Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.

Pearce, Simon H. S.; Bai, Ming; Quinn, Stephen; Kifor, Olga; Brown, Edward M.; Thakker, Rajesh

doi:10.1172/jci118987

Cited by 176 publications

(150 citation statements)

References 33 publications

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“…The Thr54Met, Leu135Gln, and Ile200del loss‐of‐function GNA11 mutations are all associated with a mild FHH phenotype characterized by serum adjusted‐calcium concentrations <2.80 mmol/L, and these clinical findings are in keeping with our in vitro studies that have shown FHH2‐associated mutations to induce only minor disturbances of CaSR signal transduction 11. Indeed, the FHH2 mutants were associated with around a 30% increase in the EC 50 values of HEK293‐CaSR cells used in this study, whereas CaSR mutations leading to FHH1 generally cause a >50% increase in the EC 50 value 3, 27. The milder shift in the Ca 2+ o set point of cells expressing FHH2‐associated Gα 11 mutants indicates that the CaSR may promote Ca 2+ i signaling by Gα 11 ‐independent mechanisms, such as via the related Gα q protein.…”

Section: Discussionsupporting

confidence: 89%

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

Gorvin

Cranston

Hannan

et al. 2016

Journal of Bone and Mineral Research

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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 been reported. FHH3 is the result of loss‐of‐function mutations of the adaptor protein‐2 σ‐subunit (AP2σ), 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 AP2σ 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 Gα11 variant was assessed by homology modeling of the related Gαq protein and by measuring the CaSR‐mediated intracellular calcium (Ca2+ i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca2+ o) using flow cytometry. Three‐dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα11 helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild‐type and the mutant Gα11 in HEK293 cells stably expressing CaSR demonstrate that the Ca2+ i responses after stimulation with Ca2+ o of the mutant Met54 Gα11 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 Gα11 mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα11 interdomain interface in CaSR signaling and Ca2+ o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

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Section: Discussionsupporting

confidence: 89%

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

Gorvin

Cranston

Hannan

et al. 2016

Journal of Bone and Mineral Research

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“…receptors with impaired glycosylation or disulfide bond formation are retained in the ER, resulting in reduced cell surface expression and/or total protein abundance (23,30,31). Biochemical studies on loss-or gain-of-function CaR mutants identified in patients suggest that signaling changes may result from differences in cell surface expression (30,32,33). In the present study, we asked whether there is a conformational or functional checkpoint in the biogenesis of CaR using several complementary strategies.…”

Section: Discussionmentioning

confidence: 98%

Rescue of Calcium-sensing Receptor Mutants by Allosteric Modulators Reveals a Conformational Checkpoint in Receptor Biogenesis

Huang

Breitwieser

2007

Journal of Biological Chemistry

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“…The changes in intracellular Ca 2ϩ levels were expressed as a normalized response. The magnitude of the transient response peak after an individual stimulus was added to the sum of the counts from the previous stimulus and expressed as a proportion of the total counts in the concentration-response curves, as described by Pearce et al (18). Individual EC 50 values (the effective concentration of extracellular Ca 2ϩ giving half of the maximal response) for each incremental, normalized concentration-response curve were plotted using the Sigma Plot software (SPSS, Chicago, IL).…”

Section: Measurement Of Intracellular Ca 2ϩ By Fluorimetry In Individmentioning

confidence: 99%

Functional Characterization of a Calcium-Sensing Receptor Mutation in Severe Autosomal Dominant Hypocalcemia with a Bartter-Like Syndrome

Vargas‐Poussou¹,

Huang²,

Hulin³

et al. 2002

Journal of the American Society of Nephrology

311

195

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Abstract. The extracellular Ca 2ϩ -sensing receptor (CaSR) plays an essential role in extracellular Ca 2ϩ homeostasis by regulating the rate of parathyroid hormone (PTH) secretion and the rate of calcium reabsorption by the kidney. Activation of the renal CaSR is thought to inhibit paracellular divalent cation reabsorption in the cortical ascending limb (cTAL) both directly and indirectly via a decrease in NaCl transport. However, in patients with autosomal dominant hypocalcemia (ADH), caused by CaSR gain-of-function mutations, a defect in tubular NaCl reabsorption with renal loss of NaCl has not been described so far. This article describes a patient with ADH due to a gain-of-function mutation in the CaSR, L125P, associated with a Bartter-like syndrome that is characterized by a decrease in distal tubular fractional chloride reabsorption rate and negative NaCl balance with secondary hyperaldosteronism and hypokalemia. The kinetics of activation of the L125P mutant receptor expressed in HEK-293 cells, assessed by measuring CaSR-stimulated changes in intracellular Ca 2ϩ and ERK activity, showed a dramatic reduction in the EC 50 for extracellular Ca 2ϩ compared with the wild-type and a loss-offunction mutant CaSR (I40F). This study describes the first case of ADH associated with a Bartter-like syndrome. It is herein proposed that the L125P mutation of the CaSR, which represents the most potent gain-of-function mutation reported so far, may reduce NaCl reabsorption in the cTAL sufficiently to result in renal loss of NaCl with secondary hyperaldosteronism and hypokalemia.

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Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells.

Abstract: The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that plays a key role in extracellular calcium ion homeostasis. We have engineered 11 CaR mutants that have been described in the disorders familial benign hypercalcemia (

Cited by 176 publications

References 33 publications

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

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

Rescue of Calcium-sensing Receptor Mutants by Allosteric Modulators Reveals a Conformational Checkpoint in Receptor Biogenesis

Functional Characterization of a Calcium-Sensing Receptor Mutation in Severe Autosomal Dominant Hypocalcemia with a Bartter-Like Syndrome

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