title: Correlating RANK ligand/RANK binding kinetics with osteoclast formation and function.
abstract: The interaction between Receptor Activator of NF-κB Ligand (RANKL) and its receptor RANK is essential for the differentiation and bone resorbing capacity of the osteoclast. Osteoprotegerin (OPG), a soluble homodimer, acts as a decoy receptor for RANKL and thus inhibits osteoclastogenesis. An imbalance in the RANKL/RANK/OPG axis, with decreased OPG and/or increased RANKL, is associated with diseases that favor bone loss, including osteoporosis. Recently, we established a yeast surface display system and screened libraries of randomly mutated RANKL proteins to identify mutations that abolish binding to OPG while preserving recognition of RANK. These efforts yielded several RANKL variants possessing substantially higher affinity for RANK compared to their wild-type (WT) counterpart. Using recombinant RANKL mutant proteins, we find those with increased affinity for RANK produce more robust signaling in osteoclast lineage cells and have greater osteoclastogenic potential. Our results are the first to document gain of function RANKL mutations. They indicate that the physiological RANKL/RANK interaction is not optimized for maximal signaling and function, perhaps reflecting the need to maintain receptor specificity within the tumor necrosis factor superfamily (TNFSF). Instead, we find, a biphasic relationship exists between RANKL/RANK affinity and osteoclastogenic capacity. In our panel of RANKL variants, this relationship is driven entirely by manipulation of the kinetic off-rate. Our structure-based and yeast surface display-derived insights into manipulating this critical signaling axis may aid in the design of novel anti-resorptive therapies as well as provide a paradigm for design of other receptor-specific TNF superfamily ligand variants.
authors: Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
authors: Julia T
authors: JT
authors: Warren
authors: Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
authors: Wei
authors: W
authors: Zou
authors: Department of Orthopaedic Surgery, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
authors: Corinne E
authors: CE
authors: Decker
authors: Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
authors: Nidhi
authors: N
authors: Rohatgi
authors: Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
authors: Christopher A
authors: CA
authors: Nelson
authors: Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
authors: Daved H
authors: DH
authors: Fremont
authors: Department of Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, Missouri.|Department of Medicine, Division of Bone and Mineral Diseases, Washington University in St. Louis School of Medicine, St. Louis, Missouri.
authors: Steven L
authors: SL
authors: Teitelbaum
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D007700:Kinetics; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism; D053244:Osteoprotegerin / Q000378:metabolism; D011485:Protein Binding; D053245:RANK Ligand / Q000235:genetics* / Q000378:metabolism*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D011994:Recombinant Proteins; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse
keywords: BONE; KINETICS; OSTEOCLAST; RANK; RANKL; RECEPTOR; SIGNAL TRANSDUCTION; TNF-SUPERFAMILY