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Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss-of-Function Gα11 Mutation - PubMed

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Cinacalcet Rectifies Hypercalcemia in a Patient With Familial Hypocalciuric Hypercalcemia Type 2 (FHH2) Caused by a Germline Loss-of-Function Gα11 Mutation

Caroline M Gorvin et al. J Bone Miner Res. 2018 Jan.

Abstract

G-protein subunit α-11 (Gα11 ) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca2+i ) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα11 , which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα11 protein to impair CaSR-mediated Ca2+i and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα11 cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 Gα11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα11 hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Keywords: CELL/TISSUE SIGNALING; DISORDERS OF CALCIUM/PHOSPHATE METABOLISM; ENDOCRINE PATHWAYS; PARATHYROID-RELATED DISORDERS; THERAPEUTICS.

© 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

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Figures

Figure 1
Figure 1

Identification and structural assessment of a Phe220Ser Gα11 mutation in a family with familial hypocalciuric hypercalcemia type 2. (A) The family comprised of 5 affected and 3 unaffected members (top). The proband (individual II.1) is indicated by an arrow. A heterozygous T‐to‐C transition at nucleotide c.659, predicted to result in a missense substitution of the WT Phe to a mutant (m) Ser at codon 220 of the Gα11 protein, was identified in the proband and confirmed to cosegregate with hypercalcemia by Sanger DNA sequence analysis. (B) Multiple protein sequence alignment of residues comprising the β3‐α2 loop, α2‐helix, and α2‐β4 loop that form switch II (red box) of Gα11 orthologs (top) and Gα‐subunit paralogs (bottom). Conserved residues are shown in gray, and WT (Phe, F) and mutant (Ser, S) residues are shown in red. (C) Structural model showing that Phe220 of Gα11 is part of a hydrophobic cluster of amino acids (yellow) within switch II and the α3‐helix, which stabilize the region in a conformation that facilitates effector binding. (D) Close‐up view of Phe220 residue, within the switch II region of the GTPase domain of Gα11, in complex with phospholipase C‐β3 (brown space‐filling model), with directly interacting residues of the α2 and α3 helices colored blue.

Figure 2
Figure 2

Effect of the mutant Ser220 Gα11 on Ca2+ i and ERK signaling in HEK‐CaSR cells. (A) Ca2+ i responses to changes in [Ca2+]e of HEK‐CaSR cells expressing WT (Phe220) or mutant (Ser220) Gα11 proteins shown as the mean ± SE of 4 to 11 transfections. The Ser220 Gα11 mutant caused a rightward shift in the Ca2+ i concentration‐response curve with significantly increased EC50 value compared to WT cells (***p < 0.0001, F‐test). (B) [Ca2+]e‐induced NFAT reporter responses, which are activated by elevations of Ca2+ i, were assessed in HEK‐CaSR cells expressing WT or mutant Gα11 proteins. Responses at each [Ca2+]e are expressed as a fold‐change of basal [Ca2+]e responses, and shown as mean ± SE of 12 transfections. NFAT luciferase reporter activity increased in all cells in a concentration‐dependent manner, and was significantly reduced in Ser220 cells compared to WT cells. (C) Ca2+ e‐induced pERK responses of cells expressing WT or mutant Gα11 protein, expressed as a ratio to total ERK levels, and shown as mean ± SE of 8 transfections. (D) Ca2+ e‐induced SRE reporter responses of cells expressing WT or mutant Gα11 protein, expressed compared to basal SRE reporter activity, and shown as mean ± SE of 8 transfections. The Ser220 Gα11 mutant was associated with significantly decreased pERK and SRE reporter responses compared to WT. **p < 0.01 and ****p < 0.0001, by a 2‐way ANOVA with Tukey's multiple‐comparisons test.

Figure 3
Figure 3

Effect of Gα11 residue 220 hydrophobicity on PLC‐mediated signaling. (A) Histograms showing Ca2+ e‐induced Ca2+ i EC50 values with 95% confidence intervals (CIs) for cells expressing WT Phe220 or residue 220 mutant (m) Gα11 proteins from 6 to 12 transfections. Statistical analyses were performed using the F‐test. The Arg220 Gα11 mutant reduced the EC50 value, whereas the Glu220 and Ser220 Gα11 mutants elevated the EC50 value. (B) Ca2+ e‐induced NFAT reporter responses, which assess Ca2+ i signaling, of cells expressing WT or residue 220 mutant Gα11 proteins. Responses at each [Ca2+]e are expressed as a fold‐change of basal [Ca2+]e responses, and shown as mean ± SE of 4 to 12 transfections. NFAT luciferase reporter activity increased in all cells in a concentration‐dependent manner, however responses were significantly elevated in Arg220 Gα11 mutant, and reduced in Glu220 Gα11 mutant cells, compared to WT cells. (C) Ca2+ e‐induced pERK responses of cells expressing WT or mutant residue 220 Gα11 proteins, expressed as a ratio to total ERK levels. The Arg220 mutant was associated with significantly increased responses, and the Glu220 and Ser220 mutants were associated with decreased responses, when compared to WT. Data are shown as mean ± SE of 4 to 8 independent transfections. (D) Ca2+ e‐induced SRE reporter responses of cells expressing WT or mutant (m) residue 220 Gα11 proteins, expressed compared to basal SRE reporter activity. The engineered Arg220 mutant was associated with significantly increased, and the engineered Glu220 and FHH2‐associated Ser220 mutants resulted in decreased responses, when compared to WT. Data are shown as mean ± SE of 4 to 8 independent transfections. Statistical analyses performed using 2‐way ANOVA with Tukey's multiple‐comparisons test comparing WT with Gα11 mutants Ala220, Arg220, Glu220, and Ser220. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4
Figure 4

Effect of mutating residues within the Gα11 switch II‐α3 hydrophobic cluster on PLC‐mediated signaling. (A) Histograms showing Ca2+ e‐induced Ca2+ i EC50 values with 95% CI for cells expressing WT and alanine (Ala) Gα11 mutants (m) of residues 217, 223, and 263 Gα11 proteins from 4 to 8 independent transfections. Only the Ala217 Gα11 mutant increased the EC50 value, when compared to WT cells. Statistical analyses were performed using the F‐test. (B, C) Ca2+ e‐induced (B) NFAT and (C) SRE luciferase reporter responses of cells expressing WT or alanine (Ala) Gα11 mutants. Responses at each [Ca2+]e are expressed as a fold‐change of basal [Ca2+]e responses, and shown as mean ± SE of 4 to 8 transfections. NFAT and SRE reporter responses were significantly reduced in Ala217‐expressing cells compared to WT cells. (D) Histograms showing Ca2+ e‐induced Ca2+ i EC50 values with 95% CI for cells expressing WT and glutamic acid (Glu) Gα11 mutants (m) of residues 217, 223, and 263 from 4 to 8 independent transfections. All Glu Gα11 mutants increased the EC50 value, when compared to WT cells. Statistical analyses were performed using the F‐test. (E, F) Ca2+ e‐induced (E) NFAT and (F) SRE luciferase reporter responses of cells expressing WT or glutamic acid (Glu) Gα11 mutants. Responses at each [Ca2+]e are expressed as a fold‐change of basal [Ca2+]e responses, and shown as mean ± SE of 4 to 8 transfections. NFAT and SRE reporter responses were significantly reduced in cells expressing all 3 glutamic acid mutants compared to WT cells. Statistical analyses for B, C, E, and F were performed using 2‐way ANOVA with Tukey's multiple‐comparisons test comparing WT to mutant 217 (green), 223 (red), and 263 (gray) Gα11 proteins. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 5
Figure 5

Effect of cinacalcet on the loss‐of‐function and hypercalcemia caused by the Phe220Ser Gα11 mutation. (A) Effect of cinacalcet on Ca2+ i responses to changes in [Ca2+]e of HEK‐CaSR cells transfected with WT or Ser220 Gα11 mutant (m). The Ca2+ i responses to changes in [Ca2+]e are expressed as a percentage of the maximum normalized responses and shown as the mean ± SE of 6 to 12 independent transfections. The Ser220 Gα11 mutant led to a rightward shift in the concentration‐response curve. The addition of 30nM cinacalcet, a concentration previously shown to rectify the loss‐of‐function associated with the reported Leu135Gln Gα11 FHH2 mutant in vitro,10 had no effect on Ser220 responses. However, addition of 100nM cinacalcet rectified the rightward shift of the Ser220 Gα11 mutant, such that it was not different to WT responses. (B, C) Histograms showing (B) NFAT and (C) SRE luciferase reporter activity in response to 5mM Ca2+ e in HEK‐CaSR cells expressing WT or Ser220 mutant constructs, treated with vehicle or cinacalcet (CIN). Data are shown as mean ± SE of 4 independent transfections; 100nM cinacalcet was required to significantly increase NFAT and SRE reporter activity to WT levels. The responses of cells expressing WT or mutant Gα11 proteins were compared using the F‐test for A and 2‐way ANOVA with Tukey's multiple‐comparisons test for B and C. **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Effect of cinacalcet on the serum ionized calcium concentrations of the FHH2 proband (individual II.1, Fig. 1 A) with the Phe220Ser Gα11 mutation. Arrows indicate initiation of oral cinacalcet at 30 mg/day (CIN 30), at 60 mg/day (CIN 60), and its discontinuation. Cinacalcet treatment decreased serum concentrations of ionized calcium and PTH from 98 ng/L (pretreatment) to 58 ng/L (posttreatment) (normal range, 8 to 73 ng/L). Shaded area indicates normal range.

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