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1.
Artigo em Inglês | MEDLINE | ID: mdl-31820785

RESUMO

CONTEXT: Autosomal dominant hypocalcemia types 1 and 2 (ADH1 and ADH2) are caused by germline gain-of-function mutations of the calcium-sensing receptor (CaSR) and its signaling partner, the G-protein subunit α11 (Gα11), respectively. Over 70 different gain-of-function CaSR mutations, but only 6 different gain-of-function Gα11 mutations are reported to date. METHODS: We ascertained two additional ADH families and investigated them for CaSR and Gα11 mutations. The effects of identified variants on CaSR signaling were evaluated by transiently transfecting wild-type (WT) and variant expression constructs into HEK293 cells stably expressing CaSR (HEK-CaSR), and measuring intracellular calcium (Ca2+i) and MAPK responses following stimulation with extracellular calcium (Ca2+e). RESULTS: CaSR variants were not found, but two novel heterozygous germline Gα11 variants, p.Gly66Ser and p.Arg149His, were identified. Homology modelling of these revealed that the Gly66 and Arg149 residues are located at the interface between the Gα11 helical and GTPase domains, which is involved in guanine nucleotide binding, and this is the site of three other reported ADH2 mutations. The Ca2+i and MAPK responses of cells expressing the variant Ser66 or His149 Gα11 proteins were similar to WT cells at low Ca2+e, but significantly increased in a dose-dependent manner following Ca2+e stimulation, thereby indicating that the p.Gly66Ser and p.Arg149His variants represent pathogenic gain-of-function Gα11 mutations. Treatment of Ser66- and His149-Gα11 expressing cells with the CaSR negative allosteric modulator NPS 2143 normalized Ca2+i and MAPK responses. CONCLUSION: Two novel ADH2-causing mutations that highlight the Gα11 interdomain interface as a hotspot for gain-of-function Gα11 mutations have been identified.

2.
J Bone Miner Res ; 34(7): 1324-1335, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30830987

RESUMO

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4+/M149T ) mice, when compared with wild-type (Brd4+/+ ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4+/M149T and Brd4+/+ mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4+/M149T mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.

3.
J Bone Miner Res ; 34(3): 497-507, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30395686

RESUMO

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2+ / Y265X ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2+/+ ). The Polg2+/Y265X and Polg2+ / + mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2+ / Y265X kidneys was reduced compared to Polg2+/+ mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2+ / Y265X mice, compared to Polg2+ / + littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

4.
Hum Mol Genet ; 28(6): 1023-1037, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445560

RESUMO

Prolactinomas are the most frequent type of pituitary tumors, which represent 10-20% of all intracranial neoplasms in humans. Prolactinomas develop in mice lacking the prolactin receptor (PRLR), which is a member of the cytokine receptor superfamily that signals via Janus kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) or phosphoinositide 3-kinase-Akt (PI3K-Akt) pathways to mediate changes in transcription, differentiation and proliferation. To elucidate the role of the PRLR gene in human prolactinomas, we determined the PRLR sequence in 50 DNA samples (35 leucocytes, 15 tumors) from 46 prolactinoma patients (59% males, 41% females). This identified six germline PRLR variants, which comprised four rare variants (Gly57Ser, Glu376Gln, Arg453Trp and Asn492Ile) and two low-frequency variants (Ile76Val, Ile146Leu), but no somatic variants. The rare variants, Glu376Gln and Asn492Ile, which were in complete linkage disequilibrium, and are located in the PRLR intracellular domain, occurred with significantly higher frequencies (P < 0.0001) in prolactinoma patients than in 60 706 individuals of the Exome Aggregation Consortium cohort and 7045 individuals of the Oxford Biobank. In vitro analysis of the PRLR variants demonstrated that the Asn492Ile variant, but not Glu376Gln, when compared to wild-type (WT) PRLR, increased prolactin-induced pAkt signaling (>1.3-fold, P < 0.02) and proliferation (1.4-fold, P < 0.02), but did not affect pSTAT5 signaling. Treatment of cells with an Akt1/2 inhibitor or everolimus, which acts on the Akt pathway, reduced Asn492Ile signaling and proliferation to WT levels. Thus, our results identify an association between a gain-of-function PRLR variant and prolactinomas and reveal a new etiology and potential therapeutic approach for these neoplasms.

5.
Hum Mol Genet ; 27(21): 3720-3733, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30052933

RESUMO

The calcium-sensing receptor (CaSR) is a homodimeric G-protein-coupled receptor that signals via intracellular calcium (Ca2+i) mobilisation and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) to regulate extracellular calcium (Ca2+e) homeostasis. The central importance of the CaSR in Ca2+e homeostasis has been demonstrated by the identification of loss- or gain-of-function CaSR mutations that lead to familial hypocalciuric hypercalcaemia (FHH) or autosomal dominant hypocalcaemia (ADH), respectively. However, the mechanisms determining whether the CaSR signals via Ca2+i or ERK have not been established, and we hypothesised that some CaSR residues, which are the site of both loss- and gain-of-function mutations, may act as molecular switches to direct signalling through these pathways. An analysis of CaSR mutations identified in >300 hypercalcaemic and hypocalcaemic probands revealed five 'disease-switch' residues (Gln27, Asn178, Ser657, Ser820 and Thr828) that are affected by FHH and ADH mutations. Functional expression studies using HEK293 cells showed disease-switch residue mutations to commonly display signalling bias. For example, two FHH-associated mutations (p.Asn178Asp and p.Ser820Ala) impaired Ca2+i signalling without altering ERK phosphorylation. In contrast, an ADH-associated p.Ser657Cys mutation uncoupled signalling by leading to increased Ca2+i mobilization while decreasing ERK phosphorylation. Structural analysis of these five CaSR disease-switch residues together with four reported disease-switch residues revealed these residues to be located at conformationally active regions of the CaSR such as the extracellular dimer interface and transmembrane domain. Thus, our findings indicate that disease-switch residues are located at sites critical for CaSR activation and play a role in mediating signalling bias.


Assuntos
Mutação com Ganho de Função , Hipercalciúria/genética , Hipocalcemia/genética , Hipoparatireoidismo/congênito , Mutação com Perda de Função , Receptores de Detecção de Cálcio/genética , Transdução de Sinais , Sequência de Aminoácidos , Sinalização do Cálcio , Análise Mutacional de DNA , Células HEK293 , Humanos , Hipercalciúria/metabolismo , Hipocalcemia/metabolismo , Hipoparatireoidismo/genética , Hipoparatireoidismo/metabolismo , Conformação Proteica , Receptores de Detecção de Cálcio/metabolismo , Alinhamento de Sequência
6.
Vascul Pharmacol ; 108: 15-22, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29654907

RESUMO

G-Protein coupled receptors (GPCRs) activate intracellular signalling pathways by coupling to heterotrimeric G-proteins that control many physiological processes including blood pressure homeostasis. The Regulator of G-Protein Signalling-1 (RGS1) controls the magnitude and duration of downstream GPCR signalling by acting as a GTPase-activating protein for specific Gα-proteins. RGS1 has contrasting roles in haematopoietic and non-haematopoietic cells. Rgs1-/-ApoE-/- mice are protected from Angiotensin II (Ang II)-induced aortic aneurysm rupture. Conversely, Ang II treatment increases systolic blood pressure to a greater extent in Rgs1-/-ApoE-/- mice than ApoE-/- mice, independent of its role in myeloid cells. However the precise role of RGS1 in hypertension and vascular-derived cells remains unknown. We determined the effects of Rgs1 deletion on vascular function in ApoE-/- mice. Rgs1 deletion led to enhanced vasoconstriction in aortas and mesenteric arteries from ApoE-/- mice in response to phenylephrine (PE) and U46619 respectively. Rgs1 was shown to have a role in the vasculature, with endothelium-dependent vasodilation being impaired, and endothelium-independent dilatation to SNP being enhanced in Rgs1-/-ApoE-/- mesenteric arteries. To address the downstream signalling pathways in vascular smooth muscle cells (VSMCs) in response to Ang II-stimulation, we assessed pErk1/2, pJNK and pp38 MAPK activation in VSMCs transiently transfected with Rgs1. pErk1/2 signalling but not pJNK and pp38 signalling was impaired in the presence of Rgs1. Furthermore, we demonstrated that the enhanced contractile response to PE in Rgs1-/-ApoE-/- aortas was reduced by a MAPK/Erk (MEK) inhibitor and an L-type voltage gated calcium channel antagonist, suggesting that Erk1/2 signalling and calcium influx are major effectors of Rgs1-mediated vascular contractile responses, respectively. These findings indicate RGS1 is a novel regulator of blood pressure homeostasis and highlight RGS1-controlled signalling pathways in the vasculature that may be new drug development targets for hypertension.


Assuntos
Angiotensina II , Pressão Sanguínea , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas RGS/metabolismo , Vasoconstrição , Vasodilatação , Animais , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatologia , Pressão Sanguínea/genética , Sinalização do Cálcio , Linhagem Celular , Modelos Animais de Doenças , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Camundongos Knockout para ApoE , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/fisiopatologia , Fosforilação , Proteínas RGS/deficiência , Proteínas RGS/genética , Receptor Tipo 1 de Angiotensina/metabolismo
7.
J Mol Endocrinol ; 61(1): R1-R12, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29599414

RESUMO

The calcium-sensing receptor (CASR) is a class C G-protein-coupled receptor (GPCR) that detects extracellular calcium concentrations, and modulates parathyroid hormone secretion and urinary calcium excretion to maintain calcium homeostasis. The CASR utilises multiple heterotrimeric G-proteins to mediate signalling effects including activation of intracellular calcium release; mitogen-activated protein kinase (MAPK) pathways; membrane ruffling; and inhibition of cAMP production. By studying germline mutations in the CASR and proteins within its signalling pathway that cause hyper- and hypocalcaemic disorders, novel mechanisms governing GPCR signalling and trafficking have been elucidated. This review focusses on two recently described pathways that provide novel insights into CASR signalling and trafficking mechanisms. The first, identified by studying a CASR gain-of-function mutation that causes autosomal dominant hypocalcaemia (ADH), demonstrated a structural motif located between the third transmembrane domain and the second extracellular loop of the CASR that mediates biased signalling by activating a novel ß-arrestin-mediated G-protein-independent pathway. The second, in which the mechanism by which adaptor protein-2 σ-subunit (AP2σ) mutations cause familial hypocalciuric hypercalcaemia (FHH) was investigated, demonstrated that AP2σ mutations impair CASR internalisation and reduce multiple CASR-mediated signalling pathways. Furthermore, these studies showed that the CASR can signal from the cell surface using multiple G-protein pathways, whilst sustained signalling is mediated only by the Gq/11 pathway. Thus, studies of FHH- and ADH-associated mutations have revealed novel steps by which CASR mediates signalling and compartmental bias, and these pathways could provide new targets for therapies for patients with calcaemic disorders.


Assuntos
Cálcio/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Homeostase/fisiologia , Humanos , Hipercalciúria/metabolismo , Hipocalcemia/metabolismo , Hipoparatireoidismo/congênito , Hipoparatireoidismo/metabolismo , Receptores de Detecção de Cálcio/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
8.
Cell Rep ; 22(4): 1054-1066, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29420171

RESUMO

Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gαq/11 and Gαi/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.


Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Sinalização do Cálcio/genética , Endossomos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Detecção de Cálcio/genética , Humanos , Mutação
9.
Sci Signal ; 11(518)2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29463778

RESUMO

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals through Gq/11 and Gi/o to stimulate cytosolic calcium (Ca2+i) and mitogen-activated protein kinase (MAPK) signaling to control extracellular calcium homeostasis. Studies of loss- and gain-of-function CASR mutations, which cause familial hypocalciuric hypercalcemia type 1 (FHH1) and autosomal dominant hypocalcemia type 1 (ADH1), respectively, have revealed that the CaSR signals in a biased manner. Thus, some mutations associated with FHH1 lead to signaling predominantly through the MAPK pathway, whereas mutations associated with ADH1 preferentially enhance Ca2+i responses. We report a previously unidentified ADH1-associated R680G CaSR mutation, which led to the identification of a CaSR structural motif that mediates biased signaling. Expressing CaSRR680G in HEK 293 cells showed that this mutation increased MAPK signaling without altering Ca2+i responses. Moreover, this gain of function in MAPK activity occurred independently of Gq/11 and Gi/o and was mediated instead by a noncanonical pathway involving ß-arrestin proteins. Homology modeling and mutagenesis studies showed that the R680G CaSR mutation selectively enhanced ß-arrestin signaling by disrupting a salt bridge formed between Arg680 and Glu767, which are located in CaSR transmembrane domain 3 and extracellular loop 2, respectively. Thus, our results demonstrate CaSR signaling through ß-arrestin and the importance of the Arg680-Glu767 salt bridge in mediating signaling bias.


Assuntos
Membrana Celular/metabolismo , Hipercalciúria/fisiopatologia , Hipocalcemia/fisiopatologia , Hipoparatireoidismo/congênito , Sistema de Sinalização das MAP Quinases , Mutação , Receptores de Detecção de Cálcio/metabolismo , Sais/metabolismo , beta-Arrestinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Membrana Celular/química , Saúde da Família , Feminino , Humanos , Hipercalciúria/genética , Hipocalcemia/genética , Hipoparatireoidismo/genética , Hipoparatireoidismo/fisiopatologia , Masculino , Modelos Moleculares , Linhagem , Conformação Proteica , Receptores de Detecção de Cálcio/química , Receptores de Detecção de Cálcio/genética , Sais/química , Homologia de Sequência de Aminoácidos
10.
Hum Mol Genet ; 27(5): 901-911, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29325022

RESUMO

Mutations of the sigma subunit of the heterotetrameric adaptor-related protein complex 2 (AP2σ) impair signalling of the calcium-sensing receptor (CaSR), and cause familial hypocalciuric hypercalcaemia type 3 (FHH3). To date, FHH3-associated AP2σ mutations have only been identified at one residue, Arg15. We hypothesized that additional rare AP2σ variants may also be associated with altered CaSR function and hypercalcaemia, and sought for these by analysing >111 995 exomes (>60 706 from ExAc and dbSNP, and 51 289 from the Geisinger Health System-Regeneron DiscovEHR dataset, which also contains clinical data). This identified 11 individuals to have 9 non-synonymous AP2σ variants (Arg3His, Arg15His (x3), Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) with 3 of the 4 individuals who had Arg15His and Met117Ile AP2σ variants having mild hypercalcaemia, thereby indicating a prevalence of FHH3-associated AP2σ mutations of ∼7.8 per 100 000 individuals. Structural modelling of the novel eight AP2σ variants (Arg3His, Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) predicted that the Arg3His, Thr112Met, Glu122Gly and Glu142Lys AP2σ variants would disrupt polar contacts within the AP2σ subunit or affect the interface between the AP2σ and AP2α subunits. Functional analyses of all eight AP2σ variants in CaSR-expressing cells demonstrated that the Thr112Met, Met117Ile and Glu142Lys variants, located in the AP2σ α4-α5 helical region that forms an interface with AP2α, impaired CaSR-mediated intracellular calcium (Cai2+) signalling, consistent with a loss of function, and this was rectified by treatment with the CaSR positive allosteric modulator cinacalcet. Thus, our studies demonstrate another potential class of FHH3-causing AP2σ mutations located at the AP2σ-AP2α interface.


Assuntos
Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Mutação , Receptores de Detecção de Cálcio/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades sigma do Complexo de Proteínas Adaptadoras/metabolismo , Cinacalcete/farmacologia , Bases de Dados Genéticas , Exoma , Feminino , Humanos , Hipercalcemia/tratamento farmacológico , Hipercalcemia/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Conformação Proteica , Transdução de Sinais , Sequenciamento Completo do Exoma
11.
J Bone Miner Res ; 33(1): 32-41, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28833550

RESUMO

G-protein subunit α-11 (Gα11 ) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca2+i ) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα11 , which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα11 protein to impair CaSR-mediated Ca2+i and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα11 cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 Gα11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα11 hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.


Assuntos
Cinacalcete/uso terapêutico , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Mutação em Linhagem Germinativa/genética , Hipercalcemia/congênito , Hipercalcemia/tratamento farmacológico , Hipercalcemia/genética , Mutação com Perda de Função/genética , Adulto , Sequência de Aminoácidos , Cinacalcete/farmacologia , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipercalcemia/enzimologia , Hipercalcemia/patologia , Lactente , Recém-Nascido , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , Transdução de Sinais , Fosfolipases Tipo C/metabolismo
12.
JCI Insight ; 2(20)2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29046478

RESUMO

Loss-of-function mutations of GNA11, which encodes G-protein subunit α11 (Gα11), a signaling partner for the calcium-sensing receptor (CaSR), result in familial hypocalciuric hypercalcemia type 2 (FHH2). FHH2 is characterized by hypercalcemia, inappropriately normal or raised parathyroid hormone (PTH) concentrations, and normal or low urinary calcium excretion. A mouse model for FHH2 that would facilitate investigations of the in vivo role of Gα11 and the evaluation of calcimimetic drugs, which are CaSR allosteric activators, is not available. We therefore screened DNA from > 10,000 mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for GNA11 mutations and identified a Gα11 variant, Asp195Gly (D195G), which downregulated CaSR-mediated intracellular calcium signaling in vitro, consistent with it being a loss-of-function mutation. Treatment with the calcimimetic cinacalcet rectified these signaling responses. In vivo studies showed mutant heterozygous (Gna11+/195G) and homozygous (Gna11195G/195G) mice to be hypercalcemic with normal or increased plasma PTH concentrations and normal urinary calcium excretion. Cinacalcet (30mg/kg orally) significantly reduced plasma albumin-adjusted calcium and PTH concentrations in Gna11+/195G and Gna11195G/195G mice. Thus, our studies have established a mouse model with a germline loss-of-function Gα11 mutation that is representative for FHH2 in humans and demonstrated that cinacalcet can correct the associated abnormalities of plasma calcium and PTH.


Assuntos
Cinacalcete/uso terapêutico , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/efeitos dos fármacos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Hipercalcemia/tratamento farmacológico , Mutação/efeitos dos fármacos , Administração Oral , Animais , Cálcio/sangue , Cálcio/urina , Cinacalcete/administração & dosagem , Modelos Animais de Doenças , Etilnitrosoureia/farmacologia , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Moleculares , Hormônio Paratireóideo/sangue , Hormônio Paratireóideo/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Albumina Sérica , Transdução de Sinais
13.
Endocrinology ; 158(8): 2486-2502, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28575322

RESUMO

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor that plays a pivotal role in extracellular calcium homeostasis. The CaSR is also highly expressed in pancreatic islet α- and ß-cells that secrete glucagon and insulin, respectively. To determine whether the CaSR may influence systemic glucose homeostasis, we characterized a mouse model with a germline gain-of-function CaSR mutation, Leu723Gln, referred to as Nuclear flecks (Nuf). Heterozygous- (CasrNuf/+) and homozygous-affected (CasrNuf/Nuf) mice were shown to have hypocalcemia in association with impaired glucose tolerance and insulin secretion. Oral administration of a CaSR antagonist compound, known as a calcilytic, rectified the glucose intolerance and hypoinsulinemia of CasrNuf/+ mice and ameliorated glucose intolerance in CasrNuf/Nuf mice. Ex vivo studies showed CasrNuf/+ and CasrNuf/Nuf mice to have reduced pancreatic islet mass and ß-cell proliferation. Electrophysiological analysis of isolated CasrNuf/Nuf islets showed CaSR activation to increase the basal electrical activity of ß-cells independently of effects on the activity of the adenosine triphosphate (ATP)-sensitive K+ (KATP) channel. CasrNuf/Nuf mice also had impaired glucose-mediated suppression of glucagon secretion, which was associated with increased numbers of α-cells and a higher α-cell proliferation rate. Moreover, CasrNuf/Nuf islet electrophysiology demonstrated an impairment of α-cell membrane depolarization in association with attenuated α-cell basal KATP channel activity. These studies indicate that the CaSR activation impairs glucose tolerance by a combination of α- and ß-cell defects and also influences pancreatic islet mass. Moreover, our findings highlight a potential application of targeted CaSR compounds for modulating glucose metabolism.


Assuntos
Hiperglicemia/tratamento farmacológico , Hiperglicemia/genética , Indanos/farmacologia , Fenilpropionatos/farmacologia , Receptores de Detecção de Cálcio/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Composição Corporal , Cálcio/metabolismo , Proliferação de Células , Intolerância à Glucose , Células HEK293 , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Camundongos , Camundongos Knockout , Mutação , Receptores de Detecção de Cálcio/antagonistas & inibidores , Receptores de Detecção de Cálcio/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética
14.
JCI Insight ; 2(3): e91079, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28194446

RESUMO

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.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Hipercalciúria/genética , Hipocalcemia/genética , Hipoparatireoidismo/congênito , Mutação , Naftalenos/administração & dosagem , Peptídeos Cíclicos/administração & dosagem , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Hipercalciúria/tratamento farmacológico , Hipercalciúria/metabolismo , Hipocalcemia/tratamento farmacológico , Hipocalcemia/metabolismo , Hipoparatireoidismo/tratamento farmacológico , Hipoparatireoidismo/genética , Hipoparatireoidismo/metabolismo , Camundongos , Naftalenos/farmacologia , Hormônio Paratireóideo/metabolismo , Peptídeos Cíclicos/farmacologia , Receptores de Detecção de Cálcio , Receptores Acoplados a Proteínas-G/metabolismo
15.
JCI Insight ; 2(3): e91103, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28194447

RESUMO

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.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Hipercalciúria/tratamento farmacológico , Hipocalcemia/tratamento farmacológico , Hipoparatireoidismo/congênito , Mutação , Naftalenos/administração & dosagem , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Cálcio/sangue , Modelos Animais de Doenças , Células HEK293 , Humanos , Hipercalciúria/genética , Hipercalciúria/metabolismo , Hipocalcemia/genética , Hipocalcemia/metabolismo , Hipoparatireoidismo/tratamento farmacológico , Hipoparatireoidismo/genética , Hipoparatireoidismo/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Naftalenos/farmacologia , Hormônio Paratireóideo/sangue , Receptores de Detecção de Cálcio
16.
JBMR Plus ; 1(1): 3-15, 2017 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-29479578

RESUMO

The adaptor protein-2 sigma subunit (AP2σ), encoded by AP2S1, forms a heterotetrameric complex, with AP2α, AP2ß, and AP2µ subunits, that is pivotal for clathrin-mediated endocytosis, and AP2σ loss-of-function mutations impair internalization of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor, and cause familial hypocalciuric hypercalcemia type-3 (FHH3). Mice with AP2σ mutations that would facilitate investigations of the in vivo role of AP2σ, are not available, and we therefore embarked on establishing such mice. We screened >10,000 mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for Ap2s1 mutations and identified 5 Ap2s1 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide gt to gc. Three-dimensional modeling and cellular expression of the missense Ap2s1 variants did not reveal them to alter AP2σ structure or CaSR-mediated signaling, but investigation of the donor splice site variant revealed it to result in an in-frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of the AP2σ α1-helix, α1-ß3 loop, and ß3 strand. Heterozygous mutant mice (Ap2s1+/del17 ) were therefore established, and these had AP2σ haplosufficiency but were viable with normal appearance and growth. Ap2s1+/del17 mice, when compared with Ap2s1+/+ mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)2) concentrations. However, homozygous Ap2s1del17/del17 mice were non-viable and died between embryonic days 3.5 and 9.5 (E3.5-9.5), thereby indicating that AP2σ likely has important roles at the embryonic patterning stages and organogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2σ.

17.
Am J Med Genet A ; 170(11): 2988-2992, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27540713

RESUMO

The aim of this study was to identify the causative mutation in a family with an unusual presentation of autosomal dominant osteopetrosis (OPT), proximal renal tubular acidosis (RTA), renal stones, epilepsy, and blindness, a combination of features not previously reported. We undertook exome sequencing of one affected and one unaffected family member, followed by targeted analysis of known candidate genes to identify the causative mutation. This identified a missense mutation (c.643G>A; p.Gly215Arg) in the gene encoding the chloride/proton antiporter 7 (gene CLCN7, protein CLC-7), which was confirmed by amplification refractory mutation system (ARMS)-PCR, and to be present in the three available patients. CLC-7 mutations are known to cause autosomal dominant OPT type 2, also called Albers-Schonberg disease, which is characterized by osteosclerosis, predominantly of the spine, pelvis and skull base, resulting in bone fragility and fractures. Albers-Schonberg disease is not reported to be associated with RTA, but autosomal recessive OPT type 3 (OPTB3) with RTA is associated with carbonic anhydrase type 2 (CA2) mutations. No mutations were detected in CA2 or any other genes known to cause proximal RTA. Neither CLCN7 nor CA2 mutations have previously been reported to be associated with renal stones or epilepsy. Thus, we identified a CLCN7 mutation in a family with autosomal dominant osteopetrosis, RTA, renal stones, epilepsy, and blindness. © 2016 Wiley Periodicals, Inc.


Assuntos
Acidose Tubular Renal/diagnóstico , Acidose Tubular Renal/genética , Canais de Cloreto , Genes Dominantes , Estudos de Associação Genética , Mutação , Osteopetrose/diagnóstico , Osteopetrose/genética , Alelos , Pré-Escolar , Análise Mutacional de DNA , Exoma , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem por Ressonância Magnética , Masculino , Linhagem , Fenótipo , Radiografia
19.
J Biol Chem ; 291(20): 10876-85, 2016 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-26994139

RESUMO

Germline loss- and gain-of-function mutations of G-protein α-11 (Gα11), which couples the calcium-sensing receptor (CaSR) to intracellular calcium (Ca(2+) 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 Ca(2+) 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 Ca(2+) i and ERK phosphorylation responses measured by flow-cytometry and Alphascreen immunoassay following exposure to extracellular Ca(2+) (Ca(2+) o) and allosteric modulators. Cinacalcet and NPS-2143 rectified the Ca(2+) 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 Ca(2+) 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.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hipercalcemia/metabolismo , Hipocalcemia/metabolismo , Mutação de Sentido Incorreto , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Substituição de Aminoácidos , Cinacalcete/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Hipercalcemia/genética , Hipocalcemia/genética , Naftalenos/farmacologia , Receptores de Detecção de Cálcio/genética
20.
J Bone Miner Res ; 31(6): 1200-6, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26729423

RESUMO

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 (Ca(2+) i ) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca(2+) 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 Ca(2+) i responses after stimulation with Ca(2+) 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 Ca(2+) o homeostasis. © 2016 American Society for Bone and Mineral Research.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP , Hipercalcemia , Mutação de Sentido Incorreto , Idoso , Substituição de Aminoácidos , Sinalização do Cálcio/genética , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Hipercalcemia/genética , Hipercalcemia/metabolismo , Hipercalcemia/patologia , Domínios Proteicos , Receptores de Detecção de Cálcio/genética , Receptores de Detecção de Cálcio/metabolismo
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