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

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 1

GNA11R6OC mutant increases Ca2+i responses, which can be normalized by the calcilytic NPS 2143.

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GNA11R6OC mutant increases Ca2+i responses, which can be normalized by t...
(A) Immunofluorescent images of HEK-CASR cells transiently transfected with WT (Arg60) or mutant (Cys60) pBI-CMV2-GNA11 constructs. All constructs had similar expression of GFP. Scale bars: 10μm. (B) Western blot analysis of lysates from HEK-CASR cells used for flow cytometry experiments. All cells expressed Gα11 and GFP at similar levels. Calnexin was used as a housekeeping protein. (C) Concentration-response curves showing normalized Ca2+i responses to increasing doses of [Ca2+]o in HEK-CASR cells expressing WT or GNA11R60C constructs. Mutant (Cys60) had a leftward shift in the concentration-response curve (blue, solid line, open circles) when compared with WT (Arg60; black, solid line, closed circles). Treatment of GNA11R60C expressing cells with increasing doses of the negative allosteric modulator NPS 2143 (red, dotted lines) led to a rightward shift in the dose response curves toward WT (Arg60) levels. (D) The GNA11R60C mutant (blue bar) was associated with reduced EC50 values compared with WT Gα11 (black bar). Treatment of GNA11R60C-expressing cells led to an increase in EC50 values, which was normalized to WT (Arg60) levels with a 20 nM dose. (E) The GNA11R60C mutant (blue bar) and NPS 2143–treated cells had similar maximal responses (Emax) to WT (Arg60) cells. (F) The GNA11R60C mutant (blue bar) and NPS 2143–treated cells had similar Hill coefficient values to WT (Arg60) cells. Data is expressed as mean ± SEM. **P < 0.02. Experiments were performed in 4 independent transfections.