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IL-17 and its receptor are founding members of a novel inflammatory cytokine family. To date, only one IL-17 receptor subunit has been identified, termed IL-17RA. All known cytokine receptors consist of a complex of multiple subunits. Although IL-17-family cytokines exist as homodimers, the configuration and stoichiometry of the IL-17R complex remain unknown. We used fluorescence resonance energy transfer (FRET) to determine whether IL-17RA subunits multimerize, and, if so, whether they are preassembled in the plasma membrane. HEK293 cells coexpressing IL-17RA fused to cyan or yellow fluorescent proteins (CFP or YFP) were used to evaluate FRET before and after IL-17A or IL-17F treatment. In the absence of ligand, IL-17RA molecules exhibited significant specific FRET efficiency, demonstrating that they exist in a multimeric, preformed receptor complex. Strikingly, treatment with IL-17A or IL-17F markedly reduced FRET efficiency, suggesting that IL-17RA subunits within the IL-17R complex undergo a conformational change upon ligand binding.
Interleukin 17A (IL-17 or CTLA8) and its receptor (IL-17R or IL-17RA) are the founding members of a novel family of proinflammatory cytokines (1). Most cells are potential targets of IL-17A, since IL-17RA is ubiquitously expressed, but the molecular biology of this receptor family is still poorly understood (2). Produced almost exclusively by effector memory T cells (3, 4), IL-17A elicits production of numerous cytokines and chemokines involved in the inflammatory response (5) and is a particularly potent activator of neutrophils (1, 6). Consequently, IL-17RA-deficient mice exhibit enhanced susceptibility to multiple infectious agents, including Klebsiella pneumoniae, Toxoplasma gondii, and Candida albicans (7–9). In contrast to its protective role in infection, IL-17A plays a pathogenic role in autoimmune disease, especially rheumatoid arthritis (RA)3 (5, 10). Blocking IL-17A in animal models of RA ameliorates symptoms (11), and other mouse models in which IL-17A production is impaired show reduced susceptibility to arthritis (12, 13). Indeed, IL-17A was recently described as the hallmark cytokine produced by a subset of “pathogenic” T cells involved in autoimmune inflammation (14). Thus, like many inflammatory cytokines, IL-17A can be protective or destructive depending on context.
All known cytokine receptors are multimeric and oligomerization of receptor subunits is essential for function. Some, like the growth hormone (GH) and erythropoietin (EPO) receptors, are homodimers, while others are heterodimers or higher order multimers. It is generally considered that cytokines trigger signal transduction by inducing association of receptor subunits, thereby bringing into proximity crucial intracellular signaling intermediates. However, there is convincing evidence that some cytokine receptors are preassembled before binding ligand. For example, crystallographic and genetic studies showed that the EPO receptor (EPOR) exists as preformed homodimer without EPO. Binding of EPO triggers structural alterations that bring together EPOR-associated JAK2 kinases, facilitating their phosphorylation and initiating signaling (15–19). Similarly, studies of the IFN-γ, Fas, and TNF receptors showed that their constituent subunits preassociate even in the absence of ligand (20–22). However, not all cytokine receptors exist as preformed multimers; for example, most evidence to date indicates that binding of GH triggers receptor dimerization (18).
Fluorescence resonance energy transfer (FRET) is a powerful tool to measure protein-protein interactions. In FRET, nonradiated energy is transferred from a donor to an acceptor fluorophore when the donor emission spectrum significantly overlaps the acceptor excitation spectrum and the fluorophores are closely approximated. FRET has been used to detect interactions between proteins that are tagged with a FRET donor/acceptor pair, such as cyan or yellow fluorescent proteins (CFP or YFP). These are generally considered to be the fluorophores of choice because they have excitation and emission wavelengths favorable for FRET, appropriate extinction coefficients and quantum yields, and CFP has high photostability (23).
Surprisingly little is currently known about the configuration of the IL-17R-binding complex. In mice, only one IL-17A-binding subunit has been identified, termed IL-17RA, which also binds weakly to IL-17F (2, 24). Since all known cytokine receptors contain multiple peptides, we hypothesized that IL-17RA also signals as part of a multisubunit complex. In this study, we provide evidence that the IL-17R-binding complex contains multiple IL-17RA subunits, which are preassembled in the plasma membrane before ligand binding.
Murine IL-17RA constructs were generated by RT-PCR and fused to FLAG and enhanced YFP and CFP (BD Clontech) (see Fig. 1). The 24p3 promoter-luciferase construct was previously described (25). TNFRΔp60/YFP (hemagglutinin tagged) was a gift from F. Chan (University of Massachusetts Medical School, Worcester, MA). HEK293, ST2, and IL-17R-deficient fibroblasts (derived from mice provided by Amgen) were maintained in α-MEM (Sigma-Aldrich) with 10% FBS (Gemini Bioproducts). Cells were transfected with calcium phosphate or Fugene 6 (Roche). IL-17A, IL-17F, and TNF-α were obtained from R&D Systems.
IL-17R-deficient MF cells were transfected with indicated constructs and normalized to an internal Renilla-luciferase control. Six hours after stimulation, standard luciferase assays were performed in triplicate as described previously (26).
For FACS, 0.25–1.5 × 106 cells were incubated on ice with 3.4 μg mAb M750 (rat anti-mouse IL-17R, IgG2a, provided by Amgen) followed by goat anti-rat-PE (BD Pharmingen). Data were analyzed on a FACSCalibur with CellQuest software (BD Biosciences). FRET data were obtained from three channel images using the macro of LSM FRET tool software (Zeiss AIM software) as previously described (27). FRET efficiencies were calculated by the FRETN and N-FRET methods, with similar results (28, 29).
To assess IL-17RA multimerization in live, single cells by FRET, murine IL-17RA with an N-terminal FLAG tag was fused to CFP or YFP (Fig. 1A). The IL-17RA has an unusually long cytoplasmic tail that could potentially increase nonspecific receptor-receptor interactions in FRET (15, 23). Therefore, for these studies, we examined FRET in both full-length and truncated forms of IL-17RA. It was previously shown that the N-terminal FLAG tag does not interfere with ligand binding or signal transduction (30), and we confirmed that the full-length IL-17R/CFP and IL-17R/YFP constructs activated signaling equivalently to wild-type IL-17RA. Specifically, IL-17R/CFP and/YFP directed transcriptional activation of the 24p3 target gene promoter linked to a luciferase reporter (Fig. 1B). However, no signals were activated by the IL-17R (1-441) truncation, because this receptor lacks key cytoplasmic signaling domains (data not shown and Ref. 31). Although the truncated receptor does not signal, it can be readily detected at the cell surface with an IL-17R-specific mAb, indicating that it appears to be conformationally intact (Fig. 1C).
To assess receptor interactions by FRET, HEK293 cells were transfected with these IL-17RA constructs. All receptors were expressed efficiently at the plasma membrane, although there was also significant expression within the cytoplasm, which is typical of ectopically expressed cytokine receptors (Figs. 1C and 2 and data not shown). As a negative control, we paired a truncated TNF receptor (TNFRΔp60/YFP) (21) with IL-17R/CFP constructs (Fig. 1A and Ref 21). The sensitized emission method was used to analyze FRET efficiency. Using the multi-track and line-scanning mode of a laser-scanning confocal microscope, cells were simultaneously recorded in the CFP, YFP, or FRET channels. We first obtained bleed-through (cross-talk) coefficients by analyzing images from cells expressing individual YFP- or CFP-tagged constructs and then obtained FRET efficiency and FRET images from cells expressing both constructs. FRET efficiencies were calculated for each pixel using methods to correct for donor and acceptor concentrations.
A major advantage of using confocal microscopy to evaluate FRET is that individual regions of interest (ROI) within a cell can be selectively examined for FRET fluorescence. In contrast, flow cytometric approaches can only assess total cellular FRET, which often includes high concentrations of fluorophores in intracellular compartments. Therefore, since we were interested in IL-17RA interactions at the cell surface, only the plasma membrane region of the cell was used as a ROI. Although the IL-17RA and TNF receptors both localize to the cell membrane, they do not associate as a receptor complex, and thus were used to determine baseline FRET efficiencies. Strikingly, cells coexpressing either full-length or truncated IL-17R/CFP and IL-17R/YFP constructs showed a marked enhancement of FRET (images for the truncated receptor are shown here, Fig. 2, C and D). Surprisingly, however, FRET between IL-17RA subunits disappeared following treatment with IL-17A (Fig. 2, B–D, and data not shown). Treatment of cells with IL-17F, another ligand for IL-17RA (24), also reduced FRET efficiency, although to a lesser extent. This result is likely due to the 10- to 30-fold reduced binding affinity of IL-17F to the IL-17RA complex (S. Levin, personal communication). Quantitative analyses of FRET efficiencies indicated that there is a statistically significant increase in FRET in cells coexpressing full-length or truncated IL-17R/CFP and IL-17R/YFP constructs in the absence of ligand compared with cells treated with IL-17A, IL-17F, or to the negative control (Fig. 2D). Together, these data show that IL-17RA subunits multimerize at the plasma membrane in the absence of ligand. Moreover, binding to IL-17RA by either ligand induces a conformational change in the receptor complex, causing a loss of FRET signal.
Neutralization of inflammatory mediators to reduce progression of RA has been used successfully for several cytokines, particularly TNF-α (32). IL-17A is also an important mediator of RA pathology, as blockade of IL-17A in rodent arthritis models reduces joint inflammation and bone erosion (10, 11, 33). Therefore, understanding the positioning of IL-17RA subunits relative to one another, both in the presence and absence of ligand, is likely to aid in the design of agents to inhibit inflammatory IL-17 signaling, since knowing the distance between subunits may allow for accurate prediction of the structural properties of putative receptor antagonists.
The data presented here show that IL-17RA subunits preassemble as multimers in the plasma membrane. In some cases, FRET has been used to calculate the distance between the donor and acceptor and thereby infer the distance between the molecules to which they are linked. It is difficult to make meaningful measurements between IL-17RA subunits, since no structural information about the cytoplasmic tail of IL-17RA is available, and therefore the relative orientations of CFP and YFP are unknown. Although a free rotation of CFP or YFP is assumed in calculating FRET efficiencies, this is probably not actually the case when fluorophores are tethered to larger proteins. However, FRET between CFP and YFP is detectable only in the range of 10–100 Å, and FRET efficiency decreases exponentially as the distance between fluorophores increases (23). Thus, these data indicate that CFP and YFP are held in extremely close proximity through their association with IL-17RA. Upon ligand stimulation, we observed a decrease in FRET efficiency similar to that of the negative control. This unexpected result suggests that ligand binding induces a conformational change in the IL-17R complex, causing the cytoplasmic tails to move further than 100 Å apart.
The loss of FRET following interaction with IL-17A or IL-17F suggests several alternative mechanisms of IL-17R signaling. Although it is formally possible that the IL-17RA subunits completely dissociate, it is more likely that the receptor complex is preserved but the C-terminal ends of the receptor tails become repositioned. For example, the receptor may be conformationally rigid, such that rotation of the receptor mediated by ligand causes changes in the relative orientations of CFP and YFP, thus reducing energy transfer (Fig. 3, A and B). It is also possible that the juxtamembrane regions of the IL-17R are pushed apart (Fig. 3C). It is known in the EPOR that alterations in the orientation or spacing of the extracellular domain can be transmitted to the cytoplasmic tails and their associated signaling molecules (17, 34–36). Alternatively, the recruitment of an intracellular signaling molecule or additional receptor subunit might physically separate IL-17RA tails (Fig. 3, D and E). Results from this study do not preclude the possibility of an additional subunit in the IL-17R complex, which has been postulated based on a discrepancy between IL-17R affinity and concentrations of IL-17A needed for biological responsiveness (2). The loss of FRET could also be due to receptor internalization and degradation, although we only see minimal loss of receptor upon short-term treatment with IL-17 (our unpublished data). Finally, these models are not mutually exclusive, and some or all may come into play upon interaction of IL-17A or IL-17F with the receptor.
These data predict the existence of a pre-ligand assembly domain in IL-17RA, analogous to a similar domain in the TNFR superfamily (21). This domain not only dictates receptor assembly, but has been shown to be a possibly useful therapeutic for inflammatory arthritis (37). Although there is no obvious homology between the extracellular domains of the TNFR and IL-17RA receptors, the pre-ligand assembly domains are quite degenerate (F. Chan, personal communication). However, these experiments set the stage for the identification of such a domain within IL-17RA.
Because IL-17A and IL-17F exist as homodimers, it has long been presumed that their cognate receptors would also be homodimeric. Consistent with this, human IL-17RD (or similar expression of fibroblast growth factor receptor, SEF) was recently shown to form a homodimer in an overexpression system (38). Although the composition and stoichiometry of the IL-17A-binding complex remains to be fully elucidated, this study demonstrates that it contains at least two identical IL-17RA subunits that are preassembled in the plasma membrane, and a conformational change in the receptor appears to occur following ligand binding.
We thank Dr. W. Sigurdson (University at Buffalo Confocal and Imaging Facility) and Dr. S. Pierce (National Institute of Allergy and Infectious Diseases) for helpful suggestions. We are grateful to Dr. F. K. M. Chan (University of Massachusetts) for TNFRp60/YFP and Drs. J. Tocker and J. Peschon (Amgen, Seattle, WA) for IL-17R-deficient mice, anti-IL-17R mAbs, and valuable comments. We also thank Dr. X. Xu, Dr. J. Fang, and Dr. C. Mullin and M. Gaffen for valuable contributions.
1This study was supported by National Institutes of Health Grants to J.M.K. (DE014831) and S.L.G. (AR050458), the Arthritis Foundation (to S.L.G.), and National Institute of Allergy and Infectious Diseases intramural funding (to T.J., L.Y., and X.J.).
3Abbreviations used in this paper: RA, rheumatoid arthritis; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein, EPO, erythropoietin, EPOR, erythropoietin receptor; GH, growth hormone; ROI, region of interest.
G. Gaffen owns stock in Amgen Incorporated.