QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 November 2005
doi: 10.1242/jcs.02651

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, Y. Articles by Bu, G. Search for Related Content PubMed PubMed Citation Articles by Li, Y. Articles by Bu, G.

Mesd binds to mature LDL-receptor-related protein-6 and antagonizes ligand binding

Yonghe Li^1,*,, Jianglei Chen², Wenyan Lu¹, Lynn M. McCormick¹, Jianjun Wang² and Guojun Bu^1,3

¹ Department of Pediatrics, St Louis Children's Hospital, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA
² Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, 224 Neckers Hall, Carbondale, IL 62901, USA
³ Departments of Cell Biology and Physiology, St Louis Children's Hospital, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA

View larger version (18K): [in a new window]   Fig. 1. Mesd binds to mature LRP6 at the cell surface with high affinity. (A) Time course of 125I-Mesd (5 nM) binding to LRP6-transduced HT1080 cells and the control cells. Assay was carried out for the indicated periods at 4°C in the absence (total) or presence of 500 nM Mesd (non-specific). (B) Saturation binding of 125I-Mesd to LRP6-transduced HT1080 cells and the control cells. Assay was carried out at indicated concentrations for 3 hours at 4°C in the absence (total) or presence (non-specific) of 500 nM Mesd. (C) Scatchard plots of data in B. All values are the average of triple determinations with the s.d. indicated by error bars.

View larger version (38K): [in a new window]   Fig. 2. Mesd binds to mature LRP5/6 but not significantly to other members of the LDLR family. (A) Binding of 125I-Mesd (5 nM) to HEK293 cells transient transfected with human HA-tagged LDLR, Myc-tagged LRP5, Myc-tagged LRP6 or control vector. Lower panel, western blot analysis for the expression of the LDLR, LRP5 and LRP6. Equal amounts of cell lysate were applied for each lane. (B) Binding of 125I-Mesd (5 nM) to LRP-null CHO cells stably transfected with LRP minireceptor mLRP4, LRP1B minireceptor mLRP1B4, VLDLR, apoER2 or empty pcDNA3 vector only. (C) Binding of 125I-Mesd (5 nM) to wild-type murine embryonic fibroblasts (MEF-1) or MEF cell lines genetically deficient in LRP (MEF-2), LDLR (MEF-3) or both (MEF-4). Lower panel, western blot analysis of LRP and the LDLR expression in MEF cell lines. (D) Binding of 125I-Mesd (5 nM) to human breast cancer cell line MCF-7, human glioblastoma cell line U87 and human aortic smooth muscle cells (SMC). Lower panel, western blot analysis of LRP expression in these cell lines. Assays were carried out for 4 hours at 4°C in the absence (total) or presence of 500 nM Mesd. Values are the means of triple determinations with the s.d. indicated by error bars. *P<0.01 indicates a significant difference compared with control cells transfected with empty pcDNA3 vector.

View larger version (26K): [in a new window]   Fig. 3. The C-terminal region of Mesd is required for interaction with LRP6. (A) Schematic representation of Mesd mutants. (B) Binding analyses of 125I-Mesd and its mutants (5 nM) to LRP6-transduced HT1080 cells. Assays were carried out for 3 hours at 4°C in the absence or presence of 500 nM Mesd or its mutants as indicated. Values are the means of triple determinations with the s.d. indicated by error bars.

View larger version (11K): [in a new window]   Fig. 4. The C-terminal region of Mesd is necessary and sufficient for LRP6 binding. (A) Binding analyses of 125I-Mesd and its mutant Mesd (150-195) (5 nM) to LRP6-transduced HT1080 cells and control cells. (B) Binding analyses of 125I-Mesd and its mutant Mesd (150-195) (5 nM) to LRP6-transduced HT1080 cells. Assays were carried out for 3 hours at 4°C in the absence or presence of 500 nM Mesd or its mutant. Values are the means of triple determinations with the s.d. indicated by error bars.

View larger version (26K): [in a new window]   Fig. 5. The C-terminal region of Mesd is required for LRP6 folding. (A) HEK293 cells were transiently transfected with the indicated cDNAs. Cell lysates were analyzed by SDS-PAGE under reducing conditions and western blotted with anti-FLAG or anti-HA antibodies as indicated. (B) HEK293 cells were cotransfected with LRP6, MESD, MesdC or empty pcDNA3 vector and a TCF/LEF transcriptional activity reporter plasmid (TOP-FLASH). The luciferase activity was measured 48 hours after transfection. Values are the means of triple determinations with the s.d. indicated by error bars.

View larger version (12K): [in a new window]   Fig. 6. LRP6 is not a constitutively active endocytosis receptor. (A) Anti-HA IgG binding to cell surface HA-tagged LRP6. Binding of 125I-anti-HA IgG (1 nM) to LRP6-transduced HT1080 cells and the control cells was carried out for 90 minutes at 4°C. (B) LRP6 endocytosis. LRP6-transduced HT1080 cells, mLRP4-transfected CHO cells and mLRP4tailess-transfected CHO cells were incubated with 1 nM 125I-anti-HA IgG at 4°C for 90 minutes, and then incubated at 37°C for the indicated times. The amount of internalized anti-HA IgG was determined. Values are the means of triple determinations with the s.d. indicated by error bars.

View larger version (14K): [in a new window]   Fig. 7. LRP6 exhibits a limited level of Mesd degradation. (A) LRP6-mediated 125I-Mesd (5 nM) degradation in LRP6-transduced HT1080 cells and control cells was carried out for 4 hours at 37°C in the absence or presence of 500 nM Mesd. (B) 125I-Mesd (5 nM) binding to LRP6-transduced HT1080 cells and the control cells was carried out for 4 hours at 4°C in the absence or presence of 500 nM Mesd. Values are the means of triple determinations with the s.d. indicated by error bars.

View larger version (17K): [in a new window]   Fig. 8. RAP binds to LRP6 and partially competes for Mesd binding. (A) RAP binds to mature LRP6 at the cell surface. Binding of 125I-RAP (5 nM) to LRP6-transduced HT1080 cells and the control cells was carried out for 4 hours at 4°C in the absence (total) or presence of 500 nM RAP, or 500 nM Mesd. (B) Binding of 125I-Mesd (5 nM) to LRP6-transduced HT1080 cells was carried out for 2 hours at 4°C in the absence (total) or presence of various concentrations of RAP or 500 nM Mesd. (C) LRP6-mediated 125I-Mesd (5 nM) degradation was carried out for 4 hours at 37°C in the absence or presence of 500 nM Mesd or 500 nM RAP. Values are the means of triple determinations with the s.d. indicated by error bars.

View larger version (52K): [in a new window]   Fig. 9. Mesd inhibits DKK1 binding to LRP6. (A-C) Serum-free conditioned medium was harvested from HEK293 cells transiently transfected with cDNA for human Myc-DKK1 and allowed to bind to LRP6-transduced HT1080 cells (B,C) and control cells (A) in the absence (A,B) or presence (C) of 1 µM Mesd. Cell-surface-bound Myc-tagged DKK proteins were fixed and detected by immunofluorescence staining with anti-Myc antibody. (D) DKK1 binding to cell surface LRP6 is inhibited by Mesd. Binding of 125I-DKK1 (5 nM) to LRP6-transduced HT1080 cells or the control cells was carried out for 3 hours at 4°C in the absence (total) or presence of 500 nM RAP or 500 nM Mesd. (E) LRP6-mediated DKK1 degradation is inhibited by Mesd. LRP6-mediated 125I-Mesd (5 nM) degradation was carried out for 4 hours at 37°C in the absence or presence of 500 nM RAP or 500 nM Mesd. Values are the means of triple determinations with the s.d. indicated by error bars. Bar, 10 µm.