IL-17 has come into prominence with its identification as the signature cytokine of the Th17 lineage (32
). However, to date the signaling mechanisms mediated by IL-17 are surprisingly poorly defined. The IL-17 receptor family is striking in that it bears little resemblance to better known cytokine families, and thus it has not been possible to infer how this receptor mediates signals based merely on homology with other systems (6
). An important clue came with the discovery of the SEFIR domain and its homology to TIR domains (8
), which was consistent with the similar panels of pro-inflammatory gene targets activated by IL-17 and TIR/IL-1R ligands (reviewed in (27
)). Despite this homology, the SEFIR domain is functionally and structurally distinct from TIRs. First, unlike TIR-containing receptors, IL-17R does not engage the prototypical innate immune adaptors MyD88 or TRIF (9
). Second, SEFIR domains do not encode the “BB-loop” structure found in TIR domains that is required to mediate specificity of homotypic TIR-TIR interactions (25
). Empirical structure-function studies of IL-17RA showed that, while the SEFIR is necessary for signaling, an extended region beyond the SEFIR is also required (12
When IL-17RC was shown to be a key component of the IL-17R signaling cascade, this raised important questions about its mechanisms of action with respect to signal transduction. Given the genetic requirement for IL-17RC in transducing IL-17 signals (13
) and the IL-17F-bound IL-17RA crystal structure suggesting a IL-17RA/IL-17RC heterodimeric receptor (14
), we wished to determine the functional significance of the IL-17RC receptor in the IL-17 signaling axis. Using IL-17RC truncations, our data indicate that the IL-17RC cytoplasmic tail is not required for its association with IL-17RA (). Furthermore, the IL-17RC SEFIR as well as an additional downstream 23 amino acids are required for IL-17-dependent IL-6 and 24p3 expression, and for a ligand-dependent association with a phosphorylated Act1 isoform (–). We have dubbed this a “SEFEX” domain, for SEFIR extension. We further show that IL-17RC is required for IL-17-dependent host defense against oropharyngeal candidiasis (). Taken together, these results demonstrate that IL-17RC is indispensable for IL-17-dependent immune responses, and identifies key structural and biochemical requirements responsible for signaling downstream of the IL-17RC receptor.
The stoichiometry of how IL-17RC associates with IL-17RA is currently undefined. FRET studies of IL-17RA suggested that pre-assembled homodimers exist on the cell surface. However, the addition of IL-17 causes a conformational shift to occur, which could be explained by dissociation of IL-17RA homodimers or recruitment of additional subunits (24
). Subsequent genetic data, demonstrating that IL-17RC is required for the IL-17-dependent production of CXCL1, supported the idea that association with IL-17RC is involved in this process (13
). Our present data indicate that a basal level of IL-17RA and IL-17RC may exist as pre-associated complexes, but that addition of IL-17A or IL-17F enhances this association (). While our results must be interpreted in the context of an overexpression system, these findings are supported by recent biochemical studies of IL-17RA, which indicate that ligand-binding increases the affinity of the IL-17RA/IL-17RC interaction (14
). Notably, we also find that IL-17 treatment induces IL-17RC to preferentially associate with a larger IL-17RA isoform. Tunicamycin treatment leads to the disappearance of the larger IL-17RA isoform, suggesting that this is a glycosolated IL-17RA isoform. Various N-linked glycosylated forms of IL-17RA have been reported, and the E3 ubiquitin ligases Act1 and TRAF6 have been shown to be associated with ubiquitinated forms of IL-17RA (7
). The relevance of these glycosylated species IL-17RA is unclear at present, but its ligand-dependent association argues that it may be critical for signaling.
IL-17RC has a higher affinity for IL-17F than IL-17A (16
). However, our data and others (13
) demonstrate that IL-17RC is nonetheless required for IL-17A-mediated signaling. IL-17RC also exists in multiple splice forms, some of which have differential recognition of IL-17-family ligands (16
). In these studies we only evaluated the unspliced (full length) IL-17RC isoform, but it will be interesting to determine in the future whether there are differences in how other splice forms of IL-17RC participate in signaling.
Previous reports indicate that the IL-17RC cytoplasmic tail, which contains a SEFIR domain, is integral for functional IL-17 responses (8
). We previously found that IL-17RA uses a SEFIR extension to mediate signaling (12
). Analogously, we show here that IL-17RC uses both its SEFIR domain and an additional downstream sequence of 23 amino acids to activate signals such as IL-6 and 24p3 expression (). Consistent with this result, Hu et al.
find that the IL-17RC SEFIR is required for functional IL-17 signal transduction both in vitro and in vivo (37
). Bioinformatic database searches and sequence alignments, however, indicate that the extended sequence downstream of the IL-17RC SEFIR lacks homology to the sequence downstream of the IL-17RA SEFIR, the Act1 SEFIR, or other known receptors or signaling intermediates (AWH, unpublished observations). Thus, the extended IL-17RC SEFEX domain may contribute a novel signaling function. Indeed, a chimeric receptor construct composed of the IL-17RA extracellular domain fused to the complete IL-17RC cytoplasmic tail cannot rescue IL-17-dependent signaling in IL-17RA−/− cells (R. Onishi and J. Park, unpublished data), implying that the role of IL-17RC is probably not simply to supply another SEFIR domain to more efficiently recruit additional Act1 molecules. Therefore, despite the fact that the IL-17RC cytoplasmic tail is much shorter than the IL-17RA intracellular domain (214 versus 521 amino acids), IL-17RC may possess novel signaling functions distinct from IL-17RA.
Act1 is an essential signaling component downstream of IL-17RA and is required for IL-17-dependent immune responses (9
). We now show that IL-17RC also associates with Act1 even in the absence of IL-17RA (). Interestingly, IL-17 treatment leads to the association of IL-17RC with a larger phosphorylated form of Act1. This result indicates for the first time that Act1 is phosphorylated, and that phospho-Act1 associates with IL-17RC in a ligand-dependent manner. Even more striking, the only IL-17RC deletions that associate with this phospho-Act1 isoform are those that retain function, i.e.
mutants that contain the IL-17RC SEFEX motif. It is possible that the SEFEX domain recruits other signaling intermediates needed to create a stable signaling scaffold to permit Act1 to efficiently transduce downstream signals, analogous to other systems such as the co-receptors of the T cell receptor (38
Although good biochemical and genetic evidence supports a role for IL-17RC within the IL-17R complex, it is still not fully established whether IL-17RC is required for all aspects of IL-17 signal transduction in vivo
. To evaluate the physiological function of IL-7RC, we used an oral fungal infection model (23
) that we and others previously showed to be strongly IL-17/IL-17RA-dependent (22
). Here, we demonstrate that IL-17RC is required for host defense against infection with the yeast Candida albicans
. (). Moreover, susceptibility to OPC was identical between IL-17RC−/− and IL-17RA−/− mice (and also IL-23p19−/− mice (22
)). This finding also suggests, albeit indirectly, that there is no additional role for IL-25 in this process, since IL-17RA but not IL-17RC participates in the IL-25 receptor complex (40
). In line with these findings, IL-17RC was recently shown to participate in the pathogenesis of autoimmune inflammation of the central nervous system, an event highly dependent on the IL-17 signaling axis (37
In conclusion, our results show that IL-17RC is critically important for IL-17-dependent signaling and immune responses. IL-17RC mediates signaling via an extended SEFIR domain, which is required for a ligand-dependent association with a phosphorylated Act1 isoform to promote downstream signaling. These studies provide the first report of a signaling intermediate directly downstream of the IL-17RC receptor, and are the first to define important structural sequence elements within this receptor. Lastly, like IL-17RA, IL-17RC is required for host defense against oral fungal infections caused by Candida albicans. It will be interesting in future studies to link IL-17RC signaling pathways directly to biological signals and ultimately exploit this knowledge to improve treatments for a host of human diseases.