The polycystin complex mediates Wnt/Ca2+ signalling

Abstract

WNT ligands induce Ca2+ signalling on target cells. PKD1 (polycystin 1) is considered an orphan, atypical G-protein-coupled receptor complexed with TRPP2 (polycystin 2 or PKD2), a Ca2+-permeable ion channel. Inactivating mutations in their genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases. Here, we show that WNTs bind to the extracellular domain of PKD1 and induce whole-cell currents and Ca2+ influx dependent on TRPP2. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or reduce TRPP2 channel activity suppress activation by WNTs. Pkd2−/− fibroblasts lack WNT-induced Ca2+ currents and are unable to polarize during directed cell migration. In Xenopus embryos, pkd1, Dishevelled 2 (dvl2) and wnt9a act within the same pathway to preserve normal tubulogenesis. These data define PKD1 as a WNT (co)receptor and implicate defective WNT/Ca2+ signalling as one of the causes of ADPKD.

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Figure 1: WNT9B binds to the extracellular domain of PKD1.
Figure 2: WNT9B activates PKD1/TRPP2.
Figure 3: Pathogenic mutations in PKD1 or TRPP2 suppress activation by WNT9B.
Figure 4: TRPP2 mediates WNT9B-induced whole-cell currents in MEFs.
Figure 5: TRPP2 mediates WNT3A-induced whole-cell currents in MEFs.
Figure 6: Interaction of DVL1 and DVL2 with PKD1 in MEFs and transiently transfected HEK293T cells.
Figure 7: TRPP2 mediates WNT9B-induced cell migration.
Figure 8: Cooperativity between pkd1 and dvl2 in Xenopus.

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Acknowledgements

We would like to thank A. Pioszak, R. Janknecht, M. Ahmad, D. Sherry and L. Rothblum for comments on the manuscript, J. Yang for mouse Flag-tagged PKD1, F. Qian for human HA-tagged PKD1 and E4 mouse monoclonal antibody against PKD1, F. Cong for ZNRF3 plasmid, and E. Petsouki and V. Gerakopoulos in the Tsiokas laboratory for help with confocal imaging and immunoblotting in S2 cells. This work was supported by grant number 81270098 from NSFC (H.N.), DK080745 from NIH (O.W.), DK59599 from NIH (L.T.), Oklahoma Center for the Advancement of Science and Technology (L.T.), Oklahoma Center for Adult Stem Cell Research (L.T.), and the John S. Gammill Endowed Chair in Polycystic Kidney Disease (L.T.). These studies used reagents provided by the NIDDK-sponsored Baltimore Polycystic Kidney Disease Research and Clinical Core Center, P30DK090868.

Author information

Affiliations

Authors

Contributions

S.K. performed protein–protein interaction and cell migration assays; H.N and V.N. performed electrophysiological experiments; U.T and O.W. performed experiments in Xenopus embryos; P.O. and T.W. provided mouse embryonic fibroblasts from wild-type and Pkd1- and Pkd2-null mice; C.B. performed Ca2+ imaging experiments; J.K. and D. M. analysed expression profile of FZDs, DVLs and other WNT (co)receptors in wild-type and Pkd2-null cells. S.K. and H.N. contributed equally to this study. L.T. supervised the study, analysed data and wrote the paper with the help of S.K. and H.N. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Leonidas Tsiokas.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Several WNTs bind to the extracellular domains of PKD1.

(a) HEK293T cells were co-transfected with indicated plasmids. Flag-tagged proteins were immunoprecipiated with rabbit α-Flag and immunoblotted with α-WNT5A (upper panel) or a combination of mouse α-Flag and mouse α-PKD1 (7e12) (middle panel). (b) HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-WNT5A (upper panel) or α-Fc (middle panel). (c) HEK293 cells stably expressing Fc, LRR-WSC-Fc, LDL-A-Fc, were co-cultured with HEK293 cells stably transfected with myc-tagged WNT5A for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-WNT5A (upper panel) or α-Fc (middle panel). Red or blue arrow heads indicate Fc fusions or WNT5A, respectively. (d) HEK293T cells were transiently transfected with F-BAP (lane 1) or full length F-PKD1 and TRPP2 (lane 2). Cells were separately transfected with WNT5A and co-cultured with F-BAP- or F-PKD1/TRPP2-transfected cells for 48 h. Flag-tagged proteins were immunoprecipitated with rabbit α-Flag and immunoblotted with α-WNT5A (lanes 1 and 2). Input WNT5A is shown in lanes 3 and 4. (e) Interaction of WNT4 with the LRR-WSC and LDL-A domains of PKD1. HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-WNT4 (upper panel) or α-Fc (middle panel). (f) Interaction of Flag-tagged WNT3A with the LRR-WSC domain of PKD1. HEK293T cells were transfected with indicated plasmids and conditioned for 48 h. Fc fusions were captured from conditioned media and immunoblotted with α-Flag (upper panel) or α-Fc (middle panel). (g) Myc-tagged human TRPP2 was purified from transiently transfected HEK293T cells using α-Myc and incubated (30 ng) with 1 μg ml−1 of purified WNT9B. Immobilized myc–TRPP2 was immunoblotted with α-WNT9B. Experiments shown in (a) and (e) were done once. Experiments shown in (d) and (g) were successfully repeated twice. Experiments shown in (b), (c), and (f) were repeated 3 times. Uncropped western blots are provided in Supplementary Fig. 8.

Supplementary Figure 2 Compromised cell surface expression of PKD1S99I.

Expression levels of several pathogenic mutants of PKD1 in transiently transfected HEK293T cells. Cells were transfected with human PKD1 cDNA tagged at its C-terminus with 3xHA (hPKD1-HA, lane 1), hPKD1C47S-HA (lane 2), hPKD1S75F-HA (lane 3), hPKD1S99I-HA (lane 4), hPKD1W139C-HA (lane 5), or pcDNA3 (mock, lane 6). HA-tagged wild type or mutant PKD1 was immunoprecipitated with a mouse α-HA and complexes were immunoblotted with a rabbit α-HA. (b) Compromised cell surface expression of PKD1S99I. HEK293T cells were transfected with (GFP, mock, lane 1), wild type PKD1 (PKD1WT) plus TRPP2 (lane 2) or PKD1S99I plus TRPP2 (lane 3). Cells were labeled with cell impermeant biotin, lysed, and biotinylated proteins were captured with streptavidin beads. Biotinylated proteins were immunoblotted by α-PKD1 (7e12, upper panel). Expression levels of PKD1 constructs in lysates are shown in lower panel. (c) Cell surface expression of TRPP2 is not affected by PKD1S99I. Biotinylated proteins (prepared as described in b) were immunoblotted by α-TRPP2 (G20, 1:500, Santa Cruz Biotechnology) (upper panel). Expression levels of TRPP2 in total lysates are shown in lower panel. TRPP2 migrated as a high molecular weight species (aggregated form) and a monomeric species (100 kDa), because lysates were boiled for 5 min, before loading onto SDS PAGE. (d) Pkd1+/+ or Pkd1−/− MEFs were labeled with cell impermeant biotin and biotinylated proteins were immunoblotted with α-TRPP2 (G20, 1:500, lanes 2 and 3). Expression levels of TRPP2 in Pkd2−/−, Pkd1+/+, and Pkd1−/− cells are shown in lanes 1, 4, and 5, respectively. Note that a non-specific band detected by α-TRPP2 (indicated by an asterisk) is only present in total cell lysates, but not in the biotinylated protein pool. Experiments were successfully repeated 3 times. Uncropped western blots are provided in Supplementary Fig. 8.

Supplementary Figure 3 Expression levels of transfected proteins in CHO-K1 cells and biochemical characterization of TRPP2Kv1.3 in HEK293T cells.

(a) CHO-K1 cells were transiently transfected with (pCDNA3, lane 1), wild-type HA-tagged PKD1 plus wild type TRPP2 (lane 2), wild type HA-PKD1 plus HA-tagged TRPP2D511V (lane 3), wild type HA-PKD1 plus TRPP2Kv1.3 (lane 4), wild type HA-PKD1 plus wild type TRPP2 plus HA-tagged ZNRF3 (lane 5), or HA-PKD1D99I plus wild type TRPP2 (lane 6). PKD1 was immunoprecipitated from lysates pooled from three plates using mouse monoclonal 7e12 and blotted with the same antibody (upper panel). TRPP2 constructs were detected in straight lysates using goat α-TRPP2 (middle panel) and the blot was sequentially probed with α-HA to detect ZNRF3 (lower panel). (b) Results from one representative out of three independent experiments all done in sextuplicates showing the effectiveness of ZNRF3 in WNT3A-induced canonical signaling in CHO-K1 cells. Statistical significance was determined One Way ANOVA Neuman-Keuls posthoc test, t-test, indicates P < 0.001. Data are shown as mean ± s.e.m. Statistics source data for three independent experiments are available in Supplementary Table 3. (c) TRPP2Kv1.3 homodimerizes and interacts with wild type TRPP2. HEK293T cells were transiently co-transfected with HA-tagged TRPP2 (HA-TRPP2) and myc-tagged TRPC3 (TRPC3-myc, negative control, lane 1), HA-TRPP2 and TRPP2Kv1.3-myc (lane 2), HA-TRPP2 and TRPP2-myc (positive control, lane 3), HA-TRPP2Kv1.3 and TRPC3-myc (negative control, lane 4), HA-TRPP2Kv1.3 and TRPP2Kv1.3-myc (lane 5), or HA-TRPP2Kv1.3 and TRPP2-myc (lane 6). HA-tagged proteins were immunoprecipitated with mouse α-HA and immunoblotted with rabbit α-myc (upper panel) or rabbit α-HA (lower panel). Expression levels of myc-tagged proteins are shown in middle panel. (d) TRPP2Kv1.3 interacts with PKD1. HEK293T cells were transfected with indicated plasmids and F-PKD1 was immunoprecipitated with mouse monoclonal α-Flag. Imunocomplexes were immunoblotted with rabbit polyclonal α-myc (upper panel) or rabbit α-Flag (middle panel). Expression levels of TRPP2-myc or TRPP2Kv1.3-myc are shown in lower panel. An irrelevant lane shown by a dotted line was deleted between lanes 3 and 4. Experiments were successfully repeated 3 times, except in (a), where experiments were done once. Uncropped original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 4 Functional expression of human PKD1 and mouse TRPP2 in Drosophila S2 cells.

(a) WNT9B binds to the surface of Drosophila S2 cells transfected with human PKD1 and mouse TRPP2. Confocal z-sections of non-permeabilized, untrasfected S2 cells incubated with 1 μg ml−1 of purified WNT9B, PKD1/TRPP2-transfected but not incubated with purified WNT9B, or PKD1/TRPP2-transfected incubated with 1 μg ml−1 of WNT9B, labeled with goat α-WNT9B detected with donkey α-goat coupled to Alexa-488. Insets show additional images of WNT9B-labeled cells from other fields. Scale bar, 5 μm (b) Expression levels of PKD1, TRPP2, or CD8α in transiently transfected S2 cells 48 h after the addition of CuSO4 (700 μM) in the cultures to induce expression of PKD1, TRPP2 and CD8α. Experiments were successfully repeated three times (a and b). (c) Time course, step currents and IV curves of WNT9B-induced whole cell currents in untrasfected (n = 9 cells pooled from 4 independent experiments) or PKD1/TRPP2-transfected S2 (n = 10 cells experiments) cells 48 h after the addition of CuSO4. Step-currents represent steady-state currents at 5 min time point just before the addition of La3+. IV curves were taken before the addition of 500 ng ml−1 WNT9B (black lines) and 4 min after the addition of WNT9B just before the addition of La3+ (red lines). Statistical tests were performed using paired Student’s t-test, P < 0.05, P < 0.01. Data are shown as mean ± s.e.m.

Supplementary Figure 5 Status of canonical WNT/β-catenin and MAPK pathways and expression levels of Fzd and Ryk mRNAs and ROR2 in Pkd2+/+ and Pkd2−/− MEFs.

Wild type or Pkd2-null MEFs were stimulated for 20 min with WNT9B (500 ng ml−1) and phosphorylation and total levels of β-catenin (a), P38 MAPK (b) or LRP6 (c) were determined by immunoblotting. Experiments were done once. (d) Expression levels of DVL1 and DVL2 in Pkd1−/−, Pkd2−/− cells, and wild type MEFs derived from littermate control animals (Pkd1+/+or Pkd2+/+). Experiments were done 3 times. (e) Relative expression of Fzd1, Fzd2, Fzd6, Fzd7, Fzd8, and Ryk genes in Pkd2+/+ and Pkd2−/− cells determined by real time RT-PCR. Experiments were done three times in triplicates and data are shown as mean ± s.e.m. (f) Expression levels of ROR2 in both cell types determined by immunoblotting. Experiment was done once. (g) Status of canonical Wnt pathway in Pkd2+/+ and Pkd2−/− cells using a TCF-based transcription assay (TOP-Flash assay). (h) Suppression of canonical Wnt pathway activity by ZNRF3 in wild type (Pkd2+/+) MEFs. (i) WNT9B did not activate the canonical Wnt pathway in wild type MEFs. Data from one representative out of three independent experiments all done in sextuplicates are shown in (gi). Statistical analysis was performed using one-way ANOVA followed by Neuman-Keuls post hoc test. N.S means non-significant, indicates P < 0.001. Data are shown as mean ± s.e.m. Statistics source data for all three independent experiments are available in Supplementary Table 3. Uncropped original scans of blots are shown in Supplementary Fig. 8.

Supplementary Figure 6 ATP induces similar currents in Pkd2+/+ and Pkd2−/− MEFs and WNT9B does not induce Ca2+ release in these cells.

Time course (a,d), step-currents (b,e), and IV curves (c,f) of ATP (100 μM)—induced whole cell currents in 100 nM of intracellular Ca2+ concentration in Pkd2+/+ (n = 7 cells pooled from 3 independent experiments) and Pkd2−/− MEFs (n = 9 cells pooled from 3 independent experiments). Step-currents represent maximum currents. IV curves were taken before the addition of ATP (black lines) and at maximum currents (red lines). Representative images of time courses (a,d) and step currents (b,e) are shown from 7, 9 cells, respectively. (g) Time course of WNT9B-induced Δ[Ca2+]i (shown as fluorescence ratio 340/380) in Pkd2+/+ (n = 47 pooled from 3 independent experiments) and Pkd2−/− cells (n = 37 cells pooled from 3 independent experiments) bathed in Ca2+ free extracellular solution (0.5 mM EGTA). (h) Summary data of ATP-induced Ca2+ release transients (peak levels) in Pkd2+/+ and Pkd2−/− MEFs. Box and whiskers graph (line: median, box: distribution of 50% of values, whiskers: minimum to maximum). Statistical test was performed using unpaired Student’s t-test, N.S means non-significant. Data are shown as mean ± s.e.m.

Supplementary Figure 7 Characterization of a second antisense morpholino oligomer targeting Pkd1 mRNA splicing (Pkd1-sMO2).

Xenopus embryos were injected with 3.2 pMol Pkd1-sMO2 into all blastomeres at the 2- to 4-cell stage and cultured until sibling control embryos reached stage 35, 40, or 43. (ac) 3D reconstruction of pronephric kidneys by immunofluorescence staining using the 3G8 and 4A6 antibodies at stage 40. Representative images are shown in panels (a,b) and results are summarized in (c). (d) Quantification of embryos displaying edema at stage 43. (e) RT-PCR of Pkd1 mRNA in uninjected and Pkd1s-MO2-injected embryos at stage 35. PCR products representing correctly spliced mRNA (200 bp) in uninjected embryos or Pkd1 mRNA retaining the intron (850 bp) in Pkd1-sMO2-injected embryos are indicated. All experiments were performed using three independent biological replicates. The number of embryos analyzed is indicated above the bars in (c,d).

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Kim, S., Nie, H., Nesin, V. et al. The polycystin complex mediates Wnt/Ca2+ signalling. Nat Cell Biol 18, 752–764 (2016). https://doi.org/10.1038/ncb3363

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