pubmed.ncbi.nlm.nih.gov

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

️Mon Jan 01 2018

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

Caroline M Gorvin et al. Sci Signal. 2018.

Abstract

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through G_q/11 and G_i/o to stimulate cytosolic calcium (Ca²⁺_i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-function CASR mutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca²⁺_i responses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSR^R680G in HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca²⁺_i responses. Moreover, this gain of function in MAPK activity occurred independently of G_q/11 and G_i/o and was mediated instead by a noncanonical pathway involving β-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced β-arrestin signaling by disrupting a salt bridge formed between Arg⁶⁸⁰ and Glu⁷⁶⁷, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through β-arrestin and the importance of the Arg⁶⁸⁰-Glu⁷⁶⁷ salt bridge in mediating signaling bias.

PubMed Disclaimer

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

**Fig. 1. Identification of an R680G CaSR mutation in a family with autosomal dominant hypocalcemia type 1 (ADH1)**
(A) Pedigree of family with 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. (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 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 HEK293 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 2-3.5mM [Ca²⁺]_e. Data are shown as the mean±SEM from 4-7 transfections, and 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 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 (0.1mM) [Ca²⁺]_e responses and presented as mean±SEM of 4 transfections. *p<0.05, **p<0.01 ****p<0.0001 using 2-way ANOVA with Tukey’s multiple-comparisons 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 ERK1/2 (pERK1/2) in HEK293 cells expressing wild-type (WT) or ADH1-associated CaSR mutants (R680G or L173F). (B) Densitometric analysis of Western blot data in panel 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 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 shows mean±SEM values for N=4-20 independent transfections. **p<0.01, ***p<0.001, ****p<0.0001 for CaSR^R680G versus CaSR^WT; ^$$$p<0.001, ^$$$$p<0.0001 for CaSR^L173F versus CaSR^WT in panels B, D and F. ^§§p<0.01, ^§§§p<0.001 for NPS-2143-treated cells compared to respective untreated cells in panel H. Two-way ANOVA with Tukey’s multiple-comparisons test.

**Fig. 4. The CaSR R680G mutation does not affect G_q/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 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 Gα_q/11 inhibitors YM-254890 (YM) and UBO-QIC (UBO), on SRE reporter activity. (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 shows mean±SEM for 8-12 independent transfections. *p<0.05, **p<0.01 ****p<0.0001 for untreated cells expressing CaSR^WT versus untreated (black) or YM- or UBO-QIC–treated (blue) cells expressing CaSR^R690G in panels D and E. ^$p<0.05, ^$$p<0.01, ^$$$$p<0.0001 for untreated cells expressing CaSR^WT versus untreated (black), or YM- or UBO-QIC–treated (red) cells expressing CaSR^L173F in panels B, D and E (2-way ANOVA with Tukey’s multiple-comparisons test).

**Fig. 5. The CaSR R680G mutation does not affect G_i/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 assessment of 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 IC₅₀ with 95% confidence intervals 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 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 shows mean±SEM for 4-12 independent transfections. *p<0.05, ****p<0.0001 for CaSR^R680G compared to CaSR^WT in panels E-F. ^$p<0.05, ^$$$$p<0.0001 for CaSR^L173F compared to CaSR^WT in panels B, C, E and F (2-way ANOVA with Tukey’s multiple-comparisons test).

**Fig. 6. Increased MAPK responses in cells expressing CaSR^R680G involve a G-protein–independent, β-arrestin–dependent pathway**
(**A-B**) Western blot analysis of HEK293 cells expressing wild-type (WT) or ADH1-associated CaSR mutants (R680G or L173F) and treated with scrambled siRNA (-) or siRNAs targeting (A) β-arrestin1 (βarr1) or (B) β-arrestin2 (βarr2). Calnexin was used as a loading control. (**C-D**) [Ca²⁺]_e-induced SRE reporter responses in cells expressing CaSR^R680G and treated with a scrambled siRNA or with siRNAs targeting (C) βarr1or (D) βarr2 or a scrambled siRNA. (**E-F**) [Ca²⁺]_e-induced SRE reporter responses in cells expressing CaSR^L173F 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 the respective cells treated with scrambled siRNA using a 2-way ANOVA with Tukey’s multiple-comparisons test. ^$p<0.05 and ^$$p<0.0001 for scrambled vs siRNA for cells expressing wild-type (black) or R680G (blue) CaSR; *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001 for scrambled siRNA treatment of cells expressing wild-type CaSR vs mutant CaSR (blue) and scrambled siRNA treatment of cells expressing wild-type CaSR vs targeted (β-arrestin) siRNA treated mutant CaSR (black). Data is shown as mean±SEM for 8-16 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 bi-lobed venus flytrap domain (VFTD), seven transmembrane domains (TMDs 1-7) with extracellular loops 1–3 (ECL1–3) and intracellular loops 1–3 (ICL1–3), 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 transmembrane domains of mGluR1 derived from the published crystal structure (44) and a model of the CaSR transmembrane regions based on homology to mGluR1. TMDs 1-7 are numbered; e and i indicate the extracellular and cytoplasmic components of the plasma membrane, respectively. (C) Close-up view of the mGluR1 binding pocket for the negative allosteric modulator 4-fluoro-N-(4-(6-isopropylamino)pyrimidin-4-yl)thiazol-2-yl)-N-methylbenzamide (FITM) (magenta and purple molecule), and (D) the 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-B**) Western blot analysis of HEK293 cells expressing wild-type or engineered mutant forms of CaSR (E767R or E837R) and treated with siRNAs targeting (A) β-arrestin1 (βarr1) or (B) β-arrestin2 (βarr2). (**C-D**) [Ca²⁺]_e-induced SRE reporter responses in cells expressing CaSR^E767R or CaSR^E837R and treated with a scrambled siRNA or with siRNAs targeting (C) βarr1or (D) βarr2. (**E-F**) Western blot analysis of HEK293 cells expressing wild-type CaSR, E767R CaSR, or a double mutant (dm) form of CaSR incorporating both the R680E and E767R mutations and treated with a scrambled siRNA or siRNA targeting (E) βarr1 or (F) βarr2. These cells were used to assess SRE reporter activity following knockdown of βarr1 or βarr2. (**G-H**) [Ca²⁺]_e-induced SRE reporter responses in cells expressing the double mutant CaSR^R680E-E767R and treated with a scrambled siRNA or with siRNAs targeting (G) βarr1or (H) βarr2. Data shows mean±SEM for 8-16 independent transfections. *p<0.05, **p<0.001, ****p<0.0001 for E767R versus WT CaSR in panels C-D; ^$p<0.05, ^$$p<0.01, ^$$$$p<0.0001 for targeted versus scrambled siRNAs for wild-type (black) or E767R (blue) CaSR in panels C-D; ^$$p<0.01, ^$$$p<0.001, ^$$$$p<0.0001 for a comparison between Glu⁶⁸⁰-Arg⁷⁶⁷ or wild-type CaSR-expressing cells treated with targeted siRNA and respective cells treated with scrambled siRNA in panels G-H (2-way ANOVA with Tukey’s multiple-comparisons).

Cited by

The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases.
Hannan FM, Kallay E, Chang W, Brandi ML, Thakker RV. Hannan FM, et al. Nat Rev Endocrinol. 2018 Dec;15(1):33-51. doi: 10.1038/s41574-018-0115-0. Nat Rev Endocrinol. 2018. PMID: 30443043 Free PMC article. Review.
Heterogeneity of G protein activation by the calcium-sensing receptor.
Abid HA, Inoue A, Gorvin CM. Abid HA, et al. J Mol Endocrinol. 2021 Jun 21;67(2):41-53. doi: 10.1530/JME-21-0058. J Mol Endocrinol. 2021. PMID: 34077389 Free PMC article.
Calcium-sensing receptor signaling - How human disease informs biology.
Gorvin CM. Gorvin CM. Curr Opin Endocr Metab Res. 2021 Feb;16:10-28. doi: 10.1016/j.coemr.2020.06.007. Epub 2020 Jul 2. Curr Opin Endocr Metab Res. 2021. PMID: 34141952 Free PMC article.
Calcium-sensing receptor-mediated NLRP3 inflammasome activation in rheumatoid arthritis and autoinflammation.
Werner LE, Wagner U. Werner LE, et al. Front Physiol. 2023 Jan 6;13:1078569. doi: 10.3389/fphys.2022.1078569. eCollection 2022. Front Physiol. 2023. PMID: 36685206 Free PMC article. Review.
Biased Signaling and Allosteric Modulation at the FSHR.
Landomiel F, De Pascali F, Raynaud P, Jean-Alphonse F, Yvinec R, Pellissier LP, Bozon V, Bruneau G, Crépieux P, Poupon A, Reiter E. Landomiel F, et al. Front Endocrinol (Lausanne). 2019 Mar 13;10:148. doi: 10.3389/fendo.2019.00148. eCollection 2019. Front Endocrinol (Lausanne). 2019. PMID: 30930853 Free PMC article. Review.

References

1. Brown EM. Role of the calcium-sensing receptor in extracellular calcium homeostasis. Best practice & research. Clinical endocrinology & metabolism. 2013;27:333–343. - PubMed
1. Chang W, Chen TH, Pratt S, Shoback D. Amino acids in the second and third intracellular loops of the parathyroid Ca2+-sensing receptor mediate efficient coupling to phospholipase C. The Journal of biological chemistry. 2000;275:19955–19963. - PubMed
1. Goolam MA, Ward JH, Avlani VA, Leach K, Christopoulos A, Conigrave AD. Roles of intraloops-2 and -3 and the proximal C-terminus in signalling pathway selection from the human calcium-sensing receptor. FEBS letters. 2014;588:3340–3346. - PubMed
1. Hofer AM, Brown EM. Extracellular calcium sensing and signalling. Nature reviews Molecular cell biology. 2003;4:530–538. - PubMed
1. Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, Brown EM. Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol. 2001;280:F291–302. - PubMed

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