β-Arrestin Interacts with the Beta/Gamma Subunits of Trimeric G-Proteins and Dishevelled in the Wnt/Ca²⁺ Pathway in Xenopus Gastrulation

<div><p>β-Catenin independent, non-canonical Wnt signaling pathways play a major role in the regulation of morphogenetic movements in vertebrates. The term non-canonical Wnt signaling comprises multiple, intracellularly divergent, Wnt-activated and β-Catenin independent signaling cascades including the Wnt/Planar Cell Polarity and the Wnt/Ca²⁺ cascades. Wnt/Planar Cell Polarity and Wnt/Ca²⁺ pathways share common effector proteins, including the Wnt ligand, Frizzled receptors and Dishevelled, with each other and with additional branches of Wnt signaling. Along with the aforementioned proteins, β-Arrestin has been identified as an essential effector protein in the Wnt/β-Catenin and the Wnt/Planar Cell Polarity pathway. Our results demonstrate that β-Arrestin is required in the Wnt/Ca²⁺ signaling cascade upstream of Protein Kinase C (PKC) and Ca²⁺/Calmodulin-dependent Protein Kinase II (CamKII). We have further characterized the role of β-Arrestin in this branch of non-canonical Wnt signaling by knock-down and rescue experiments in Xenopus embryo explants and analyzed protein-protein interactions in 293T cells. Functional interaction of β-Arrestin, the β subunit of trimeric G-proteins and Dishevelled is required to induce PKC activation and membrane translocation. In Xenopus gastrulation, β-Arrestin function in Wnt/Ca²⁺ signaling is essential for convergent extension movements. We further show that β-Arrestin physically interacts with the β subunit of trimeric G-proteins and Dishevelled, and that the interaction between β-Arrestin and Dishevelled is promoted by the beta/gamma subunits of trimeric G-proteins, indicating the formation of a multiprotein signaling complex.</p></div

Arrb2 functionally interacts with Dvl in Wnt/Ca²⁺ signaling.

<p>Xenopus embryos were injected with 500 pg <i>pkcα-gfp</i> RNA and co-injected as indicated above the images. Animal Caps were prepared at stage 10 and immunostained as indicated. Nuclei were stained with Hoechst 33258 (blue). Images show representative results from at least two independent experiments with a minimum of six Animal Caps per experiment. Scale bars: 50 µm. Fzd7 induced PKCα-GFP translocation (A) was impaired by a triple knock-down of Dvl1, Dvl2 and Dvl3 (B). (C) Co-expression of Arrb2 partially rescued PKCα-GFP translocation in the triple Dvl knock-down. (D) Triple Dvl knock-down inhibited elongation of Keller open face explants. Co-injection of PCKα or Arrb2 mRNA rescued the CE phenotype of triple Dvl morphant explants. The average percentage of explants showing full (75-100%, light grey), partial (25-50%, medium grey) or no elongation (<25%, dark grey) from at least three independent experiments are shown. Asterisks indicate statistically significant deviations in the percentage of fully elongated explants (* p>0.95, t-test).</p

Arrb2 physically interacts with Gβ and Dvl.

<p>Epitope-tagged proteins were overexpressed, immunoprecipitated and detected by Western Blotting as indicated. (A) Flag-Arrb2 was co-expressed with a combination of HA-Gβ1 and HA-Gγ2 to allow the formation of Gβγ heterodimers (Gβγ). Co-expression of Dvl1, Dvl2 or Dvl3 enhanced the interaction between Arrb2 and Gβ1 in co-immunoprecipitation experiments from HEK 293T cells. (B) Endogenous Gβ and Dvl2 were detected in immunoprecipitates of endogenous Arrb2 from unstimulated and Wnt-stimulated HEK 293T cells. (C) Binding of Dvl2 to Arrb2 was also observed in the absence of exogenous Gβ. Myc-Dvl2 co-precipitated equally well with Flag-Arrb2 when Gβ1 and Gγ2 were overexpressed (Gβγ) as in the presence of the Gβ-sequestering β-ARKct in HEK 293T cells. By contrast, binding of Gβ1 to Arrb2 was impaired by β-ARKct and partially restored by co-expression of Dvl2. (D) When myc-Dvl2 was precipitated, the amount of Flag-Arrb2 and that of Gβ1 that co-precipitated with Dvl2 was significantly reduced by the co-expression of β-ARKct.</p

STAT1 and ERK1/2 activation by FGFR3 mutants.

<p>(A) The N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants used in this study all cause FGFR3-skeletal dysplasias and signal through ERK MAP kinase in contrast to STAT1, that is activated mostly by the K650M and K650E-FGFR3 mutants. (B) According to the study compiling the clinical data of 591 patients suffering from FGFR3-related skeletal dysplasia <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003961#pone.0003961-PassosBueno1" target="_blank">[1]</a>, the STAT1-activating K650M and K650E account for as little as 4.9% of cases. It is therefore unlikely that activation of STAT1 plays a central role in FGFR3-related skeletal dysplasias as currently believed, but rather represents a signaling feature unique to small subset of patients carrying the K650M and K650E mutations.</p

The effect of FGFR3 mutants on RCS chondrocyte proliferation in context of STAT1 and ERK1/2 activation.

<p>(A) RCS chondrocytes transfected with vectors expressing wild-type FGFR3, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650E and K650M), and kinase-inactive mutant K508M were grown for 48 hours and analyzed for the indicated molecules by western blotting. Note differential STAT1 and ERK activation by the activating FGFR3 mutants. Cells transfected with empty plasmid (pcDNA3) serve as negative control for transfection. (B) RCS chondrocytes were transfected as described in (A), grown for 72 hours and counted. Note the inhibition of RCS growth by wild-type FGFR3 as well as the activating mutants, as compared to cells transfected either by kinase-inactive K508M-FGFR3 or an empty plasmid. The data represent an average from four individually transfected wells with indicated standard deviation. The cell count difference compared between cells transfected with wild-type FGFR3 and empty plasmid, as well as the cell count difference between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's <i>t</i>-test, <i>p</i><0.01). (C) The experiment shown on (B) was repeated five times to eliminate the variance associated with differential transfection efficiency. The differences in percentages of growth compared between cells transfected with wild-type FGFR3 and empty plasmid, and between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's <i>t</i>-test, <i>p</i><0.01).</p

STAT1 activation by FGFR3 mutants in a cell-free kinase assay.

<p>Full-length wild-type FGFR3 or its activating mutants N540K, G380R, R248C, Y373C, K650M and K650E were expressed in CHO cells, activated by brief FGF2 treatment and purified by immunoprecipitation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003961#s3" target="_blank">Materials and Methods</a>. Immunocomplexes were subjected to kinase assay with recombinant STAT1 as a substrate. Cells transfected with GFP vector serve as negative control for immunoprecipitation. Samples utilizing recombinant FGFR3 tyrosine kinase domain (TK) or those with omitted ATP serve as positive or negative control for kinase assay, respectively. Note that only K650M and K650E-FGFR3 mutants cause STAT1 phosphorylation, as evidenced by western blotting with antibody recognizing STAT1 only when phosphorylated at Y701 (P-Y701-STAT1). FGFR3 and STAT1 western blottings serve as controls for kinase or substrate quantity. Note the appearance of both immature and mature (glycosylated) FGFR3 forms expressed by CHO cells.</p

STAT5 activation by FGFR3 mutants.

<p>RCS chondrocytes transfected with vectors expressing wild-type FGFR3, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M and K650E), and kinase-inactive mutant K508M were grown for 24 hours and analyzed for the indicated molecules by western blotting. Note the significant STAT5(Y694) phosphorylation induced by K650M and K650E-FGFR3. The membrane used for P-STAT5-Y694 detection was reprobed with antibody recognizing STAT5 regardless of its phosphorylation. Arrow indicates P-STAT5(Y694) or STAT5 signal. FGFR3 and ACTIN western blottings serve as loading controls. Cells transfected by GFP vector serve as negative control for transfection.</p

BCRP and MRP1 inhibitors suppress efflux function in ascites treated ID8 cells.

<p>(<b>A</b>). ID8 cells from normal culture, ascites pre-treatment culture, or ascites (<i>in vivo</i> cells) were treated with or without inhibitors targeting MDR1, MRP1 or BCRP for 10 min and then incubated with eFFlux ID Green dye for 30 min. fluorescence intensity of each condition was measured by flow cytometry. Multidrug resistance activity factor (MAF) was calculated for each sample, and mean MAF (+/- SD from triplicate samples) is shown for each condition in B. MAF values falling below background are indicated by gray border. Statistically significant increases in MAF between ascites-treated and <i>in vivo</i> ascites cells (compared to untreated cells) are indicated. Error bars represent SDs from three independent experiments. * indicates p<0.05, Student’s t-test.</p

Ascites treatment increases expression of MDR1a/b and BCRP in ID8 cells.

<p>Total RNA was harvested from ID8 cells in normal culture as well as from ID8 cells pre-treated with ascites for 7 days. Expression levels of the indicated genes (relative to beta actin) were determined by real-time PCR(<b>A</b>). Fold increases in gene expression (ascites treated ID8 cells over normal ID8 cells) are shown in <b>B</b>. Three independent experiments were performed and error bar represents SD. * indicates p<0.05 and *** p<0.001, Student’s t-test.</p

Human ovarian cancer cells derived from ascites exhibit increased efflux compared to cells derived from the primary tumor.

<p><b>A and B.</b> Immortalized human ovarian cancer cells derived from patient ascites (HA1 and HA2 cells) or primary tumor site (TD cells) were incubated with eFFLux ID green dye for 40 min. Fluorescence intensity for each condition was measured by flow cytometry. Histogram of each cancer cell line derived from patient ascites (HA1 or HA2; blue) is overlaid with histogram of the primary tumor-derived line (TD; red)). <b>B.</b> Mean fluorescence intensity (MFI) was calculated for each line from three independent experiments. Fold decrease in geometric mean fluorescence intensity (GMFI) (relative to tumor-derived line) was calculated for each cell line in three independent experiments. Results are reported as the mean fold decrease from three independent trials treated (+/- SD). <b>C.</b> Total cellular extracts were obtained from a primary tumor-derived human ovarian cancer cell line (TD) and from each of two ascites-derived human ovarian cancer cell lines (HA1, HA2). Equivalent amounts of extracted proteins were subjected to SDS-PAGE and immunblotted with antibodies specific for MDR1, MRP1, BCRP, or beta actin, followed by the appropriate species IRdye-conjugated secondary antibody. Protein bands were detected by Odyssey infrared imaging. Protein bands were quantified using Image J software (NIH). Ratios of the indicated protein to beta actin are shown. <b>D and E.</b> Ascites-derived and tumor-derived human cell lines were incubated with or without inhibitors targeting MDR1, MRP or BCRP for 10 min and then incubated with eFFlux ID Green dye for 30 min. Samples were analyzed by flow cytometry. Histograms (+ inhibitor vs – inhibitor) were overlaid for each line (<b>D</b>). <b>E.</b> Multidrug resistance activity factor (MAF) was calculated for each sample, and mean MAF (+/- SD) from three independent experiments is shown for each condition in E. MAF values falling below background are indicated in gray. Statistically significant increases in MAF between ascites-treated and <i>in vivo</i> ascites cells (compared to untreated cells) are indicated. * indicates p<0.05. ** indicates p<.01, Student’s t-test.</p