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* Department of Oral Biology,
Department of Microbiology and Immunology, and
Confocal Facility, University at Buffalo, State University of New York, Buffalo, NY 14214;
Department of Inflammation Research, Amgen Inc., Seattle WA 98119; and
¶ Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 S.L.G. was supported by the National Institutes of Health Grants AR05048 and AI49329. J.M.K. was supported by National Institutes of Health Grant DE14831. J.P.M. was supported by National Institutes of Health Training Grant DE07034 to the Department of Oral Biology. N.I. and T.J. are supported by National Institute of Allergy and Infectious Diseases intramural funding.
2 Address correspondence and reprint requests to Dr. Sarah L. Gaffen, 3435 Main Street, Buffalo, NY 14214. E-mail address: sgaffen{at}buffalo.edu
3 Abbreviations used in this paper: PLAD, pre-ligand assembly domain; CFP, cyan fluorescent protein; co-IP, coimmunoprecipitation; ECD, extracellular domain; FL, full length; FN, fibronectin III-like; FRET, fluorescence resonance energy transfer; HA, hemagglutinin; N-FRET, normalized FRET; YFP, yellow fluorescent protein; Y2H, yeast two-hybrid.
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