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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.

Original publication

DOI

10.1002/jbmr.3241

Type

Journal article

Journal

J Bone Miner Res

Publication Date

18/08/2017

Keywords

CELL/TISSUE SIGNALING, DISORDERS OF CALCIUM/PHOSPHATE METABOLISM, ENDOCRINE PATHWAYS, PARATHYROID-RELATED DISORDERS, THERAPEUTICS