recombinant human arg 179 gln fgf23  (R&D Systems)

Journal: bioRxiv

Article Title: Structural factors of RGS14 regulating hormone-dependent phosphate transport mediated by NPT2A

doi: 10.1101/2024.03.29.586499

Figure Lengend Snippet: RGS14 truncation mutants were generated to identify functional domains suppressing hormone-regulated phosphate transport. A . Serial deletions from the N-terminus of RGS14 were generated, as outlined in Experimental Procedures. Previously characterized functional domains are depicted in the full-length (WT) RGS14 and the truncation mutants ( - ). Regulator of G protein Signaling (RGS) domain, Ras-binding domains 1 and 2 (R1, R2), G protein regulatory (Go-Loco) motif, and -DSAL PDZ ligand are depicted. B . Transfected WT-RGS14 and truncation mutants (constructs1-4) expressed in HEK293 cells. Molecular weight markers (kDa) are presented for reference. Empty vector-transfected cell lysate (V) served as a negative control. C . Hormone-sensitive phosphate uptake was measured in OK cells transfected with WT-RGS14 or the indicated truncation mutant. Phosphate transport was normalized to baseline phosphate uptake under control conditions (100%). Where indicated, cells were treated with 100nM FGF23 or PTH before phosphate uptake measurements. n =6. ****p<0.001 vs control.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25-251 (herein referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (2604-FG-025).

Techniques: Generated, Functional Assay, Binding Assay, Transfection, Molecular Weight, Plasmid Preparation, Negative Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein

doi: 10.1016/j.jbc.2022.101836

Figure Lengend Snippet: RGS14 knockdown unmasks PTH-sensitive phosphate uptake. A , siRNA ablation of RGS14 in primary human patient cells ( upper panel , quantification normalized to actin in lower panel ). Normalized RGS14 levels and statistical analysis of siRNA knockdown in six patient samples are depicted (paired two-tailed t test; ∗∗ p < 0.0001). B , siRNA knockdown of RGS14 unmasks PTH-sensitive Pi transport. Because patient samples gave comparable results with siRNA knockdown, they were combined to achieve sufficient material for transport experiments. Results were normalized for each experiment, where phosphate uptake under control untreated conditions was defined as 100%. Data represent the mean ± SE (error bars); n = 6 (two-way ANOVA with Bonferroni post hoc test; F [1,10] = 15.84, p < 0.005, ∗∗ p < 0.001). C , expression and siRNA knockdown on RGS14 in HPCT cells did not alter protein levels of other markers of phosphate transport. D , siRNA knockdown of RGS14 in HPCT cells reveals FGF23-sensitive and PTH-sensitive phosphate transport, mean ± SE; n = 6 (two-way ANOVA with Bonferroni post hoc test; F [6,45] = 7.956, p < 0.0001, ∗ p < 0.0001). One hundred micrometers PF-06869206 (PF) inhibited phosphate transport by greater than 95% (two-way ANOVA with Tukey’s test for multiple comparisons ( F [10,15] = 4.030, p < 0.01). FGF23, fibroblast growth factor 23; HPCT, human proximal convoluted tubule; PTH, parathyroid hormone; RGS14, regulator of G protein signaling 14.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25–251 (referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (catalog no.: 2604-FG-025).

Techniques: Two Tailed Test, Expressing

Journal: The Journal of Biological Chemistry

Article Title: RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein

doi: 10.1016/j.jbc.2022.101836

Figure Lengend Snippet: RGS14 suppresses hormone-regulated Pi transport. A , RGS14 inhibits PTH-sensitive and FGF23-sensitive phosphate uptake. Human RPTECs were transfected with empty vector (control) or human RGS14. After 48 h, cells were treated for 2 h with 10 nM PTH(1–34) or 100 nM FGF23. Phosphate uptake was measured as detailed in the section. Data represent n = 6 independent experiments performed in triplicate. ∗∗∗∗ p < 0.0001 (paired t test). Data were normalized for each experiment, where phosphate uptake under untreated control conditions was defined as 0% inhibition. B , RGS14 forms a ternary complex with NHERF1 and NPT2A. RPTEC cells were transfected with HA-NPT2A, FLAG-NHERF1, and untagged hRGS14 as indicated. The NPT2A–NHERF1–RGS14 ternary complex was identified by serial immunoprecipitation (IP) of HA-NPT2A with agarose-conjugated monoclonal-anti-HA antibody, elution with 2 mg/ml HA peptide, followed by IP of FLAG-NHERF1 with subsequent immunodetection of RGS14. NPT2A was immunoprecipitated with agarose-conjugated monoclonal-anti-HA antibody, elution with 2 mg/ml HA peptide, followed by IP of FLAG-NHERF1 and subsequent detection of Ezrin by immunoblotting ( top three panels ). Input controls for each protein are shown in the bottom three panels (lysate). Duplicate experiments yielded identical results. FGF23, fibroblast growth factor 23; HA, hemagglutinin; NHERF1, sodium hydrogen exchanger regulatory factor-1; NPT2A, sodium phosphate cotransporter 2a; PTH, parathyroid hormone; RGS14, regulator of G protein signaling 14; RPTEC, renal proximal tubule epithelial cell.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25–251 (referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (catalog no.: 2604-FG-025).

Techniques: Transfection, Plasmid Preparation, Inhibition, Immunoprecipitation, Immunodetection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein

doi: 10.1016/j.jbc.2022.101836

Figure Lengend Snippet: RGS14 stabilizes the NHERF1–NPT2A complex. OK cells were transfected with FLAG-RGS14 or empty vector. About 48 h later, cells were treated for 1 h with 100 nM PTH or FGF23, and NHERF1 was immunoprecipitated. The presence of NPT2A and RGS14 in the IP sample was analyzed by immunoblot. A , representative example of OK cells. B , summary of four independent experiments where the amount of RGS14 or NPT2A immunoprecipitating with NHERF1 is quantified. The data show that neither FGF23 nor PTH dissociates NHERF1 and NPT2A in the presence of RGS14, whereas in its absence, hormone treatment dissociates NPT2A from NHERF1 (two-way ANOVA with Bonferroni post hoc test F [2,18] = 36.46, p < 0.0001). ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.00001. FGF23, fibroblast growth factor 23; IP, immunoprecipitation; NHERF1, sodium hydrogen exchanger regulatory factor-1; NPT2A, sodium phosphate cotransporter 2a; OK, opossum kidney; PTH, parathyroid hormone; RGS14, regulator of G protein signaling 14.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25–251 (referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (catalog no.: 2604-FG-025).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: RGS14 regulates PTH- and FGF23-sensitive NPT2A-mediated renal phosphate uptake via binding to the NHERF1 scaffolding protein

doi: 10.1016/j.jbc.2022.101836

Figure Lengend Snippet: Working model for RGS14 regulation of hormone-sensitive phosphate uptake by human proximal tubule kidney cells. NHERF1 binding to NPT2A stabilizes the NHERF1–NPT2A complex at the plasma membrane to facilitate phosphate uptake. PTH and FGF23 each negatively regulate NPT2A phosphate uptake. PTH acting through the PTHR–Gs-cAMP–PKA pathway phosphorylates NHERF1 causing it to dissociate from NPT2A and block phosphate uptake. Similarly, FGF23 acting through the FGFR1c/αKLOTHO receptor–ERK–SGK1 pathway phosphorylates NHERF1 to uncouple it from NPT2A and also block phosphate uptake. Human RGS14, via its PDZ motif, binds directly to the PDZ2 domain of NHERF1 to stabilize the NHERF1–NPT2A complex, thus blocking PTH and FGF23 actions and inhibiting phosphate uptake. ERK, extracellular signal–regulated kinase; FGF23, fibroblast growth factor 23; NHERF1, sodium hydrogen exchanger regulatory factor-1; NPT2A, sodium phosphate cotransporter 2a; PTH, parathyroid hormone; RGS14, regulator of G protein signaling 14; SGK1, serum and glucocorticoid-regulated kinase 1.

Article Snippet: Recombinant human Arg 179 Gln-FGF23 25–251 (referred to as FGF23), which is resistant to furin cleavage and inactivation, was obtained from R&D Systems (catalog no.: 2604-FG-025).

Techniques: Binding Assay, Membrane, Blocking Assay