A G‐protein Subunit‐α11 Loss‐of‐Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

ABSTRACT Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous disorder with three variants, FHH1 to FHH3. FHH1 is caused by loss‐of‐function mutations of the calcium‐sensing receptor (CaSR), a G‐protein coupled receptor that predominantly signals via G‐protein subunit alpha‐11 (Gα11) to regulate calcium homeostasis. FHH2 is the result of loss‐of‐function mutations in Gα11, encoded by GNA11, and to date only two FHH2‐associated Gα11 missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss‐of‐function mutations of the adaptor protein‐2 σ‐subunit (AP2σ), which plays a pivotal role in clathrin‐mediated endocytosis. We describe a 65‐year‐old woman who had hypercalcemia with normal circulating parathyroid hormone concentrations and hypocalciuria, features consistent with FHH, but she did not have CaSR and AP2σ mutations. Mutational analysis of the GNA11 gene was therefore undertaken, using leucocyte DNA, and this identified a novel heterozygous GNA11 mutation (c.161C>T; p.Thr54Met). The effect of the Gα11 variant was assessed by homology modeling of the related Gαq protein and by measuring the CaSR‐mediated intracellular calcium (Ca2+ i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca2+ o) using flow cytometry. Three‐dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα11 helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild‐type and the mutant Gα11 in HEK293 cells stably expressing CaSR demonstrate that the Ca2+ i responses after stimulation with Ca2+ o of the mutant Met54 Gα11 led to a rightward shift of the concentration‐response curve with a significantly (p < 0.01) increased mean half‐maximal concentration (EC50) value of 3.88 mM (95% confidence interval [CI] 3.76–4.01 mM), when compared with the wild‐type EC50 of 2.94 mM (95% CI 2.81–3.07 mM) consistent with a loss‐of‐function. Thus, our studies have identified a third Gα11 mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα11 interdomain interface in CaSR signaling and Ca2+ o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).


Introduction
F amilial hypocalciuric hypercalcemia (FHH) is characterized by lifelong elevations of serum calcium concentrations in association with normal or mildly raised serum parathyroid hormone (PTH) concentrations in 80% of patients and low urinary calcium excretion (urinary calcium-to-creatinine clearance ratio <0.01) in 80% of patients. (1,2) FHH may be inherited as an autosomal dominant condition, and it is a genetically heterogeneous disorder with three recognized variants, FHH1-3. FHH1 (OMIM #145980) is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR), a G-protein coupled receptor (GPCR) (3) that initiates activation of the G-protein subunit aq/11 (Ga q/11 ) family, leading to enhancement of phospholipase C (PLC) activity (4) and elevation of inositol 1,4,5trisphosphate (IP 3

) with rapid increase in intracellular calcium
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(Ca 2þ i ) concentrations. (5,6) These signal transduction events allow the parathyroid CaSR to respond to small fluctuations in the prevailing extracellular calcium concentration ([Ca 2þ ] o ) by inducing alterations in PTH secretion through mechanisms that likely involve effects on PTH mRNA stability (7) and PTH granule exocytosis from the apical pole of parathyroid cells. (8) Moreover, the kidney CaSR is considered to influence urinary calcium excretion by modulating expression of claudin proteins that mediate the paracellular reabsorption of calcium in the renal thick ascending limb. (9,10) FHH2 (OMIM #145981) is the result of loss-of-function mutations in the G-protein subunit-a11 (Ga 11 ), encoded by GNA11, and to date only two FHH2-associated Ga 11 missense mutations have been reported (Fig. 1). (11) These two FHH2-causing Ga 11 mutations comprise a Leu135Gln missense substitution and an in-frame isoleucine deletion at codon 200 (Ile200del), which are located in the Ga-subunit helical and GTPase domains, respectively. (11) Both of these FHH2-causing Ga 11 mutations, which are predicted to disrupt G-protein activation, have been shown to impair CaSR signal transduction. (11) FHH3 (OMIM #600740) is caused by loss-of-function mutations of the adaptor protein-2 s-subunit (AP2s), encoded by the AP2S1 gene. AP2s has a pivotal role in clathrin-mediated endocytosis of GPCRs such as the CaSR, and to date more than 50 FHH3 patients with AP2s mutations, which are all missense mutations involving the Arg15 residue (Arg15Cys, Arg15His and Arg15Leu), have been reported. (12)(13)(14)(15)(16) Approximately 65% of FHH patients will have a CaSR mutation, 5% an AP2s mutation, <1% a Ga 11 mutation, and the remaining $30% of FHH patients are considered to have involvement of a genetic abnormality that remains to be identified. Here we report the identification of a novel Ga 11 mutation in a patient with FHH in whom CaSR and AP2s mutations had been previously excluded.

Case report
The patient, a 65-year-old woman of Indian origin, presented with poor mobility and recurrent falls. She underwent investigation and was found to have hypercalcemia (serum adjusted-calcium concentration ¼ 2.77 mmol/L, normal range ¼ 2.20-2.60 mmol/L) in association with normal serum concentrations of phosphate (0.85 mmol/L; normal range ¼ 0.70-1.40 mmol/L), creatinine (45 mmol/L; normal range ¼ 40-130 mmol/L), PTH (5.9 pmol/L; normal range ¼ 1.0-7.0 pmol/L), and serum 25-hydroxyvitamin D (52 nmol/L; normal >50 nmol/L). Urinary calcium-to-creatinine clearance ratio was low at 0.01 (normal >0.02). These findings are consistent with a diagnosis of FHH, although there appeared to be an absence of a family history of hypercalcemia, based on the patient not having knowledge of any relatives who suffered from symptomatic hypercalcemia and the relatives not being available for medical assessment. DNA sequence analyses of the CASR and AP2S1 genes had not identified any abnormalities. Informed consent was obtained for the study using protocols approved by the Multi-Centre Research Ethics Committee (UK) (MREC/02/2/93).
DNA sequence analysis DNA sequence analyses of GNA11 exons 1-7 and their adjacent splice sites (NM_002067) (Fig. 1) was performed using leucocyte DNA and gene-specific primers (Sigma-Aldrich, St. Louis, MO, USA), as previously reported. (11) Publicly accessible databases, including dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000 genomes (http://browser.1000genomes.org), the National Heart, Lung and Blood Institute (NHLBI) Exome Sequencing Project (http://evs.gs.washington.edu/EVS/, EVS data release ESP6500SI) representing the exomes of approximately 6500 individuals, and the Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org) representing exomes of 60,706 unrelated individuals, were examined for the presence of sequence variants, and any potential pathogenic sequence abnormality identified within the patient DNA was confirmed by restriction endonuclease analyses, as described. (12) Protein sequence alignment and three-dimensional modeling of Ga 11 structure Protein sequences of Ga 11 paralogs were aligned using ClustalOmega (http://www.ebi.ac.uk/Tools/msa/clustalo/). (17) Ga 11 three-dimensional modeling was undertaken using the is connected to the helical domain (encoded by the 3' portion of exon 2, exon 3, and 5' portion of exon 4) by the linker 1 (L1) and linker 2 (L2) peptides. The locations of the P-loop (P) (red line), three flexible switch regions (S1 to S3) (red line), and the interdomain interface (comprising portions of the a1, aF, a5, b1, P-loop, and L1 peptide motifs) (blue line) are shown below the GNA11 exons. The previously reported loss-of-function Leu135Gln and Ile200del mutations (11) are located in the helical and GTPase domains, respectively, whereas the Thr54Met mutation (bold), identified by this study, is located at the interdomain interface. Coding regions are shaded gray and untranslated regions are represented by open boxes.
reported three-dimensional structure of Ga q in complex with the small molecule inhibitor YM-254890 (Protein Data Bank accession no. 3AH8). (18) The Ga q protein, which shares 90% identity at the amino acid level with Ga 11 , (11) was used because crystal structures of Ga 11 are not available. Molecular modeling was performed using The PyMOL Molecular Graphics System (Version 1.2r3pre, Schr€ odinger, LL Pymol). (11) Cell culture and transfection Wild-type and mutant GNA11 (pBI-CMV2-GNA11) expression constructs were generated as described, (11) and transiently transfected into HEK293 cells stably expressing CaSR (HEK293-CaSR) (12) using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). The bidirectional pBI-CMV2 cloning vector was used because it facilitated the co-expression of Ga 11 and GFP, (11,12) and site-directed mutagenesis was used to generate the mutant GNA11 construct using the Quikchange Lightning Site-directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) and gene-specific primers (Sigma-Aldrich), as described. (19) Cells were maintained in DMEM-Glutamax media (Thermo-Fisher, Waltham, MA, USA) with 10% fetal bovine serum (Gibco, Thermo-Fisher) and 400 mg/mL geneticin (Thermo-Fisher) at 37°C, 5% CO 2 . Successful transfection was confirmed by visualizing GFP fluorescence using an Eclipse E400 fluorescence microscope with a Y-FL Epifluorescence attachment and a triband 4,6-diamidino-2-phenylindole-FITC-Rhodamine filter, and images captured using a DXM1200C digital camera and NIS Elements software (Nikon, Tokyo, Japan). (11)(12)(13) The expression of Ga 11 and CaSR proteins was also determined by Western blot analyses using anti-Ga 11 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-GFP (Santa Cruz), anticalnexin (Millipore, Billerica, MA, USA) or anti-CaSR (AbCam, Cambridge, UK) antibodies. The Western blots were visualized using an Immuno-Star Western C kit (Bio-Rad, Hercules, CA, USA) on a Bio-Rad Chemidoc XRSþ system. (11) Intracellular calcium measurements The Ca 2þ i responses of HEK293-CaSR cells expressing wild-type or mutant Ga 11 proteins were assessed by a flow cytometrybased assay, as reported. (11)(12)(13) In brief, HEK293-CaSR cells were cultured in T75 flasks and transiently transfected 24 hours later with 16 mg DNA. (11) Forty-eight hours after transfection, the cells were detached, resuspended in calcium (Ca 2þ )-and magnesium (Mg 2þ )-free Hanks' buffered saline solution (HBSS), and loaded with 1 mg/mL Indo-1-acetoxymethylester (Indo-1-AM) for 1 hour at 37°C. After removal of free dye, cells were resuspended in Ca 2þ -and Mg 2þ -free HBSS and maintained at 37°C. Transfected cells, in suspension, were stimulated by sequentially adding Ca 2þ to the Ca 2þ -and Mg 2þ -free HBSS to increase the [Ca 2þ ] o in a stepwise manner from 0 to 15 mM and then analyzed on a MoFlo modular flow cytometer (Beckman Coulter, Indianapolis, IN, USA) by simultaneous measurements of GFP expression (at 525 nm), Ca 2þ i -bound Indo-1AM (at 410 nm), and free Indo-1AM (ie, not bound to Ca 2þ i ) (at 485 nm), using a JDSU Xcyte UV laser (Coherent Radiation, Santa Clara, CA, USA), on each cell at each [Ca 2þ ] o , as described (11,12) . The peak mean fluorescence ratio of the Ca 2þ i transient response after each individual stimulus was measured using Cytomation Summit software (Beckman Coulter) and expressed as a normalized response, as described. (11,12)

Results
Identification of a novel Thr54Met Ga 11 mutation in an FHH proband DNA sequence analyses of the GNA11 coding regions and adjacent splice sites (Fig. 1) identified a heterozygous C-to-T transition at nucleotide c.161, in the FHH patient (Figure 2A). This C-to-T transition (ACG to ATG) resulted in a missense substitution, Thr54Met, of the Ga 11 protein (Fig. 2B). The sequence alteration also led to the gain of an NspI and loss of a BsiHKAI restriction endonuclease site (Fig. 2B), which were used to confirm the presence of the mutation in the patient (Fig. 2C, D). Bioinformatic analyses using SIFT and MutationTasting software (20,21) predicted the variant to be damaging and likely disease-causing (SIFT score 0, MutationTasting score 0.99). In addition, the absence of this DNA sequence abnormality in >6500 exomes from the NHLBI-ESP cohort and >60,700 exomes from the ExAC cohort, together with evolutionary conservation of the Thr54 residue in vertebrate Ga-subunit paralogs (Fig. 3A), indicated that the Thr54Met abnormality likely represented a pathogenic GNA11 mutation rather than a benign polymorphic variant.

Structural characterization of the Thr54Met Ga 11 mutant protein
The Thr54Met mutation is located within the Ga 11 a-1 helix ( Fig. 1 and Fig. 3A, B), which comprises part of the interface at which the GTPase and helical domains interact to bind GDP and GTP. (22) In contrast, the previously reported FHH2-causing Iledel200 and Leu135Gln mutations, (11) which affect the GTPase and helical domains of Ga 11 , respectively, are situated away from the guanine-nucleotide binding site ( Fig. 1 and Fig. 3B). The Thr54 Ga 11 residue is located next to the phosphate-binding loop (P-loop) (Fig. 3A), which is a highly conserved nucleotidebinding peptide motif that plays a critical role in binding GDP. (23)(24)(25) Three-dimensional homology modeling of the Ga 11 protein revealed the wild-type Thr54 residue to form polar contacts with the ribose and b-phosphate moieties of GDP within the interdomain interface (Fig. 3B, C) and to interact with the a-F helix of the helical domain, which also mediates GDP binding (Fig. 3C). (22) These findings are consistent with the reported role of the a-1 helix as a structural hub that mediates interactions between the GTPase and helical domains to ensure GDP binding, thereby maintaining the Ga-subunit in an inactive conformation. (26) The Ga 11 Met54 mutant is predicted to disrupt these interdomain contacts and alter GDP binding (Fig. 3C).

Functional characterization of the Thr54Met Ga 11 mutant protein
To determine the effects of the predicted changes in Ga 11 structure (Fig. 3B, C) on CaSR-mediated signaling, Ca 2þ i responses to alterations in [Ca 2þ ] o were assessed in HEK293-CaSR cells that were transiently transfected with either the pBI-CMV2 empty vector or pBI-CMV2 expressing the wild-type (Thr54) or mutant (Met54) Ga 11 proteins. The Ca 2þ i responses of cells expressing the Met54 Ga 11 mutant were also compared with cells transiently transfected with the reported FHH2associated Gln135 Ga 11 mutant protein. (11) Expression of CaSR, Ga 11 and GFP was confirmed by fluorescence microscopy and/or Western blot analyses (Fig. 4A, B). Calnexin was used as a loading control in Western blot analyses, and Ga 11 expression was demonstrated to be similar in cells transiently transfected with wild-type or mutant Ga 11 proteins and greater than that of cells transfected with the empty pBI-CMV2 vector (Fig. 4B). The Ca 2þ i responses in wild-type and mutant Ga 11 -expressing cells were shown to increase in a dose-dependent manner after stimulation with increasing concentrations of Ca 2þ o between 0-15 mM. However, exposure to a significantly greater [Ca 2þ ] o was required to achieve half-maximal (EC 50 ) Ca 2þ i responses for cells expressing either the Met54 or Gln135 mutant Ga 11 proteins compared with wild-type-expressing cells. (Fig. 4C, D). Thus, the Met54 or Gln135 mutant-expressing cells showed rightward shifts in the concentration-response curves, with significantly elevated mean EC 50 values (p < 0.01) of 3.88 mM (95% confidence interval [CI] 3.76-4.01 mM) and 3.65 mM (95% CI 3.57-3.74 mM), respectively, compared with 2.94 mM (95% CI 2.81-3.07 mM) for wild-type expressing cells and consistent with the Ga 11 mutants leading to an impairment of CaSR signal transduction (Fig. 4C, D). The Hill coefficients did not significantly differ between wild-type and mutant Ga 11 -expressing cells (Fig. 4E). However, cells expressing the Met54 mutant had significantly reduced maximal signaling responses compared with cells expressing either wild-type or Gln135 mutant Ga 11 proteins (p < 0.05) (Fig. 4F).

Discussion
Our studies have identified a novel heterozygous germline GNA11 mutation in a patient with FHH, which resulted in an impairment of Ca 2þ i signaling similar to the loss-of-function previously reported for the FHH2-associated Leu135Gln and Ile200del GNA11 mutations. (11) The Thr54Met mutation represents only the third loss-of-function GNA11 mutation to be reported, and thus these findings provide further support for a critical role of the Ga 11 protein in parathyroid gland function and Ca 2þ o homeostasis, and highlight the importance of GNA11 gene analyses in FHH patients that do not harbor CASR or AP2S1 mutations. The Thr54Met, Leu135Gln, and Ile200del loss-offunction GNA11 mutations are all associated with a mild FHH phenotype characterized by serum adjusted-calcium concentrations <2.80 mmol/L, and these clinical findings are in keeping with our in vitro studies that have shown FHH2-associated mutations to induce only minor disturbances of CaSR signal transduction. (11) Indeed, the FHH2 mutants were associated with around a 30% increase in the EC 50 values of HEK293-CaSR cells used in this study, whereas CaSR mutations leading to FHH1 generally cause a >50% increase in the EC 50 value. (3,27) The milder shift in the Ca 2þ o set point of cells expressing FHH2associated Ga 11 mutants indicates that the CaSR may promote Ca 2þ i signaling by Ga 11 -independent mechanisms, such as via the related Ga q protein. Indeed, reported studies that selectively ablated Ga q and/or Ga 11 in the parathyroid glands of mice have highlighted the importance of both of these G-proteins for CaSR function, (28,29) and in the setting of FHH1, CaSR mutations likely impair Ca 2þ i responses via both Ga 11 and Ga q , thus leading to a greater loss-of-function than Ga 11 mutations that cause FHH2.
The Ga 11 -subunit consists of a Ras-like GTPase domain that binds GDP and GTP, and a smaller helical domain that acts as a clasp to secure these bound guanine-nucleotides. (30) Threedimensional modeling indicated the Thr54Met mutation to be located at the interdomain interface, which represents a highly conserved and critical region containing the P-loop motif that binds GDP (23,24) and also facilitates interactions between the helical and GTPase domains that maintain Ga-subunits in an inactive GDP-bound conformation. (22) The Thr54Met mutation likely alters GDP binding, but in contrast to the other reported Ga 11 mutations (Arg60Cys, Arg60Leu, and Arg181Gln), (11,31,32) which are also located at the interdomain interface (Fig. 3B, C) and cause Ga 11 gain-of-function that is associated with the clinical disorder of autosomal dominant hypocalcemia type-2 (ADH2), the Thr54Met Ga 11 mutation causes loss-of-function and FHH2. Thus, it seems that mutations involving the Ga 11 interdomain interface may result in Ga 11 loss-of-function or gain-of-function. Crystal structures of Ga 11 proteins are not available to evaluate the structure-function effects of the Thr54Met mutation at the interdomain interface; however, the introduction of the mutant Met54 residue may sterically impair G-protein function, as highlighted by a previous crystallography study of a loss-of-function G-protein alpha-i (Ga i ) P-loop mutation, which revealed the mutant Ga i residue to sterically hinder conformational changes of the flexible "switch" regions during Ga-subunit activation. (33) Moreover, interdomain interface mutations that disrupt guanine-nucleotide binding may also result in an "empty-pocket" mutant Ga-subunit that exerts dominant-negative effects by binding and sequestering partner GPCRs. (34) These findings may also help to provide an explanation for the observed differences in the maximal signaling responses of the FHH2-causing Met54 and Gln135 Ga 11 mutants (Fig. 4F). Thus, the Met54 Ga 11 mutant, but not the Gln135 Ga 11 mutant, led to a significant reduction in the maximal signaling response of CaSR-expressing cells (Fig. 4F), even though both Ga 11 mutants increase the Ca 2þ o set point of CaSR-expressing cells to a similar degree, as illustrated by their EC 50 values (Fig. 4D). The maximal signaling response of a GPCR is influenced by the ability of the receptor to couple with its cognate G-protein, (35) and thus it is possible that the Met54 Ga 11 mutant impairs coupling and/or dissociation of Ga 11 from the CaSR by influencing guanine-nucleotide binding at the The helical (blue) and GTPase (green) domains are connected by the L1 and L2 peptides (gray). The GTPase domain contains the P-loop (pink), which binds GDP (black) at the interdomain interface (comprising parts of a1, a5, aF, P-loop, L1, and b1 peptide motifs). The reported Leu135Gln and Ile200del mutations (11) are shown in purple, and the Thr54Met mutation is shown in red. (C) Closeup views of the Ga 11 interdomain interface, which show the wild-type Thr54 residue, located in the GTPase domain, form polar contacts (hatched black lines) with GDP and the Arg181 residue (yellow) of the helical domain aF helix. Substitution of the wild-type Thr54 residue with the mutant Met54 residue (orange) is predicted to disrupt these polar contacts, thereby impairing interdomain interactions and the binding of GDP.
interdomain interface, (34) whereas the Gln135 Ga 11 mutant, which is located in the Ga 11 helical domain and not predicted to influence CaSR-Ga 11 coupling, may potentially diminish CaSR signal transduction by influencing the interaction of Ga 11 with downstream effectors. (36) In conclusion, we have identified a novel Ga 11 loss-of-function mutation, Thr54Met, that causes FHH2 and which provides new insights into the critical role of the Ga 11 interdomain interface in CaSR signaling.

Disclosures
All authors state that they have no conflicts of interest. i responses are shown as the mean AE SEM of 4 independent transfections. The FHH2-associated Ga 11 mutants (Met54 and Gln135) led to a rightward shift of the concentrationresponse curves (blue and red, respectively) when compared with WT Ga 11 (black), which harbors Thr and Leu residues at codons 54 and 135, respectively. (D) The Met54 and Gln135 mutants (shaded bars) were associated with significantly increased EC 50 values compared with cells expressing WT Ga 11 (black bar). (E) The Hill coefficients of the wild-type and mutant Ga 11 proteins were similar (ie, not significantly different). (F) The Met54 mutant was associated with a significantly reduced maximal signaling response compared with WT and mutant Gln135 Ga 11 proteins. Ã p < 0.05, ÃÃ p < 0.01.