Fig. 5.
Knockdown of LRP6 impairs the targeting of Gαs to the plasma membrane. (A) Localization of Gαs and Gβ1 to the plasma membrane is impaired by knockdown of LRP6. Stable HEK 293 cell lines expressing GFP-Gαs, GFP-Gβ1, or CFP-PTH1R were transfected with siRNAs specific for LRP5 or LRP6 or with siRNA containing a random sequence (Ctrl). Green fluorescence was visualized by fluorescence microscopy. (B) Cells from (A) in which Gαs, Gβ1, or PTH1R was localized primarily at the plasma membrane or in the cytosol were quantified. For each treatment, 100 cells on three different slides were analyzed. The number of cells in which fluorescent protein was localized to either the plasma membrane (black bars) or the cytosol (white bars) divided by the total number of cells was calculated and expressed as a percentage ± SD. *P < 0.05 relative to siRNA control cells. (C) Localization of Gαs to the plasma membrane is impaired by deletion of LRP6 in osteoblasts from mouse femur. Immunohistochemical analysis of the amount of Gαs in sections of femurs from 2-month-old male lrp6-floxed (WT) and lrp6-floxed;OC-Cre (KO) mice. Photos are representative of tissue sections stained for Gαs and counterstained with methyl green. Gαs-containing osteoblasts were stained brown. Three random high-power fields per specimen and a total of six specimens in each group were analyzed. The image shown is from one of these specimens. (D) Osteoblasts from (C) in which Gαs was localized to the plasma membrane or to the cytosol were counted in a blinded fashion from three random high-power fields per specimen in a 2-mm square, 1 mm distal to the lowest point of the growth plate in the secondary spongiosa; a total of six specimens in each group were used. The osteoblasts in which Gαs was localized either to the plasma membrane (black bars) or to the cytosol (white bars) are presented as a percentage of the total number of osteoblasts. *P < 0.05 relative to WT samples. (E and F) The interaction between Gαs and PTH1R is attenuated by knockdown of LRP6. HEK 293 cells were transfected with siRNA specific for GFP or LRP6. Reciprocal immunoprecipitations were performed to identify the interaction between PTH1R and Gαs. Data are representative of three experiments.