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A G-protein Subunit-?11 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 (G?11 ) to regulate calcium homeostasis. FHH2 is the result of loss-of-function mutations in G?11 , encoded by GNA11, and to date only two FHH2-associated G?11 missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss-of-function mutations of the adaptor protein-2 ?-subunit (AP2?), 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 AP2? 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 G?11 variant was assessed by homology modeling of the related G?q protein and by measuring the CaSR-mediated intracellular calcium (Ca(2+) i ) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca(2+) o ) using flow cytometry. Three-dimensional modeling revealed the Thr54Met mutation to be located at the interface between the G?11 helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild-type and the mutant G?11 in HEK293 cells stably expressing CaSR demonstrate that the Ca(2+) i responses after stimulation with Ca(2+) o of the mutant Met54 G?11 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 G?11 mutation (Thr54Met) causing FHH2 and reveal a critical role for the G?11 interdomain interface in CaSR signaling and Ca(2+) o homeostasis. © 2016 American Society for Bone and Mineral Research.

SUBMITTER: Gorvin CM

PROVIDER: S-EPMC4949650 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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

Gorvin Caroline M CM Cranston Treena T Hannan Fadil M FM Rust Nigel N Qureshi Asjid A Nesbit M Andrew MA Thakker Rajesh V RV

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20160206 6

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 ...[more]

PMID: 26729423

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Project description:G-protein subunit ?-11 (G?11 ) 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 G?11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous G?11 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 G?11 , 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 G?11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 G?11 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 G?11 cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 G?11 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 G?11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel G?11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the G?11 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.

Dataset Information

A G-protein Subunit-?11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2).

Publications

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

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