Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy

Abstract

Heterozygous germline gain-of-function mutations of G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) — which has a germline hypermorphic Gα11 mutation, Ile62Val — may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα11 mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.

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

“…The ERK protein represents a major component of the MAPK signaling cascade, which has previously been shown to be activated by PLC . We assessed the effect of the Gα 11 Phe220Ser mutation on ERK phosphorylation by measuring changes in pERK in response to increases in Ca 2+ e in WT and mutant Ser220 Gα 11 ‐expressing cells using the AlphaScreen assay, and also by assessing serum‐response element (SRE)‐mediated transcriptional activity, which is regulated by ERK signaling . The pERK responses of the Ser220 Gα 11 mutant were shown to be significantly reduced.…”

“…Cells were incubated in serum‐free media for 12 hours, followed by treatment of cells for 4 hours with 0.1mM to 10mM CaCl 2 . Cells were lysed and assays performed with Dual‐Glo luciferase (Promega, Madison, WI, USA) on a Veritas Luminometer (Promega), as described . Luciferase:renilla ratios were expressed as fold‐changes relative to responses at basal CaCl 2 concentrations.…”

“…Luciferase:renilla ratios were expressed as fold‐changes relative to responses at basal CaCl 2 concentrations. For studies with cinacalcet (Cambridge Bioscience, Cambridge, UK), the drug was added to cells, as described . All assays were performed using four biological replicates on up to three independent occasions.…”

“…Transfected cells were incubated in serum‐free media 12 hours prior to treatment of cells with 0.1mM to 10mM CaCl 2 . Cells were then lysed in Surefire lysis buffer, and AlphaScreen Surefire ERK assays (PerkinElmer, Beaconsfield, UK) measuring phosphorylated and total proteins were performed, as described . The fluorescence signal in both assays was measured using the PHERAstar FS microplate reader (BMG Labtech) .…”

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

“…Mutations were introduced into the pEGFP-N1-CaSR WT construct by site-directed mutagenesis using the Quikchange Lightning Kit (Agilent Technologies) and gene-specific primers (SigmaAldrich) as described (46, 47), Engineered mutations were verified using dideoxynucleotide sequencing with the BigDye Terminator v3.1 cycle sequencing kit (Life Technologies) and an automated detection system (ABI3730 automated capillary sequencer; Applied Biosystems) as previously reported (48). Wild-type and mutant CaSR pEGFP-N1 constructs, and luciferase reporter constructs (pGL4.30-NFAT and pGL4.33-SRE, Promega) were transiently transfected into HEK293 cells using Lipofectamine 2000 (LifeTechnologies) 48 hours before experiments, as described (49). Single siRNAs targeted to β-arrestin1 (Catalog No: 6218S, Cell Signalling Technology) or β-arrestin2 (Catalog No: sc29208, SantaCruz Biotechnology), or scrambled siRNA (Catalog No: SR301839, Origene) were transfected 24 hours before experiments at a concentration of 100nM.…”