Figure 3.

DEP domain of Dishevelled (Dvl) is required for degradation of Dishevelled by Gβγ. (A) Dishevelled interacts with all isoforms of Gβ except for Gβ₃. HA-tagged human Dv-1 was co-transfected with all isoforms of Gβ into HEK293T cells. Immunoprecipitation followed by western blotting was performed. (B) The level of DEP domain deleted Dishevelled was not reduced by Gβ₂γ₂. Various deletion constructs of Dishevelled were co-transfected with Gβ₂γ₂ into HEK293T cells and western blotting was performed. (C) The data from luciferase assay confirm the result obtained in (B). Luciferase assay was performed using superTOPFlash and pTK-renilla luciferase after co-transfection of various deletion constructs of Dishevelled and Gβ₂γ₂. (D) DEP domain of Dishevelled is required for interaction with Gβ₂γ₂. Various deletion constructs of Dishevelled with Gβ₂γ₂ were co-transfected into HEK293T cells and immunoprecipitation followed by western blotting was performed. Arrow indicates ΔDEP-Dishevelled. (E) Membrane localization of ΔDEP-Dishevelled via myristoylation/ palmitoylation signal is not sufficient for the down-regulation of Dishevelled by Gβ₂γ₂. ΔDEP-Dishevelled and myr-ΔDEP, which has a myristoylation/ palmitoylation signal sequence to the N-terminal of ΔDEP-Dishevelled, were transfected alone or with Gβ₂γ₂ into HEK293T cells. After fractionation, western blotting was performed using the membrane fraction (left panel). Indirect immunofluorescence analysis with anti-HA antibody shows myr-ΔDEP-Dishevelled is highly localized in plasma membrane (right panel). (F) Luciferase assay using superTOPFlash was conducted to confirm the result obtained in (E).