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.