Fig. 3.

PTH promotes the association of LRP6 and Gα_s. (A) Gα_s interacts with the cytoplasmic domain of LRP5 (LRP5C) and LRP6 (LRP6C). GST, GST-LRP5C, or GST-LRP6C was incubated with HEK 293 cell lysates. Bound Gα_s was detected by Western blotting analysis with an antibody against Gα_s (upper panel). Gα_s in cell lysates was detected by Western blotting analysis with an antibody against Gα_s (middle panel). The GST proteins were visualized by Coomassie brilliant blue staining (lower panel). (B) PTH does not affect the interaction between Gα_s and LRP5. HEK 293 cells were transfected with plasmid encoding HA-tagged LRP5, deprived of serum for 14 hours to avoid protein phosphorylation by serum-derived hormones or growth factors, and treated with PTH(1–34) (50 nM). LRP5-associated Gα_s was detected by Western blotting analysis of samples that were subjected to immunoprecipitation (IP) with an antibody against HA. Immunoprecipitation with a mouse preimmune antibody (Pre) was also performed as a negative control. WCL, whole-cell lysate. (C) PTH increases the extent of the interaction between Gα_s and LRP6. HEK 293 cells were transfected with plasmid encoding VSVG-tagged LRP6, deprived of serum for 14 hours, and treated with PTH(1–34) (50 nM). LRP6-associated Gα_s was detected by Western blotting analysis of samples immunoprecipitated with antibody against VSVG. (D) The interactions of LRP6 with Gα_s and Gβ₁ are blocked by AlF₄^–. HEK 293 cells were transfected with plasmid encoding VSVG-tagged LRP6 together with plasmids encoding FLAG-tagged Gα_s, HA-tagged Gβ₁, and Gγ₂ and were lysed in lysis buffer containing AlF₄^– (100 μM AlCl₃, 10 mM NaF). LRP6-associated Gα_s and Gβγ were detected by Western blotting analysis of samples immunoprecipitated with antibody against VSVG. (E) LRP6 does not bind to Gα_o or Gα_i2. HEK 293 cells were transfected with plasmid encoding VSVG-tagged LRP6. LRP6-associated Gα_s, Gα_o, and Gα_i2 were detected by Western blotting analysis of samples immunoprecipitated with antibodies against Gα_s, Gα_o, and Gα_i2. Immunoprecipitation with a mouse preimmune antibody (Pre) was also performed as a negative control. (F) The PTH-induced interaction between Gα_s and LRP6 was not affected by knockdown of axin. HEK 293 cells were transfected with scrambled control siRNA or siRNA specific for axin together with the indicated plasmids and treated with PTH(1–34) (50 nM). LRP6-associated Gα_s was detected by Western blotting analysis of samples immunoprecipitated with antibody against VSVG.