期刊
JOURNAL OF CELLULAR BIOCHEMISTRY
卷 108, 期 5, 页码 1066-1075出版社
WILEY-LISS
DOI: 10.1002/jcb.22335
关键词
LRP5; LRP6; MesD; KREMEN-2; HIGH BONE MASS; BINDING ASSAY
Dkk1 is a secreted antagonist of the LRP5-mediated Writ 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 beta-propellers plus the ligand binding domain, or the first P-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 P-propellers plus the ligand binding domain, or the first beta-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. (C) 2009 Wiley-Liss, Inc.
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