Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor - PubMed (original) (raw)

Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor

Jill M Kramer et al. J Immunol. 2007.

Abstract

IL-17 is the hallmark cytokine of the newly described "Th17" lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a "pre-ligand assembly domain" (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1

FIGURE 1

The FN2 domain of IL-17RA mediates ligand-independent assembly. A, Predicted structure of IL-17RA FN domains. PHYRE predicted two FN domains within the mouse IL-17RA ECD. Yellow, _β_-Sheets; red, _α_-helices; green, unstructured loops; blue, turns. Sequences of each domain are shown. B, Schematic diagram of IL-17RA FRET and Y2H constructs. SEFIR is a major signaling domain in IL-17RA (22). AD, Activation domain; BD, binding domain. C, FN2linker drives ligand-independent association in cells. HEK293 cells expressing the indicated combinations of IL-17RA constructs fused to CFP or YFP were assayed for N-FRET in the absence (open bars) or presence of IL-17 (filled bars) or IL-17F (gray bar) for 10 min. Significance was assessed by t test, p < 0.05; n.s., not significant. D, Representative images of IL-17RAΔ/CFP plus IL-17RAΔFN2linker/YFP. CFP, YFP, and FRET emissions are shown. Unstim., Unstimulated. E, IL-17RA can co-IP with IL-17RAΔFN2linker. HEK293 cells expressing IL-17RAΔFN2linker/YFP were transiently transfected with IL-17RA.HA. Cells were lysed and immunoprecipitated (IP) with anti-HA or M177 anti-IL-17RA Abs and immunoblotted with Abs to GFP (which cross-react with YFP) or HA. Note that we always observe slight cross-reactivity of anti-HA Abs with YFP and CFP for unknown reasons. W, Western blot.

FIGURE 2

FIGURE 2

Requirements for IL-17 signaling and binding. A, The FN1 domain is dispensable but the linker is required for IL-17-dependent signaling. IL-17RA−/−fibroblasts were transfected in triplicate with the indicated IL-17RA constructs (with FL cytoplasmic tails) and 24p3-Luc (18). Cells were stimulated with IL-17 (200 ng/ml) and/or TNF-α (2 ng/ml). After 6 h, luciferase activity was determined and normalized to Renilla luciferase. B and C, IL-17RA-neutralizing Abs bind the FN2linker domain. B, HEK293 cells stably transfected with IL-17RΔ/CFP (top) or transiently transfected with IL-17RAΔFN2linker/CFP (bottom) were incubated with a non-neutralizing (clone M177) or neutralizing Ab (clone M750) to murine IL-17RA. Filled histograms are isotype controls. C, Whole cell lysates from HEK293 cells stably expressing IL-17RAΔ/CFP or IL-17RAΔFN2linker/YFP were immunoprecipitated (IP) with M750 or M177 as indicated and blotted with anti-GFP Abs. D and E, FN2linker has a reduced affinity for IL-17. HEK293 cells stably expressing IL-17RAΔ/CFP or IL-17RAΔFN2linker/YFP were stained with various concentrations of IL17.Fc after blocking endogenous human IL-17RA (D) or with M750 (E). F, The linker is required but not sufficient for IL-17 binding. HEK293 cells transiently transfected with IL-17RAΔ/YFP, IL-17RAΔFN1linker/YFP, or IL-17RAΔFN2/YFP were stained with IL17.Fc and gated on YFP-positive cells.

FIGURE 3

FIGURE 3

Model of IL-17RA subunit dynamics. Data suggest that the cytoplasmic tails of IL-17RA are held in proximity before ligand binding (A), but separate upon binding IL-17 (B). Based on FRET, at least one FN1 domain is sufficient to mediate ligand-induced subunit reconfiguration (C). However, in the absence of both FN1 domains the cytoplasmic tails show increased association (D). IL-17 requires the FN2 and linker regions to bind the receptor but may also contact FN1 (B_–_D).

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