A cell-based Dkk1 binding assay reveals roles for extracellular domains of LRP5 in Dkk1 interaction and highlights differences between wild-type and the high bone mass mutant LRP5(G171V)
| Autor: | Jeanne J. Matteo, Veronique Damagnez, Valerie E. Coleburn, Kristina Allen, Wei Chen, Ramesh A. Bhat, Frederick J. Bex, Richard J. Murrills, Peter V.N. Bodine, Bheem M. Bhat |
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| Rok vydání: | 2009 |
| Předmět: |
musculoskeletal diseases
Mutant Receptors Cell Surface Biology medicine.disease_cause Biochemistry Bone and Bones Cell Line medicine Extracellular Humans Protein Interaction Domains and Motifs Receptor Molecular Biology LDL-Receptor Related Proteins Mutation Binding Sites Ligand binding assay Wnt signaling pathway Wild type Cell Biology Transfection Molecular biology Low Density Lipoprotein Receptor-Related Protein-5 Amino Acid Substitution Receptors LDL Low Density Lipoprotein Receptor-Related Protein-6 Intercellular Signaling Peptides and Proteins Biological Assay Molecular Chaperones Protein Binding |
| Zdroj: | Journal of cellular biochemistry. 108(5) |
| ISSN: | 1097-4644 |
| Popis: | Dkk1 is a secreted antagonist of the LRP5-mediated Wnt signaling pathway that plays a pivotal role in bone biology. Because there are no well-documented LRP5-based assays of Dkk1 binding, we developed a cell-based assay of Dkk1/LRP5 binding using radioactive 125I-Dkk1. In contrast to LRP6, transfection of LRP5 alone into 293A cells resulted in a low level of specific binding that was unsuitable for routine assay. However, co-transfection of LRP5 with the chaperone protein MesD (which itself does not bind Dkk1) or Kremen-2 (a known Dkk1 receptor), or both, resulted in a marked enhancement of specific binding that was sufficient for evaluation of Dkk1 antagonists. LRP5 fragments comprising the third and fourth β-propellers plus the ligand binding domain, or the first β-propeller, each inhibited Dkk1 binding, with mean IC50s of 10 and 196 nM, respectively. The extracellular domain of Kremen-2 (“soluble Kremen”) was a weaker antagonist (mean IC50 806 nM). We also found that cells transfected with a high bone mass mutation LRP5(G171V) had a subtly reduced level of Dkk1 binding, compared to wild type LRP5-transfected cells, and no enhancement of binding by MesD. We conclude that (1) LRP5-transfected cells do not offer a suitable cell-based Dkk1 binding assay, unless co-transfected with either MesD, Kremen-2, or both; (2) soluble fragments of LRP5 containing either the third and fourth β-propellers plus the ligand binding domain, or the first β-propeller, antagonize Dkk1 binding; and (3) a high bone mass mutant LRP5(G171V), has subtly reduced Dkk1 binding, and, in contrast to LRP5, no enhancement of binding with MesD. J. Cell. Biochem. 108: 1066–1075, 2009. © 2009 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
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