Sarah Howles

Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

BACKGROUND Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide-binding protein (G-protein)-coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia. METHODS We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells. RESULTS The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2-associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2-associated mutations increased cell sensitivity. CONCLUSIONS Gα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.).

Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3.

Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis, which internalizes plasma membrane constituents such as G protein–coupled receptors (GPCRs). AP2, a heterotetramer of α, β, μ and σ subunits, links clathrin to vesicle membranes and binds to tyrosine- and dileucine-based motifs of membrane-associated cargo proteins. Here we show that missense mutations of AP2 σ subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone. We found AP2S1 mutations in >20% of cases of FHH without mutations in calcium-sensing GPCR (CASR), which cause FHH1. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular calcium and reduced CaSR endocytosis, probably through loss of interaction with a C-terminal CaSR dileucine-based motif, whose disruption also decreased intracellular signaling. Thus, our results identify a new role for AP2 in extracellular calcium homeostasis.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype–phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca2+o) homeostasis. To elucidate the role of AP2σ2 in Ca2+o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype–phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype–phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Label-free quantitative proteomics reveals differentially regulated proteins influencing urolithiasis

Urinary proteins have been implicated as inhibitors of kidney stone formation (urolithiasis). As a proximal fluid, prefiltered by the kidneys, urine is an attractive biofluid for proteomic analysis in urologic conditions. However, it is necessary to correct for variations in urinary concentration. In our study, individual urine samples were normalized for this variation by using a total protein to creatinine ratio. Pooled urine samples were compared in two independent experiments. Differences between the urinary proteome of stone formers and nonstone-forming controls were characterized and quantified using label-free nano-ultraperformance liquid chromatography high/low collision energy switching analysis. There were 1063 proteins identified, of which 367 were unique to the stone former groups, 408 proteins were unique to the control pools, and 288 proteins were identified for comparative quantification. Proteins found to be unique in stone-formers were involved in carbohydrate metabolism pathways and associated with disease states. Thirty-four proteins demonstrated a consistent >twofold change between stone formers and controls. For ceruloplasmin, one of the proteins was shown to be more than twofold up-regulated in the stone-former pools, this observation was validated in individuals by enzyme-linked immunosorbent assay. Moreover, in vitro crystallization assays demonstrated ceruloplasmin had a dose-dependent increase on calcium oxalate crystal formation. Taken together, these results may suggest a functional role for ceruloplasmin in urolithiasis.

Allosteric Modulation of the Calcium-sensing Receptor Rectifies Signaling Abnormalities Associated with G-protein α-11 Mutations Causing Hypercalcemic and Hypocalcemic Disorders

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca2+i) signaling, lead to familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2), respectively, whereas somatic Gα11 mutations mediate uveal melanoma development by constitutively up-regulating MAPK signaling. Cinacalcet and NPS-2143 are allosteric CaSR activators and inactivators, respectively, that ameliorate signaling disturbances associated with CaSR mutations, but their potential to modulate abnormalities of the downstream Gα11 protein is unknown. This study investigated whether cinacalcet and NPS-2143 may rectify Ca2+i alterations associated with FHH2- and ADH2-causing Gα11 mutations, and evaluated the influence of germline gain-of-function Gα11 mutations on MAPK signaling by measuring ERK phosphorylation, and assessed the effect of NPS-2143 on a uveal melanoma Gα11 mutant. WT and mutant Gα11 proteins causing FHH2, ADH2 or uveal melanoma were transfected in CaSR-expressing HEK293 cells, and Ca2+i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca2+ (Ca2+o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca2+i responses of FHH2- and ADH2-associated Gα11 loss- and gain-of-function mutations, respectively. ADH2-causing Gα11 mutations were demonstrated not to be constitutively activating and induced ERK phosphorylation following Ca2+o stimulation only. The increased ERK phosphorylation associated with ADH2 and uveal melanoma mutants was rectified by NPS-2143. These findings demonstrate that CaSR-targeted compounds can rectify signaling disturbances caused by germline and somatic Gα11 mutations, which respectively lead to calcium disorders and tumorigenesis; and that ADH2-causing Gα11 mutations induce non-constitutive alterations in MAPK signaling.

Cinacalcet for Symptomatic Hypercalcemia Caused by AP2S1 Mutations

The authors show that cinacalcet, which mediates allosteric modulation of the calcium-sensing receptor, corrects loss of function of AP2S1 mutations that cause familial hypocalciuric hypercalcemia type 3 and ameliorates symptomatic hypercalcemia.

Identification of a G-Protein Subunit-α11 Gain-of-Function Mutation, Val340Met, in a Family With Autosomal Dominant Hypocalcemia Type 2 (ADH2).

Autosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain‐of‐function mutations of the G‐protein coupled calcium‐sensing receptor (CaSR), and ADH2 caused by germline gain‐of‐function mutations of G‐protein subunit α‐11 (Gα11). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole‐exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three‐dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C‐terminal α5 helix, which may affect G‐protein coupled receptor binding and G‐protein activation. In vitro functional expression of wild‐type (Val340) and mutant (Met340) Gα11 proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration‐response curve with a significantly (p < 0.0001) reduced mean half‐maximal concentration (EC50) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild‐type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain‐of‐function mutation. A novel His403Gln variant in transforming growth factor, beta‐induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain‐of‐function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C‐terminal region for G‐protein function and CaSR signal transduction.

Mutational analysis of the adaptor protein 2 sigma subunit (AP2S1) gene: search for autosomal dominant hypocalcemia type 3 (ADH3).

CONTEXT Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-α11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2σ 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an additional form of ADH, which we designated ADH3. OBJECTIVE The objective of the study was to investigate the hypothesis that gain-of-function AP2S1 mutations may cause ADH3. DESIGN The sample size required for the detection of at least one mutation with a greater than 95% likelihood was determined by binomial probability analysis. Nineteen patients (including six familial cases) with hypocalcemia in association with low or normal serum PTH concentrations, consistent with ADH, but who did not have CASR or GNA11 mutations, were ascertained. Leukocyte DNA was used for sequence and copy number variation analysis of AP2S1. RESULTS Binomial probability analysis, using the assumption that AP2S1 mutations would occur in hypocalcemic patients at a prevalence of 20%, which is observed in FHH patients without CASR or GNA11 mutations, indicated that the likelihood of detecting at least one AP2S1 mutation was greater than 95% and greater than 98% in sample sizes of 14 and 19 hypocalcemic patients, respectively. AP2S1 mutations and copy number variations were not detected in the 19 hypocalcemic patients. CONCLUSION The absence of AP2S1 abnormalities in hypocalcemic patients, suggests that ADH3 may not occur or otherwise represents a rare hypocalcemic disorder.

Lack of effectiveness of botulinum neurotoxin A on isolated detrusor strips and whole bladders from mice and guinea-pigs in vitro.

To differentiate between the effects of parasympathetic and sensorimotor stimulation of isolated mouse and guinea‐pig bladders in vitro by measuring the pressure increases to electrical field stimulation (EFS) and then comparing the effects of botulinum neurotoxin A (BoNT‐A) applied either to the lumen or to the external bathing medium.

Gα11 mutation in mice causes hypocalcemia rectifiable by calcilytic therapy

Heterozygous germline gain-of-function mutations of G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in autosomal dominant hypocalcemia type 2 (ADH2). ADH2 may cause symptomatic hypocalcemia with low circulating parathyroid hormone (PTH) concentrations. Effective therapies for ADH2 are currently not available, and a mouse model for ADH2 would help in assessment of potential therapies. We hypothesized that a previously reported dark skin mouse mutant (Dsk7) — which has a germline hypermorphic Gα11 mutation, Ile62Val — may be a model for ADH2 and allow evaluation of calcilytics, which are CaSR negative allosteric modulators, as a targeted therapy for this disorder. Mutant Dsk7/+ and Dsk7/Dsk7 mice were shown to have hypocalcemia and reduced plasma PTH concentrations, similar to ADH2 patients. In vitro studies showed the mutant Val62 Gα11 to upregulate CaSR-mediated intracellular calcium and MAPK signaling, consistent with a gain of function. Treatment with NPS-2143, a calcilytic compound, normalized these signaling responses. In vivo, NPS-2143 induced a rapid and marked rise in plasma PTH and calcium concentrations in Dsk7/Dsk7 and Dsk7/+ mice, which became normocalcemic. Thus, these studies have established Dsk7 mice, which harbor a germline gain-of-function Gα11 mutation, as a model for ADH2 and have demonstrated calcilytics as a potential targeted therapy.