title: Manipulation of receptor oligomerization as a strategy to inhibit signaling by TNF superfamily members.
abstract: Signaling by receptor activator of nuclear factor κB (RANK) in response to its ligand RANKL, which is a member of the tumor necrosis factor (TNF) superfamily of cytokines, stimulates osteoclast formation and bone resorption. Thus, this ligand-receptor pair is a therapeutic target for various disorders, such as osteoporosis and metastasis of cancer to bone. RANKL exists as a physiological homotrimer, with each monomer recognizing a single molecule of RANK or the decoy receptor osteoprotegerin (OPG), which inhibits osteoclastogenesis. We engineered a RANKL protein in which all three monomers of RANKL were encoded as a single polypeptide chain, which enabled us to independently control receptor binding at each binding interface. To generate an effective RANK inhibitor, we used an unbiased forward genetic approach to identify mutations in RANKL that had a 500-fold increased affinity for RANK but had decreased affinity for the decoy receptor OPG. Incorporating mutations that blocked receptor binding into this high-affinity RANKL variant generated a mutant RANKL that completely inhibited wild-type RANKL-induced osteoclastogenesis in vitro and bone resorption in mice. Our approach may be generalized to enable the inhibition of other TNF receptor signaling systems, which are implicated in a wide range of pathological conditions.
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Julia T
authors.initials: JT
authors.lastname: Warren
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Nelson
authors.affiliation: Department of Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Corinne E
authors.initials: CE
authors.lastname: Decker
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zou
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA. fremont@pathology.wustl.edu teitelbs@wustl.edu.
authors.forename: Daved H
authors.initials: DH
authors.lastname: Fremont
authors.affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA. Division of Bone and Mineral Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. fremont@pathology.wustl.edu teitelbs@wustl.edu.
authors.forename: Steven L
authors.initials: SL
authors.lastname: Teitelbaum
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001862:Bone Resorption / Q000235:genetics; D006801:Humans; D051379:Mice; D020125:Mutation, Missense; D010010:Osteoclasts / Q000378:metabolism*; D053244:Osteoprotegerin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000235:genetics*; D053245:RANK Ligand / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse