Bone Abstracts

Twelve different mutations in TNFRSF11A (encoding the RANK receptor) have been associated with osteoclast-poor autosomal recessive osteopetrosis in patients. Two truncated RANK proteins resulting from substitution mutations (W434X and G280X), identified in two infants, cause loss of the intracellular oligomerisation motif and in the case of the G280X mutation the TRAF6 binding domain. A third mutation was identified in a 10-year-old patient and is a frameshift mutation encoding a protein that is truncated within the extracellular, N-terminal domain (R110Pfs52X) raising the possibility that translation of the C-terminal region of the protein from alternative translation initiation sites may occur. We have been investigating the effects of these mutations on receptor processing and downstream activation of NFκβ to inform on the molecular cause for this condition.

W434X-RANK and G280X-RANK proteins were overexpressed in Hela cells and immunoprecipitation revealed that, unlike wild-type-RANK, the W434X–RANK and G280X-RANK mutant proteins failed to oligomerise. In addition, whereas wild-type-RANK and W434X-RANK interact with TRAF6, G280X-RANK does not. p65 translocation experiments revealed that RANKL activates NFκβ in cells transfected with wild-type-RANK or W434X-RANK, but not G280X-RANK. These results strongly suggest that, whilst the TRAF6 domain is critical for activation of the RANK signalling pathway, the oligomerisation motif is not essential.

For the R110Pfs52X mutation, in vitro osteoclast cultures surprisingly demonstrated some osteoclast formation in the absence of RANK ligand. We generated seven myc-tagged expression constructs representing the N-terminal and potential C-terminal translation products. p65 translocation experiments demonstrated that the R110Pfs52X product does not support ligand-dependent or ligand-independent activation of NFκβ, whereas the putative C-terminal products of the alternative translation start sites induced only ligand-independent activation of NFκβ which may explain the ligand-independent osteoclast formation observed in osteoclast cultures.

Taken together, these results provide molecular insights into the regulation of RANK signalling underlying the patient phenotypes associated with each mutation.