Premium
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)
Author(s) -
Murrills Richard J.,
Matteo Jeanne J.,
Bhat Bheem M.,
Coleburn Valerie E.,
Allen Kristina M.,
Chen Wei,
Damagnez Veronique,
Bhat Ramesh A.,
Bex Frederick J.,
Bodine Peter V.N.
Publication year - 2009
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.22335
Subject(s) - dkk1 , lrp5 , transfection , wnt signaling pathway , mutant , microbiology and biotechnology , chemistry , extracellular , receptor , binding domain , biology , biochemistry , binding site , signal transduction , gene
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 125 I‐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 IC 50 s of 10 and 196 nM, respectively. The extracellular domain of Kremen‐2 (“soluble Kremen”) was a weaker antagonist (mean IC 50 806 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.