title: Functional dissection of osteoprotegerin and its interaction with receptor activator of NF-kappaB ligand.
abstract: The receptor activator of NF-kappaB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nM. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of approximately 3 microM, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction.
authors.affiliation: Department of Biochemistry and Biophysics and Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. schneewe@mail.med.upenn.edu
authors.forename: Lumelle A
authors.initials: LA
authors.lastname: Schneeweis
authors.forename: Derril
authors.initials: D
authors.lastname: Willard
authors.forename: Marcos E
authors.initials: ME
authors.lastname: Milla
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D001681:Biological Assay; D002352:Carrier Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D016207:Cytokines / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008956:Models, Chemical; D009154:Mutation; D010010:Osteoclasts / Q000378:metabolism; D053244:Osteoprotegerin; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014780:Viruses / Q000235:genetics
chemical_list: D002352:Carrier Proteins; D016207:Cytokines; D004220:Disulfides; D006023:Glycoproteins; D008024:Ligands; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D011506:Proteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C505686:TNFRSF11A protein, human; C506030:TNFRSF11B protein, human; C505688:TNFSF11 protein, human; D006493:Heparin