FIG. 5.

NHERF2 couples LPA₂ to PLC-β3 specifically. (A) LPA₂ forms a molecular complex with PLC-β3 in a NHERF2-dependent manner. The NHERF2 constructs (NHERF2, NHERF2 WT; NHERF2 ΔPDZ2, the second PDZ domain-deleted form) were cotransfected in combination with either Flag-PLC-β3 (left) or Flag-PLC-β1 (right) as indicated. After 2 days, COS-7 cells were washed and incubated with PBS containing 0.5 mM DSP (Pierce), which is a cell-permeable cross-linker, for 30 min. After the residual DSP was blocked with PBS containing 50 mM Tris buffer, the cleared lysates were subjected to a pull-down assay using GST-LPA₂-CT immobilized onto GSH beads. After a washing step, the resulting precipitates were subjected to SDS-PAGE and then analyzed by Western blot analysis with anti-Flag antibody (top) and anti-NHERF2 antibody (middle) or by Ponceau S staining (bottom) as indicated. The results are representative of three independent experiments. (B) Specific coimmunoprecipitation of PLC-β3 and not of PLC-β1 with Flag LPA₂. HeLa cells (at a density of 6 × 10⁶ cells/150-mm dish) were infected with either the recombinant Flag-LPA₂ adenovirus or control empty virus at an MOI of 10. At 24 h postinfection, the infected cells were incubated with PBS containing 0.5 mM DSP for 30 min. After the cell lysates were blocked with 50 mM Tris buffer, they were immunoprecipitated with anti-Flag antibody. After a washing step, the resulting precipitates were analyzed with the specific antibodies as indicated. (C) Gene silencing of PLC-β isoforms with specific siRNAs. HeLa cells were transfected with siRNAs directed against PLC-β1 or PLC-β3, along with control siRNA (Luciferase), as described in Materials and Methods. After 3 days, the HeLa cells were lysed and then analyzed by Western blot analysis with specific antibodies, as indicated. (D) PLC-β3 is functionally coupled to LPA₂. At 24 h after siRNA transfection, the cells were split at a density of 2 × 10⁵ cells/35-mm well and infected with recombinant adenovirus expressing Flag-LPA₂ for 4 h. At 12 h after viral infection, the infected cells were labeled with 1 μCi of [³H]inositol/ml for 12 h and treated with either 0.1% BSA only (−) or 1 μM LPA-BSA conjugates (+). The generation of [³H]IPs was analyzed as described in Materials and Methods. The results are presented as means ± the SE of three experiments performed in duplicate.