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

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.

Calcimimetic and calcilytic therapies for inherited disorders of the calcium‐sensing receptor signalling pathway

“…The effectiveness of cinacalcet in rectifying the FHH2‐associated loss‐of‐function abnormalities caused by the downstream Gα 11 protein has been assessed by in vitro and in vivo studies. Thus, recent in vitro studies have shown that cinacalcet improves the signalling responses of HEK293 cells stably expressing the CaS receptor (HEK–CaS receptor), which have been transiently transfected with FHH2‐associated Gα 11 mutants (Babinsky et al ., ; Gorvin et al ., ). Indeed, nanomolar doses of cinacalcet were demonstrated to rectify the impaired Ca 2+ i responses associated with loss‐of‐function Gα 11 mutations (Table ) (Babinsky et al ., ; Gorvin et al ., ).…”

“…Thus, recent in vitro studies have shown that cinacalcet improves the signalling responses of HEK293 cells stably expressing the CaS receptor (HEK–CaS receptor), which have been transiently transfected with FHH2‐associated Gα 11 mutants (Babinsky et al ., ; Gorvin et al ., ). Indeed, nanomolar doses of cinacalcet were demonstrated to rectify the impaired Ca 2+ i responses associated with loss‐of‐function Gα 11 mutations (Table ) (Babinsky et al ., ; Gorvin et al ., ). Moreover, siRNA knockdown studies showed that cinacalcet enhanced the signalling mediated by the FHH2 mutant Gα 11 proteins rather than by exerting indirect effects on endogenously expressed wild‐type Gα 11 proteins (Babinsky et al ., ).…”

“…A patient with FHH2 due to a heterozygous germline Phe 220 Ser Gα 11 mutation has been treated with cinacalcet (Table ). The patient had hypercalcaemia in association with headaches, constipation and pruritus and was initially commenced on cinacalcet 30 mg daily, which failed to normalize his elevated serum calcium concentrations over a period of 3 months (Gorvin et al ., ). However, the hypercalcaemia of this FHH2 patient did normalize following treatment with 60 mg daily of cinacalcet (Gorvin et al ., ).…”

“…The patient had hypercalcaemia in association with headaches, constipation and pruritus and was initially commenced on cinacalcet 30 mg daily, which failed to normalize his elevated serum calcium concentrations over a period of 3 months (Gorvin et al ., ). However, the hypercalcaemia of this FHH2 patient did normalize following treatment with 60 mg daily of cinacalcet (Gorvin et al ., ). These findings are in keeping with cellular studies, which showed that a higher (100 nM) cinacalcet concentration was required to rectify the loss of function of the Phe 220 Ser Gα 11 mutation in vitro compared with other FHH2‐causing mutations, which have responded to 10–40 nM concentrations of cinacalcet (Babinsky et al ., ; Gorvin et al ., ).…”

“…However, the response of FHH2‐associated mutant cells to cinacalcet treatment may be influenced by the location of the mutation within the Gα 11 protein. Thus, FHH2‐causing mutations located within regions of the Gα 11 GTPase domain involved in GPCR binding (Ile200del) or PLC coupling (Phe 220 Ser) have been shown to require higher (40–100 nM) cinacalcet concentrations for rectifying responses (Babinsky et al ., ; Gorvin et al ., ) than the FHH2‐causing Leu 135 Gln mutation, which is located in the guanine nucleotide‐stabilizing Gα 11 helical domain and which responded to treatment with 10 nM cinacalcet (Babinsky et al ., ).…”