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).
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 beta-propellers plus the ligand binding domain, or the first beta-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 beta-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.
Murrills
Richard J
RJ
Department of Osteoporosis and Frailty, Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, Pennsylvania 19426, USA. murrills@aol.com
Matteo
Jeanne J
JJ
Bhat
Bheem M
BM
Coleburn
Valerie E
VE
Allen
Kristina M
KM
Chen
Wei
W
Damagnez
Veronique
V
Bhat
Ramesh A
RA
Bex
Frederick J
FJ
Bodine
Peter V N
PV
D019943:Amino Acid Substitution; D001665:Binding Sites; D001681:Biological Assay; D001842:Bone and Bones / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D026502:LDL-Receptor Related Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism*
C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; C486076:KREMEN2 protein, human; D026502:LDL-Receptor Related Proteins; C555482:LRP5 protein, human; C555475:LRP6 protein, human; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C506348:MESD protein, human; D018832:Molecular Chaperones; D011956:Receptors, Cell Surface; D011973:Receptors, LDL