Journal: Journal of Biological Chemistry

Article Title: Both LRP5 and LRP6 Receptors Are Required to Respond to Physiological Wnt Ligands in Mammary Epithelial Cells and Fibroblasts

doi: 10.1074/jbc.m112.362137

Figure Lengend Snippet: FIGURE 7. Evidence for LRP5-LRP6 complexes. A, the IFAST method for immunoprecipitation (36). Microchannel devices contain 3 wells connected by oil. Antibody, paramagnetic particles (PMPs), and cell lysate are combined in the input well (left-hand side), and complexes are pulled through into the wash well and the final elution well (right-hand side) by means of a magnet (according to the “Experimental Procedures”). B, specificity control. Cell lysate (300 g; wild type MEFs) was incubated with non-immune IgG and paramagnetic particles and processed. Lanes are labeled “Unbound” (input after pull-through of paramagnetic particles) and “Bound” (output after elution). C, evaluation of co-immunoprecipitation with LRP5. MEFs were treated with no ligand (UT), Wnt3a (20 ng/ml), Wnt9b (400 ng/ml), or Wnt5a (40 ng/ml) for 2 h, lysed, and immunoprecipitated with anti-LRP5 (1.2 g) and paramagnetic particles. The analysis of whole cell lysate (WCL; before IP) is shown on the left-hand side together with unbound and bound fractions. The efficiency of pull-through was calculated as described under “Experimental Procedures” (and presented here as a percentage). The same or parallel blots were probed for LRP6, phospho-LRP6, Axin1, and two specificity controls, EGF receptor (EGFR) and vinculin. D, evaluation of co-immunoprecipitation with Axin1. E, evaluation of co-immunoprecipitation with phospho-LRP. Results shown are representative of three separate MEF batches.

Article Snippet: Recombinant mouse Wnt3a (100 ng/ml unless otherwise indicated), Wnt5a (40 ng/ml), and Wnt9b (400 ng/ml) were purchased from R&D Systems.

Techniques: Immunoprecipitation, Control, Incubation, Labeling

Journal: Journal of Biological Chemistry

Article Title: Both LRP5 and LRP6 Receptors Are Required to Respond to Physiological Wnt Ligands in Mammary Epithelial Cells and Fibroblasts

doi: 10.1074/jbc.m112.362137

Figure Lengend Snippet: FIGURE 8. Model of LRP interactions. A and B, evidence for interactions derived from IFAST IP. Proteins associated into complexes are shown in the uninduced (basal level) MEFs (A) and for MEFs treated with Wnt3a (B). C, a model depicting LRP signaling activities in response to different Wnt ligands. Left-hand side, binding of Wnt1/Wnt9b ligands is not effective when LRP5 or LRP6 are present alone. Shown here is one potential explanation: the presence of an inhibitor (INH) in the adult mammary gland/MEF culture that blocks assembly of LRP-Fzd complexes by E1-E2 ligands. Under the same conditions, Wnt3a (shown here binding at a separate domain, E3-E4) can effectively signal via LRP6 (or LRP5; center). Signaling by the Wnt1 class of ligands requires complexation of LRP5 and LRP6 (right-hand side). This complex is proposed to be inhibitor-resistant and may have different co-receptor/signaling component associations.

Article Snippet: Recombinant mouse Wnt3a (100 ng/ml unless otherwise indicated), Wnt5a (40 ng/ml), and Wnt9b (400 ng/ml) were purchased from R&D Systems.

Techniques: Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) Schematic diagram of the full length PC1 construct employed in these studies. The positions of the Flag tag, HA tag, GPS site and tethered agonist (TA) are indicated, as is the position of the lysine residues whose availability for biotinylation is assessed in 1e. (b) Immunofluorescence detection and quantification of surface PC1 NTF following 3 hrs treatment with Wnt9b. Flag staining (NTF) was performed under non-permeabilizing conditions. HA staining, following permeabilization, revealed total cell quantity of CTF and was used to normalize surface staining. Scale bar=5μM. Graph depicts ratio of Flag to HA staining, normalized to the mean of this value obtained after vehicle treatment. (c) PC1 and 2 expressing-HEK293 cells were treated with Wnt9b or subjected to an alkaline stripping protocol and surface biotinylation was performed. Proteins recovered by streptavidin pull-down were probed with anti-Flag. Cell lysates were probed with anti-HA antibodies. Surface NTF is detected by anti-Flag antibody, and total cell-associated PC1 is represented by the HA signal. The graph depicts ratio of Flag to HA signal, normalized to the mean of this value obtained after vehicle treatment. (d) Western blot detection of total PC1 NTF using anti-Flag antibody. Cells were treated with vehicle or Wnt9b overnight. Bar graphs represent Flag/HA signal ratio. (e) Western blot detection of biotinylated PC1 CTF in experiment where cell surface biotinylation was followed by HA pulldown and streptavidin blotting. Results show increased accessibility of the Lys residue in the PC1-CTF TM6-7 extracellular loop (extracellular loop 3) upon Wnt9b treatment or following alkaline stripping. Total cell-associated PC1 is represented by the HA signal. The graph depicts ratio of Streptavidin to HA signal, normalized to the mean of this value obtained after vehicle treatment. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed for all of the panels except for the time course depicted in 1b, for which a paired Student t-Test was employed. P values <0.05 were considered significant. *=P<0.05; **=P<0.01; ***=P<0.001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Construct, FLAG-tag, Immunofluorescence, Staining, Expressing, Stripping Membranes, Western Blot, Residue, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) GSK3β co-immunoprecipitates with PC1 following NTF removal by exposure to high pH (strip) or Wnt9b. GSK3β associated with PC1 after 5 minutes of Wnt9b treatment. (b) GSK3β co-immunoprecipitates with PC1 from lysates prepared from Tg248 Pkd1 BAC-transgenic mouse kidneys. (c) Fluorescent biosensor assay of endogenous GSK3β kinase activity in HEK293 cells. PC1 and 2 inhibit GSK3β and this effect is enhanced by 3 hrs treatment with 13nM Wnt9b. Scale bar=100 μM. Graph depicts fluorescence intensity measured in each condition normalized to mean of that measured in cells that express GFP alone. (d) GSK3β kinase assay employing a peptide that electrophoretically migrates in agarose faster when phosphorylated. Lysates from PC1-expressing mouse kidney were compared to similar aged WT kidney lysates. Graph shows mean of the ratio of GSK3β-phosphorylated peptide (PP) to unphosphorylated peptide (P) under each condition. (e) Detection of S9 phosphorylated GSK3β in lysates from un-induced, pre-cystic and cystic kidneys. Blots were probed with anti pan-GSK3β, anti pS9-GSK3β and anti-actin antibodies. Graph depicts the mean of the ratio of the pS9-GSK3β signal to the pan GSK3β signal for each condition. (f) Fluorescent biosensor assay of endogenous GSK3β kinase activity in immortalized murine M113 cells. Untransfected cells were compared to cells expressing PC1 and PC2 as well as to PC1 and 2 expressing cells treated with Wnt9b for 3hrs Scale bar=100 μM. Graph depicts fluorescence intensity measured in each condition normalized to mean of that measured in cells that express GFP alone. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant. *=P<0.05; **=P<0.01; ***=P<0.001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Stripping Membranes, Transgenic Assay, Biosensor Assay, Activity Assay, Fluorescence, Kinase Assay, Expressing, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) PC1 ΔNTF co-immunoprecipitates with GSK3β to the same extent as Wnt9b-stimulated full length PC1. Less GSK3β co-immunoprecipitates with PC1 ΔNTF+ΔTA , which lacks a tethered agonist. The graph depicts the ratio of GSK3β signal to the HA signal, normalized to the value of this ratio for the HEK 1+2 condition. (b) GSK3β biosensor shows that PC1 ΔNTF inhibits GSK3β to the same extent as a maximal concentration (10 μM) of GSK3β inhibitor SB-216763. PC1 ΔNTF+ΔTA does not inhibit GSK3β. The graph depicts the ratio of GFP fluorescence in each condition, normalized to the mean of this value obtained with cells that express GFP alone. (c) Confocal Z-stack imaging of PC1 ΔNTF and PC1 ΔNTF+ΔTA detected with anti-HA antibody added to the media bathing unfixed and non-permeabilized cells shows that both proteins expressed in the absence of PC2 reach the surfaces of HEK293 cells. (d) PC1 ΔNTF+ΔTA , lacking the Stachel sequence, does not inhibit the GSK3β in the fluorescence biosensor assay. The ability of PC1 ΔNTF+ΔTA to inhibit GSK3β can be partially restored by addition of soluble Stachel peptide (300 μM). Scrambled Stachel sequence (scrm) did not produce this effect. Versions of the PC1 ΔNTF protein that carry successive groups of alanine substitutions in the sequences of their tethered agonists (PC1 ΔNTF -Ala1-4) do not inhibit GSK3β. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated. In panel 3d the values obtained for each condition were compared to those obtained in the GFP alone condition (indicated by the symbols above each bar) and pairwise comparisons of the values obtained in a subset of conditions related by a single experimental manipulation are indicated by the brackets above the bars.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Concentration Assay, Fluorescence, Imaging, Sequencing, Biosensor Assay, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) GSK3β activity measured +/− Gα13 or RhoA or control (Ctrl) siRNAs, or ROCK inhibitor Y27632. Suppressing Gα13, RhoA or ROCK dampened GSK3β inhibition by Wnt9b-stimulated PC1 or PC1ΔNTF. 10 μM SB-216763 (SB) treatment demonstrates fluorescence detected with maximal GSK3β inhibition. Graph depicts means of ratios of GFP signals normalized to mean of value obtained with cells transfected with GFP and Ctrl RNAi. (b) GTP-bound RhoA abundance was assessed by rotekin RhoA-GTP binding domain bead pull-down, followed by western blotting for RhoA. Wnt9b stimulation of PC1, or PC1ΔNTF expression, increased GTP-bound RhoA. Graph depicts means of ratios of RhoA-GTP signal, normalized to mean of value obtained with empty vector (EV) transfection. The quantity of RhoA-GTP is also substantially higher in lysates prepared from the kidneys of Tg248 Pkd1 BAC-transgenic mice as compared to that present in lysates of wild type mouse kidneys. (c) and (d) Detection of in vitro (c) and in vivo (d) quantity of Gα13 present in anti-HA precipitates of PC1 was assessed by western blotting. Wnt9b treatment modestly but significantly increased quantity of PC1-associated Gα13 PC1 (c). Graphs in c and d depict means of ratios of signal detected in Wnt9b-treated condition, normalized to mean of value obtained with vehicle-treated cells. Graph depicts the intensity of the Gα13 signal in each condition normalized to the corresponding HA signal. (e) Biosensor assay to detect importance of G-protein binding site of PC1 for regulation of GSK3β. PC1 or PC1 ΔNTF lacking its G-protein binding site, or PC1 carrying a mutation within the G-protein binding site sequence (ΔL4132), does not inhibit GSK3β. Similarly, soluble Stachel peptide does not induce GSK3β inhibition in cells expressing PC1 ΔNTF+ΔTA+ΔL4132 . Graph depicts ratio of GFP fluorescence, normalized to mean of value obtained with cells expressing GFP alone. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values ≤ 0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated. In panels 4a and 4f the values obtained for each condition were compared to those obtained in the GFP alone condition (indicated by the symbols above each bar) and pairwise comparisons of the values obtained in a subset of conditions related by a single experimental manipulation are indicated by the brackets above the bars.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Activity Assay, Control, Inhibition, Fluorescence, Transfection, Binding Assay, Western Blot, Expressing, Plasmid Preparation, Transgenic Assay, In Vitro, In Vivo, Biosensor Assay, Protein Binding, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) Immunofluorescent localization of the full length PC1 (green) in the primary cilia of LLCPK1+2 cells treated with wnt9b, or with a combination of Wnt9b and barbadin. Cilia are detected by immunostaining for Arl13b (orange) (b) Immunofluorescent localization of PC1 ΔNTF and PC1 ΔNTFΔTA in the primary cilia of LLCPK cells. Barbadin treatment results in localization of PC1 ΔNTF to cilia, while treatment with soluble TA peptide causes loss of PC1 ΔNTF+ΔTA from the cilia. (c) Immunofluorescence detection of the full length PC1 in the primary cilia of cells treated with mechanical stimulation, with medium from cells preconditioned by mechanical stimulation or with ATP added to the medium. Barbadin and Apyrase treatment blocked the effects of mechanical stimulation and ATP on PC1 ciliary localization. Data are shown as mean ±SEM, n≥3 for all experiments. Scale bar = 5μ. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Immunostaining, Immunofluorescence, Control

Journal: bioRxiv

Article Title: Polycystin-1 acts as an atypical adhesion GPCR that responds to novel Wnt signaling and mechanical stimuli

doi: 10.1101/2025.05.21.655326

Figure Lengend Snippet: (a) Immunofluorescent detection of the localizations of PC1S3164A and PC1S3164D to the primary cilia in LLCPK cells. ATP treatment did not alter the ciliary distribution of PC1S3164A, whereas the ciliary exclusion of PC1 S3163D was reversed by Barbadin treatment. PKA inhibitor H89A treatment prevented the departure of PC1 from primary cilia that is induced by either ATP or Wnt9b treatment. (b) Biosensor assay was used to assess the importance of PC1 PKA phosphorylation site S3164 for PC1 activation-dependent inhibition of GSK3β kinase in HEK293 cells. Data are shown as mean ±SEM, n≥3 for all experiments. To assess statistical significance unpaired Student t-Test analysis was performed. P values <0.05 were considered significant *=P<0.05; **=P<0.01; ***=P<0.001.; ****=P<0.0001 Differences between mean values obtained for experimental conditions versus control conditions that produce minimal and/or maximal signals were evaluated. In panel 3d the values obtained for each condition were compared to those obtained in the GFP alone condition (indicated by the symbols above each bar) and pairwise comparisons of the values obtained in a subset of conditions related by a single experimental manipulation are indicated by the brackets above the bars.

Article Snippet: Recombinant Wnt9b (0.5ng/μL) (R&D Systems, 3669) was applied for 3 hrs or overnight.

Techniques: Biosensor Assay, Phospho-proteomics, Activation Assay, Inhibition, Control