Interleukin-17 receptor subunit dynamics
Kramer, Jill M.
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Inflammation is mediated by many factors, principle among them pro-inflammatory cytokines. While inflammation is critical for host defense and resolution of infection, it is often pathologic in its unregulated state. Importantly, IL-17 (IL-17A) has been identified as an essential player in many inflammatory diseases. IL-17A is secreted by a novel subset of T cells, termed Th17, and binds to the IL-17 receptor (IL-17RA). Initially, one subunit was identified for this receptor, although it was likely that IL-17RA signaled in the context of a larger complex, as is characteristic for all known cytokine receptors to date. Therefore, we assessed IL-17RA homodimerization through a chimeric receptor approach. The IL-17RA extracellular domain (ECD) was fused to the transmembrane (TMD) and cytoplasmic (CD) domains of the IL-2Rβ and γ c subunits, respectively. These constructs were expressed in an IL-2-dependent cell line, HT-2, and homodimerization was assessed by measuring an IL-2-specific signal. Although this study suggested IL-17RA did not self-associate, this result may have been related to the nature of the chimeras themselves, and therefore we pursued our hypothesis using a different approach. Accordingly, we used fluorescence resonance energy transfer (FRET) to assess IL-17RA self-association. Here, we fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to the IL-17RA to assess subunit interactions. Results from this study revealed that the IL-17RA is pre-assembled in the absence of ligand in a multimeric manner, and addition of ligand results in a conformational change in the receptor that causes the cytoplasmic tails to move apart. Then, we identified two putative domains that may mediate IL-17RA oligomerization through computational modeling. Deletion of one of these domains abrogated receptor self-association in a yeast two-hybrid (Y2H) approach. Furthermore, FRET studies confirmed this result, demonstrating the presence of a unique IL-17RA pre-ligand assembly domain (PLAD). In addition, we identified a region of the receptor that is crucial for binding IL-17A by functional assays and binding analysis with an IL-17A fusion protein. Identification of the IL-17RA PLAD and ligand binding domain has important therapeutic potential, as therapeutics that disrupt the receptor oligomerization and signaling will mitigate inflammatory signaling and ameliorate inflammatory pathology.