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Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors
Kelly L. Roszko, … , Thomas Gardella, Michael Mannstadt
Kelly L. Roszko, … , Thomas Gardella, Michael Mannstadt
Published February 9, 2017
Citation Information: JCI Insight. 2017;2(3):e91079. https://doi.org/10.1172/jci.insight.91079.
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Research Article Endocrinology

Knockin mouse with mutant Gα11 mimics human inherited hypocalcemia and is rescued by pharmacologic inhibitors

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Abstract

Heterotrimeric G proteins play critical roles in transducing extracellular signals generated by 7-transmembrane domain receptors. Somatic gain-of-function mutations in G protein α subunits are associated with a variety of diseases. Recently, we identified gain-of-function mutations in Gα11 in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated human ADH2. They also exhibited low bone mineral density and increased skin pigmentation. Treatment with NPS 2143, a negative allosteric modulator of the calcium-sensing receptor (CASR), increased PTH and calcium concentrations in WT and mutant mice, suggesting that the gain-of-function effect of GNA11R6OC is partly dependent on coupling to the CASR. Treatment with the Gα11/q-specific inhibitor YM-254890 increased blood calcium in heterozygous but not in homozygous GNA11R60C mice, consistent with published crystal structure data showing that Arg60 forms a critical contact with YM-254890. This animal model of ADH2 provides insights into molecular mechanism of this G protein–related disease and potential paths toward new lines of therapy.

Authors

Kelly L. Roszko, Ruiye Bi, Caroline M. Gorvin, Hans Bräuner-Osborne, Xiao-Feng Xiong, Asuka Inoue, Rajesh V. Thakker, Kristian Strømgaard, Thomas Gardella, Michael Mannstadt

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Figure 8

YM-254890 fully inhibited parathyroid hormone–induced (PTH-induced) stimulation of Ca2+i signaling via the PTH1 receptor in HEK cells transfected with GNA11WT but not with GNA11R6OC.

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YM-254890 fully inhibited parathyroid hormone–induced (PTH-induced) stim...
Fura-2–treated HEK-293–derived cells in which the genes encoding the endogenous Gαq and Gα11 were knocked out were cotransfected with plasmids encoding either Gα11R60C (A) or Gα11WT (B), along with the PTHR1. Forty-eight hours after transfection, cells were pretreated for 45 minutes with Fura-2,AM at a concentration of 5 × 10–6 M, together with either DMSO (0.1%) or DMSO containing YM-254890 at the indicated concentration and then treated with vehicle (Veh.) or PTH(1-34) (1 × 10–7 M) and analyzed for changes in Ca2+i signaling: blue line, PTH + DMSO; green line, PTH + 1 × 10-8 M YM-254890; red line, PTH + 1 × 10-7 M YM-254890; purple line, PTH + 1 × 10–6 M YM-254890; orange line, PTH + 1 × 10–5 M YM-254890; aqua line, PTH + 1 × 10–4.5 M YM-254890; black line, Vehicle + DMSO. Ratiometric Fura-2 fluorescence was measured using an Envision plate reader. Data are means (± SEM) of duplicate wells from a single experiment; 2 other replicate experiments yielded comparable results. (C) Dose-response curves for YM-254890 generated by plotting the AUC for each PTH-induced response observed in the absence or presence of inhibitor in the experiments of panels A and B. (D) Model of Gαq in complex with YM-254890. The orientation on the left shows the close proximity of Arg60, mutated in ADH2, with YM-254890 (red), and the orientation on the right shows the close proximity of Arg183, at which more strongly activating oncogenic mutations occur, with bound guanine nucleotide GDP (cyan). The side chain nitrogens of Arg60 and Arg180 are colored blue; the RAS and helical portions of the G protein are shaded dark and light gray, respectively, and helix-5 of the RAS domain, which participates in GPCR interaction, is shaded aqua. The model was prepared using the x-ray coordinate file PDB.3AH8 (23).

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