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CELL GROWTH AND DEVELOPMENT

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

Yong-Seok Oh, Nam Won Jo, Jung Woong Choi, Hyeon Soo Kim, Sang-Won Seo, Kyung-Ok Kang, Jong-Ik Hwang, Kyun Heo, Sun-Hee Kim, Yun-Hee Kim, In-Hoo Kim, Jae Ho Kim, Yoshiko Banno, Sung Ho Ryu, Pann-Ghill Suh
Yong-Seok Oh
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Nam Won Jo
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Jung Woong Choi
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Hyeon Soo Kim
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Sang-Won Seo
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Kyung-Ok Kang
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Jong-Ik Hwang
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Kyun Heo
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Sun-Hee Kim
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Yun-Hee Kim
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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In-Hoo Kim
2Division of Basic Sciences, National Cancer Center, Ilsan-Gu, Goyang-Si, Gyeonggi-do 411-764
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Jae Ho Kim
3Department of Physiology, College of Medicine, Pusan National University, Suh-Gu, Busan 620-739, Republic of Korea
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Yoshiko Banno
4Department of Biochemistry and Internal Medicine, Gifu University School of Medicine, Gifu 500-8705, Japan
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Sung Ho Ryu
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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Pann-Ghill Suh
1Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784
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  • For correspondence: pgs@postech.ac.kr
DOI: 10.1128/MCB.24.11.5069-5079.2004
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    FIG. 1.

    LPA2 interacts with NHERF2 in an isoform-specific manner. (A) NHERF2 binds to the CT of LPA2. The GST-fused CTs of three LPA receptor isoforms (GST-LPA1-CT, GST-LPA2-CT, and GST-LPA3-CT) and GST (4 μg) were immobilized onto glutathione beads, and incubated with lysates (3 mg) from rat kidney. The resulting precipitates were subjected to SDS-PAGE and analyzed by Western blot analysis with anti-NHERF2 antibody (upper panel) or by Ponceau S staining (lower panel). (B) Flag-LPA2, but not Flag-LPA1, coimmunoprecipitates NHERF2. Flag-LPA1 or Flag-LPA2 was cotransfected with NHERF2 into COS-7 cells, as indicated. The cell lysates were immunoprecipitated with anti-Flag antibody. The resulting precipitates were analyzed by Western blot analysis with anti-NHERF2 antibody (upper panel). The same blot was reprobed with anti-Flag antibody to show the amount of precipitated LPA receptors (middle panel). The NHERF2 expression levels in the total cell lysates are also shown (bottom panel). (C) Preferential interaction of GST-fused NHERF2 to LPA2-CT. MBP-fused LPA2-CT (MBP-LPA2-CT) was incubated with GST or GST-fused NHERF isoforms (GST-NHERF1 and GST-NHERF2), which were immobilized on GSH beads. The amounts of MBP-LPA2-CT bound to the GST-fused proteins were measured by SDS-PAGE and after Western blot analysis with anti-MBP antibody (upper) or Ponceau S staining (lower). (D) HA-NHERF2, but not HA-NHERF1, is coimmunoprecipitated with LPA2. Either HA-NHERF1 or HA-NHERF2 was transiently coexpressed with Flag-LPA2 in COS-7 cells. The cell lysates were immunoprecipitated with anti-Flag antibody. After being washed, the resulting precipitates and total lysates were subjected to SDS-PAGE after immunoblot analysis with anti-HA antibody or anti-Flag antibody, as indicated. These results are representative of at least two independent experiments.

  • FIG. 2.
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    FIG. 2.

    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.

  • FIG. 3.
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    FIG. 3.

    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.

  • FIG. 4.
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    FIG. 4.

    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).

  • FIG. 5.
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    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.

  • FIG. 6.
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    FIG. 6.

    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.

  • FIG. 7.
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    FIG. 7.

    Schematic view of the NHERF2-dependent regulation of LPA2-mediated PLC-β3 signaling. Prior to agonist stimulation, both LPA2 and PLC-β3 directly interact with NHERF2, which is localized to the plasma membrane. NHERF2, which is multimerized by a PDZ-PDZ interaction, clusters LPA2 and PLC-β3 in close proximity, thereby creating spatially compact signaling complexes beneath the plasma membrane. Consequently, the LPA2-NHERF2-PLC-β3 complex enables LPA2 to transduce its signal to PLC-β3 with efficiency and specificity.

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NHERF2 Specifically Interacts with LPA2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-β3 Activation
Yong-Seok Oh, Nam Won Jo, Jung Woong Choi, Hyeon Soo Kim, Sang-Won Seo, Kyung-Ok Kang, Jong-Ik Hwang, Kyun Heo, Sun-Hee Kim, Yun-Hee Kim, In-Hoo Kim, Jae Ho Kim, Yoshiko Banno, Sung Ho Ryu, Pann-Ghill Suh
Molecular and Cellular Biology May 2004, 24 (11) 5069-5079; DOI: 10.1128/MCB.24.11.5069-5079.2004

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NHERF2 Specifically Interacts with LPA2 Receptor and Defines the Specificity and Efficiency of Receptor-Mediated Phospholipase C-β3 Activation
Yong-Seok Oh, Nam Won Jo, Jung Woong Choi, Hyeon Soo Kim, Sang-Won Seo, Kyung-Ok Kang, Jong-Ik Hwang, Kyun Heo, Sun-Hee Kim, Yun-Hee Kim, In-Hoo Kim, Jae Ho Kim, Yoshiko Banno, Sung Ho Ryu, Pann-Ghill Suh
Molecular and Cellular Biology May 2004, 24 (11) 5069-5079; DOI: 10.1128/MCB.24.11.5069-5079.2004
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KEYWORDS

Cytoskeletal Proteins
Isoenzymes
Receptors, G-Protein-Coupled
Type C Phospholipases

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