NHERF2 Specifically Interacts with LPA₂ Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-β3 Activation

The interaction between LPA₂ and NHERF2 requires both the second PDZ domain of NHERF2 and the C-terminal PDZ-binding motif of LPA₂. (A) Six His-NHERF2 (WT) and its fragments (P1, the first PDZ domain; P2, the second PDZ domain; and C, the C-terminal fragment of NHERF2) were immobilized on Ni²⁺-affinity resin and subsequently incubated with the GST-LPA₂-CT. The amounts of GST-LPA₂-CT bound to the immobilized proteins were measured by SDS-PAGE, followed by Western blot analysis with anti-GST antibody. (B) Flag-LPA₂ was cotransfected with either wild-type NHERF2 (NHERF2 WT) or the second PDZ-deleted form (NHERF2 ΔPDZ2), as indicated. The cell lysates were immunoprecipitated with anti-Flag antibody. The resulting precipitates were analyzed by Western blot analysis with the anti-NHERF2 antibody (left, upper) to detect the precipitated NHERF2 WT or NHERF2 ΔPDZ2. The same blot was reprobed with anti-Flag antibody (left, lower) to show the amount of precipitated LPA₂. The expression levels of NHERF2 WT and of NHERF2 ΔPDZ2 within the total cell lysates are also shown (right). (C) NHERF2 was cotransfected with wild-type Flag-LPA₂ (Flag-LPA₂ WT) or its mutant form (Flag-LPA₂ Mut) into COS-7 cells, as indicated. The transfected cells were serum deprived for 24 h prior to treatment with 1 μM LPA in 0.1% BSA for the indicated times. After being washed with ice-cold phosphate-buffered saline (PBS), the cells were lysed and immunoprecipitated with anti-Flag antibody. The resulting precipitates (top, middle) and total lysates (bottom) were analyzed by Western blot analysis with either anti-NHERF2 antibody or anti-Flag antibody, as indicated. These results are representative of three independent experiments.

NHERF2 increases LPA-induced IP generation and subsequent Ca²⁺ mobilization in Rat-1 cells. (A) Rat-1 cells were stably transfected with vector alone (Vector), NHERF2 WT, and the second PDZ domain-deleted mutant (NHERF2 ΔPDZ2), individually. The NHERF2 (upper) and NHERF1 (lower) expression levels were determined by Western blot analysis with the specific antibodies against each isoform of NHERF, as indicated. (B) Rat-1 cell lines were split at a density of 3 × 10⁵ cells/well, which were then loaded with 1 μCi of [³H]inositol/ml for 24 h at 37°C and stimulated with LPA at the indicated concentrations. The generation of [³H]IPs was determined as described in Materials and Methods. The data are presented as means ± the standard errors (SE) of three separate experiments performed in duplicate. (C) LPA-induced Ca²⁺ mobilization in Rat-1 cell lines. Rat-1 cells were loaded with 1 μM Fluo-3/AM for 30 min at 37°C, washed twice with prewarmed Ca²⁺-free Locke's solution, and then stabilized for 5 min under a microscope. Fluorescence confocal microscopic images of the cytosolic Ca²⁺ mobilization of Rat-1 cell lines were captured sequentially before (−) and after (+) the addition of 10 μM LPA (magnification, ×200) and in the presence or absence of 5 μM U73122, as indicated. Ca²⁺-sensitive fluorescence images of each of the Rat-1 cells were subjected to densitometry over time (right). The results shown are those of a single experiment that is representative of three experiments performed with independent preparations.

NHERF2 is required for the efficient LPA₂-mediated PLC-β activation in HeLa cells. (A) Gene silencing of either the NHERF1 or NHERF2 with siRNAs. HeLa cells were transfected with siRNAs directed against NHERF1 or NHERF2 or with a control siRNA (Luciferase GL3), as described in Materials and Methods. One day after the siRNA transfection, the cells were split at a density of 2 × 10⁵ cells/well. After 24 h, the cells were infected with the recombinant adenovirus expressing Flag-LPA₂. After 24 h postinfection, the HeLa cells were lysed and analyzed by Western blot with the specific antibodies as indicated. These results were representative of three independent experiments. (B) Effects of NHERF2 gene silencing on LPA₂-mediated PLC-β activation in HeLa cells. After 12 h postinfection, the infected cells were labeled with 1 μCi of [³H]inositol/ml for 12 h at 37°C, treated with either 0.1% BSA only (−) or 1 μM LPA-BSA conjugates (+), and then analyzed for [³H]IPs as described in Materials and Methods. The data are presented as means ± the SE of three separate experiments performed in duplicate. (C) The effects of a mutation at the C-terminal PDZ-binding motif of LPA₂ on LPA-induced IPs generation in HeLa cells. HeLa cells were transfected with the indicated constructs (Vector, control vector; LPA₂ WT, wild-type LPA₂; LPA₂ Mut, mutant LPA₂). At 24 h posttransfection, the cells were labeled with [³H]inositol for 12 h and treated with either 0.1% BSA only (−) or 1 μM LPA-BSA conjugates (+). The generation of IPs was analyzed as described previously. The results are presented as means ± SE obtained from three separate experiments performed in duplicate. In addition, the expressions of Flag-LPA₂ WT and Flag-LPA₂ Mut were analyzed by immunoprecipitation and subsequent Western blot analysis with anti-Flag antibody (inside the graph).

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.

NHERF2 potentiates LPA-induced ERK activation and COX-2 induction. (A) Rat-1/vector and Rat-1/NHERF2 WT cells were serum deprived for 24 h and then treated with the LPA-BSA conjugates at the indicated concentrations for 10 min. Independently, Rat-1/NHERF2 WT cells were pretreated with dimethyl sulfoxide (−) or 5 μM U73122 (+) for 20 min and then treated with 1 μM LPA in 0.1% BSA for 10 min. The cell lysates (20 μg) were subjected to SDS-PAGE and analyzed with either anti-pERK antibody or anti-ERK antibody. The results shown are representative of three independent experiments. (B) Serum-deprived Rat-1 cell lines (vector, NHERF2 WT, and NHERF2 ΔPDZ2) were treated with 5 μM LPA or with 50 ng of EGF/ml for 10 h at 37°C. In an independent experiment, Rat-1/NHERF2 WT cells were pretreated with dimethyl sulfoxide (−) or 30 μM PD98059 (+) for 30 min and then treated with 5 μM LPA for 10 h at 37°C. The cell lysates (20 μg) were subjected to SDS-PAGE and analyzed by Western blot with anti-COX-2 antibody or anti-actin antibody as indicated. The results shown are those of a single experiment, which is representative of three experiments performed with independent preparations.