FIG 4.

PCP pathway-specific DEP domain functional units are dispensable for the rescue of the cellular response to Wnt3a. (A) Schematic representation of the DVL1 DEP domain mutants used for the rescue experiments. The alignment of the sequences of individual human DVL isoforms with the Drosophila melanogaster (DROME) Dsh sequence and a summary of previously published Wnt/β-catenin or PCP pathways phenotypes of individual mutants (9, 10, 12, 19–21) are provided. All mutants were generated in the DVL1(1–502) mutant background. (B) (Left) Relocalization of individual DVL mutants to the membrane after cotransfection with Frizzled 5 (FZD5). (Right) Three categories of DVL1 localization after FZD5 coexpression were established: strong, weak, and no membrane localization of DVL1. One hundred cells were counted per condition (n = 3). All mutants showed reduced membrane recruitment compared to that for the control, the DVL1(1–502) mutant. Bar, 7.5 μm. (C to E) HEK293 cells were transfected as indicated and analyzed by the TopFlash reporter assay (C, D) (n = 4 for panel C, n = 3 for panel D) or WB (Ei, blots; Eii, quantification; n = 3). In the assay whose results are presented in panels D and E, cells were pretreated with R-spondin 1 (R-SPO1) and the DIDI mutant was transfected in a 100-ng dose so that its level equaled the protein levels of the other DVL1 mutants. All DVL1 DEP domain mutants except for the ΔLPDSG and DIDI mutants rescued the response to Wnt3a to an extent comparable to that for the DVL1(1–502) mutant. (F and G) Analysis of DEP mutants in DVL KO T-REx cells. All mutants except the ΔLPDSG and DIDI mutants were able to restore Wnt/β-catenin-dependent transcription, as analyzed by the TopFlash reporter assay (n = 4) (F) and S1490-LRP6 phosphorylation (G). Cells for which the results are shown in panels F and G were pretreated with R-spondin1 (R-SPO1). (H) The pattern of localization of full-length (FL) DVL1 and the individual DVL mutants resembles that of the DVL1(1–502) mutant. Colocalization with axin1 was determined by immunocytofluorescence. All mutants except DIDI colocalized with axin1. (Top) DVL1 only (green); (bottom) DVL1 (green) and axin1 (red). ctrl, control. Bars, 7.5 μM. (I) Coimmunoprecipitation of DVL1 mutants in the pulldown of endogenous axin1 and CK1ε. Cells were treated as indicated; unspecific IgG was used as a negative control. Analysis for statistically significant differences was performed by paired Student's t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).