Table 1.
Yeast Two-Hybrid Screen of IL-17RC cytoplasmic domain.
Figure 1.
Neither Rrad nor CFD mediate IL-17 signal transduction. A. Rrad-deficient fibroblasts mediate normal IL-17 induction of IL-6 secretion.
Multiple Rrad−/− cell lines (derived from adult tail biopsies of Rrad−/− mice) or ST2 stromal cell lines were treated with IL-17 (200 /ng/ml), IL-17F (200/ ng/ml) or suboptimal TNFα (2 ng/ml) for 24 h. IL-6 was measured in culture supernatants in triplicate by ELISA. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ‡p<0.05 by Chi Square comparing experimental replicates. B. Rrad-deficient fibroblasts mediate normal IL-17 induction of target gene expression. A representative Rrad−/− cell line or IL-17RC−/− fibroblasts were treated with IL-17 or TNFα as outlined in panel A for 24 h. Expression of the indicated genes was assessed by qPCR in triplicate. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls of each cell line. ‡p<0.05 by Chi Square comparing experimental replicates. C. Complement deficient cell lines mediate normal IL-17 signaling. Fibroblast cell lines from CFD−/−, C3−/− or IL-17RC−/− mice were treated with IL-17 and TNFα as described in panel A, and IL-6 concentrations in culture supernatants were assessed by ELISA. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ‡p<0.05 by Chi Square comparing experimental replicates. ¶concentration values above standard curve for ELISA detection. All data are representative of at least two independent experiments.
Figure 2.
AnapC7 binds to IL-17R but does not impact IL-17 signaling.
A. Schematic diagram of IL-17RA and IL-17RC mutants. ECD, extracellular domain. SEFIR and SEFEX domain approximate boundaries are indicated [22], [24]. CBAD, C/EBPβ activation domain. B. AnapC7 associates with IL-17RA and IL-17RC. HEK293T cells were transfected with AnapC7 tagged with HA and IL-17RA or IL-17RC tagged with Myc, as indicated. Lysates were immunoprecipitated with anti-Myc Abs and immunoblotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. C. RNA silencing of AnapC7 does not alter IL-17-dependent signaling. ST2 cells were transfected with the indicated siRNAs, treated with IL-17 (black bars) for 24 h and IL-6 in culture supernatants assessed by ELISA. n.s., not significant. *p<0.05 by ANOVA and post-hoc Tukey’s test relative to unstimulated controls. ‡p<0.05 by Chi Square comparing experimental replicates.
Figure 3.
AnapC5 binds to the IL-17 receptor and restricts IL-17-mediated signal transduction. A. AnapC5 associates with IL-17RA, IL-17RC and AnapC7.
HEK293T cells were transfected with AnapC5 (tagged with HA) together with IL-17RA, IL-17RC or AnapC7 (tagged with Myc), as indicated. Lysates were subjected to IP with anti-Myc Abs and immunoblotted with anti-HA or anti-Myc. Arrows indicate identity of each Myc-tagged protein. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. B. AnapC5 associates with IL-17RA through the inhibitory CBAD domain. HEK293T cells were transfected with AnapC5 and the indicated IL-17RA deletion constructs. Lysates were subjected to co-IP with anti-Myc and blotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-Myc. Migration of protein size markers is indicated. C. AnapC5 associates with IL-17RC in the SEFIR domain. HEK293T cells were transfected with AnapC5 and the indicated IL-17RC deletion constructs. Lysates were subjected to co-IP with anti-Myc and blotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. D. Knockdown of AnapC5 enhances IL-17 signaling. ST2 cells were transfected with the indicated siRNAs, stimulated with IL-17 for 24 h, and IL-6 in culture supernatants assessed by ELISA. n.s., not significant. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ‡p<0.05 by Chi Square comparing experimental replicates. E. Efficient knockdown of AnapC5 and AnapC7. mRNA from the samples in panel D were assessed for AnapC5 and AnapC7 expression by qPCR. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ‡p<0.05 by Chi Square comparing experimental replicates. Data are representative of at least 2 independent experiments.
Figure 4.
The IL-17R-A20 signaling complex involves AnapC5 rather than Itch and TAXBP1. A. AnapC5 but not AnapC7 associates with A20.
HEK293T cells were co-transfected with AnapC5 [short (s) and long (l) forms] or AnapC7 (both Myc-tagged) together with A20. Lysates were subjected to IP with anti-Myc Abs and blotted for A20 or Myc. WCL were blotted for A20. Migration of protein size markers is indicated. B. AnapC5 associates with endogenous A20. HEK293T cells were transfected with Myc-tagged AnapC5. Lysates were subjected to IP with anti-Myc Abs and blotted for A20 or Myc. WCL were blotted for β-tubulin as a loading control. C. Knockdown of Itch and TAXBP1 does not impact IL-17R mediated signaling. ST2 cells were transfected with the indicated siRNAs, stimulated with IL-17 for 24 h, and mRNA expression was assessed by qPCR. n.s., not significant. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ‡p<0.05 by Chi Square comparing experimental replicates. D. Efficient knockdown of Itch and TAXBP1. mRNA from the samples in panel B were assessed for Itch and TAXBP1 expression by qPCR. *p<0.05 by ANOVA and post-hoc Tukey’s test compared to mock-transfected controls. ‡p<0.05 by Chi Square comparing experimental replicates. All data are representative of at least two independent experiments.
Figure 5.
Schematic model of AnapC5 and AnapC7 in IL-17R signaling.
The data in this paper support a model in which AnapC5 serves an adaptor or scaffold protein to facilitate A20 recruitment to the CBAD domain of IL-17RA. Although AnapC7 binds to both IL-17RA and IL-17RC, its functional role is still unclear.