A G-protein Subunit-11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)
A G-protein Subunit-11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)
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DOI：
10.1002/jbmr.2778
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发表时间：
2016-06-01
影响因子：
6.2
通讯作者：
Thakker, Rajesh V.
中科院分区：
文献类型：
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作者：
Gorvin, Caroline M.;Cranston, Treena;Thakker, Rajesh V.
家族性低钙高钙血症（FHH）是一种遗传异质性疾病，有三种变体，FHH1到FHH3。fh1是由钙敏感受体（CaSR）的功能缺失突变引起的，CaSR是一种G蛋白偶联受体，主要通过G蛋白亚基α -11 （G(11)）发出信号来调节钙稳态。FHH2是由GNA11编码的G（11）中功能缺失突变的结果，迄今为止仅报道了两个FHH2相关的G（11）错义突变（Leu135Gln和Ile200del）。FHH3是接头蛋白-2西格玛亚基（AP2 sigma）功能缺失突变的结果，AP2西格玛在网格蛋白介导的内吞作用中起关键作用。我们描述了一位65岁的女性，她患有高钙血症和正常的循环甲状旁腺激素浓度和低钙尿，特征与FHH一致，但她没有CaSR和AP2 sigma突变。因此，利用白细胞DNA对GNA11基因进行了突变分析，发现了一种新的杂合GNA11突变（c.161C>T; p.Thr54Met）。通过对相关G(q)蛋白的同源性建模，以及使用流式细胞术测量稳定表达CaSR的HEK293细胞对细胞外钙（Ca-o(2+)）改变的CaSR介导的细胞内钙（Ca-i(2+)）反应，来评估G（11）变异的影响。三维模型显示，Thr54Met突变位于G（11）螺旋结构域和GTPase结构域之间的界面，并可能损害GDP结合和结构域间的相互作用。在稳定表达CaSR的HEK293细胞中表达野生型和突变体G（11）表明，突变体Met54 G（11）在Ca-o（2+）刺激后的Ca-i（2+）反应导致浓度-反应曲线向右移动，平均半最大浓度（EC50）值显著（p < 0.01）增加3.88mM(95%置信区间[CI] 3.76-4.01mM)，而野生型EC50值为2.94mM (95% CI 2.81-3.07mM)，与功能丧失一致。因此，我们的研究已经确定了导致FHH2的第三个G（11）突变（Thr54Met），并揭示了G（11）域间界面在CaSR信号传导和Ca-o（2+）稳态中的关键作用。(c) 2016年美国骨与矿物研究学会。
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 sigma-subunit (AP2 sigma), 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 sigma 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-i(2+)) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca-o(2+)) 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-i(2+) responses after stimulation with Ca-o(2+) 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 3.88mM (95% confidence interval [CI] 3.76-4.01mM), when compared with the wild-type EC50 of 2.94mM (95% CI 2.81-3.07mM) 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-o(2+) homeostasis. (c) 2016 American Society for Bone and Mineral Research.