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

First published online September 30, 2004
doi: 10.1242/10.1242/dev.01318

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 Cong, F. Articles by Varmus, H. Search for Related Content PubMed PubMed Citation Articles by Cong, F. Articles by Varmus, H.

Wnt signals across the plasma membrane to activate the ß-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP

Feng Cong^*,, Liang Schweizer and Harold Varmus

Cancer Biology and Genetics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA

View larger version (17K): [in a new window]   Fig. 1. Synergistic effects of Wg and Frizzled on LEF-dependent transcription. S2 cells were transfected with indicated plasmids and plasmids as described in the Materials and methods. Fold-activation values were measured relative to the levels of luciferase activity in cells transfected with an empty vector and normalized by Renilla luciferase activities. All experiments were carried out in triplicate and error bars represent standard deviation.

View larger version (36K): [in a new window]   Fig. 2. Activation of Wnt/ß-catenin signaling by rat Fz1 and human FZ5 point mutants. (A) Schematic representation of rat Fz1 double-alanine-scanning mutants. Amino acids residues of transmembrane segments, three intracellular loops and the N-terminal section of the intracellular tail were shown. In these rat Fz1 mutants, two neighboring amino acid were changed to Ala, and a GFP tag was fused to the C termini of all mutants. The majority of mutants cover the intracellular regions, while two mutants reside in the extracellular loops. The activities of wild-type and mutant rat Fz1 to stimulate LEF-dependent transcription were measured in S2 cells. The activities of mutants rat Fz1 relative to that of wild-type rat Fz1 are indicated by color. Amino acid residues essential for the signaling activity of rat Fz1 and human FZ5 and studied in detail in later experiments are indicated by asterisks. (B) Single point mutations abolish signaling activity of rat Fz1. Activities of rat Fz1, rat Fz1 R340A, rat Fz1 L524A and rat Fz1 K619A were measured in S2 cells. The membrane localization of wild-type and mutant rat Fz1 was examined by membrane biotinylation, precipitation with avidin-agarose beads and immunoblotting with anti-GFP antibodies (insert). (C) Single point mutations abolish signaling activity of human FZ5. Activities of human FZ5, human FZ5 R263A, human FZ5 L443A and human FZ5 K525A were measured in S2 cells. All human FZ5 constructs were fused with a GFP epitope at their C termini. The membrane localizations of wild-type and mutant human FZ5 were examined by the membrane biotinylation assay (insert). (D) The Trp residue in the Lys-Thr-x-x-x-Trp motif can be substituted by Tyr. The Trp residue in the Lys-Thr-x-x-x-Trp motif (W624 in rat Fz1 and W530 in human FZ5) was mutated to either Ala or Tyr, and activities of these mutants were measured in S2 cells.

View larger version (59K): [in a new window]   Fig. 3. Point mutations decrease the interaction between Dsh and HFz5. (A) Recruitment of Dsh to the plasma membrane by wild-type and mutant human FZ5. Myc-tagged Dsh and GFP-tagged human FZ5 were co-expressed in COS cells. Cells were stained by anti-Myc 9E10 monoclonal antibodies and Texas Red-conjugated goat anti-mouse secondary antibodies, and examined by fluorescence microscopy. (B) Physical interaction between Dsh and wild-type or mutant human FZ5. Myc-tagged Dsh and GFP-tagged human FZ5 were co-expressed in 293 cells. Cell lysates were immunoprecipitated with anti-Myc antibodies, and immunoprecipitates were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-GFP antibodies (upper panel). Expression of Dsh and human FZ5 in total cell lysates was examined by immunoblotting with anti-Myc and anti-GFP antibodies (lower panels).

View larger version (35K): [in a new window]   Fig. 4. Stimulation of the signaling activity of LRP6 by oligomerization. (A) Schematic representation of wild-type and mutant LRP6. Signal peptide (SP), EGF repeats (E1-E4), LDLR repeats (L1-3) and transmembrane domain (TM) are indicated. In LRP6EGF, all EGF repeats were deleted. In LRP6N, the whole extracellular domain was deleted. Two copies of FKBPv were tethered to the C terminus of LRP6EGF to form LRP6EGF-FKBP. The extracellular domain of LRP6 was replaced by the extracellular domain of TrkC or human FZ5 to form TrkN-LRP6C and HFz5N-LRP6C. (B) AP20187 increases the stimulatory effect of LRP6EGF-FKBP on LEF-dependent transcription in 293 cells. 293 cells were transfected with the indicated plasmids and CMV-LEF1, CMV-Renilla luciferase and a LEF-luciferase reporter. Cells were either treated with vehicles or 50 nM AP20187 for 36 hours. The levels of luciferase activities were normalized for Renilla luciferase activities. (C) AP20187 increases LRP6EGF-FKBP-induced ß-catenin stabilization in 293 cells. 293 cells were transfected with the indicated plasmids and treated with vehicles or 50 nM AP20187 for 36 hours. Cells were subjected to subcellular fractionation, and cytosolic ß-catenin was determined by immunoblotting. (D) AP20187 increases the binding of Axin to LRP6EGF-FKBP during ß-catenin stabilization in 293 cells. 293 cells were transfected with the indicated plasmids and treated with vehicles or 50 nM AP20187 for 36 hours. Proteins were immunoprecipitated from cell lysates with anti-HA antibodies, and immunoprecipitates were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-Myc antibodies (upper panel). Expression of Axin and LRP6EGF or LRP6EGF-FKBP in total cell lysates was examined by immunoblotting with anti-Myc and anti-HA antibodies (lower panels). (E) NT3 increases the signaling activity of TrkN-LRP6C in S2 cells. S2 cells were transfected with the indicated plasmids and treated with 200 ng/ml NT-3 (Upstate) for 48 hours. Cells were lysed and the luciferase activities were normalized for Renilla luciferase activity. (F) The signaling activity of LRP6, with or without inducible oligomerization, is Dsh independent. S2 cells were treated with dsRNAs, transfected with the indicated effector plasmids and treated with 200 ng/ml NT3 for 48 hours. Cells were lysed and luciferase activities were measured.

View larger version (30K): [in a new window]   Fig. 5. The MESD chaperone protein increases the membrane localization and signaling activity of LRP6. (A) MESD increases the membrane localization of LRP6. LRP6 and LRP6N were fused with YFP at their C termini, and expressed in 293 cells with or without co-expression of MESD. The subcellular localization of wild-type and mutant LRP6 was examined by confocal fluorescence microscopy. (B) MESD increases the signaling activity of LRP6. 293 cells were transfected with the indicated plasmids together with TOP-FLASH and CMV-Renilla. The luciferase activities were normalized for Renilla luciferase activities.

View larger version (19K): [in a new window]   Fig. 6. Functional substitution of the extracellular domain of LRP6 with the extracellular domain of human FZ5. S2 cells were treated with the indicated dsRNAs and transfected with the indicated effector plasmid together with LEF-1 reporter plasmids. Forty-eight hours after transfection, cells were lysed and luciferase activities were measured.

View larger version (23K): [in a new window]   Fig. 7. Strong synergy between human FZ5 and LRP6 in Wnt/ß-catenin signaling by fusing the intracellular domain of LRP6 to human FZ5. (A) Schematic representation of human FZ5, LRP6 and the HFz5-LRP6 chimera. The intracellular domain of LRP6 was fused to the C terminus of human FZ5 to form HFz5-LRP6C. The C terminus of HFz5-LRP6C was fused with GFP. (B) Hyperactivity of HFz5-LRP6C. S2 cells were treated with control or Dsh-dsRNA and transfected with the indicated plasmids. As control, Arg263, Leu443 or Lys525 of human FZ5 was mutated in HFz5-LRP6C. Membrane expressions of HFz5-LRP6C and its mutants were determined by the membrane biotinylation assay (insert).

View larger version (21K): [in a new window]   Fig. 8. Strong synergy between TrkN-LRP6C and NT3-HFz5 in Wnt/ß-catenin signaling. (A) Schematic representation of human FZ5, LRP6 and the NT3-HFz5 chimera. NT3 was fused to the N terminus of human FZ5 to form NT3-HFz5. (B) Synergetic effect of TrkN-LRP6C and NT3-HFz5 on LEF-dependent transcription. S2 cells were transfected with the indicated plasmids. Luciferase activities were measured 48 hours after transfection.

View larger version (32K): [in a new window]   Fig. 9. Requirement of Wg can be by-passed by fusing Wg to human FZ5 or LRP6 and mutating the palmitoylation site of Wg reduces the activity of both chimeric receptors. (A) Schematic representation of human FZ5, TrkN-LRP6C, Wg-HFz5, Wg-TrkN-LRP6C. Wg was fused to the N terminus of human FZ5 to form Wg-HFz5. Both human FZ5 and Wg-HFz5 were fused with GFP at their C termini. Wg was also fused to the N terminus of TrkN-LRP6C to form Wg-TrkN-LRP6C. (B) Hyperactivity of Wg-HFz5 requires both Arrow and Dsh. S2 cells were treated with control-, Arrow- or Dsh-dsRNA, and transfected with the indicated plasmids. Luciferase activities were measured 48 hours after transfection. (C) Mutating C93, the potential palmitoylation site of Wg, reduces the signaling activity of Wg-HFz5. The membrane expressions of human FZ5, Wg-HFz5 and WgC93S-HFz5 were determined by membrane biotinylation and immunoblotting with anti-GFP antibodies (insert). (D) Wg-TrkN-LRP6C is a Dsh-dependent hyperactive chimeric receptor and mutating the palmitoylation site of Wg reduces the activity of this chimera. The membrane expressions of TrkN-LRP6C, Wg-TrkN-LRP6C and WgC93S-TrkN-LRP6C were determined by membrane biotinylation and immunoblotting with anti-TrkC antibodies (insert).

View larger version (40K): [in a new window]   Fig. 10. Physical interactions between Wnt3A and its palmitoylation site mutant with the extracellular domain of LRP6 and mouse Fz8. Conditioned medium of Rat2 cells expressing HA-tagged Wnt3A or Wnt3A C77S was mixed with conditioned medium of 293 cells expressing LDLR-IgG (A,B), LRP6N-IgG (A), mFz8CRD-IgG (B), and incubated protein G-agarose beads. Precipitates were washed and fractionated by SDS-PAGE, and immunoblotted with anti-HA antibodies (upper panel). Input of Wnt3A in this pull down assay was determined by immunoblotting with anti-HA antibodies (lower panel).

View larger version (10K): [in a new window]   Fig. 11. A model for transducing the Wnt signal by Frizzled and LRP (see Discussion).