دورية أكاديمية
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).
| العنوان: | 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). |
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| المؤلفون: | Murrills RJ; Department of Osteoporosis and Frailty, Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, Pennsylvania 19426, USA. murrills@aol.com, Matteo JJ, Bhat BM, Coleburn VE, Allen KM, Chen W, Damagnez V, Bhat RA, Bex FJ, Bodine PV |
| المصدر: | Journal of cellular biochemistry [J Cell Biochem] 2009 Dec 01; Vol. 108 (5), pp. 1066-75. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Wiley-Liss Country of Publication: United States NLM ID: 8205768 Publication Model: Print Cited Medium: Internet ISSN: 1097-4644 (Electronic) Linking ISSN: 07302312 NLM ISO Abbreviation: J Cell Biochem Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: <2004>- : Hoboken, NJ : Wiley-Liss Original Publication: New York : Liss, c1982- |
| مواضيع طبية MeSH: | Intercellular Signaling Peptides and Proteins/*metabolism , LDL-Receptor Related Proteins/*metabolism , Molecular Chaperones/*metabolism , Receptors, Cell Surface/*metabolism , Receptors, LDL/*metabolism, Amino Acid Substitution ; Binding Sites ; Biological Assay ; Bone and Bones/metabolism ; Cell Line ; Humans ; LDL-Receptor Related Proteins/chemistry ; LDL-Receptor Related Proteins/genetics ; Low Density Lipoprotein Receptor-Related Protein-5 ; Low Density Lipoprotein Receptor-Related Protein-6 ; Molecular Chaperones/genetics ; Mutation ; Protein Binding ; Protein Interaction Domains and Motifs ; Receptors, Cell Surface/genetics |
| مستخلص: | 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. |
| المشرفين على المادة: | 0 (DKK1 protein, human) 0 (Intercellular Signaling Peptides and Proteins) 0 (KREMEN2 protein, human) 0 (LDL-Receptor Related Proteins) 0 (LRP5 protein, human) 0 (LRP6 protein, human) 0 (Low Density Lipoprotein Receptor-Related Protein-5) 0 (Low Density Lipoprotein Receptor-Related Protein-6) 0 (MESD protein, human) 0 (Molecular Chaperones) 0 (Receptors, Cell Surface) 0 (Receptors, LDL) |
| تواريخ الأحداث: | Date Created: 20090912 Date Completed: 20100809 Latest Revision: 20191028 |
| رمز التحديث: | 20221213 |
| DOI: | 10.1002/jcb.22335 |
| PMID: | 19746449 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1097-4644 |
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| DOI: | 10.1002/jcb.22335 |