Research ArticleG Protein Signaling

The G protein α subunit variant XLαs promotes inositol 1,4,5-trisphosphate signaling and mediates the renal actions of parathyroid hormone in vivo

See allHide authors and affiliations

Science Signaling  25 Aug 2015:
Vol. 8, Issue 391, pp. ra84
DOI: 10.1126/scisignal.aaa9953
  • Fig. 1 XLαs is located in the renal proximal tubules and distal tubules at early postnatal stages.

    (A) Immunofluorescence staining of XLαs in kidneys taken from P2 wild-type (WT) and XLKO (KO) mice. XLαs is shown in red, phalloidin (to stain F-actin) is in green, and nuclei are in blue. Asterisks indicate glomeruli. Scale bars, 20 μm. (B) Immunofluorescence staining of XLαs and the Ca2+-binding protein calbindin D-28K in kidneys taken from P2 WT and XLKO mice. XLαs is shown in red, calbindin D-28K is shown in green, and nuclei are in blue. Arrows indicate distal tubules that are positive for calbindin D-28K staining. Scale bars, 40 μm. Images in all panels are representative of three independent experiments.

  • Fig. 2 P2 XLKO mice have increased amounts of Cyp27b1 and Cyp24a1 mRNAs and Npt2a protein.

    (A and B) Whole kidneys from WT and XLKO mice were processed and then analyzed by qRT-PCR to determine the relative amounts of Cyp27b1 (A) and Cyp24a1 (B) mRNAs. Data are means ± SEM of littermates from three independent litters (n = 12 mice per group). **P < 0.01, ***P < 0.001. (C) Renal brush border membranes isolated from WT and XLKO mice were subjected to Western blotting analysis to detect Npt2a protein. Villin was used as a loading control. Western blots are representative of three independent experiments. (D) Densitometric analysis of the relative abundance of Npt2a protein normalized to that of villin in WT and XLKO from the experiments represented in (C). Data are means ± SEM of seven mice from each group. **P < 0.01.

  • Fig. 3 P2 XLKO mice exhibit resistance to PTH.

    (A and B) P2 WT and XLKO littermate mice were injected subcutaneously with vehicle (10 to 12 mice per group) or PTH (50 nmol/kg; six mice per group). Two hours later, the mice were subjected to qRT-PCR analysis of the relative abundance of Cyp27b1 mRNA in P2 kidneys (A) and analysis of the serum concentrations of phosphate (B). Data are means ± SEM of four independent experiments. **P < 0.01. NS, not significant. (C) P2 WT and XLKO littermate mice were injected subcutaneously with vehicle or PTH (50 nmol/kg). Fifteen minutes later, renal brush border membranes were isolated and subjected to Western blotting analysis of Npt2a protein. Villin was used as a loading control. Western blots are representative of four independent experiments. (D) Densitometric analysis of the relative abundance of Npt2a protein normalized to that of villin in WT and XLKO mice from the experiments represented in (C). Data are means ± SEM of six or seven mice from each group. *P < 0.05, **P < 0.01. (E) Extent of the PTH-induced reduction in Npt2a abundance in the indicated WT and XLKO mice relative to that in vehicle-treated mice. Data are means ± SEM of three independent experiments. *P < 0.05.

  • Fig. 4 s abundance in proximal tubules and PTH-induced concentrations of urinary cAMP are increased in P2 XLKO mice.

    (A) P2 XLKO and WT littermate mice were injected subcutaneously with vehicle or PTH (50 nmol/kg). Fifteen minutes later, the concentrations of cAMP in the urine of the mice were determined and expressed relative to the amounts of urinary creatinine. Data are means ± SEM of six to nine mice per group from three independent experiments. *P < 0.05; ***P < 0.001. (B) Whole kidneys from P2 WT and XLKO mice were subjected to qRT-PCR analysis to determine the relative amounts of Gαs mRNA. Data are means ± SEM of 12 mice per group from four independent experiments. **P < 0.01. (C) Renal brush border membranes isolated from WT and XLKO mice were subjected to Western blotting analysis with an antibody that recognizes both Gαs and XLαs, as indicated. Villin was used as a loading control. (D) Densitometric analysis of the relative abundance of Gαs protein normalized to that of villin in WT and XLKO mice from the experiments represented in (C). Data are means ± SEM of four mice from each group. **P < 0.01.

  • Fig. 5 Basal and PTH-stimulated Gq/11 signaling is suppressed in P2 XLKO mice.

    (A) Proximal tubule–enriched renal cortices isolated from P2 WT and XLKO littermate mice were left untreated or were treated with the indicated concentrations of PTH for 30 min. IP1 concentrations were then determined as described in Materials and Methods. Data are means ± SEM of eight mice per group from two independent experiments. (B) Proximal tubule–enriched cortices isolated from P2 WT or XLKO mice were left untreated or were treated with PTH for 30 min. Samples were then subjected to fractionation, and the indicated membrane and cytosolic fractions were subjected to Western blotting analysis with antibodies specific for the indicated proteins. Western blots are representative of three independent experiments that combined samples from four to five pups of each genotype. (C to E) Densitometric analysis of the relative abundances of PKCδ (C), PKCα (D), and PKCζ (E) proteins in the membrane and cytosolic fractions of the indicated mice from the experiments represented in (B). Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Transgenic overexpression of XLαs specifically in proximal tubules rescues the phenotype of P2 XLKO mice.

    (A) Whole kidneys from P2 WT and rptXLαs (Tg) littermate mice were analyzed by qRT-PCR to determine the relative abundance of XLαs mRNA. Data are means ± SEM of four independent experiments. (B) Serum phosphate concentrations in P2 WT, XLKO, rptXLαs (Tg), and XLKO:rptXLαs (Tg + KO) littermate mice. Data are means ± SEM of 12 to 15 mice per group from seven litters. (C to E) Kidneys from the indicated P2 littermate mice were subjected to qRT-PCR analysis to determine the relative abundances of Cyp27b1 (C), Cyp24a1 (D), and Kid1 (E) mRNAs. Data are means ± SEM of six mice per group from three litters. (F) Kidneys from the indicated P2 littermate mice were subjected to subcellular fractionation and Western blotting analysis with antibodies against the indicated proteins. Western blots are representative of three independent experiments. (G to I) Densitometric analysis of the relative abundances of PKCδ (G), PKCα (H), and PKCζ (I) proteins in the membrane and cytosolic fractions of the indicated mice from the experiments represented in (F). Data are means ± SEM of four to five mice per group combined from three independent experiments.

  • Fig. 7 Overexpression of XLαs induces basal and agonist-stimulated generation of IP3.

    (A) Proximal tubule–enriched cortices isolated from P2 WT and rptXLαs (Tg) littermate mice were left untreated or were treated with the indicated concentrations of PTH before being analyzed to determine the amounts of IP1. Data are means ± SEM of eight mice per group from three independent experiments. (B) HEK 293 cells transfected with control plasmid (pcDNA) or with increasing concentrations of plasmid encoding XLαs were analyzed to determine their IP1 concentrations under unstimulated (basal) conditions. Data are means ± SEM of eight experiments. #P < 0.05 between group 1 and group 2, &P < 0.05 between group 2 and groups 3 and 4, $P < 0.05 between group 5 and groups 3 and 4. (C) HEK 293 cells stably expressing PTHR were transfected with control plasmid (pcDNA) or with plasmid encoding XLαs and then were left untreated or were treated with the indicated concentrations of PTH for 30 min. Samples were analyzed to determine the amounts of IP1. Data are means ± SEM of eight samples per group from four experiments. Inset: The same data presented with a different y-axis scale for the lower concentrations of PTH. (D) HEK 293 cells transfected with control plasmid or plasmid encoding XLαs were left untreated or were treated with thrombin (1 U/ml) for 30 min before being subjected to IP1 analysis. Data are means ± SEM of eight samples per group from four experiments. (E and F) HEK 293 cells transfected with control plasmid or with plasmids encoding WT XLαs or the XLαs-R543H mutant were analyzed to determine the relative amounts of cAMP (E) and IP1 (F) under basal conditions. Data are means ± SEM of 12 samples per group from four independent experiments. #P < 0.05 when comparing between pcDNA-transfected cells and cells expressing WT XLαs, &P < 0.05 when comparing between cells expressing WT XLαs and cells expressing XLαs-R543H. (G and H) WT HEK 293 cells (G) and Gαq/11−/− HEK 293 cells (H) were transiently transfected with control plasmid (pcDNA) or with plasmids encoding XLαs, Gαq, or Gα11, as indicated. The cells were then left unstimulated or were stimulated with thrombin (1 U/ml) for 30 min before being analyzed to determine their amounts of IP1. Data are means ± SEM of 12 samples per group from four independent experiments. **P < 0.01, ***P < 0.001.

  • Table 1 Comparison of serum biochemistries.

    Analysis of the serum concentrations of phosphate, Ca2+, PTH, 1,25(OH)2D, and FGF23 in P2 wild-type (WT) and XLKO mice. Data are means ± SEM of 18 to 27 (WT) or 14 to 20 (XLKO) mice combined from four to seven litters. *P < 0.05, **P < 0.01, ***P < 0.001.

    WTXLKO
    Phosphate (mg/dl)10.32 ± 0.2411.42 ± 0.41*
    Ca2+ (mM)1.48 ± 0.011.42 ± 0.02*
    PTH (pg/ml)65.25 ± 1.34138.10 ± 4.99*
    1,25(OH)2D (pM)183.42 ± 4.64378.02 ± 6.86***
    FGF23 (pg/ml)524.11 ± 11.64158.86 ± 9.29***

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/8/391/ra84/DC1

      Fig. S1. XLαs is located in the kidney at early postnatal stages, and XLKO mice do not have defects in renal structure.

      Fig. S2. The abundances of Npt2a protein and Slc20a2 mRNA are similar between P2 wild-type and XLKO mice.

      Fig. S3. Increased renal Gαs abundance in P2 XLKO kidneys.

      Fig. S4. The abundances of PKCβII, PKCθ, phosphorylated ERK1/2, and phosphorylated substrates of PKA are not decreased in the proximal tubules of P2 XLKO mice.

      Table. S1. Sequences of the primer pairs used in qRT-PCR assays.

    • Supplementary Materials for:

      The G protein α subunit variant XLαs promotes inositol 1,4,5- trisphosphate signaling and mediates the renal actions of parathyroid hormone in vivo

      Qing He, Yan Zhu, Braden A. Corbin, Antonius Plagge, Murat Bastepe*

      *Corresponding author. E-mail: bastepe{at}helix.mgh.harvard.edu

      This PDF file includes:

      • Fig. S1. XLαs is located in the kidney at early postnatal stages, and XLKO mice do not have defects in renal structure.
      • Fig. S2. The abundances of Npt2a protein and Slc20a2 mRNA are similar between P2 wild-type and XLKO mice.
      • Fig. S3. Increased renal Gαs abundance in P2 XLKO kidneys.
      • Fig. S4. The abundances of PKCβII, PKCθ, phosphorylated ERK1/2, and phosphorylated substrates of PKA are not decreased in the proximal tubules of P2 XLKO mice.
      • Table. S1. Sequences of the primer pairs used in qRT-PCR assays.

      [Download PDF]

      Technical Details

      Format: Adobe Acrobat PDF

      Size: 912 KB


      Citation: Q. He, Y. Zhu, B. A. Corbin, A. Plagge, M. Bastepe, The G protein α subunit variant XLαs promotes inositol 1,4,5-trisphosphate signaling and mediates the renal actions of parathyroid hormone in vivo. Sci. Signal. 8, ra84 (2015).

      © 2015 American Association for the Advancement of Science

    Navigate This Article