Searchable abstracts of presentations at key conferences on calcified tissues
Bone Abstracts (2014) 3 PP158 | DOI: 10.1530/boneabs.3.PP158

ECTS2014 Poster Presentations Cell biology: osteoclasts and bone resorption (22 abstracts)

Study of the molecular effects of disease-causing mutations in RANK using human protein expression models

Subhajit Das 1 , Janice Bramham 2 , Angela Duthie 1 , Susan Clark 1 & Julie Crockett 1

1University of Aberdeen, Aberdeen, UK; 2University of Edinburgh, Edinburgh, UK.

The interaction of Receptor Activator of NFkB ligand (RANKL) with its cognate receptor RANK is crucial for osteoclast formation. We studied eight point mutations within human RANK associated with rare forms of osteopetrosis to gain mechanistic insights into the regulation of RANK signalling.

We investigated the role of the oligomerisation domain within the cytoplasmic region of RANK studying two mutations (W434X and G280X) identified in rare cases of osteopetrosis. Immunoprecipitation showed that, by contrast to WT RANK, W434X and G280X prevented ligand-independent but not ligand-dependent oligomerisation and, in the W434X mutant with an intact TRAF6 binding domain, NFkB signalling was normal. Hence, removal of the oligomerisation motif did not interfere with ligand-induced trimerisation and subsequent NFkB signalling.

The structural importance of the four extracellular, cysteine-rich domains (CRD1-4) in RANK has previously been highlighted in the crystal structure of the mouse RANK–RANKL complex. We studied six disease-associated point mutations within this region of human RANK. RANKL-dependent NFkB activation could only be detected in cells overexpressing WT, but not mutant proteins suggesting altered sensitivity to RANKL. Recombinant, WT and mutant his-tagged RANK proteins (residues 26–210) were expressed in mammalian cells and purified by Ni2+-affinity, followed by size-exclusion chromatography. Surface plasmon resonance analysis confirmed the very high affinity interaction between human RANKL and WT RANK (Kd < 1 nM). RANKL showed slightly weaker binding to four mutants (A134V, D148V and R170G in CRD3; and C175R in CRD4) and no binding to two mutants, G53R (in CRD1) and R129C (in the linker between CRD2 and CRD3), indicating the crucial role of the latter two amino acids in human RANK. This study shows the effect of disease-associated mutations in RANK on the biophysical interaction of human RANK protein with human RANKL.

Taken together, this work provides novel insights into factors influencing RANKL–RANK interaction and signalling.

Volume 3

European Calcified Tissue Society Congress 2014

Prague, Czech Republic
17 May 2014 - 20 May 2014

European Calcified Tissue Society 

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