Structural and Functional Attributes of the Interleukin-36 Receptor

Abstract

Signal transduction by the IL-36 receptor (IL-36R) is linked to several human diseases. However, the structure and function of the IL-36R is not well understood. A molecular model of the IL-36R complex was generated and a cell-based reporter assay was established to assess the signal transduction of recombinant subunits of the IL-36R. Mutational analyses and functional assays have identified residues of the receptor subunit IL-1Rrp2 needed for cytokine recognition, stable protein expression, disulfide bond formation and glycosylation that are critical for signal transduction. We also observed that, overexpression of ectodomain (ECD) of Il-1Rrp2 or IL-1RAcP exhibited dominant-negative effect on IL-36R signaling. The presence of IL-36 cytokine significantly increased the interaction of IL-1Rrp2 ECD with the co-receptor IL-1RAcP. Finally, we found that single nucleotide polymorphism A471T in the Toll-interleukin 1 receptor domain (TIR) of the IL-1Rrp2 that is present in ∼2% of the human population, down-regulated IL-36R signaling by a decrease of interaction with IL-1RAcP.

Keywords: cytokine; immunology; membrane protein; psoriasis; receptor structure-function; signal transduction.

FIGURE 1.

Molecular models of the IL-36 receptor and its interaction with IL-36 cytokines. A, schema of the domain organization for IL-1Rrp2 and co-receptor IL-1RAcP. The N-terminal signal peptides are colored gray. The three Ig-like domains and the Toll/interleukin-1 receptor domains are labeled as D1, D2, D3, and TIR, respectively. The numbers above the schematics denote the approximate residues demarcating the domains. B, ribbon structures of the model of IL-1Rrp2 ectodomain in complex with IL-1RAcP ectodomain and IL-36γ. The TIR domains are not shown. C, surface charge of the IL-1Rrp2 and IL-1RAcP complex bound to IL-36γ (green ribbon structure). D, molecular models of D1 of IL-1Rrp2 that contacts IL-36α, IL-36β, and IL-36γ.

FIGURE 2.

A cell-based reporter assay to examine the function of IL-1Rrp2 in IL-36R signaling. A, HEK human embryonic kidney 293T cells express an undetectable level of IL-1Rrp2. The mRNA levels of IL-1Rrp2 and IL-1RAcP in 293T cells were quantified using real-time RT-PCR and adjusted to the levels from NCI/ADR-RES cells. The data were normalized to the GAPDH mRNA control from each sample. B, transfection of IL-1Rrp2 plasmid triggered NF-κΒ promoter activation upon IL-36 agonist addition. 293T cells were transfected to express IL-1Rrp2 and reporter constructs and then mock-treated or stimulated with 2 ng/ml of IL-36γ overnight. The luciferase activities are plotted as fold induction relative to cells transfected with the vector. The white and black bars denote the mock-treated and IL-36γ-stimulated activity. All results shown represent the means and standard deviations from at least three independent samples. C, effects of different ligands on the NF-κΒ activity in cells transfected to express IL-1Rrp2. D, effect of a FLAG-tagged IL-1Rrp2 on protein accumulation and signal transduction in 293T cells. Equal amounts of IL-1Rrp2 and FLAG-tagged IL-1Rrp2 plasmids were transfected into 293T cells, and the protein level was determined by Western blotting analysis probed with either goat anti-IL-1Rrp2 or rat anti-FLAG antibody. The NF-κΒ activation was determined as described above. E, siRNA knockdown of endogenous MyD88 in 293T cells significantly decreased IL-36γ mediated NF-κB activation. 293T cells were first transfected with 40 nm of either control siRNAs or MyD88 siRNAs for 48 h and then transfected to express IL-1Rrp2 for 24 h. The cells were mock-treated or treated with 2 ng/ml of IL-36γ. A significant reduction of MyD88 protein by the siRNA knockdown was confirmed by Western blotting analysis. The asterisk denotes that the two samples differed by a p value of < 0.05 in the Student's t test. F, effect of knockdown of endogenous Tollip on IL-36R signaling.

FIGURE 3.

Effects of cysteine to alanine substitutions in the IL-1Rrp2 ectodomain on protein accumulation and signaling. A, sequence alignment of cysteine residues in the IL-1R family. The IL-1Rrp2 ECD sequence is shown on the top, and residues in the IL-1R family that have conserved cysteine are marked in red. Cys pairs that are predicted to form disulfide bonds are connected by lines below the sequences. B, molecular model of potential disulfide bonds in three Ig-like domains of IL-1Rrp2. The cysteine residues, including the side chains, are in green. C, effects of cysteine mutations on IL-36R signaling and protein expression. Equal amounts of either WT IL-1Rrp2 or mutants were transfected into 293T cells along with the reporter plasmids as described. The cells were mock-treated or stimulated with 2 ng/ml of either IL-36α, IL-36β, IL-36γ, or IL-1β. The data were plotted relative to WT IL-1Rrp2 and expressed as a mean of three independent samples and the range for standard error. The levels of accumulated WT IL-1Rrp2 and cysteine mutants were determined by Western blotting analysis. β-Actin from each sample was detected as a loading control. The relative amount of protein was quantified by ChemiDoc software (Bio-Rad).

FIGURE 4.

N-linked glycosylation in IL-1Rrp2 is required for IL-36R signaling. A, IL-1Rrp2 contains N-linked glycans. 293T cells were transfected with IL-1Rrp2 for 24 h, and the cell lysate was mock-treated or treated with PNGase F or O-glycosidase. The lysate was separated using SDS-PAGE, and IL-1Rrp2 was detected by Western blotting analysis. B, effects of tunicamycin treatment on NF-κΒ activation. 293T cells were transfected with IL-1Rrp2 for 6 h, and treated with increasing concentrations of tunicamycin for an additional 18 h, followed by stimulation with IL-36γ. The data were plotted as NF-κB fold induction relative to mock-treated sample. The IL-1Rrp2 level in the cell lysate was determined by Western blotting analysis. C, effects of swainsonine on NF-κB activation. D, effects of PNGase F or O-glycosidase treatment on the migration of endogenous IL-1Rrp2 in NCI cells. The level of IL-1Rrp2 in the cell lysate was detected by Western blotting analysis. E, effects of tunicamycin on IL-36R signaling in NCI cells. NCI cells treated with increasing concentrations of tunicamycin for 24 h were stimulated with IL-36γ. The IL-6 level in the medium (solid line) was determined by ELISA. Cell proliferation (dashed line) was assessed by WST-1 assay according to the manufacturer's protocol. IL-1Rrp2 accumulation was detected by Western blotting analysis. F, effects of swainsonine on IL-36R signaling in NCI cells. The solid line and dashed line denoted IL-6 level and cell proliferation, respectively. The Western blotting analysis showed the effects of swainsonine on IL-1Rrp2 accumulation.

FIGURE 5.

N-linked glycosylation in IL-1Rrp2 is critical for signaling and trafficking to the cell surface. A, sequence alignment of predicted N-linked glycosylation sites in IL-1Rrp2 and orthologs from other species. Residues in IL-1Rrp2 predicted to be glycosylated are shown above the IL-1Rrp2 sequence. B, effects of mutations in the predicted N-linked glycosylation residues on the electrophoretic migration of the resultant IL-1Rrp2 proteins. C, effects of substitution of predicted glycosylation sites in the IL-1Rrp2 on signal transduction by all three IL-36 cytokines. 293T cells were transfected with equal amounts of the plasmids expressing WT or mutant IL-1Rrp2. The cells were mock-treated or stimulated with 2 ng/ml of the IL-36 agonists. The data were plotted relative to WT IL-1Rrp2 from two independent experiments, each performed with three independent samples. D, model of the localization of glycosylation sites in the IL-1Rrp2 ECD. The asparagine residues are marked in blue. E, cellular location of WT or mutant IL-1Rrp2 was determined by flow cytometry. Total amount of IL-1Rrp2 was determined in detergent-permeabilized cells while IL-1Rrp2 present on the cell surface was detected in non-permeablilized cells. Flow cytometry was performed as described in “Experimental Procedures.” The data were presented as averages of four independent samples.

FIGURE 6.

Residues in the IL-1Rrp2 ectodomain can differentially recognize three IL-36 cytokines. A, locations of the residues in IL-1Rrp2 that had differential response in signaling by the three IL-36 cytokines. The locations of the side chains are shown in blue in the ribbon diagram. The inset shows the overall electrostatic charges are shown as the envelope, with basic areas in blue and acidic ones in red. B, effects of increasing concentrations of the N41A mutant on IL-36R signaling. 293T cells were transfected with increasing concentrations of either WT or N41A along with constant amounts of reporter plasmids for 24 h and stimulated with 2 ng/ml of IL-36α, IL-36β, or IL-36γ. Signal transduction level was assessed using the NF-κΒ promoter that drives firefly luciferase expression. The transfection of 0.5 ng of plasmid of WT IL-1Rrp2 was plotted as 100%. The protein level was determined by Western blotting analysis. C, effects of increasing concentrations of the C42A mutant on IL-36R signaling. D, effects of the C118A mutant on IL-36R signaling. The level of C118A protein produced in the cells was shown in the Western blotting analysis.

FIGURE 7.

Overexpression of ECDs of IL-1Rrp2 and IL-1RAcP exhibit dominant-negative effect on IL-36R signaling. A, schematic of ECDs of IL-1RAcP and IL-1Rrp2. The IL-1RAcP ECD₃₆₇ contains the signal peptide at the N terminus and a FLAG tag at the C terminus, whereas the full-length and truncated IL-1Rrp2 ECDs contain the HA tags at the C terminus of the constructs. B, transient expression of IL-1RAcP ECD₃₆₇ and IL-1Rrp2 ECD₃₃₅. Western blotting analysis shows the abundances of ECD₃₃₅ in the cell lysate and in the medium of cell culture. C, overexpression of IL-1RAcP ECD₃₆₇ can inhibit IL-36R signaling induced by IL-36 cytokines. HEK293T cells were co-transfected with constant amount of IL-1Rrp2 plasmid along with increasing concentrations of IL-1RAcP ECD₃₆₇. The cells were then treated with 2 ng/ml of either IL-1β, IL-36α, IL-36β, or IL-36γ and the data were plotted as percent of IL-1RAcP ECD₃₆₇ mock-transfected control. D, overexpression of ECD₃₃₅ inhibits IL-36R signaling induced by IL-36 cytokines. E, effects of expression of IL-1Rrp2 ECD₂₅₈ and ECD_126–211 on IL-36R signaling. F, levels of IL-1Rrp2 ECD₃₃₅ and ECD₂₅₈ in the culture medium. The secreted ECDs in medium were detected as input control.

FIGURE 8.

Interaction between IL-1Rrp2 and IL-1RAcP is increased in the presence of the IL-36 cytokine. A, molecular model of the interaction between the IL-1RAcP ECD and the IL-1Rrp2 ECD. B, chemical basis for the interaction between the ECDs of IL-1RAcP and IL-1Rrp2. The two molecules are shown with the positively charged residues in blue and the negatively charged residues in red. The blue arrow shows the position of the IL-1Rrp2 domain 3 in the absence of interaction with IL-1RAcP. C, IL-1RAcP forms a complex with the IL-1Rrp2. Upper panel: results of the co-IP assay with full-length IL-1Rrp2 and IL-1RAcP. Lower panel: results of a co-IP assay with IL-1Rrp2 ECD₃₃₅ and IL-1RAcP. Where present, IL-36γ was added to cells at 20 ng/ml. D, interaction of IL-1RAcP ECD₃₆₇ with IL-1Rrp2 ECD₃₃₅. The cells were co-transfected with IL-1RAcP ECD₃₆₇ along with either the full-length IL-1Rrp2 ECD₃₃₅, truncated ECD₂₅₈ or ECD_126–211. After treated with IL-36γ, the complex in the culture medium was immunoprecipitated with anti-FLAG antibody and the interaction was detected by anti-HA antibody.

FIGURE 9.

Effects of IL-1Rrp2 SNPs on signal transduction. A, locations of three non-synonymous SNPs in the IL-1Rrp2. B, properties of the IL-1Rrp2 SNPs. SNP data were derived from NCBI SNP database. C, comparisons of the effects of SNPs on IL-36R signaling in 293T cells. Cells were transfected with plasmids encoding WT IL-1Rrp2 or the SNPs. The cells were mock-treated or stimulated with three IL-36 ligands, respectively. The data were plotted relative to WT IL-1Rrp2. D, analysis of SNP expression in 293T cells. E, molecular modeling the TIR domain of IL-1Rrp2 and its interaction with TIR domain of MyD88 and IL-1RAcP. S1 and S2 denote the two grooves in the IL-1Rrp2 TIR domain that are modeled to interact, respectively, with the IL-1RAcP TIR and the MyD88 TIR. Ala-471 is located in the S1 groove. F, immunoprecipitation to examine the interaction between the FLAG-tagged WT or A471T IL-1Rrp2 TIR domain with MyD88. 293T cells were co-transfected with MyD88 along with either the WT IL-1Rrp2 or A471T for 24 h and then mock-treated or stimulated by IL-36γ for 5 h. The antibody used for IP was targeted against MyD88. MyD88 levels in the lysate and immunoprecipitated complex were detected as the control. G, immunoprecipitation assay to examine the interaction between the FLAG-tagged WT and A471T IL-1Rrp2 TIR domain with IL-1RAcP. Immunoprecipitation used the anti-IL-1RAcP antibody. The TIR domain of IL-1Rrp2 was detected with an anti-FLAG antibody. The data represent three independent experiments with consistent results.