytic domain, but cytoplasmic-region is needed for substrate recognition. This is sup- also the disintegrin domain are able to inhibit shedding of the ported by the finding that both molecules (DCat-ADAMDcyto and wild-type molecule (Fig. 4A), as described for DCat-ADAM17 [25]. DCat-ADAM-GPI) inhibit the proteolytic activity of the wild-type Furthermore, the soluble membrane-proximal domain is able to re- molecule to the same extent (Fig. 4A). Though, it does not exclude duce the proteolytic activity of ADAM17 (Supplementary Fig. 2A). that the transmembrane- and cytoplasmic-region have accessory The soluble membrane-proximal domain is expressed and secreted functions and that the transmembrane region ensures a correct ori- at low levels and therefore the local concentration is expected to be entation of the extracellular domains. This is supported by the low, whereas the GPI anchored variant accumulates on the plasma observation that a variant of ADAM17 attached to the plasma membrane. As a consequence, the GPI anchored membrane-proxi- membrane by a GPI anchor is proteolytically inactive [9], whereas mal domain is more efficient in inhibition than the soluble domain. ADAM17 lacking only the cytoplasmic portion is active [5]. Further studies should clarify which amino-acid residues of the Variants lacking the catalytic- (DCat-ADAM17-GPI) or addition- membrane-proximal domain are involved in the interaction be- ally the disintegrin-domain (MPD17-GPI) were both able to bind tween substrate and enzyme. In addition, it would be interesting IL-6R and IL-1RII (Fig. 2C, D and 3B–E), corroborating that solely to study whether this is a direct interaction or mediated by acces- the membrane-proximal domain is needed for substrate binding sory molecules. and that neither the catalytic- nor the disintegrin-domain is essen- The presented data demonstrate that the membrane-proximal tial for this purpose. The importance of the membrane-proximal domain of ADAM17 is the minimal requirement for substrate rec- domain in substrate recognition is in agreement with reports from ognition of type-I transmembrane proteins, like IL-1RII and IL-6R. Tape et al. and Reddy et al., who demonstrated that blockade of the Interestingly, this does not hold true for TNF-a. This difference is non-catalytic extracellular domains leads to reduced shedding not unexpected, since distinct modes of processing for the type-II activity [17] and that the membrane-proximal domain of ADAM17 transmembrane protein TNF-a and type-I transmembrane pro- is essential for shedding of the IL-1RII [5]. teins, like IL-6R, were described [30,31]. A peptide representing The latter observation could be verified with the ADAM the cleavage site of TNF-a is processed very efficiently by ADAM17, 17-EGF22 chimeric molecule, which is completely inactive. Similar whereas cleavage of the corresponding IL-6R peptide was not results were obtained using a ‘‘cat-dis ADAM17/EGF ADAM-10B’’ detectable [31], implicating that two distinct mechanisms of recog- chimera by Reddy et al. [5]. The two chimera differ in two main as- nition exist; one independent of the non-catalytic extracellular do- pects, first the choice of the domain borders and second in the mains and one dependent on the membrane-proximal domain of presence/absence of the cytoplasmic tail. ADAM17. Further analysis should address the recognition of The domain borders of ADAM17-EGF22 are based on the previously type-II and GPI-anchored substrates and study if co-localization published structures of ADAM10 [11] and ADAM22 [12].Hencethe within the cell is sufficient for these proteins to be shed. disintegrin and membrane-proximal domains of ADAM17 are sepa- rated between residue 580 and 581, which differs only slightly from Acknowledgements the ADAM17-ADAM10B chimera of Reddy et al., where the cut is lo- cated between 573 and 574 [5] and proceeded by the corresponding The authors would like to thank Britta Hansen, Jessica Schneider ADAM10 sequence, but lacking the cytoplasmic tail [5]. In contrast, and Sina Viehweg for their excellent technical assistance. Athena in the ADAM17-EGF22 chimera solely the membrane-proximal do- Chalaris for DCat-ADAM17 and ADAM17ex/ex mEFs; Michael Martin main was replaced (Supplementary Fig. 1A). The exclusive and exact for the IL-1RII plasmid and Andrea Rittger for the alkaline phospha- exchange of the membrane-proximal domain results in an ADAM17- tase cDNA. This study has been supported by the Deutsche EGF22 chimera which still comprisestheamino-acidresidues located Forschungsgemeinschaft (SFB 877, A1, A2, A6, Z3) and the Cluster C-terminal to this domain, including the unstructured stalk region as of Excellence ‘Inflammation at Interfaces’. well as the transmembrane region and cytoplasmic tail of ADAM17. Therefore the loss of activity in the ADAM17-EGF22 chimera is exclu- Appendix A. Supplementary data sively due to the exchange of the membrane-proximal domain and not to an alteration in the regions located C-terminally of the membrane- Supplementary data associated with this article can be found, in proximal domain. the online version, at http://dx.doi.org/10.1016/j.febslet.2012.03.012. Differences in the localization of the chimera within microdo- mains of the plasma membrane should be considered since References ADAM17 is known to be located in lipid rafts [24], whereas ADAM10 is supposed to be a non-raft protein [28]. The presence [1] Scheller, J., Chalaris, A., Garbers, C. and Rose-John, S. (2011) ADAM17: a of the C-terminal parts downwards from the membrane-proximal molecular switch to control inflammation and tissue regeneration. Trends Immunol. 32, 380–387. domain of ADAM17, e.g. the transmembrane region, might ensure [2] Black, R.A. et al. (1997) A disintegrin that releases tumour- a proper sorting of ADAM17-EGF22 to the lipid rafts. necrosis factor-alpha from cells. Nature 385, 729–733. The second difference between the two molecules is the pres- [3] Moss, M.L. et al. (1997) Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385, 733–736. ence of the cytoplasmic tail in ADAM17-EGF22 which is absent [4] Müllberg, J. et al. (1993) Differential shedding of the two subunits of the in ADAM17-ADAM10B [5]. For both [22,29] it has been interleukin-6 receptor. FEBS Lett. 332, 174–178. suggested that steric hindrances within the cytoplasmic region is [5] Reddy, P. et al. (2000) Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme. J. Biol. Chem. 275, 14608–14614. a regulatory property of these enzymes, resulting in a reduced [6] Peschon, J.J. et al. (1998) An essential role for ectodomain shedding in activity by lowering the accessibility of the proteases to their sub- mammalian development. Science 282, 1281–1284. strates. Whereas ADAM17-EGF22 is completely inactive minor [7] Soond, S.M., Everson, B., Riches, D.W. and Murphy, G. (2005) ERK-mediated phosphorylation of Thr735 in TNFalpha-converting enzyme and its potential shedding activity was detected for ADAM17-ADAM10B [5]. This role in TACE protein trafficking. J. Cell. Sci. 118, 2371–2380. might be due to the absence of the cytoplasmic tail in ADAM17- [8] Maretzky, T., Zhou, W., Huang, X.Y. and Blobel, C.P. (2011) A transforming Src ADAM10B and consequently the loss of its inhibitor potential. mutant increases the bioavailability of EGFR ligands via stimulation of the cell- Nevertheless one should be careful comparing these data because surface metalloproteinase ADAM17. Oncogene 30, 611–618. [9] Li, X., Pérez, L., Pan, Z. and Fan, H. (2007) The transmembrane domain of TACE the performed shedding assays are different. regulates protein ectodomain shedding. Cell Res. 17, 985–998. The demonstrated importance of the ADAM17 membrane-prox- [10] Takeda, S. (2009) Three-dimensional domain architecture of the ADAM family imal domain for substrate recognition is further underlined by the proteinases. Semin. Cell. Dev. Biol. 20, 146–152. 1100 I. Lorenzen et al. / FEBS Letters 586 (2012) 1093–1100

[11] Janes, P.W. et al. (2005) Adam meets Eph: an ADAM substrate recognition [21] Chalaris, A. et al. (2007) Apoptosis is a natural stimulus of IL6R shedding and module acts as a molecular switch for ephrin cleavage in trans. Cell 123, 291– contributes to the pro-inflammatory trans-signaling function of neutrophils. 304. Blood 110, 1748–1755. [12] Liu, H., Shim, A.H. and He, X. (2009) Structural characterization of the [22] Li, X., Perez, L. and Fan, H. (2011) Inhibitory role of TACE/ADAM17 cytotail in ectodomain of a disintegrin and metalloproteinase-22 (ADAM22), a neural protein ectodomain shedding. World J. Biol. Chem. 2, 246–251. adhesion receptor instead of metalloproteinase: insights on ADAM function. J. [23] Chalaris, A. et al. (2010) Critical role of the disintegrin metalloprotease Biol. Chem. 284, 29077–29086. ADAM17 for intestinal inflammation and regeneration in mice. J. Exp. Med. [13] Takeda, S., Igarashi, T., Mori, H. and Araki, S. (2006) Crystal structures of VAP1 207, 1617–1624. reveal ADAMs’ MDC domain architecture and its unique C-shaped scaffold. [24] Tellier, E., Canault, M., Rebsomen, L., Bonardo, B., Juhan-Vague, I., Nalbone, G. EMBO J. 25, 2388–2396. and Peiretti, F. (2006) The shedding activity of ADAM17 is sequestered in lipid [14] Milla, M.E. et al. (1999) Specific sequence elements are required for the rafts. Exp. Cell. Res. 312, 3969–3980. expression of functional tumor necrosis factor-alpha-converting enzyme [25] Solomon, K.A., Pesti, N., Wu, G. and Newton, R.C. (1999) Cutting edge: a (TACE). J. Biol. Chem. 274, 30563–30570. dominant negative form of TNF-alpha converting enzyme inhibits proTNF and [15] Wang, Y., Herrera, A.H., Li, Y., Belani, K.K. and Walcheck, B. (2009) Regulation TNFRII secretion. J. Immunol. 163, 4105–4108. of mature ADAM17 by redox agents for L-selectin shedding. J. Immunol. 182, [26] Garbers, C. et al. (2011) Species Specificity of ADAM10 and ADAM17 Proteins 2449–2457. in Interleukin-6 (IL-6) Trans-signaling and Novel Role of ADAM10 in Inducible [16] Willems, S.H., Tape, C.J., Stanley, P.L., Taylor, N.A., Mills, I.G., Neal, D.E., IL-6 Receptor Shedding. J. Biol. Chem. 286, 14804–14811. McCafferty, J. and Murphy, G. (2010) Thiol negatively regulate the [27] Schlöndorff, J., Becherer, J.D. and Blobel, C.P. (2000) Intracellular maturation cellular shedding activity of ADAM17. Biochem. J. 428, 439–450. and localization of the tumour necrosis factor alpha convertase (TACE). [17] Tape, C.J., Willems, S.H., Dombernowsky, S.L., Stanley, P.L., Fogarasi, M., Biochem. J. 347, 131–138. Ouwehand, W., McCafferty, J. and Murphy, G. (2011) Cross-domain inhibition [28] Harris, B., Pereira, I. and Parkin, E. (2009) Targeting ADAM10 to lipid rafts in of TACE ectodomain. Proc. Natl. Acad. Sci. U S A 108, 5578–5583. neuroblastoma SH-SY5Y cells impairs amyloidogenic processing of the [18] Suthaus, J., Tillmann, A., Lorenzen, I., Bulanova, E., Rose-John, S. and Scheller, J. amyloid precursor protein. Brain Res. 1296, 203–215. (2010) Forced homo- and heterodimerization of all gp130-type receptor [29] Janes, P.W. et al. (2009) Cytoplasmic relaxation of active Eph controls ephrin complexes leads to constitutive ligand-independent signaling and cytokine- shedding by ADAM10. PLoS Biol. 7, e1000215. independent growth. Mol. Biol. Cell. 21, 2797–2807. [30] Althoff, K., Reddy, P., Peschon, J., Voltz, N., Rose-John, S. and Müllberg, J. (2000) [19] Lorenzen, I., Trad, A. and Grötzinger, J. (2011) Multimerisation of A Disintegrin Shedding of interleukin-6 receptor and tumor necrosis factor alpha. And Metalloprotease protein-17 (ADAM17) is mediated by its EGF-like Contribution of the stalk sequence to the cleavage pattern of domain. Biochem. Biophys. Res. Commun. 415, 330–336. transmembrane proteins. Eur. J. Biochem. 267, 2624–2631. [20] Trad, A., Hedemann, N., Shomali, M., Pawlak, V., Grötzinger, J. and Lorenzen, I. [31] Mohan, M.J. et al. (2002) The tumor necrosis factor-alpha converting enzyme (2011) Development of sandwich ELISA for detection and quantification of (TACE): a unique metalloproteinase with highly defined substrate selectivity. human and murine a disintegrin and metalloproteinase17. J. Immunol. Biochemistry 41, 9462–9469. Methods 371, 91–96.