Research ArticleGPCR SIGNALING

A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin–biased signaling

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Science Signaling  20 Feb 2018:
Vol. 11, Issue 518, eaan3714
DOI: 10.1126/scisignal.aan3714
  • Fig. 1 Identification of an R680G CaSR mutation in a family with ADH1.

    (A) Pedigree of family with autosomal dominant hypocalcemia type 1 (ADH1). The proband (individual II-3) is indicated by an arrow. (B) A heterozygous C-to-G transition at nucleotide c.2038 was identified in the proband and his father by Sanger DNA sequencing and confirmed to cosegregate with hypocalcemia. WT, wild type. (C) This C-to-G transition changes a CGC codon to GGC and is predicted to result in a missense amino acid substitution from Arg to Gly at position 680 in the calcium-sensing receptor (CaSR) protein. (D) Multiple sequence alignment of residues surrounding the Arg680 (R) residue encompassing extracellular loop 1 (ECL1) and transmembrane domain 3 (TMD3). The Arg680 (R) residue, which is evolutionarily conserved, is located within TMD3, and the mutant Gly680 (G) residue is shown in red. Conserved residues are shaded in gray.

  • Fig. 2 The CaSR R680G mutation does not affect intracellular Ca2+ signaling.

    (A) Western blot analysis of human embryonic kidney (HEK) 293 cells expressing wild-type (WT) or ADH1-associated mutant (R680G and L173F) CaSRs. Calnexin is a loading control. (B) Intracellular Ca2+ (Ca2+i) responses to changes in extracellular Ca2+ concentration ([Ca2+]e) in cells expressing the indicated wild-type or mutant CaSRs in the absence or presence of the allosteric CaSR inhibitor NPS-2143. Inset shows magnification of the curves between 2 and 3.5 mM [Ca2+]e. Data are means ± SEM from four to seven transfections, and half-maximal effective concentration (EC50) values with 95% confidence intervals (CIs) are provided (F test). (C) Histogram showing EC50 values with 95% CIs for cells expressing wild-type or R680G or L173F mutant CaSRs in the absence or presence of NPS-2143. (D) Western blot analysis of cells expressing the indicated wild-type and mutant forms of CaSR and used for assessment of nuclear factor of activated T cell (NFAT) reporter responses. (E) [Ca2+]e-induced NFAT reporter responses of cells expressing wild-type or mutant CaSRs. Responses at each [Ca2+]e are shown as a fold change of basal (0.1 mM) [Ca2+]e responses and presented as means ± SEM of four transfections. *P < 0.05, **P < 0.01, and ****P < 0.0001 using two-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests versus cells expressing wild-type CaSR at each [Ca2+]e.

  • Fig. 3 The CaSR R680G mutation increases downstream MAPK signaling.

    (A) Western blot analysis showing Ca2+e-induced phosphorylation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) in HEK293 cells expressing wild-type or ADH1-associated CaSR mutants (R680G or L173F). (B) Densitometric analysis of Western blot data in (A). (C) Western blot analysis showing transgenic expression of the indicated forms of CaSR in cells used to assess Ca2+e-induced phosphorylation of ERK1/2 by AlphaScreen analysis. Calnexin is a loading control. (D) Ca2+e-induced ERK1/2 phosphorylation in CaSR-expressing cells as measured by AlphaScreen analysis, shown as the ratio of phosphorylated ERK1/2 (pERK) to total ERK. (E) Western blot analysis showing transgenic expression of the indicated forms of CaSR in cells used to assess Ca2+e-induced serum-response element (SRE) reporter activity. (F) Ca2+e-induced SRE reporter activity in CaSR-expressing cells. (G) Western blot analysis showing transgenic expression of CaSR in cells used to assess the effects of NPS-2143 on Ca2+e-induced SRE responses. (H) SRE reporter activity in CaSR-expressing cells in the absence or presence of the allosteric CaSR inhibitor. Data are means ± SEM values for n = 4 to 20 independent transfections. **P < 0.01, ***P < 0.001, and ****P < 0.0001 for CaSRR680G versus CaSRWT; $$$P < 0.001 and $$$$P < 0.0001 for CaSRL173F versus CaSRWT in (B), (D), and (F). §§P < 0.01 and §§§P < 0.001 for NPS-2143–treated cells compared to respective untreated cells in (H) (two-way ANOVA with Tukey’s multiple comparison test).

  • Fig. 4 The CaSR R680G mutation does not affect Gq/11-mediated signaling.

    (A) Western blot analysis of HEK293 cells expressing wild-type (WT) or ADH1-associated CaSR mutants (R680G or L173F). These cells were used for the assessment of IP1 responses. Calnexin is a loading control. (B) Ca2+e-induced IP1 fold change in cells expressing the indicated forms of CaSR. (C) Western blot analysis of cells used to assess the effect of the Gαq/11 inhibitors YM-254890 (YM) and UBO-QIC (UBO) on SRE reporter activity. Veh, vehicle. (D) [Ca2+]e-induced SRE reporter activity in cells expressing the indicated forms of CaSR in the presence or absence of YM. (E) [Ca2+]e-induced SRE reporter in cells expressing the indicated forms of CaSR in the presence or absence of UBO-QIC. Data are means ± SEM for 8 to 12 independent transfections. *P < 0.05, **P < 0.01, and ****P < 0.0001 for untreated cells expressing CaSRWT versus untreated (black) or YM- or UBO-QIC–treated (blue) cells expressing CaSRR680G in (D) and (E). $P < 0.05, $$P < 0.01, and $$$$P < 0.0001 for untreated cells expressing CaSRWT versus untreated (black) or YM- or UBO-QIC–treated (red) cells expressing CaSRL173F in (B), (D), and (E) (two-way ANOVA with Tukey’s multiple comparison test).

  • Fig. 5 The CaSR R680G mutation does not affect Gi/o-mediated signaling.

    (A) Western blot analysis of HEK293 cells expressing wild-type (WT) or ADH1-associated CaSR mutants (R680G or L173F). These cells were used for the assessment of cyclic adenosine monophosphate (cAMP) responses. Calnexin is a loading control. (B) Ca2+e-induced fold change in cAMP abundance in cells expressing the indicated forms of CaSR. (C) Histograms showing the cAMP half-maximal inhibitory concentration (IC50) with 95% CIs for cells expressing the indicated forms of CaSR. (D) Western blot analysis of cells expressing the indicated forms of CaSR in the presence of pertussis toxin (PTx) or vehicle (Veh). These cells were used to assess the effect of PTx on SRE reporter activity. (E) Fold change in [Ca2+]e-induced SRE reporter activity in cells expressing the indicated forms of CaSR in the absence or presence of PTx. (F) Histograms showing area under the curve (AUC) of SRE reporter responses in vehicle- or PTx-treated cells expressing the indicated forms of CaSR. Data are means ± SEM for 4 to 12 independent transfections. *P < 0.05, **P < 0.01, and ****P < 0.0001 for CaSRR680G compared to CaSRWT in (E) and (F). $P < 0.05 and $$$$P < 0.0001 for CaSRL173F compared to CaSRWT in (B), (C), (E), and (F) (two-way ANOVA with Tukey’s multiple comparison test).

  • Fig. 6 Increased MAPK responses in cells expressing CaSRR680G involve a G protein–independent, β-arrestin–dependent pathway.

    (A and B) Western blot analysis of HEK293 cells expressing wild-type (WT) or ADH1-associated CaSR mutants (R680G or L173F) and treated with scrambled small interfering RNA (siRNA) (−) or siRNAs targeting β-arrestin1 (βarr1) (A) or β-arrestin2 (βarr2) (B). Calnexin was used as a loading control. (C and D) [Ca2+]e-induced SRE reporter responses in cells expressing CaSRR680G and treated with a scrambled siRNA or with siRNAs targeting βarr1 (C) or βarr2 (D) or a scrambled siRNA. (E and F) [Ca2+]e-induced SRE reporter responses in cells expressing CaSRL173F and treated with siRNAs targeting βarr1 (E) or βarr2 (F) or a scrambled siRNA. The responses of cells treated with siRNAs targeting β-arrestin were compared to those of the respective cells treated with scrambled siRNA using a two-way ANOVA with Tukey’s multiple comparison test. $P < 0.05 and $$P < 0.0001 for scrambled versus siRNA for cells expressing CaSRWT (black) or CaSRR680G (blue); *P < 0.05, **P < 0.01, ***P < 0.001, and ***P < 0.0001 for scrambled siRNA–treated cells expressing CaSRWT versus mutant CaSR (blue) and scrambled siRNA–treated cells expressing CaSRWT versus targeted (β-arrestin) siRNA-treated mutant CaSR (black). Data are means ± SEM for 8 to 16 independent transfections.

  • Fig. 7 The Arg680 residue of CaSR forms a salt bridge with either Glu767 or Glu837.

    (A) Schematic diagram of a CaSR monomer showing the extracellular bilobed venus flytrap domain (VFTD), seven TMDs (TMD1 to TMD7) with ECL1 to ECL3 and intracellular loops 1 to 3 (ICL1 to ICL3), and the cytoplasmic domain. The locations of Arg680 in TMD3 (R680, blue), Glu767 in ECL2 (E767, red), and Glu837 in TMD7 (E837, magenta) are indicated. (B) Ribbon diagram showing the TMDs of metabotropic glutamate receptor 1 (mGluR1) derived from the published crystal structure (44) and a model of the CaSR transmembrane regions based on homology to mGluR1. TMD1 to TMD7 are numbered; “e” and “i” indicate the extracellular and intracellular aspects of the plasma membrane, respectively. (C) Close-up view of the mGluR1 binding pocket for the negative allosteric modulator FITM [4-fluoro-N-(4-(6-isopropylamino)pyrimidin-4-yl)thiazol-2-yl)-N-methylbenzamide] (magenta and purple molecule). (D) Corresponding region in the CaSR. Distances between selected atoms are indicated with dashed lines.

  • Fig. 8 Disruption of the Arg680-Glu767 salt bridge leads to increased β-arrestin–mediated MAPK signaling.

    (A and B) Western blot analysis of HEK293 cells expressing wild-type (WT) or engineered mutant forms of CaSR (E767R or E837R) and treated with siRNAs targeting βarr1 (A) or βarr2 (B). (C and D) [Ca2+]e-induced SRE reporter responses in cells expressing CaSRE767R or CaSRE837R and treated with a scrambled siRNA or with siRNAs targeting βarr1 (C) or βarr2 (D). (E and F) Western blot analysis of HEK293 cells expressing CaSRWT, CaSRE767R, or a double-mutant form of CaSR incorporating both the R680E and E767R mutations and treated with a scrambled siRNA or siRNA targeting βarr1 (E) or βarr2 (F). These cells were used to assess SRE reporter activity after knockdown of βarr1 or βarr2. (G and H) [Ca2+]e-induced SRE reporter responses in cells expressing the double mutant CaSRR680E+E767R and treated with a scrambled siRNA or with siRNAs targeting βarr1 (G) or βarr2 (H). Data are means ± SEM for 8 to 16 independent transfections. *P < 0.05, **P < 0.001, and ****P < 0.0001 for CaSRE767R versus CaSRWT in (C) and (D); $P < 0.05, $$P < 0.01, $$$P < 0.001, and $$$$P < 0.0001 for targeted versus scrambled siRNAs for CaSRWT (black) or CaSRE767R (blue) in (C) and (D); $$P < 0.01 and $$$$P < 0.0001 for a comparison between Glu680-Arg767 or CaSRWT-expressing cells treated with targeted siRNA and respective cells treated with scrambled siRNA in (G) and (H) (two-way ANOVA with Tukey’s multiple comparison test).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/518/eaan3714/DC1

    Fig. S1. Plasma membrane and cytoplasmic CaSR in HEK293 cells.

    Fig. S2. Abundance of CaSR in plasma membrane fractions.

    Fig. S3. ERK phosphorylation in the ADH1-associated R680G and L173F mutant CaSRs used for densitometry analysis.

    Fig. S4. siRNA-mediated knockdown of β-arrestin1 and β-arrestin2.

    Fig. S5. Effect of NPS-2143 on β-arrestin–mediated MAPK signaling after stimulation with 10 mM [Ca2+]e.

    Fig. S6. Effect of the engineered mutants E767R and E837R CaSRs on MAPK signaling.

    Fig. S7. ERK phosphorylation in engineered E767R and E837R CaSR mutants used for densitometry analysis.

    Fig. S8. Analysis of the CaSR Glu837 residue by homology modeling using the structure of mGluR1.

    Fig. S9. Western blots to assess ERK phosphorylation in the engineered Glu680-Arg767 double CaSR mutant used for densitometry analysis.

    Table S1. Clinical and biochemical findings in the parents and proband with the R680G CaSR mutation.

  • Supplementary Materials for:

    A calcium-sensing receptor mutation causing hypocalcemia disrupts a transmembrane salt bridge to activate β-arrestin–biased signaling

    Caroline M. Gorvin, Valerie N. Babinsky, Tomas Malinauskas, Peter H. Nissen, Anders J. Schou, Aylin C. Hanyaloglu, Christian Siebold, E. Yvonne Jones, Fadil M. Hannan,* Rajesh V. Thakker*

    *Corresponding author. Email: rajesh.thakker{at}ndm.ox.ac.uk (R.V.T.); fadil.hannan{at}liverpool.ac.uk (F.M.H.)

    This PDF file includes:

    • Fig. S1. Plasma membrane and cytoplasmic CaSR in HEK293 cells.
    • Fig. S2. Abundance of CaSR in plasma membrane fractions.
    • Fig. S3. ERK phosphorylation in the ADH1-associated R680G and L173F mutant CaSRs used for densitometry analysis.
    • Fig. S4. siRNA-mediated knockdown of β-arrestin1 and β-arrestin2.
    • Fig. S5. Effect of NPS-2143 on β-arrestin–mediated MAPK signaling after stimulation with 10 mM [Ca2+]e.
    • Fig. S6. Effect of the engineered mutants E767R and E837R CaSRs on MAPK signaling.
    • Fig. S7. ERK phosphorylation in engineered E767R and E837R CaSR mutants used for densitometry analysis.
    • Fig. S8. Analysis of the CaSR Glu837 residue by homology modeling using the structure of mGluR1.
    • Fig. S9. Western blots to assess ERK phosphorylation in the engineered Glu680-Arg767 double CaSR mutant used for densitometry analysis.
    • Table S1. Clinical and biochemical findings in the parents and proband with the R680G CaSR mutation.

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    © 2018 American Association for the Advancement of Science

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