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. 2021 May 5;78(11):5015–5040. doi: 10.1007/s00018-021-03845-3

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Impact of mutations within the IRHD on ADAM17. a The conserved motif and a highly variable sequence within the non-structured stretch are represented as weblogo presentation. Noteworthy, the fact that some amino acid residues such as tryptophan and cysteine residues usually appear in low frequency in protein sequences leads to a higher scoring when they are highly conserved. Designed mutations of the murine iRhom2 are shown. The construct miR2_1 and miR2_2 were designed by mutating the region located N-terminally from the conserved motif either by replacement with a flexible linker (miR2_1) or by changing charges of three amino acid residues (miR2_2) to create a drastic change at this position. To generate miR2_3 parts of the conserved motif were replaced with a flexible linker. b–d HEK293 cells stably expressing the indicated iRhom constructs or GFP as negative control (ctr) were used for the described experiments. The transferrin receptor (TfR) served as input control. b To analyse the ADAM17 maturation, glycosylated proteins were enriched via ConA beads. Maturation was assessed by densitometric measurements and calculation of the ratio between mADAM17 and total ADAM17 derived by the sum of mADAM17 and imADAM17. n = 4. c To analyse the binding between ADAM17 and iRhom constructs, co-IPs were performed by using the iRhom constructs (with HA tag) as bait. Quantitative analysis of ADAM17 binding can be found in figures S2d, e. n = 4. d ADAM17-mediated shedding activity was assessed by performing an alkaline phosphatase (AP) assay in HEK293 cells stably expressing the indicated iRhom construct. The ADAM17 substrate IL-1R_II tagged with AP was used. Since ADAM17 activity can be upregulated by stimulation of certain G-protein coupled receptors, HEK293 cells were stimulated with the PAR1 (protease activated receptor 1) agonist TRAP6. Cells were incubated for 2 h under unstimulated condition or stimulated with TRAP6. In all cell lines with expression of iRhom2 constructs, wt_iRhom2 or control there was a significant stimulation of ADAM17-mediated shedding by TRAP6 (p < 0.01). n = 8