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Deletions in the cytoplasmic domain of iRhom1 and iRhom2 promote shedding of the TNF receptor by the protease ADAM17

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Science Signaling  03 Nov 2015:
Vol. 8, Issue 401, pp. ra109
DOI: 10.1126/scisignal.aac5356
  • Fig. 1 N truncated iRhom1-ΔN confers TNF resistance as identified by CPR screening.

    (A) Polymerase chain reaction (PCR) results from each round in the CPR screen (see Materials and Methods) showing enrichment for c-FLIP and two short versions of iRhom1 in TNF-resistant cells. (B) Systematic representation of wild-type (WT) and short versions of iRhom1 identified by CPR relative to their predicted transmembrane domain structures. (C) Immunoblotting and densitometry for T7 in lysates from L-929 cells expressing a control vector, T7-tagged WT or ΔN-iRhom1 (iR1) or iRhom2 (iR2) (n = 3). (D and E) Cell death, assessed by annexin V binding and 7-AAD staining using flow cytometry, in 1 × 105 L-929 cells transfected as indicated and treated with recombinant TNF for up to 48 hours (n ≥ 5). Data are means ± SEM from the number of experiments (n) indicated; *P < 0.05, **P < 0.01, ***P < 0.001 against ΔN; #P < 0.05, ##P < 0.01 against vector. nt, nucleotide; aa, amino acid.

  • Fig. 2 iRhom2-Δ N induces TNFR shedding.

    (A and B) Immunoblotting and densitometry for PARP (A) or total and phosphorylated NF-κB pathway proteins (B) in lysates from L-929 cells overexpressing a vector control, iRhom2-WT, or iRhom2-ΔN and exposed to recombinant TNF (2.5 ng/ml) for up to 8 hours (A) or 45 min (B) (n = 3, normalized to control and actin). (C and D) Flow cytometry analysis of TNFR1 and TNFR2 surface abundance on L-929 cells overexpressing WT or truncated iRhom2 (C) or iRhom1 (D) (n = 5 or 6, respectively). (E and F) Amount of TNFR1 and TNFR2 in supernatants from 1 × 105 L-929 cells expressing WT or truncated iRhom2 (E) or iRhom1 (F) (n = 5). Data are means ± SEM from the number of experiments (n) indicated; *P < 0.05, **P < 0.01, ***P < 0.001 against ΔN; #P < 0.05, ##P < 0.01, ###P < 0.001 against vector. RFU, relative fluorescence units.

  • Fig. 3 Effects of ΔN-iRhoms can be blocked by ADAM17 inhibitors.

    (A and B) Abundance of TNFR1 and TNFR2 in the culture supernatants from 1 × 105 L-929 cells expressing WT or truncated iRhom2 cultured in presence or absence of marimastat (MM; 20 μM) (A) or GI or GW (each 3 μM) (B) for 6 hours (n = 5 or 6, respectively). (C) As in (B) in 1 × 105 L-929 cells expressing WT or truncated iRhom1. (D) Cell death as a percentage of annexin–7-AAD+ cells in cultures (n = 3) of 1 × 105 L-929 cells expressing WT or truncated iRhom2 treated with recombinant TNF in the presence or absence of marimastat (20 μM). Data are means ± SEM from the number of experiments (n) indicated; **P < 0.01, ***P < 0.001 against WT; #P < 0.05, ###P < 0.001 against vector.

  • Fig. 4 Stable knockdown of ADAM17 prevents increased TNFR shedding and resistance to TNF.

    (A) Immunoblot for pro (P) and mature (M) ADAM17 in lysates from L-929 cells expressing full-length and ΔN-iRhom2 transfected with either a scrambled control (Scr1) or an ADAM17-targeted shRNA (ADAM17-Sh1). Blot is representative of three experiments. (B) Surface abundance of TNFR1 and TNFR2 as determined by flow cytometry on L-929 cells expressing a vector control, full-length (WT) iRhom2 or iRhom2-ΔN in the presence of either control or ADAM17 shRNA (n = 6). (C and D) Cell death as a proportion of annexin–7-AAD+ cells in cultures of 1 × 105 L-929 cells stably expressing full-length or ΔN-iRhom2 (C) or iRhom1 (D) and either scrambled or ADAM17 shRNA treated with recombinant TNF for 48 hours (n = 4 or 6, respectively). Data are means ± SEM from the number of experiments (n) indicated; *P < 0.05, ***P < 0.001. MFI, mean fluorescence intensity.

  • Fig. 5 Truncation of the cytoplasmatic tail results in increased surface expression of iRhom2.

    (A) Immunoprecipitation (IP) for T7 or ADAM17 followed by immunoblotting for the same in lysates from L-929 cells stably expressing T7-tagged WT or truncated iRhom2. Blot is representative of three experiments. (B) Immunoblotting for iRhom2 using a T7 antibody in intracellular and cell surface fractions from L-929 cells stably expressing WT or truncated iRhom2. Blot is representative of three experiments. (C) Surface abundance of iRhom2, determined using an antibody against T7, on stably transfected L-929 cells (n = 8). (D) Immunocytochemistry for iRhom2 using T7 antibodies (Cy3), phalloidin–fluorescein isothiocyanate (FITC), and Hoechst staining in stably transfected L-929 cells, fixed, and/or permeabilized as indicated (n ≥ 3 experiments). (E and F) Flow cytometry analysis of MFI of the surface abundance of iRhom2 on unpermeabilized stably transfected L-929 cells expressing either scrambled or ADAM17 shRNA (n ≥ 4 experiments) (E) or immortalized WT or Adam17 knockout (KO) mouse embryonic fibroblasts (MEFs) (n = 12 experiments) (F). Data are means ± SEM from the number of experiments (n) indicated; ***P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Na+/K+ ATPase, Na+- and K+-dependent adenosine triphosphatase.

  • Fig. 6 N-terminal mutations in iRhom2 trigger constitutive activity of ADAM17.

    (A and B) TNFR1 and TNFR2 in conditioned media of 1 × 105 L-929 cells expressing WT or ΔN-iRhom2 (A) or iRhom1 (B) cultured with PMA (100 ng/ml) in the presence (where indicated) of GI or GW (3 μM each) (n = 6). (C) KitL2 shedding in immortalized MEFs (iMEFs) genetically lacking iRhom2 transfected with alkaline phosphatase (AP)–tagged KitL2 along with MAD2 (control), iRhom2-WT, or iRhom2-ΔN, and (where indicated, +) preincubated with marimastat overnight (MM/ON), followed by washout of the inhibitor (n = 3). (D) Transforming growth factor–α (TGF-α) shedding from iMEFs genetically lacking iRhom1 and iRhom2 and transfected with AP-tagged TGF-α along with MAD2 (control), murine WT iRhom2, or iRhom2I156T/P159L (n ≥ 5). (E) Concentration of soluble TNFR1 in supernatants from 2 × 105 keratinocytes from either healthy donors (K17 cells) or tylosis patients who have mutations in IRHOM2(l186T/WT) [TYLK1 and TYLK2 treated with vehicle (dimethyl sulfoxide; DMSO) or TMI-005 ADAM17 inhibitor for 24 (left) or 48 (right) hours]. Data are means ± SEM from the number of experiments (n) indicated; *P < 0.05, **P < 0.01, ***P < 0.001 against WT (A and B) or MAD2 (D and E); #P < 0.05 against vector (A and B) or mutant construct (D and E).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/401/ra109/DC1

    Fig. S1. Cloning of CPR screen–identified iRhom versions.

    Fig. S2. TNFR1 and TNFR2 abundance in iRhom-expressing cells.

    Fig. S3. ADAM17 mediates cleavage of TNFRs.

    Fig. S4. Stable knockdown of ADAM17 prevents ΔN-iRhom–dependent TNFR shedding.

    Fig. S5. Localization patterns of truncated iRhoms and association with ADAM17.

    Fig. S6. Increased ADAM17 activity associated with N-terminal truncated iRhom2.

  • Supplementary Materials for:

    Deletions in the cytoplasmic domain of iRhom1 and iRhom2 promote shedding of the TNF receptor by the protease ADAM17

    Sathish K. Maney, David R. McIlwain, Robin Polz, Aleksandra A. Pandyra, Balamurugan Sundaram, Dorit Wolff, Kazuhito Ohishi, Thorsten Maretzky, Matthew A. Brooke, Astrid Evers, Ananda A. Jaguva Vasudevan, Nima Aghaeepour, Jürgen Scheller, Carsten Münk, Dieter Häussinger, Tak W. Mak, Garry P. Nolan, David P. Kelsell, Carl P. Blobel, Karl S. Lang, Philipp A. Lang*

    *Corresponding author. E-mail: philipp.lang{at}med.uni-duesseldorf.de

    This PDF file includes:

    • Fig. S1. Cloning of CPR screen–identified iRhom versions.
    • Fig. S2. TNFR1 and TNFR2 abundance in iRhom-expressing cells.
    • Fig. S3. ADAM17 mediates cleavage of TNFRs.
    • Fig. S4. Stable knockdown of ADAM17 prevents ΔN-iRhom–dependent TNFR shedding.
    • Fig. S5. Localization patterns of truncated iRhoms and association with ADAM17.
    • Fig. S6. Increased ADAM17 activity associated with N-terminal truncated iRhom2.

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    Citation: S. K. Maney, D. R. McIlwain, R. Polz, A. A. Pandyra, B. Sundaram, D. Wolff, K. Ohishi, T. Maretzky, M. A. Brooke, A. Evers, A. A. J. Vasudevan, N. Aghaeepour, J. Scheller, C. Münk, D. Häussinger, T. W. Mak, G. P. Nolan, D. P. Kelsell, C. P. Blobel, K. S. Lang, P. A. Lang, Deletions in the cytoplasmic domain of iRhom1 and iRhom2 promote shedding of the TNF receptor by the protease ADAM17. Sci. Signal. 8, ra109 (2015).

    © 2015 American Association for the Advancement of Science

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