Figure 6. Loss of GRK2 impairs canonical Wnt signaling.

• A
  Control and GRK2 ^−/− fibroblasts were transfected with the TOPflash Firefly (F) luciferase vector together with a control Renilla (R) luciferase vector, and the effect of Wnt3A on TOPflash transcriptional activation was determined by dual‐luciferase assay. Note the impaired response to Wnt3A in GRK2 ^−/− cells. Mean ± SEM. Welch′s t‐test; number of biological experiments is indicated.
• B, C
  Low phosphorylation (p) of Wnt3A co‐receptor LRP6 as determined by Western blot with pS1490‐ and pT1572‐LRP6 specific antibodies, reduced LRP6 and DVL2 expression, and increased steady state phosphorylation of DVL2. A representative experiment of four is shown. #, nonspecific band. (C) Densitometry of Western blot results demonstrating decreased levels of LRP6 in GRK2 ^−/− cells, lower response of GRK2 ^−/− cells to Wnt3A by LRP6 phosphorylation at S1490, and tendency toward the similar defect at T1572. Densitometry also showed less of total DVL2 levels, and increased steady state pDVL2 in GRK2 ^−/− cells. Mean ± SEM. Mann–Whitney U‐test; number of biological experiments is indicated.
• D
  Loss of interaction between Frizzled 4 (FZD4) and ß‐Arrestin 2 (ARRB2) in GRK2 ^−/− fibroblasts. Proximity ligation assay (PLA) between expressed FZD4‐GFP and ARRB2‐Flag showed significantly reduced proximity events between the two proteins in GRK2 ^−/− cells. White dashed lines outline the transfected cells. Central band, median. Box, 1^st–3^rd quartile. Whiskers, 10–90% percentile. Mann–Whitney U‐test; number of biological experiments and the total numbers of analyzed cells are indicated. Scale bar, 10 μm.

Source data are available online for this figure.