Cinacalcet corrects hypercalcemia in mice with an inactivating Gα11 mutation
Sarah A. Howles, Fadil M. Hannan, Caroline M. Gorvin, Sian E. Piret, Anju Paudyal, Michelle Stewart, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker
Sarah A. Howles, Fadil M. Hannan, Caroline M. Gorvin, Sian E. Piret, Anju Paudyal, Michelle Stewart, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker
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Research Article Endocrinology Metabolism

Cinacalcet corrects hypercalcemia in mice with an inactivating Gα11 mutation

Abstract

Loss-of-function mutations of GNA11, which encodes G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in familial hypocalciuric hypercalcemia type 2 (FHH2). FHH2 is characterized by hypercalcemia, inappropriately normal or raised parathyroid hormone (PTH) concentrations, and normal or low urinary calcium excretion. A mouse model for FHH2 that would facilitate investigations of the in vivo role of Gα11 and the evaluation of calcimimetic drugs, which are CaSR allosteric activators, is not available. We therefore screened DNA from > 10,000 mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for GNA11 mutations and identified a Gα11 variant, Asp195Gly (D195G), which downregulated CaSR-mediated intracellular calcium signaling in vitro, consistent with it being a loss-of-function mutation. Treatment with the calcimimetic cinacalcet rectified these signaling responses. In vivo studies showed mutant heterozygous (Gna11+/195G) and homozygous (Gna11195G/195G) mice to be hypercalcemic with normal or increased plasma PTH concentrations and normal urinary calcium excretion. Cinacalcet (30mg/kg orally) significantly reduced plasma albumin–adjusted calcium and PTH concentrations in Gna11+/195G and Gna11195G/195G mice. Thus, our studies have established a mouse model with a germline loss-of-function Gα11 mutation that is representative for FHH2 in humans and demonstrated that cinacalcet can correct the associated abnormalities of plasma calcium and PTH.

Authors

Sarah A. Howles, Fadil M. Hannan, Caroline M. Gorvin, Sian E. Piret, Anju Paudyal, Michelle Stewart, Tertius A. Hough, M. Andrew Nesbit, Sara Wells, Stephen D.M. Brown, Roger D. Cox, Rajesh V. Thakker

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Figure 1

Structural characterization of the Asp195Gly Gα11 mutation.

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Structural characterization of the Asp195Gly Gα11 mutation. (A) Genomic ...
(A) Genomic organization of Gna11 showing the location of reported human familial hypocalciuric hypercalcemia type 2 (FHH2) mutations (black) (3, 10) and Gna11 variants identified in N-ethyl-N-nitrosourea– mutagenized (ENU-mutagenized) mice (red). The G-protein subunit α11 (Gα11) GTPase domain (encoded by exon 1, 5′ portion of exon 2, 3′portion of exon 4 and exons 5–7) 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 2 (L2) peptides. The GTPase domain contains 3 flexible regions, termed switch regions I–III (SwI–SwIII). The Asp195Gly mutation is located within the GTPase domain and between the switch I and II regions. (B) Multiple protein sequence alignment of Gα11 residues comprising a 13–amino acid region that links the β2 strand of the switch I region with the β3 strand of the switch II region. Conserved residues are shown in gray. The WT (Asp, D) and mutant (m) (Gly, G) residues are shown in red. (C) Homology model of the GDP-bound Gα11 protein. The Gα helical (blue) and GTPase (green) domains and bound GDP nucleotide (black) are shown. Switch regions I–III are shown in orange. Previously reported residues mutated in FHH2 patients (3, 10) are shown in yellow. The mutated Asp195 residue (red) is located in a 13–amino acid region (gray) and adjacent to the β2-β3 loop. (D) Close-up view the β2-β3 hairpin loop region of WT and mutant Gα11 proteins showing the structural effects of the Asp195Gly mutant on hydrogen bonds (broken lines) within the hairpin loop. The Asp195Gly Gα11 mutant is predicted to result in a loss of a polar contact (hydrogen bond) between the Asp195 side chain and the backbone of the Glu197 (E197) residue. The one-letter amino acid codes indicate the following: D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; I, isoleucine; L, leucine; M, methionine; N, asparagine; Q, glutamine; and T, threonine.