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    Bio-Rad phosphorylated nherf1 fragments
    FIGURE 2. Structural determinants of <t>NHERF1</t> association with Npt2a. A, OKH cells were transiently transfected with empty vector (control), FLAG-NHERF1, HA-GFP-Npt2a or HA-GFP-Npt2a(L639A). 48 h after transfection, the cells were treated with vehicle or 100 nM PTH(1–34) for 2 h. FLAG-tagged proteins were immunoprecipitated with FLAG-agarose. The precipitated protein was then immunoblotted with HA antibody. Actin expression was used as a loading control. The figure is representative of three independent experiments. B, Npt2a interacts with PDZ1. OKH-HA-GFP-Npt2a cells were transiently transfected with empty vector, wild-type NHERF1, or NHERF1 harboring mutations in PDZ1 and/or PDZ2 core-binding domains (sPDZ1-NHERF1, sPDZ2-NHERF1, and sPDZ1/2- NHERF1) or truncated NHERF1 lacking the ezrin-binding domain (NHERF1-EBD). 48 h after transfection, HA-tagged proteins were immunoprecipitated with HA-agarose. The precipitated protein was then immunoblotted with NHERF1 antibody. An example of three independent experiments is shown. C, sodium- dependent Pi uptake was measured in OKH cells transiently transfected with empty vector, wild-type NHERF1, sPDZ1-NHERF1, sPDZ2-NHERF1, sPDZ1/2- NHERF1, or NHERF1-EBD and treated with vehicle or PTH(1–34) (100 nM, 2 h). Data are summarized as the mean S.E. (error bars) (n 3; **, p 0.01, versus vector). D, OKH cells were transfected with HA-PTHR, FLAG-NHERF1, FLAG-L110V-NHERF1, FLAG-R153Q-NHERF1, or FLAG-E225K-NHERF1. 48 h after transfec- tion,FLAG-taggedproteinswereimmunoprecipitated(IP).Theprecipitatedproteinwasimmunoblotted(IB)withHAantibody.Dataarerepresentativeofthree independent experiments. E, OKH cells were transfected with empty vector, wild-type NHERF1, or mutated NHERF1. Cell surface binding of [125I]PTH(1–34) was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. of triplicate determinations. F, OKH cells were transfected with wild-type NHERF1 or mutated forms of NHERF1. Cells were treated with 100 nM PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. (n 4). IP, immunoprecipitation; IB, immunoblot.
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    1) Product Images from "Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport"

    Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

    Journal: Journal of Biological Chemistry

    doi: 10.1074/jbc.m112.369405

    FIGURE 2. Structural determinants of NHERF1 association with Npt2a. A, OKH cells were transiently transfected with empty vector (control), FLAG-NHERF1, HA-GFP-Npt2a or HA-GFP-Npt2a(L639A). 48 h after transfection, the cells were treated with vehicle or 100 nM PTH(1–34) for 2 h. FLAG-tagged proteins were immunoprecipitated with FLAG-agarose. The precipitated protein was then immunoblotted with HA antibody. Actin expression was used as a loading control. The figure is representative of three independent experiments. B, Npt2a interacts with PDZ1. OKH-HA-GFP-Npt2a cells were transiently transfected with empty vector, wild-type NHERF1, or NHERF1 harboring mutations in PDZ1 and/or PDZ2 core-binding domains (sPDZ1-NHERF1, sPDZ2-NHERF1, and sPDZ1/2- NHERF1) or truncated NHERF1 lacking the ezrin-binding domain (NHERF1-EBD). 48 h after transfection, HA-tagged proteins were immunoprecipitated with HA-agarose. The precipitated protein was then immunoblotted with NHERF1 antibody. An example of three independent experiments is shown. C, sodium- dependent Pi uptake was measured in OKH cells transiently transfected with empty vector, wild-type NHERF1, sPDZ1-NHERF1, sPDZ2-NHERF1, sPDZ1/2- NHERF1, or NHERF1-EBD and treated with vehicle or PTH(1–34) (100 nM, 2 h). Data are summarized as the mean S.E. (error bars) (n 3; **, p 0.01, versus vector). D, OKH cells were transfected with HA-PTHR, FLAG-NHERF1, FLAG-L110V-NHERF1, FLAG-R153Q-NHERF1, or FLAG-E225K-NHERF1. 48 h after transfec- tion,FLAG-taggedproteinswereimmunoprecipitated(IP).Theprecipitatedproteinwasimmunoblotted(IB)withHAantibody.Dataarerepresentativeofthree independent experiments. E, OKH cells were transfected with empty vector, wild-type NHERF1, or mutated NHERF1. Cell surface binding of [125I]PTH(1–34) was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. of triplicate determinations. F, OKH cells were transfected with wild-type NHERF1 or mutated forms of NHERF1. Cells were treated with 100 nM PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. (n 4). IP, immunoprecipitation; IB, immunoblot.
    Figure Legend Snippet: FIGURE 2. Structural determinants of NHERF1 association with Npt2a. A, OKH cells were transiently transfected with empty vector (control), FLAG-NHERF1, HA-GFP-Npt2a or HA-GFP-Npt2a(L639A). 48 h after transfection, the cells were treated with vehicle or 100 nM PTH(1–34) for 2 h. FLAG-tagged proteins were immunoprecipitated with FLAG-agarose. The precipitated protein was then immunoblotted with HA antibody. Actin expression was used as a loading control. The figure is representative of three independent experiments. B, Npt2a interacts with PDZ1. OKH-HA-GFP-Npt2a cells were transiently transfected with empty vector, wild-type NHERF1, or NHERF1 harboring mutations in PDZ1 and/or PDZ2 core-binding domains (sPDZ1-NHERF1, sPDZ2-NHERF1, and sPDZ1/2- NHERF1) or truncated NHERF1 lacking the ezrin-binding domain (NHERF1-EBD). 48 h after transfection, HA-tagged proteins were immunoprecipitated with HA-agarose. The precipitated protein was then immunoblotted with NHERF1 antibody. An example of three independent experiments is shown. C, sodium- dependent Pi uptake was measured in OKH cells transiently transfected with empty vector, wild-type NHERF1, sPDZ1-NHERF1, sPDZ2-NHERF1, sPDZ1/2- NHERF1, or NHERF1-EBD and treated with vehicle or PTH(1–34) (100 nM, 2 h). Data are summarized as the mean S.E. (error bars) (n 3; **, p 0.01, versus vector). D, OKH cells were transfected with HA-PTHR, FLAG-NHERF1, FLAG-L110V-NHERF1, FLAG-R153Q-NHERF1, or FLAG-E225K-NHERF1. 48 h after transfec- tion,FLAG-taggedproteinswereimmunoprecipitated(IP).Theprecipitatedproteinwasimmunoblotted(IB)withHAantibody.Dataarerepresentativeofthree independent experiments. E, OKH cells were transfected with empty vector, wild-type NHERF1, or mutated NHERF1. Cell surface binding of [125I]PTH(1–34) was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. of triplicate determinations. F, OKH cells were transfected with wild-type NHERF1 or mutated forms of NHERF1. Cells were treated with 100 nM PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. (n 4). IP, immunoprecipitation; IB, immunoblot.

    Techniques Used: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Expressing, Binding Assay, Western Blot

    FIGURE 3. NHERF1 forms a ternary complex with Npt2a and ezrin. A, Npt2a, NHERF1, and ezrin form a ternary complex (lane 2) that dissoci- ates upon treatment with PTH (lane 6). Truncated NHERF1 lacking the EBD (NHERF1-EBD) and Npt2a in which the carboxyl-terminal PDZ-binding domain was mutated (Npt2a(L639A)) do not form a ternary complex. B, mutant NHERF1 constructs are poorly able to form a ternary complex with Npt2a and ezrin and are refractory to PTH. Shown is a representative result of OKH cells stably expressing HA-Npt2a transiently transfected with FLAG-tagged wild-type NHERF1 or L110V-, R153Q-, or E225K-NH- ERF1. 48 h after transfection, the cells were treated, and the ternary com- plex was detected as above. C, aggregate results from experiments shown in B (n 3; **, versus no PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)).
    Figure Legend Snippet: FIGURE 3. NHERF1 forms a ternary complex with Npt2a and ezrin. A, Npt2a, NHERF1, and ezrin form a ternary complex (lane 2) that dissoci- ates upon treatment with PTH (lane 6). Truncated NHERF1 lacking the EBD (NHERF1-EBD) and Npt2a in which the carboxyl-terminal PDZ-binding domain was mutated (Npt2a(L639A)) do not form a ternary complex. B, mutant NHERF1 constructs are poorly able to form a ternary complex with Npt2a and ezrin and are refractory to PTH. Shown is a representative result of OKH cells stably expressing HA-Npt2a transiently transfected with FLAG-tagged wild-type NHERF1 or L110V-, R153Q-, or E225K-NH- ERF1. 48 h after transfection, the cells were treated, and the ternary com- plex was detected as above. C, aggregate results from experiments shown in B (n 3; **, versus no PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)).

    Techniques Used: Binding Assay, Mutagenesis, Construct, Stable Transfection, Expressing, Transfection, Comparison

    FIGURE 5. NHERF1 mutants are resistant to phosphorylation upon PTH stimulation. A, PTH promotes phosphorylation of wild-type NHERF1 but not of NHERF1 mutants. Representative experiment shows PTH-stimulated phos- phorylation within the two large fragments of NHERF1 cleaved by CNBr con- taining Ser77 within PDZ1 (residues 2–156) and in a Ser-rich cluster located in the linker between PDZ2 and the EBD (residues 208–329) (Cluster). B, quanti- fication of phosphorylation results from A (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)). Error bars, S.E.; IB, immunoblot.
    Figure Legend Snippet: FIGURE 5. NHERF1 mutants are resistant to phosphorylation upon PTH stimulation. A, PTH promotes phosphorylation of wild-type NHERF1 but not of NHERF1 mutants. Representative experiment shows PTH-stimulated phos- phorylation within the two large fragments of NHERF1 cleaved by CNBr con- taining Ser77 within PDZ1 (residues 2–156) and in a Ser-rich cluster located in the linker between PDZ2 and the EBD (residues 208–329) (Cluster). B, quanti- fication of phosphorylation results from A (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)). Error bars, S.E.; IB, immunoblot.

    Techniques Used: Phospho-proteomics, Control, Comparison, Western Blot

    FIGURE 6. Functional and structural analysis of NHERF1 mutations. A and B, R153Q-NHERF1 acts as a loss-of-function mutation and not as a dominant negative inhibitor of wild-type NHERF1. OKH cells were transfected with 0.2 g of GFP-NHERF1 alone or 0–1.0 g of R153Q-NHERF1, with the balance made of empty vector (A). PTH-sensitive Pi uptake was measured as before (B) (n 3 independent determinations). C and D, affinity of wild-type NHERF1 (WT) and mutant R153Q-NHERF1 for Npt2a was determined by fluorescence polarization, and KD was derived from the calculated anisotropy (C). Affinity measurements by ITC for wild-type NHERF1 (D, left) and R153Q-NHERF1 (D, right) are shown. Error bars, S.E.; IB, immunoblot.
    Figure Legend Snippet: FIGURE 6. Functional and structural analysis of NHERF1 mutations. A and B, R153Q-NHERF1 acts as a loss-of-function mutation and not as a dominant negative inhibitor of wild-type NHERF1. OKH cells were transfected with 0.2 g of GFP-NHERF1 alone or 0–1.0 g of R153Q-NHERF1, with the balance made of empty vector (A). PTH-sensitive Pi uptake was measured as before (B) (n 3 independent determinations). C and D, affinity of wild-type NHERF1 (WT) and mutant R153Q-NHERF1 for Npt2a was determined by fluorescence polarization, and KD was derived from the calculated anisotropy (C). Affinity measurements by ITC for wild-type NHERF1 (D, left) and R153Q-NHERF1 (D, right) are shown. Error bars, S.E.; IB, immunoblot.

    Techniques Used: Functional Assay, Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Fluorescence, Derivative Assay, Western Blot

    FIGURE 7. Rescue of NHERF1 structure and function. A, rescue of loss of function of R153-NHERF1 mutant by preventing closed NHERF1 conformation. PTH-sensitive Pi uptake was measured in OKH cells transfected with the indicated construct. Results are the average S.E. (error bars) (n 4; **, versus vector, p 0.001, one-way repeated measures ANOVA (Dunnett’s post hoc multiple-comparison test)). B–E, double R153Q/L358A-NHERF1 mutation restores PTH- induced phosphorylation to NHERF1 (B and C) (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)) and inhibition by H89 and Ht31 of PTH-sensitive Pi uptake (D) (n 3; **, PTH versus St-Ht31 PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)) and reestablishes the ability to form the ternary complex even in the presence of the R153Q mutation (E). IP, immunoprecipitation; IB, immunoblot.
    Figure Legend Snippet: FIGURE 7. Rescue of NHERF1 structure and function. A, rescue of loss of function of R153-NHERF1 mutant by preventing closed NHERF1 conformation. PTH-sensitive Pi uptake was measured in OKH cells transfected with the indicated construct. Results are the average S.E. (error bars) (n 4; **, versus vector, p 0.001, one-way repeated measures ANOVA (Dunnett’s post hoc multiple-comparison test)). B–E, double R153Q/L358A-NHERF1 mutation restores PTH- induced phosphorylation to NHERF1 (B and C) (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)) and inhibition by H89 and Ht31 of PTH-sensitive Pi uptake (D) (n 3; **, PTH versus St-Ht31 PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)) and reestablishes the ability to form the ternary complex even in the presence of the R153Q mutation (E). IP, immunoprecipitation; IB, immunoblot.

    Techniques Used: Mutagenesis, Transfection, Construct, Plasmid Preparation, Comparison, Phospho-proteomics, Control, Inhibition, Immunoprecipitation, Western Blot

    FIGURE 8. Model of PTH-mediated disassembly of the Npt2a-NHERF1-ezrin ternary complex and endocytosis of Npt2a. A, in the resting state, Npt2a is presentinbrushbordersofapicalmembranes,boundtoPDZ1ofwild-typeNHERF1aspartofthespontaneouslyformedNpt2a-NHERF1-ezrinternarycomplex. The EBD of NHERF1 binds to ezrin, linking the complex to cytoskeletal elements. In this way, Npt2a is tethered to apical membranes and mediates phosphate uptake. Upon PTH exposure, PTHR stimulates cAMP formation and activates PKA. PKA regulatory (R) subunits undergo a conformational change and release the catalytic (C) subunits. Ezrin binds to PKA regulatory subunits, positioning them in close proximity to the ternary complex. PKA catalytic subunits phosphor- ylate (P) NHERF1, with subsequent dissociation of Npt2a, which is endocytosed, thereby inhibiting phosphate transport. B, in the presence of mutant forms of NHERF1, less Npt2a is present in brush border apical membranes. PTH stimulates cAMP normally. Because NHERF1 is locked in an inactive conformation, access to catalytic PKA subunits is prevented, thereby precluding phosphorylation and dissociation of tethered Npt2a from NHERF1.
    Figure Legend Snippet: FIGURE 8. Model of PTH-mediated disassembly of the Npt2a-NHERF1-ezrin ternary complex and endocytosis of Npt2a. A, in the resting state, Npt2a is presentinbrushbordersofapicalmembranes,boundtoPDZ1ofwild-typeNHERF1aspartofthespontaneouslyformedNpt2a-NHERF1-ezrinternarycomplex. The EBD of NHERF1 binds to ezrin, linking the complex to cytoskeletal elements. In this way, Npt2a is tethered to apical membranes and mediates phosphate uptake. Upon PTH exposure, PTHR stimulates cAMP formation and activates PKA. PKA regulatory (R) subunits undergo a conformational change and release the catalytic (C) subunits. Ezrin binds to PKA regulatory subunits, positioning them in close proximity to the ternary complex. PKA catalytic subunits phosphor- ylate (P) NHERF1, with subsequent dissociation of Npt2a, which is endocytosed, thereby inhibiting phosphate transport. B, in the presence of mutant forms of NHERF1, less Npt2a is present in brush border apical membranes. PTH stimulates cAMP normally. Because NHERF1 is locked in an inactive conformation, access to catalytic PKA subunits is prevented, thereby precluding phosphorylation and dissociation of tethered Npt2a from NHERF1.

    Techniques Used: Mutagenesis, Phospho-proteomics



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    FIGURE 2. Structural determinants of <t>NHERF1</t> association with Npt2a. A, OKH cells were transiently transfected with empty vector (control), FLAG-NHERF1, HA-GFP-Npt2a or HA-GFP-Npt2a(L639A). 48 h after transfection, the cells were treated with vehicle or 100 nM PTH(1–34) for 2 h. FLAG-tagged proteins were immunoprecipitated with FLAG-agarose. The precipitated protein was then immunoblotted with HA antibody. Actin expression was used as a loading control. The figure is representative of three independent experiments. B, Npt2a interacts with PDZ1. OKH-HA-GFP-Npt2a cells were transiently transfected with empty vector, wild-type NHERF1, or NHERF1 harboring mutations in PDZ1 and/or PDZ2 core-binding domains (sPDZ1-NHERF1, sPDZ2-NHERF1, and sPDZ1/2- NHERF1) or truncated NHERF1 lacking the ezrin-binding domain (NHERF1-EBD). 48 h after transfection, HA-tagged proteins were immunoprecipitated with HA-agarose. The precipitated protein was then immunoblotted with NHERF1 antibody. An example of three independent experiments is shown. C, sodium- dependent Pi uptake was measured in OKH cells transiently transfected with empty vector, wild-type NHERF1, sPDZ1-NHERF1, sPDZ2-NHERF1, sPDZ1/2- NHERF1, or NHERF1-EBD and treated with vehicle or PTH(1–34) (100 nM, 2 h). Data are summarized as the mean S.E. (error bars) (n 3; **, p 0.01, versus vector). D, OKH cells were transfected with HA-PTHR, FLAG-NHERF1, FLAG-L110V-NHERF1, FLAG-R153Q-NHERF1, or FLAG-E225K-NHERF1. 48 h after transfec- tion,FLAG-taggedproteinswereimmunoprecipitated(IP).Theprecipitatedproteinwasimmunoblotted(IB)withHAantibody.Dataarerepresentativeofthree independent experiments. E, OKH cells were transfected with empty vector, wild-type NHERF1, or mutated NHERF1. Cell surface binding of [125I]PTH(1–34) was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. of triplicate determinations. F, OKH cells were transfected with wild-type NHERF1 or mutated forms of NHERF1. Cells were treated with 100 nM PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. (n 4). IP, immunoprecipitation; IB, immunoblot.
    Phosphorylated Nherf1 Fragments, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FIGURE 2. Structural determinants of NHERF1 association with Npt2a. A, OKH cells were transiently transfected with empty vector (control), FLAG-NHERF1, HA-GFP-Npt2a or HA-GFP-Npt2a(L639A). 48 h after transfection, the cells were treated with vehicle or 100 nM PTH(1–34) for 2 h. FLAG-tagged proteins were immunoprecipitated with FLAG-agarose. The precipitated protein was then immunoblotted with HA antibody. Actin expression was used as a loading control. The figure is representative of three independent experiments. B, Npt2a interacts with PDZ1. OKH-HA-GFP-Npt2a cells were transiently transfected with empty vector, wild-type NHERF1, or NHERF1 harboring mutations in PDZ1 and/or PDZ2 core-binding domains (sPDZ1-NHERF1, sPDZ2-NHERF1, and sPDZ1/2- NHERF1) or truncated NHERF1 lacking the ezrin-binding domain (NHERF1-EBD). 48 h after transfection, HA-tagged proteins were immunoprecipitated with HA-agarose. The precipitated protein was then immunoblotted with NHERF1 antibody. An example of three independent experiments is shown. C, sodium- dependent Pi uptake was measured in OKH cells transiently transfected with empty vector, wild-type NHERF1, sPDZ1-NHERF1, sPDZ2-NHERF1, sPDZ1/2- NHERF1, or NHERF1-EBD and treated with vehicle or PTH(1–34) (100 nM, 2 h). Data are summarized as the mean S.E. (error bars) (n 3; **, p 0.01, versus vector). D, OKH cells were transfected with HA-PTHR, FLAG-NHERF1, FLAG-L110V-NHERF1, FLAG-R153Q-NHERF1, or FLAG-E225K-NHERF1. 48 h after transfec- tion,FLAG-taggedproteinswereimmunoprecipitated(IP).Theprecipitatedproteinwasimmunoblotted(IB)withHAantibody.Dataarerepresentativeofthree independent experiments. E, OKH cells were transfected with empty vector, wild-type NHERF1, or mutated NHERF1. Cell surface binding of [125I]PTH(1–34) was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. of triplicate determinations. F, OKH cells were transfected with wild-type NHERF1 or mutated forms of NHERF1. Cells were treated with 100 nM PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. (n 4). IP, immunoprecipitation; IB, immunoblot.

    Journal: Journal of Biological Chemistry

    Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

    doi: 10.1074/jbc.m112.369405

    Figure Lengend Snippet: FIGURE 2. Structural determinants of NHERF1 association with Npt2a. A, OKH cells were transiently transfected with empty vector (control), FLAG-NHERF1, HA-GFP-Npt2a or HA-GFP-Npt2a(L639A). 48 h after transfection, the cells were treated with vehicle or 100 nM PTH(1–34) for 2 h. FLAG-tagged proteins were immunoprecipitated with FLAG-agarose. The precipitated protein was then immunoblotted with HA antibody. Actin expression was used as a loading control. The figure is representative of three independent experiments. B, Npt2a interacts with PDZ1. OKH-HA-GFP-Npt2a cells were transiently transfected with empty vector, wild-type NHERF1, or NHERF1 harboring mutations in PDZ1 and/or PDZ2 core-binding domains (sPDZ1-NHERF1, sPDZ2-NHERF1, and sPDZ1/2- NHERF1) or truncated NHERF1 lacking the ezrin-binding domain (NHERF1-EBD). 48 h after transfection, HA-tagged proteins were immunoprecipitated with HA-agarose. The precipitated protein was then immunoblotted with NHERF1 antibody. An example of three independent experiments is shown. C, sodium- dependent Pi uptake was measured in OKH cells transiently transfected with empty vector, wild-type NHERF1, sPDZ1-NHERF1, sPDZ2-NHERF1, sPDZ1/2- NHERF1, or NHERF1-EBD and treated with vehicle or PTH(1–34) (100 nM, 2 h). Data are summarized as the mean S.E. (error bars) (n 3; **, p 0.01, versus vector). D, OKH cells were transfected with HA-PTHR, FLAG-NHERF1, FLAG-L110V-NHERF1, FLAG-R153Q-NHERF1, or FLAG-E225K-NHERF1. 48 h after transfec- tion,FLAG-taggedproteinswereimmunoprecipitated(IP).Theprecipitatedproteinwasimmunoblotted(IB)withHAantibody.Dataarerepresentativeofthree independent experiments. E, OKH cells were transfected with empty vector, wild-type NHERF1, or mutated NHERF1. Cell surface binding of [125I]PTH(1–34) was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. of triplicate determinations. F, OKH cells were transfected with wild-type NHERF1 or mutated forms of NHERF1. Cells were treated with 100 nM PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. (n 4). IP, immunoprecipitation; IB, immunoblot.

    Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

    Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Expressing, Binding Assay, Western Blot

    FIGURE 3. NHERF1 forms a ternary complex with Npt2a and ezrin. A, Npt2a, NHERF1, and ezrin form a ternary complex (lane 2) that dissoci- ates upon treatment with PTH (lane 6). Truncated NHERF1 lacking the EBD (NHERF1-EBD) and Npt2a in which the carboxyl-terminal PDZ-binding domain was mutated (Npt2a(L639A)) do not form a ternary complex. B, mutant NHERF1 constructs are poorly able to form a ternary complex with Npt2a and ezrin and are refractory to PTH. Shown is a representative result of OKH cells stably expressing HA-Npt2a transiently transfected with FLAG-tagged wild-type NHERF1 or L110V-, R153Q-, or E225K-NH- ERF1. 48 h after transfection, the cells were treated, and the ternary com- plex was detected as above. C, aggregate results from experiments shown in B (n 3; **, versus no PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)).

    Journal: Journal of Biological Chemistry

    Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

    doi: 10.1074/jbc.m112.369405

    Figure Lengend Snippet: FIGURE 3. NHERF1 forms a ternary complex with Npt2a and ezrin. A, Npt2a, NHERF1, and ezrin form a ternary complex (lane 2) that dissoci- ates upon treatment with PTH (lane 6). Truncated NHERF1 lacking the EBD (NHERF1-EBD) and Npt2a in which the carboxyl-terminal PDZ-binding domain was mutated (Npt2a(L639A)) do not form a ternary complex. B, mutant NHERF1 constructs are poorly able to form a ternary complex with Npt2a and ezrin and are refractory to PTH. Shown is a representative result of OKH cells stably expressing HA-Npt2a transiently transfected with FLAG-tagged wild-type NHERF1 or L110V-, R153Q-, or E225K-NH- ERF1. 48 h after transfection, the cells were treated, and the ternary com- plex was detected as above. C, aggregate results from experiments shown in B (n 3; **, versus no PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)).

    Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

    Techniques: Binding Assay, Mutagenesis, Construct, Stable Transfection, Expressing, Transfection, Comparison

    FIGURE 5. NHERF1 mutants are resistant to phosphorylation upon PTH stimulation. A, PTH promotes phosphorylation of wild-type NHERF1 but not of NHERF1 mutants. Representative experiment shows PTH-stimulated phos- phorylation within the two large fragments of NHERF1 cleaved by CNBr con- taining Ser77 within PDZ1 (residues 2–156) and in a Ser-rich cluster located in the linker between PDZ2 and the EBD (residues 208–329) (Cluster). B, quanti- fication of phosphorylation results from A (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)). Error bars, S.E.; IB, immunoblot.

    Journal: Journal of Biological Chemistry

    Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

    doi: 10.1074/jbc.m112.369405

    Figure Lengend Snippet: FIGURE 5. NHERF1 mutants are resistant to phosphorylation upon PTH stimulation. A, PTH promotes phosphorylation of wild-type NHERF1 but not of NHERF1 mutants. Representative experiment shows PTH-stimulated phos- phorylation within the two large fragments of NHERF1 cleaved by CNBr con- taining Ser77 within PDZ1 (residues 2–156) and in a Ser-rich cluster located in the linker between PDZ2 and the EBD (residues 208–329) (Cluster). B, quanti- fication of phosphorylation results from A (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)). Error bars, S.E.; IB, immunoblot.

    Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

    Techniques: Phospho-proteomics, Control, Comparison, Western Blot

    FIGURE 6. Functional and structural analysis of NHERF1 mutations. A and B, R153Q-NHERF1 acts as a loss-of-function mutation and not as a dominant negative inhibitor of wild-type NHERF1. OKH cells were transfected with 0.2 g of GFP-NHERF1 alone or 0–1.0 g of R153Q-NHERF1, with the balance made of empty vector (A). PTH-sensitive Pi uptake was measured as before (B) (n 3 independent determinations). C and D, affinity of wild-type NHERF1 (WT) and mutant R153Q-NHERF1 for Npt2a was determined by fluorescence polarization, and KD was derived from the calculated anisotropy (C). Affinity measurements by ITC for wild-type NHERF1 (D, left) and R153Q-NHERF1 (D, right) are shown. Error bars, S.E.; IB, immunoblot.

    Journal: Journal of Biological Chemistry

    Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

    doi: 10.1074/jbc.m112.369405

    Figure Lengend Snippet: FIGURE 6. Functional and structural analysis of NHERF1 mutations. A and B, R153Q-NHERF1 acts as a loss-of-function mutation and not as a dominant negative inhibitor of wild-type NHERF1. OKH cells were transfected with 0.2 g of GFP-NHERF1 alone or 0–1.0 g of R153Q-NHERF1, with the balance made of empty vector (A). PTH-sensitive Pi uptake was measured as before (B) (n 3 independent determinations). C and D, affinity of wild-type NHERF1 (WT) and mutant R153Q-NHERF1 for Npt2a was determined by fluorescence polarization, and KD was derived from the calculated anisotropy (C). Affinity measurements by ITC for wild-type NHERF1 (D, left) and R153Q-NHERF1 (D, right) are shown. Error bars, S.E.; IB, immunoblot.

    Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

    Techniques: Functional Assay, Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Fluorescence, Derivative Assay, Western Blot

    FIGURE 7. Rescue of NHERF1 structure and function. A, rescue of loss of function of R153-NHERF1 mutant by preventing closed NHERF1 conformation. PTH-sensitive Pi uptake was measured in OKH cells transfected with the indicated construct. Results are the average S.E. (error bars) (n 4; **, versus vector, p 0.001, one-way repeated measures ANOVA (Dunnett’s post hoc multiple-comparison test)). B–E, double R153Q/L358A-NHERF1 mutation restores PTH- induced phosphorylation to NHERF1 (B and C) (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)) and inhibition by H89 and Ht31 of PTH-sensitive Pi uptake (D) (n 3; **, PTH versus St-Ht31 PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)) and reestablishes the ability to form the ternary complex even in the presence of the R153Q mutation (E). IP, immunoprecipitation; IB, immunoblot.

    Journal: Journal of Biological Chemistry

    Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

    doi: 10.1074/jbc.m112.369405

    Figure Lengend Snippet: FIGURE 7. Rescue of NHERF1 structure and function. A, rescue of loss of function of R153-NHERF1 mutant by preventing closed NHERF1 conformation. PTH-sensitive Pi uptake was measured in OKH cells transfected with the indicated construct. Results are the average S.E. (error bars) (n 4; **, versus vector, p 0.001, one-way repeated measures ANOVA (Dunnett’s post hoc multiple-comparison test)). B–E, double R153Q/L358A-NHERF1 mutation restores PTH- induced phosphorylation to NHERF1 (B and C) (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)) and inhibition by H89 and Ht31 of PTH-sensitive Pi uptake (D) (n 3; **, PTH versus St-Ht31 PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)) and reestablishes the ability to form the ternary complex even in the presence of the R153Q mutation (E). IP, immunoprecipitation; IB, immunoblot.

    Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

    Techniques: Mutagenesis, Transfection, Construct, Plasmid Preparation, Comparison, Phospho-proteomics, Control, Inhibition, Immunoprecipitation, Western Blot

    FIGURE 8. Model of PTH-mediated disassembly of the Npt2a-NHERF1-ezrin ternary complex and endocytosis of Npt2a. A, in the resting state, Npt2a is presentinbrushbordersofapicalmembranes,boundtoPDZ1ofwild-typeNHERF1aspartofthespontaneouslyformedNpt2a-NHERF1-ezrinternarycomplex. The EBD of NHERF1 binds to ezrin, linking the complex to cytoskeletal elements. In this way, Npt2a is tethered to apical membranes and mediates phosphate uptake. Upon PTH exposure, PTHR stimulates cAMP formation and activates PKA. PKA regulatory (R) subunits undergo a conformational change and release the catalytic (C) subunits. Ezrin binds to PKA regulatory subunits, positioning them in close proximity to the ternary complex. PKA catalytic subunits phosphor- ylate (P) NHERF1, with subsequent dissociation of Npt2a, which is endocytosed, thereby inhibiting phosphate transport. B, in the presence of mutant forms of NHERF1, less Npt2a is present in brush border apical membranes. PTH stimulates cAMP normally. Because NHERF1 is locked in an inactive conformation, access to catalytic PKA subunits is prevented, thereby precluding phosphorylation and dissociation of tethered Npt2a from NHERF1.

    Journal: Journal of Biological Chemistry

    Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

    doi: 10.1074/jbc.m112.369405

    Figure Lengend Snippet: FIGURE 8. Model of PTH-mediated disassembly of the Npt2a-NHERF1-ezrin ternary complex and endocytosis of Npt2a. A, in the resting state, Npt2a is presentinbrushbordersofapicalmembranes,boundtoPDZ1ofwild-typeNHERF1aspartofthespontaneouslyformedNpt2a-NHERF1-ezrinternarycomplex. The EBD of NHERF1 binds to ezrin, linking the complex to cytoskeletal elements. In this way, Npt2a is tethered to apical membranes and mediates phosphate uptake. Upon PTH exposure, PTHR stimulates cAMP formation and activates PKA. PKA regulatory (R) subunits undergo a conformational change and release the catalytic (C) subunits. Ezrin binds to PKA regulatory subunits, positioning them in close proximity to the ternary complex. PKA catalytic subunits phosphor- ylate (P) NHERF1, with subsequent dissociation of Npt2a, which is endocytosed, thereby inhibiting phosphate transport. B, in the presence of mutant forms of NHERF1, less Npt2a is present in brush border apical membranes. PTH stimulates cAMP normally. Because NHERF1 is locked in an inactive conformation, access to catalytic PKA subunits is prevented, thereby precluding phosphorylation and dissociation of tethered Npt2a from NHERF1.

    Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

    Techniques: Mutagenesis, Phospho-proteomics