FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

  1. Ulrike Künzel
  2. Adam Graham Grieve
  3. Yao Meng
  4. Boris Sieber
  5. Sally A Cowley
  6. Matthew Freeman  Is a corresponding author
  1. University of Oxford, United Kingdom
11 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
FRMD8 is a novel interaction partner of iRhom1 and iRhom2.

(A) Volcano plot representing results from three iRhom2 co-immunoprecipitations. The fold change of label-free quantification values (in log2 ratio) was plotted against the p value (-log10 transformed). The grey dotted line indicates p-values <0.05 (analysed with a two-sample t-test). Benjamini-Hochberg correction was applied to adjust the p-value for multiple hypothesis testing (dark grey dotted line). (B) Lysates of HEK293T cells stably expressing human iRhom1-3xHA or iRhom2-3xHA transfected with human FRMD8-V5 (where indicated) were subjected to anti-HA and anti-V5 immunoprecipitation (HA-IP, V5–IP) and a western blot using anti-HA and anti-V5 antibodies was performed. Black arrowheads indicated the co-immunoprecipitated FRMD8-V5; white arrowheads indicated the co-immunoprecipitated iRhoms.

https://doi.org/10.7554/eLife.35012.003
Figure 1—figure supplement 1
Setup and confirmation of the mass spectrometry screen.

(A) HEK293T cells transiently transfected with human iRhom2-3xHA or UNC93B1-3xHA were stained with DAPI (blue) to label nuclei, anti-HA to label iRhom2-HA (red), and anti-calnexin to label the ER (green). Scale bar = 10 μm. (B) Lysates and anti-HA immunoprecipitation (HA-IP) from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells stably expressing iRhom2-3xHA (where indicated) were immunoblotted for HA and FRMD8. Nonspecific bands are marked with an asterisk.

https://doi.org/10.7554/eLife.35012.004
FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity.

(A) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. (B, C) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. (D) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. (E, F) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value>0.05; *=p value<0.05; ***=p value<0.001; ****=p value<0.0001.

https://doi.org/10.7554/eLife.35012.006
Figure 3 with 1 supplement
FRMD8 binds to the iRhom2 N-terminus.

(A) Schematic representation of truncated human iRhom2 constructs used in (B–E). (B, C) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently co-transfected with FRMD8-V5 and either empty vector (vect) or truncated human iRhom2-3xHA constructs were immunoblotted for V5 and HA. (D) iRhom1/2 double knockout HEK293T cells stably expressing empty vector (vect) or human iRhom2-3xHA constructs were transiently transfected with alkaline phosphatase (AP)-tagged AREG and then incubated with 200 nM PMA or with DMSO for 30 min. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed using a Mann-Whitney test. ****=p value<0.0001. (E) Lysates from iRhom1/2 double knockout HEK293T cells transiently transfected with empty vector (vect) or human iRhom2-3xHA constructs were immunoblotted for ADAM17 and HA.

https://doi.org/10.7554/eLife.35012.007
Figure 3—figure supplement 1
A) Amino acid sequence alignment of human and mouse iRhom2 N-terminal region using Clustal Omega.

The region required for FRMD8 binding is highlighted in red. Conserved phosphorylation sites that have been mutated to alanine in the iRhom2pDEAD (Figure 10—figure supplement 1) are marked in yellow. Grey residues indicate additional phosphorylation sites that have been reported on PhosphoSitePlus (www.phosphosite.org). An asterisk (*) indicates positions which have a fully conserved residue, a colon (:) indicates strongly similar properties of the amino acids, and a period (.) indicates weakly similar properties according to the Clustal Omega tool. (B) Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with FRMD8-V5 and either empty vector (vect), mouse iRhom2WT (WT) or Rhom2cub (Δ268) were immunoblotted for V5 and HA.

https://doi.org/10.7554/eLife.35012.008
iRhom2 binds to FRMD8 and ADAM17 simultaneously.

(A) Lysates, anti-HA and anti-V5 immunoprecipitations (HA-IP, V5–IP) of HEK293T cells co-expressing human iRhom2-3xHA and human FRMD8-V5 were immunoblotted for ADAM17, HA and V5. (B) Lysates of wild-type (WT) and ADAM17 knockout (KO) HEK293T cells were transiently transfected with human iRhom2-3xHA and FRMD8-V5 (where indicated), anti-HA and anti-V5 immunoprecipitated (HA-IP; V5–IP) and immunoblotted for ADAM17, HA, and V5. (C) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA were anti-HA immunoprecipitated (HA-IP) and stained for ADAM17 and HA. Nonspecific bands are indicated by an asterisk. (D) Lysates of WT and iRhom1/2 double knockout (DKO) HEK293T cells stably expressing human iRhom2WT-3xHA or iRhom2Δ201-300-3xHA were anti-V5 immunoprecipitated (V5–IP) and immunoblotted for ADAM17, HA and V5.

https://doi.org/10.7554/eLife.35012.009
FRMD8 promotes cell surface localisation of iRhom2.

(A, B) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2Δ300-3xHA and transiently transfected with FRMD8-V5 for 72 hr. Cells were stained for HA (red), V5 (green) and DAPI for DNA (blue). Single confocal sections are shown, taken through the centre of the nucleus. (C) Schematic model of the FRMD8-iRhom2Δ300 construct used in (E). (D, E) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2Δ300-3xHA or FRMD8-iRhom2Δ300-3xHA and transiently transfected with ADAM17-V5 for 72 hr. Cells were stained for HA (green), V5 (red) and DAPI for DNA (blue). Single confocal sections are shown, taken either through the centre of the nucleus (MEDIAL), or at basal regions close to the coverslip (BASAL). In all images the scale bar = 10 µm.

https://doi.org/10.7554/eLife.35012.010
FRMD8 loss leads to degradation of iRhoms and mature ADAM17 through the lysosomal pathway.

(A–D) Immunofluorescence of iRhom1/2 double knockout HEK293T cells stably expressing iRhom2-3xHA or iRhom2Δ300-3xHA treated with DMSO (CON) or 100 nM bafilomycin A1 (BAF) for 16 hr prior to fixation. Cells were stained for HA (green), the lysosomal marker LAMP1 (red) and DAPI for DNA (blue). LAMP1-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. (E, F) iRhom2Δ300-3xHA cells were treated as in (A–D), but with 72 hr expression of ADAM17-V5 and labelling of HA (green), V5 (red) and DAPI for DNA (blue). Arrows indicate colocalising puncta. Single confocal sections are shown, taken through the centre of the nucleus. HA- and V5-labelled regions (within white boxes) have been magnified. Scale bar = 10 µm. (G) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with the solvent DMSO (–), 10 µM MG-132 (MG) or 200 nM bafilomycin A1 (Baf) for 16 hr were enriched for glycosylated proteins using concanavalin A (conA) beads and immunoblotted for ADAM17 and transferrin receptor 1 (TfR). TfR was used as a loading control although it is also susceptible to bafilomycin treatment. Mature ADAM17 levels from three experiments were quantified relative to TfR levels using ImageJ.

https://doi.org/10.7554/eLife.35012.011
Figure 7 with 1 supplement
FRMD8 loss leads to the destabilisation of ADAM17 and iRhom2.

(A) Unpermeabilised WT (black) and FRMD8 KO HEK293T (cyan) cells stably expressing human iRhom2-3xHA were immunostained on ice for HA. Wild-type HEK293T cells immunostained for HA served as a negative control (grey). (B) Cells were permeabilised and stained at room temperature with an anti-HA antibody. Immunostaining with the Alexa Fluor 488-coupled secondary antibody served as a control (grey). The flow cytometry graphs shown are one representative experiment out of three experiments. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test; ns = p value>0.05; *=p value<0.05. (C) Lysates of HEK293T cells stably expressing human iRhom2-3xHA and transiently transfected with FRMD8-V5 (where indicated) were analysed by western blot for iRhom2 levels using anti-HA, anti-V5 and anti-actin immunostaining. Nonspecific bands are marked with an asterisk. (D) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA (where indicated) were immunoblotted for HA, FRMD8 and actin. An asterisk marks nonspecific bands. (E) FRMD8 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used in (D).

https://doi.org/10.7554/eLife.35012.012
Figure 7—figure supplement 1
FRMD8 stabilises iRhom levels by preventing its lysosomal degradation.

(A) iRhom1/2 double knockout HEK293T cells stably expressing iRhom2WT-3xHA, iRhom2Δ300-3xHA or FRMD8-iRhom2Δ300-3xHA were treated with 100 µg/ml cycloheximide (CHX) for the indicated time (0–8 hr) to block protein synthesis. Cell lysates were immunoblotted for HA and actin. (B) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with 10 µM MG-132 (MG), 200 nM bafilomycin A1 (Baf) or 50 mM ammonium chloride (NH4Cl) for 16 hr were immunoblotted for ADAM17, FRMD8, and actin. An asterisk marks a nonspecific band. (C) N-glycosylation of iRhom2 was analysed using EndoH and PNGase to distinguish ER/cis-Golgi (EndoH sensitive) and late Golgi localisation (EndoH resistant). Lysates of WT and FRMD8 KO HEK293T cells transiently transfected with mouse iRhom2-3xHA were deglycosylated with EndoH or PNGase and then immunoblotted for mouse iRhom2, human FRMD8 and actin. An asterisk marks a nonspecific band. (D) Lysates of HEK293T cells stably expressing human iRhom1-3xHA and transfected with FRMD8-V5 (where indicated) were immunoblotted for HA, V5, and actin. (E) Levels of ADAM17 were analysed in HEK293T-iRhom2-3xHA and HEK293T WT cells transfected with siRNAs targeting iRhom2 where indicated. Cell lysates were immunoblotted using an anti-ADAM17 or anti-actin antibody. An asterisk marks a nonspecific band.

https://doi.org/10.7554/eLife.35012.013
FRMD8 stabilises endogenous iRhom2.

(A, B) Levels of endogenously 3xHA tagged iRhom2 were analysed in HEK293T-iRhom2-3xHA cells transfected with FRMD8-V5 plasmid, siRNAs targeting iRhom2, non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA. Cell lysates were anti-HA immunoprecipitated (HA-IP) to detect endogenous iRhom2-3xHA levels and immunoblotted using anti-HA antibody. Cell lysates were immunoblotted for ADAM17, V5, and actin. (C) FRMD8 and iRhom2 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used for the experiment shown in (B) to demonstrate that the destabilisation of endogenous iRhom2 was not induced by a change in iRhom2 mRNA levels.

https://doi.org/10.7554/eLife.35012.014
Figure 9 with 2 supplements
FRMD8 is required for iRhom2/TACE regulation in human iPSC-derived macrophages and mice .

(A) Schematic representation of the differentiation protocol of iPSCs into macrophages based on (van Wilgenburg et al., 2013). Scale bars = 10 μm. (B) Lysates of iPSC-derived macrophages (on day seven after harvest from EBs) were immunoblotted for ADAM17, FRMD8, and actin. Western blots from three experiments were quantified using ImageJ with actin serving as the loading control. (C) 25,000 iPSC-derived macrophages were either left unstimulated or stimulated with 50 ng/ml LPS for 4 hr. TNFα concentration in the cell supernatants was measured by ELISA and then normalised to the protein concentration in macrophage cell lysates to adjust the cytokine release for potential differences in cell numbers. Each experiment was performed in biological triplicates. Data from three independent experiments were statistically analysed using a Mann-Whitney test; ***=p value<0.001; ****=p value<0.0001. (D, E) Lysates from tissues derived from Frmd8-/- or Rhbdf2-/- and their wild-type littermates were immunoblotted for ADAM17, FRMD8, iRhom2 and actin. Blots from three experiments using three different littermates of Frmd8-/- and Frmd8+/+ mice were quantified using ImageJ with actin serving as the loading control.

https://doi.org/10.7554/eLife.35012.015
Figure 9—figure supplement 1
Generation of FRMD8 knockout iPSCs and iPSC-derived macrophages.

(A) Sequencing of the genomic DNA isolated from clonal FRMD8 KO iPSCs shows a 1-nt insertion (clone 1) and a 7-nt and 10-nt deletion (clone 2). The targeting sequence of the sgRNA is shown in bold; small letters indicate the sequence within the intronic region; the protospacer adjacent motif (PAM) sequence underlined. (B) Parental wild-type and FRMD8 KO iPSC lines were karyotyped by SNP array. Detected copy number variations are indicated in red (DNA copy number loss in the indicated region) and green (DNA copy number increase). The AH017-13 iPSC line used was derived from a female donor, therefore the Y chromosome is marked in red (loss of Y chromosome DNA). (C) 25,000 iPSC-derived macrophages were either left unstimulated, stimulated with 50 ng/ml LPS, or with 50 ng/ml LPS and simultaneously with 2 μM GI or 2 μM GW for 4 hr. TNFα concentration in the cell supernatants was measured by ELISA and then normalised to the protein concentration in macrophage cell lysates to adjust the cytokine release for potential differences in cell numbers. Each experiment was performed in biological triplicates. Data from three independent experiments were statistically analysed using a Mann-Whitney test; ns = p value>0.05; ****=p value<0.0001. (D) TNFα mRNA levels relative to actin mRNA levels were measured by TaqMan PCR in WT and FRMD8 KO iPSC-derived macrophages without stimulation and after stimulation with 200 ng/ml LPS for 0.5 hr.

https://doi.org/10.7554/eLife.35012.016
Figure 9—figure supplement 2
Generation of Frmd8 knockout mice.

(A) Schematic representation of the insertion of a lacZ/neomycin cassette into the Frdm8 locus in the ES cells used to generate Frmd8-/- mice. (B) Offspring of Frmd8+/- × Frmd8+/- (HET x HET) crosses listed by genotype: Frmd8+/+(WT), Frmd8+/- (HET), and Frmd8-/- (KO). Two Frmd8 mouse strains were bred (both in BL6 background): one with the entire lacZ/neomycin cassette inserted and one strain in which the neomycin resistance gene has been removed from the cassette. (C) Lysates from skin derived from Frmd8-/-, Rhbdf2-/- mice and their wild-type littermate were immunoblotted for iRhom2 and actin.

https://doi.org/10.7554/eLife.35012.017
Figure 10 with 1 supplement
FRMD8 stabilises the iRhom2/ADAM17 sheddase complex at the cell surface.

Schematic representation of the role of FRMD8 in the iRhom2/ADAM17 pathway: under wild-type conditions ADAM17 and iRhom2 are stabilised by FRMD8 and thereby protected from degradation through the endolysosmal pathway.

https://doi.org/10.7554/eLife.35012.018
Figure 10—figure supplement 1
Lysates and anti-HA immunoprecipitation (HA-IP) from HEK293T cells transiently transfected with human FRMD8-V5 and mouse iRhom2WT (WT) or iRhom2pDEAD (pDEAD) were immunoblotted for V5 and HA.

Where indicated cells have been stimulated with 200 nM PMA for 30 min.

https://doi.org/10.7554/eLife.35012.019

Tables

Table 1
List of iRhom2 interaction partners identified in the mass spectrometry screen that have either shown a significant adjusted p-value or been reported previously (Adrain et al., 2012; McIlwain et al., 2012; Grieve et al., 2017).

P-values from a two-sample t-test in Perseus are listed below. P-values were adjusted for multiple hypothesis testing with the Benjamini-Hochberg correction and are listed under ‘adjusted p-values’.

https://doi.org/10.7554/eLife.35012.005
Prot. IDProtein nameGenep-valueAdjusted
p-value
Q9BZ67FERM domain-containing protein 8FRMD82.44 · 10−72.38 · 10−4
Q9BY50Signal peptidase subunit SEC11CSEC11C2.94 · 10−61.71 · 10−3
Q9NUP9Protein lin-7 homolog CLIN7C1.55 · 10−56.45 · 10−3
P11940Polyadenylate-binding protein 1;
Polyadenylate-binding protein 3
PABPC1;
PABPC3
2.10 · 10−57.66 · 10−3
Q9BXB1Leucine-rich repeat-containing GPCR 4LGR42.63 · 10−58.53 · 10−3
O00629Importin subunit alpha-3KPNA45.76 · 10−51.40 · 10−2
Q13454Tumor suppressor candidate 3TUSC37.05 · 10−51.58 · 10−2
Q9HD26GOPC/PISTGOPC8.54 · 10−51.78 · 10−2
Q9UKX7Nuclear pore complex protein Nup50NUP508.77 · 10−51.71 · 10−2
O95754Semaphorin-4FSEMA4F1.06 · 10−41.83 · 10−2
Q96EK5KIF1-binding proteinKIAA12791.47 · 10−42.26 · 10−2
P51809Vesicle-associated membrane protein 7VAMP71.55 · 10−42.26 · 10−2
Q12899Tripartite motif-containing protein 26TRIM261.93 · 10−42.68 · 10−2
O14763TRAIL receptor 2TNFRSF10B2.29 · 10−43.04 · 10−2
Q5SWA1Protein phosphatase 1 subunit 15BPPP1R15B3.35 · 10−44.25 · 10−2
Q6PJF5iRhom2RHBDF23.42 · 10−44.16 · 10−2
P61225Ras-related protein Rap-2bRAP2B4.27 · 10−44.98 · 10−2
P28482Mitogen-activated protein kinase 1MAPK13.37 · 10−30.22
P27361Mitogen-activated protein kinase 3MAPK35.34 · 10−30.32
P6225814-3-3 protein epsilonYWHAE6.61 · 10−30.35
P78536ADAM17ADAM178.07 · 10−30.40
P6310414-3-3 protein zeta/deltaYWHAZ9.14 · 10−30.41
P2734814-3-3 protein thetaYWHAQ1.20 · 10−20.45
P3194714-3-3 protein sigmaSFN2.19 · 10−20.63
Q0491714-3-3 protein etaYWHAH2.33 · 10−20.65
P6198114-3-3 protein gammaYWHAG3.15 · 10−20.75
P3194614-3-3 protein alpha/betaYWHAB6.53 · 10−21
P51812Ribosomal protein S6 kinase alpha-3RPS6KA36.53 · 10−21
Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background
(mouse)
Frmd8-/-: C57BL/6-
Frmd8tm1(KOMP)Vlcg
This paperN/AFRMD8 KO mice generated
as described in materials
and methods
Strain, strain background
(mouse)
WT control: C57BL/6This paperN/AWT control for FRMD8 KO
mice
Strain, strain background
(mouse)
Rhbdf2-/-: C57BL/6
-Rhbdl6A22
(Adrain et al., 2012)N/A
Cell line (human)HEK293T cellsFreeman labRRID:CVCL_0063
Cell line (human)HEK293T human
iRhom1WT
This paperN/AHEK293T cells transduced
with pLEX.puro-human
iRhom1WT-3xHA
Cell line (human)HEK293T human
iRhom2WT
This paperN/AHEK293T cells transduced
with pLEX.puro-human
iRhom2WT-3xHA
Cell line (human)HEK293T human
UNC93B1
This paperN/AHEK293T cells transduced
with pLEX.puro-human
UNC93B1-3xHA
Cell line (human)HEK293T FRMD8 KOThis paperN/ACRISPR/Cas9-mediated KO
cell line as described in
materials and methods
Cell line (human)HEK293T FRMD8 KO
+ human iRhom2WT
This paperN/AFRMD8 KO cells transduced
with pLEX.puro-human
iRhom2WT-3xHA
Cell line (human)HEK293T endogenous
iRhom2-3xHA
This paperN/ACRISPR/Cas9-mediated knock-in
cell line as described in
materials and methods
Cell line (human)HEK293T iRhom1/iRhom2
double-knockout (DKO)
This paperN/ACRISPR/Cas9-mediated KO
cell line as described in
materials and methods
Cell line (human)HEK293T iRhom1/iRhom2
DKO + human iRhom2WT
This paperN/ADKO cells transduced with
pLEX.puro-human
iRhom2WT-3xHA
Cell line (human)HEK293T iRhom1/iRhom2
DKO + human iRhom2Δ300
This paperN/ADKO cells transduced with
pLEX.puro-human
iRhom2Δ300-3xHA
Cell line (human)HEK293T iRhom1/iRhom2
DKO + human FRMD8-
iRhom2Δ300
This paperN/ADKO cells transduced with
pLEX.puro-human FRMD8-
iRhom2Δ300-3xHA
Cell line (human)HEK293T iRhom1/iRhom2
DKO + human iRhom2Δ
201–300
This paperN/ADKO cells transduced with
pLEX.puro-human
iRhom2Δ201-300-3xHA
Cell line (human)hiPSC line AH017-13(Fernandes et al., 2016)James Martin Stem
Cell Facility
Cell line (human)hiPSC line AH017-13 FRMD8
KO clone 1 (clone F1)
This paperN/ACRISPR/Cas9-mediated KO
cell line as described in
materials and methods
Cell line (human)hiPSC line AH017-13 FRMD8
KO clone 2 (clone G6)
This paperN/ACRISPR/Cas9-mediated KO
cell line as described in
materials and methods
Cell line (human)hiPSC line AH017-13 WT
(clone E4)
This paperN/Aunedited WT control for
FRMD8 KO iPSCs clones
Cell line (mouse)Frmd8-/- ES cells: C57BL/
6NTac-Frmd8tm1(KOMP)Vlcg
(clone 17364AC3)
KOMPRRID:IMSR_KOMP:
VG17364-1-Vlcg
Antibodyanti-β-actin-HRP, mouse
monoclonal (clone AC-15)
Sigma-AldrichCat#A3854;
RRID:AB_262011
dilution is described in
materials and methods
Antibodyanti-ADAM10, mouse
monoclonal (clone SHM14)
BioLegendCat#352702;
RRID:AB_10897813
dilution is described in
materials and methods
Antibodyanti-ADAM10, rabbit
polyclonal
Cell Signaling
Technology
Cat#14194dilution is described in
materials and methods
Antibodyanti-ADAM17, mouse
monoclonal (clone A300E)
(Yamamoto et al., 2012);
received from Stefan
Düsterhöft
N/Adilution is described in
materials and methods
Antibodyanti-ADAM17, rabbit
polyclonal
AbcamCat#ab39162;
RRID:AB_722565
dilution is described in
materials and methods
Antibodyanti-calnexin, rabbit
polyclonal
Santa CruzCat#sc-11397;
RRID:AB_2243890
dilution is described in
materials and methods
Antibodyanti-FRMD8, rabbit
polyclonal
AbcamCat#ab169933dilution is described in
materials and methods
Antibodyanti-HA, rabbit polyclonalSanta CruzCat#sc-805;
RRID:AB_631618
dilution is described in
materials and methods
Antibodyanti-HA, rat monoclonal
(clone 3F10)
RocheCat#11867423001;
RRID:AB_10094468
dilution is described in
materials and methods
Antibodyanti-HA tag, rabbit
monoclonal (C29F4)
CSTCat#3724;
RRID:AB_1549585
dilution is described in
materials and methods,
used at 1:1000 for IF
Antibodyanti-HA-HRP, rat
monoclonal (clone 3F10)
RocheCat#12013819001;
RRID:AB_390917
dilution is described in
materials and methods
Antibodyanti-iRhom2, rabbit
polyclonal
(Adrain et al., 2012)N/Adilution is described in
materials and methods
Antibodyant-LAMP1, mouse
monoclonal (H4A3)
Santa CruzCat#sc-20011;
RRID:AB_626853
dilution is described in
materials and methods,
used at 1:250 for IF
Antibodyanti-transferrin receptor 1,
mouse monoclonal
(clone H68.4)
Thermos Fisher
Scientific
Cat#13–6800;
RRID:AB_86623
dilution is described in
materials and methods
Antibodyanti-V5, goat polyclonalSanta CruzCat#sc-83849;
RRID:AB_2019670
dilution is described in
materials and methods,
used at 1:1000 for IF
Antibodyanti-goat-HRP, mouse
monoclonal
Santa CruzCat#sc-2354;
RRID:AB_628490
dilution is described in
materials and methods
Antibodyanti-mouse-HRP, goat
polyclonal
Santa CruzCat#sc-2055;
RRID:AB_631738
dilution is described in
materials and methods
Antibodyanti-rabbit-HRP, goat
polyclonal
Sigma-AldrichCat#A9169;
RRID:AB_258434
dilution is described in
materials and methods
Antibodyanti-mouse Alexa Fluor
488, donkey polyclonal
Thermos Fisher
Scientific
Cat#A-21202;
RRID:AB_141607
dilution is described in
materials and methods
Antibodyanti-rabbit Alexa Fluor
488, donkey polyclonal
Thermos Fisher
Scientific
Cat#A-21206;
RRID:AB_2535792
dilution is described in
materials and methods
Antibodyanti-rabbit Alexa Fluor
647, donkey polyclonal
Thermos Fisher
Scientific
Cat#A-31573;
RRID:AB_2536183
dilution is described in
materials and methods
Recombinant DNA
reagent
cDNA: human iRhom2
(NM_024599.2)
OrigeneCat#SC122961
Recombinant DNA
reagent
cDNA: human FRMD8
(NM_031904)
OrigeneCat#SC107202
Recombinant DNA
reagent
cDNA: human UNC93B1(Brinkmann et al., 2007)N/A
Recombinant DNA
reagent
Plasmid: pLEX.puroThermo Fisher
Scientific
Cat#OHS4735
Recombinant DNA
reagent
Plasmid: pcDNA3.1(+)Thermo Fisher
Scientific
Cat#V790-20
Recombinant DNA
reagent
Plasmid: entiviral
packaging plasmid
(Adrain et al., 2012)N/A
Recombinant DNA
reagent
Plasmid: entiviral
envelope plasmid
(Adrain et al., 2012)N/A
Recombinant DNA
reagent
Plasmid: pSpCas9(BB)
−2A-Puro (pX459)
(Ran et al., 2013)Addgene plasmid
#48139
Recombinant DNA
reagent
Plasmid: epX459(1.1)received from Joey
Riepsaame
N/A
Recombinant DNA
reagent
Plasmid: pSpCas9(BB)
−2A-Puro V2.0 (pX462 V2.0)
(Ran et al., 2013)Addgene plasmid
#62987
Transfected construct
(human)
Plasmid: pLEX.puro-human
iRhom1WT-3xHA
(Christova et al., 2013)N/A
Transfected construct
(human)
Plasmid: pLEX.puro-human
iRhom2WT-3xHA
This paperN/Acloned from human
iRhom2 (NM_024599.2)
Transfected construct
(human)
Plasmid: pLEX.puro-human
iRhom2Δ100-3xHA
This paperN/Ahuman iRhom2 lacking
amino acids 1–100
Transfected construct
(human)
Plasmid: pLEX.puro-human
iRhom2Δ200-3xHA
This paperN/Ahuman iRhom2 lacking
amino acids 1–200
Transfected construct
(human)
Plasmid: pLEX.puro-human
iRhom2Δ300-3xHA
This paperN/Ahuman iRhom2 lacking
amino acids 1–300
Transfected construct
(human)
Plasmid: pLEX.puro-human
iRhom2Δ201-300-3xHA
This paperN/Ahuman iRhom2 lacking
amino acids 1–382
Transfected construct
(human)
Plasmid: pLEX.puro-human
iRhom2Δ382-3xHA
This paperN/Ahuman iRhom2 lacking
amino acids 201–300
Transfected construct
(human)
Plasmid: pLEX.puro-human
FRMD8-iRhom2Δ300-3xHA
This paperN/Ahuman FRMD8 fused to
human iRhom2Δ300 via
a flexible linker (GSGSGS)
Transfected construct
(mouse)
Plasmid: pM6P.blast-mouse
iRhom2WT-3xHA
(Grieve et al., 2017)N/A
Transfected construct
(mouse)
Plasmid: pM6P.blast-mouse
iRhom2cub-3xHA
(Grieve et al., 2017)N/A
Transfected construct
(mouse)
Plasmid: pM6P.blast-mouse
iRhom2pDEAD-3xHA
(Grieve et al., 2017)N/A
Sequence-based
reagent
gRNA targeting exon 7
of human FRMD8 (ACCC
ATAAAACGGCAGCTCG)
This paperN/AgRNA targeting exon 7
of human FRMD8
Sequence-based
reagent
gRNA targeting exon 19 of
human RHBDF2 (AG
CGGTCAGTGCAGCACCT)
This paperN/AgRNA targeting exon 19
of human RHBDF2
Sequence-based
reagent
gRNA targeting exon 3 of
human RHBDF1 (GGAACC
ATGAGTGAGGCCCC)
This paperN/AgRNA targeting exon 3
of human RHBDF1
Sequence-based
reagent
gRNA targeting exon 3 of
human RHBDF1 (GGGTGG
CTTCTTGCGCTGCC)
This paperN/AgRNA targeting exon 3
of human RHBDF1
Sequence-based
reagent
gRNA targeting exon 10 of
human RHBDF1 (AGCCGT
GTGCATCTATGGCC)
This paperN/AgRNA targeting exon 10
of human RHBDF1
Sequence-based
reagent
gRNA targeting exon 10 of
human RHBDF1 (CCGTCTC
ATGCTGCGAGAAC)
This paperN/AgRNA targeting exon 10
of human RHBDF1
Sequence-based
reagent
gRNA targeting exon 2 of
human RHBDF2 (GCAGAG
CCGGAAGCCACCCC)
This paperN/AgRNA targeting exon 2
of human RHBDF2
Sequence-based
reagent
gRNA targeting exon 2 of
human RHBDF2 (GGGTCT
CTTTCTCGGGTGGC)
This paperN/AgRNA targeting exon 2
of human RHBDF2
Sequence-based
reagent
gRNA targeting exon 9 of
human RHBDF2 (AAACTC
GTCCATGTCATCATCACC)
This paperN/AgRNA targeting exon 9
of human RHBDF2
Sequence-based
reagent
gRNA targeting exon 9 of
human RHBDF2 (ACGGG
TGCGATGCCATACGC)
This paperN/AgRNA targeting exon 9
of human RHBDF2
Sequence-based
reagent
non-targeting siGENOME
control pool
DharmaconD-001206-13-50
Sequence-based
reagent
siGENOME SMARTpool for
human FRMD8
DharmaconM-018955-01-0010
Sequence-based
reagent
siRNA targeting human
RHBDF2 (HSS128594)
Thermo Fisher
Scientific
Cat#1299001
Sequence-based
reagent
siRNA targeting human
RHBDF2 (HSS128595)
Thermo Fisher
Scientific
Cat#1299001
Sequence-based
reagent
Human ACTB
(Hs99999903_m1)
Thermo Fisher
Scientific
Cat#4331182
Sequence-based
reagent
Human ADAM17
(Hs01041915_m1)
Thermo Fisher
Scientific
Cat#4331182
Sequence-based
reagent
Human FRMD8
(Hs00607699_mH)
Thermo Fisher
Scientific
Cat#4331182
Sequence-based
reagent
Human RHBDF2
(Hs00226277_m1)
Thermo Fisher
Scientific
Cat#4331182
Sequence-based
reagent
Human TNF
(Hs00174128_m1)
Thermo Fisher
Scientific
Cat#4331182
Commercial assay or
kit
BCA Protein Assay KitThermo Fisher
Scientific
Cat# 23225
Commercial assay
or kit
Human TNF alpha
ELISA Kit
Thermo Fisher
Scientific
Cat#88-7346-86
Commercial assay
or kit
SuperScript VILO cDNA
synthesis kit
Thermo Fisher
Scientific
Cat#11754050
Chemical compound,
drug
1,10-PhenanthrolineSigma-AldrichCat#131377–5G
Chemical compound,
drug
DSP (dithiobis(succinimidyl
propionate))
Thermos Fisher
Scientific
Cat#22585
Chemical compound,
drug
EDTA-free protease
inhibitor mix
RocheCat#11873580001
Chemical compound,
drug
GW280264X (GW)(Lorenzen et al., 2016);
received from Stefan
Düsterhöft
N/A
Chemical compound,
drug
GI254023X (GI)(Lorenzen et al., 2016);
received from Stefan
Düsterhöft
N/A
Chemical compound,
drug
LPSSigma-AldrichCat#L5668-2ML
Chemical compound,
drug
nocodazoleSigma-AldrichCat#M1404
Chemical compound,
drug
PNPP substrateThermos Fisher
Scientific
Cat#34045
Chemical compound,
drug
Rho kinase inhibitor
Y-27632
AbcamCat#ab120129
Peptide, recombinant
protein
Q5 High-Fidelity DNA
polymerase
New England BiolabsCat#M0491S
Peptide, recombinant
protein
Sequencing Grade
Trypsin
PromegaCat#V5111
Peptide, recombinant
protein
HA peptideRocheCat#I2149-.5MG
Peptide, recombinant
protein
M-CSFGibcoCat#PHC9501
Peptide, recombinant
protein
IL-3GibcoCat#PHC0033
Peptide, recombinant
protein
BMP-4InvitrogenCat#PHC9534
Peptide, recombinant
protein
VEGFPeproTechCat#100–20
Peptide, recombinant
protein
SCFMiltenyiCat#130-094-303
Peptide, recombinant
protein
RhFGF (bFGF)R and DCat#4114-TC
Otheranti-HA magnetic beadsThermo Fisher
Scientific
Cat#88837
Otheranti-V5 magnetic beadsMBL InternationalCat#M167-11
Otherconcanavalin A sepharoseSigma-AldrichCat#C9017-25ML
OtherC18 spin columnsThermo Fisher
Scientific
Cat#89873
Othervivaspin concentrator
500 (10,000 kDa MWCO)
SartoriusCat#VS0102
Othermouse IgG agaroseSigma-AldrichCat#A0919-5ML
OtherDMEMThermo Fischer
Scientific
Cat#41965039
OtherFetal bovine serumThermo Fischer
Scientific
Cat#10500064
OtherFish skin gelatinSigma-AldrichCat#G7765
OtherKnockOut -DMEMThermo Fischer
Scientific
Cat#10829
OtherKnockOut - serum
replacement
Thermo Fischer
Scientific
Cat#10828
OtherMEM Non-Essential Amino
Acids (100x)
Thermo Fischer
Scientific
Cat#11140–035
OtherGlutaMAX (100x)Thermo Fischer
Scientific
Cat#35050–038
Other2-Mercaptoethanol (1000x)Thermo Fischer
Scientific
Cat#31350–010
OtherPenicillin-Streptomycin
(P/S 100x)
Thermo Fischer
Scientific
Cat#15140–122
Other6-well ultra-low attachment
plates
CorningCat#3471
OtherX-VIVO 15LonzaCat#BE04-418
OthermTeSR1Stemcell TechnologiesCat#12491
OtherParaformaldehyde 16%Electron Microscopy
Sciences
Cat#15710
OtherhESC-qualified GeltrexThermo Fischer
Scientific
Cat#A1413302
OtherProLong Gold antifade
reagent with DAPI
Molecular ProbesCat#P36935
Software, algorithmFlowJo (version X 10.0.7r2)FlowJo, LLChttps://www.flowjo.com/
solutions/flowjo
Software, algorithmPrism (version 7)GraphPadhttps://www.graphpad.com/
scientific-software/prism/
Software, algorithmMaxQuant (version 1.5.0.35)(Cox and Mann, 2008)http://www.coxdocs.org/
doku.php?id=maxquant:start
Software, algorithmPerseus (version 1.5.5.3)(Tyanova et al., 2016)http://www.coxdocs.org/
doku.php?id=perseus:start
Software, algorithmFiji (version 2.0.0-rc-
43/1.52a)
(Schindelin et al., 2012)https://fiji.sc/
Software, algorithmClustal OmegaEMBL-EBIhttps://www.ebi.ac.uk/
Tools/msa/clustalo/

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  1. Ulrike Künzel
  2. Adam Graham Grieve
  3. Yao Meng
  4. Boris Sieber
  5. Sally A Cowley
  6. Matthew Freeman
(2018)
FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex
eLife 7:e35012.
https://doi.org/10.7554/eLife.35012