FIG. 3.

Ror2 is associated with Fz7 to mediate Wnt5a-induced Dvl2 polymerization and AP-1 activation. (A) Association of Ror2 with Fz7 requires the CRD but not intracellular region of Ror2. L cells were transfected with Fz7-CFP and Ror2-CFP (Ror2 WT or its derived mutants) as indicated. WCL or anti-HA immunoprecipitates were analyzed by immunoblotting with anti-GFP or anti-HA antibodies to detect the proteins indicated. (B) Wnt5a stimulation enhances the association of Ror2 with Fz7. L cells were transfected with Fz7-CFP and Ror2-HA as indicated and treated with the indicated concentration of Wnt5a for 15 min. WCL or anti-HA immunoprecipitates were analyzed as in panel A. (C) Association of Wnt5a with Fz7-Ror2 receptor complex. L cells were transfected with Fz7-CFP and Ror2-Flag as indicated. These cells were cocultured with L cells transfected with Wnt5a-HA (Wnt5a-HA/L; +) or mock plasmids (−). After 48 h in culture, WCL or anti-Flag immunoprecipitates were analyzed by immunoblotting to detect the proteins indicated. (D) Fz7 is required for Wnt5a-induced Dvl2 polymerization. Ror2/L were treated with either control or Fz7 siRNA (#1 and #2) for 36 h and then transfected with YFP-Dvl2. After 24 h in culture, expression levels of Fz7 or GAPDH were analyzed by RT-PCR (left). Extents of Wnt5a-induced polymerization of YFP-Dvl2 were examined as described in Fig. 2. The data are expressed as the mean percentages of cells with the indicated average number of the puncta per cell at 6, 12, and 17 min after stimulation (n = 15 [control], 9 [Fz7 siRNA #1], 4 [Fz7 siRNA #2]) (right). (E) Fz7 is required for Wnt5a-induced AP-1 activation. Ror2/L cells were treated with Fz7 siRNA for 36 h and then transfected with the AP-1 reporter plasmids. After serum starvation for 12 h, these cells were treated with Wnt5a or its vehicle for 24 h and subjected to luciferase assays.