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This study aimed to screen for a novel osteogenic peptide based on the calcium-sensing receptor (CaSR) and explore its molecular mechanism and gastrointestinal stability. In this study, a novel osteogenic peptide (Phe-Ser-Gly-Leu, FSGL) derived from bovine bone collagen hydrolysate was successfully screened by molecular docking and synthesised by solid phase peptide synthesis for further analysis. Cell experiments showed that FSGL significantly enhanced the osteogenic activity of MC3T3-E1 cells by acting on CaSR, including proliferation (152.53%), differentiation, and mineralization. Molecular docking and molecular dynamics further demonstrated that FSGL was a potential allosteric activator of CaSR, that turned on the activation switch of CaSR by closing the Venus flytrap (VFT) domain and driving the two protein chains in the VFT domain to easily form dimers. In addition, 96.03% of the novel osteogenic peptide FSGL was stable during gastrointestinal digestion. Therefore, FSGL showed substantial potential for enhancing the osteogenic activity of osteoblasts. This study provided new insights for the application of CaSR in the targeted screening of osteogenic peptides to improve bone health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Despite advancements in understanding the structure and functions of the Calcium Sensing Receptor (CaSR), gaps persist in our knowledge of the specific functions of its residues. In this study, we used phylogeny-based techniques to identify functionally equivalent orthologs of CaSR, predict residue significance, and compute specificity-determining position (SDP) scores to understand its evolutionary basis. The analysis revealed exceptional conservation of the CaSR subfamily, emphasizing the critical role of residues with high SDP scores in receptor activation and pathogenicity. To further enhance the findings, gradient-boosting trees were applied to differentiate between gain- and loss-of-function mutations responsible for hypocalcemia and hypercalcemia. Lastly, we investigated the importance of these mutations in the context of receptor activation dynamics. In summary, through comprehensive exploration of the evolutionary history of the CaSR subfamily, coupled with innovative phylogenetic methodologies, we identified activating and inactivating residues, providing valuable insights into the regulation of calcium homeostasis and its connections to associated disorders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bircan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: End-stage renal disease (ESRD) commonly manifests with disrupted calcium balance, leading to renal osteodystrophy. We posited that variations in the genetic makeup of vitamin D and calcium-sensing receptors, specifically single nucleotide polymorphisms (SNPs), could affect calcium homeostasis. This study aimed to identify the genetic predictors related to vitamin D and calcium-sensing receptors on calcium metabolism using machine learning algorithm analysis in ESRD., Patients and Methods: We conducted a cross-sectional analysis on adults with ESRD. We gathered comprehensive demographic data and medical history. Blood samples were collected to measure SNPs, and a panel of calcium metabolism biomarkers associated with the calcium-sensing receptor and vitamin D receptor. The biomarkers included calcium, phosphate, vitamin D, parathyroid hormone (PTH), sclerostin, procollagen type 1 alpha 1, osteocalcin, and bone-specific alkaline phosphatase. We utilized machine learning algorithms to pinpoint genetic markers predictive of vitamin D deficiency., Results: We found a notable decrease in serum procollagen type 1 alpha 1 levels among individuals with the CC of rs10190 (related to the calcium-sensing receptor) compared to those with the TT genotype and in those with the TT of rs739837 (pertaining to the vitamin D receptor) compared to the GG genotype. Similarly, the TT genotype of rs10190 was associated with significantly lower serum phosphate levels compared to CC and CT genotypes. Additionally, a lower serum PTH level was noted in individuals with the CT of rs1802757 (calcium-sensing receptor) compared to those with the CC genotype. Our machine learning analysis identified rs2221266 and rs1042636 as the most significant SNPs linked to vitamin D deficiency, demonstrating considerable predictive accuracy., Conclusions: Our findings indicate that specific single nucleotide polymorphisms in the vitamin D and calcium-sensing receptors significantly influence calcium metabolism biomarkers in ESRD patients. Assessing the clinical implications of these genetic variations is crucial for advancing personalized medicine in renal care.

Purpose: To evaluate the association between vitamin D receptor (VDRs) and calcium-sensitive receptor (CaSR) gene polymorphisms and calcium-containing kidney stones (CCKS) in Dai populations., Methods: A total of 160 CCKS patients and 87 healthy controls were included in this study. CCKS was confirmed using urological computed tomography (CT), plain abdominal radiograph, or surgical lithotomy. Stone samples obtained during surgery were analyzed using infrared spectroscopy. Venous blood and 24-h urine samples were collected and analyzed using Sanger sequencing and high-performance liquid chromatography, respectively. Genetic variants in the VDR gene (rs7975232, rs2228570, rs731236, and rs1544410) and CaSR gene (rs7652589, rs1801725, and rs1042636) were identified through sequence analysis., Results: Analysis of genotype and allele frequencies revealed that the rs7975232 polymorphism in the VDR gene and the rs7652589 allele in the CaSR gene were significantly associated with CCKS. Furthermore, patients carrying the AC and AA genotypes of rs7975232 showed a higher incidence of hypocitraturia compared to those with other genotypes (p < 0.05). The AA and GG genotypes of rs1042636 and the AA genotype of rs7652589 were significantly associated with hypercalciuria (p < 0.05)., Conclusion: CCKS in this study population may be closely related to hypocitraturia caused by the VDR locus rs7975232 polymorphism and hypercalciuria caused by the CaSR locus rs1042636 and rs7652589 polymorphism., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The calcium-sensing receptor (CaSR) gene encodes a cell membrane G protein-coupled receptor (GPCR) which has a key role in maintaining the extracellular Ca 2+ homeostasis. We aimed at correcting the compound heterozygous mutation in the 6th [c.1656delA, p.I554SfsX73] and 7th [c.2217 T > A, p.C739X] exons of the CASR gene which the original patient-derived iPSC line had. The mutation is associated with neonatal severe primary hyperparathyroidism of the patient. We generated and characterized a CRISP/Cas9-edited hiPSC line with the restored sequence in the sixth exon of the CASR gene, bearing only heterozygous mutation in the 7th exon. The results showed that the new genetically modified cell line has karyotype without abnormalities, typical hiPSCs morphology, characteristic expression of pluripotency markers, and ability to develop into three germ layers, and differentiates in chondrogenic, adipogenic, osteogenic directions. This new cell line will complement the existing pool of CaSR-mutated cell lines, a valuable resource for in-depth understanding of neonatal severe primary hyperparathyroidism. This will allow further exploration of the application of pharmacological drugs in the context of personalized medicine to correct Ca-homeostasis disorders., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Glucagon-like peptide-1 (GLP-1) plays a crucial role in regulating glucose homeostasis by stimulating insulin secretion and suppressing glucagon release. Our previous study observed that pea protein hydrolysate (PPH) exhibited the function of triggering GLP-1 secretion. However, the underlying mechanisms have not been revealed. Herein, the mechanisms of PPH-stimulated GLP-1 secretion were investigated in NCI-H716 cells. The PPH-induced GLP-1 secretion was reduced ( p < 0.05) after adding the sensing receptor antagonists NPS-2143 and 4-AMBA, indicating that activation of both calcium-sensing receptor (CaSR) and peptide-transporter 1 (PepT1) was involved in PPH-triggered GLP-1 release. Moreover, the intracellular Ca 2+ level increased by 2.01 times during the PPH-induced GLP-1 secretion. Similarly, the cAMP content also increased by 1.43 times after stimulation by PPH. The RT-qPCR results showed that PPH increased the gene expression of prohormone convertase 1/3 (PCSK-1) by 2.79-fold, which effectively promoted the conversion of proglucagon (GCG) to GLP-1. The specific pathway of PPH-induced GLP-1 secretion may involve both CaSR and PepT1 activation-induced Ca 2+ influx and cAMP generation, which effectively enhanced the enzyme activity of prohormone convertase 1/3 (PCSK-1) and ultimately promoted GLP-1 secretion.

Glial cells missing 2 (GCM2) has been identified as an essential factor for parathyroid differentiation, and GCM2 silencing in parathyroid cells decreases calcium-sensing receptor (CaSR) expression. However, the role of GCM2 in parathyroid differentiation from induced pluripotent stem cells (iPSCs) is unclear. Here, we investigated the role of GCM2 in parathyroid differentiation from iPSCs using the Tet-On 3 G system. We confirmed that iPS cells transfected with GCM2/TRE3G and pCMV-Tet3G vectors express GCM2 in a doxycycline-dependent manner. Though parathyroid glands derive from the endoderm and differentiate via the third pharyngeal arch (PPE), overexpression of GCM2 in iPSCs significantly abolished the suppression of OCT4 and SOX2, suggesting inhibition of endodermal differentiation. GCM2 overexpression at the stage of differentiation into the third PPE also increased the expression levels of CaSR and parathyroid hormone, and increased the number of CaSR + /EpCAM + cells. These results suggest that GCM2 regulates parathyroid differentiation after endoderm differentiation rather than at an earlier stage., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Dietary proteins could stimulate Glucagon-like peptide 1 (GLP-1) secretion. However, only a few food-derived GLP-1-secreting peptides have been identified. Herein, three GLP-1-secreting peptides were identified from pea protein hydrolysate (PPH) by calcium-sensing receptor (CaSR) activation-based molecular docking. PPH-triggered GLP-1 secretion was mediated by CaSR activation. A total of 4221 peptides were sequenced from PPH through peptidomic analysis. Subsequently, three GLP-1-secreting peptides, including RFY, FEPF, and FLFK, were screened by CaSR activation-based molecular docking, and peptide-induced GLP-1 secretion were mediated by CaSR activation. More importantly, FEPF and FLFK exhibited good digestive stability. The molecular docking suggested that binding energy between peptides and CaSR was negatively correlated with their ability to stimulate GLP-1 secretion, and some binding sites in CaSR, such as Asn102 and Tyr218, play a crucial role in stimulating GLP-1 secretion. Our findings suggest that the targeted discovery of pea protein-derived GLP-1-secreting peptides through CaSR activation-based molecular docking is an effective strategy., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work., (Copyright © 2024. Published by Elsevier Ltd.)

Luciferases catalyze a reaction that involves the emission of light, a phenomenon referred to as "bioluminescence". The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), induces characteristic signaling pathways that stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca 2+ mobilization from the endoplasmic reticulum. ERK1/2 causes an activation of the serum response element (SRE), whereas Ca 2+ causes an activation of the nuclear factor of activated T-cells response element (NFAT-RE). Transfection of cells with a vector containing a firefly luciferase reporter gene under the control of the SRE or NFAT-RE allows the monitoring of ERK1/2 activation and Ca 2+ mobilization, respectively, by measuring luminescence. In a dual luciferase assay, firefly luminescence is normalized by co-transfecting an internal control vector, which contains a constitutively active promoter driving the expression of a second luciferase, namely, Renilla luciferase, whose activity can be quantified within the same sample. Here, a protocol for the analysis of CaSR signaling using dual luciferase assays in HEK293 cells is provided. The assays can, for example, be used to investigate functional consequences of mutations in the CaSR gene., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The calcium-sensing receptor (CaSR) has a critical role in maintaining serum calcium concentrations within the normal physiological range, and mutations in the receptor, or components of its signaling and trafficking pathway, cause disorders of calcium homeostasis. Inactivating mutations cause neonatal severe hyperparathyroidism or familial hypocalciuric hypercalcemia (FHH), while gain-of-function mutations cause autosomal dominant hypocalcemia (ADH). Characterizing the functional impact of mutations of the CaSR, and components of the CaSR-signaling pathway, is clinically important to enable correct diagnoses of FHH and ADH, optimize management, and prevent inappropriate parathyroidectomy or vitamin D supplementation. CaSR signals predominantly by activating the G-alpha subunit-11 to mobilize calcium release from intracellular stores. Thus, measurement of CaSR-induced intracellular calcium (Ca 2+ i ) signaling is the gold standard method to investigate the pathogenicity of CaSR genetic variants. This protocol describes a method to assess CaSR-induced Ca 2+ I signaling using the Indo-1 calcium indicator dye and flow cytometry. This method has been used to assess multiple genetic variants in CaSR and components of its signaling and trafficking pathway in HEK293 cells., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The American Association of Endocrine Surgeons suggests screening for familial hypocalciuric hypercalcemia for 24-hour urine calcium <100 mg and for calcium to creatinine clearance ratio <1%. We explored the biochemical and clinical profiles of genetically tested patients to determine the usefulness of these recommendations., Methods: This was a retrospective review of patients who underwent analysis of the calcium-sensing receptor gene., Results: In total, 401 patients were identified between 2005 and 2024; 332 (83%) were negative for a mutation, 44 (11%) were positive, and 25 (6%) had variants of unknown significance. Median serum calcium was lower in patients who were negative (negative 10.1 mg/dL, variants of unknown significance 10.6 mg/dL, positive 10.9 mg/dL). Median urine calcium was lower in patients who were positive (negative 110 mg, variants of unknown significance 149 mg, positive 82 mg). Median parathyroid hormone levels were comparable between groups (negative 65 pg/mL, variants of unknown significance 58 pg/mL, positive 60 pg/mL). Eight of 21 (38%) patients who tested positive had urine calcium >100 mg and 5 of 21 (24%) had urine calcium >200 mg. Eight of 35 (23%) patients who were positive had a calcium to creatinine clearance ratio >1%. Urine calcium and calcium to creatinine clearance ratio receiver operating characteristic curves showed poor performance in identifying patients with familial hypocalciuric hypercalcemia (area under the curve [95% confidence interval]): 0.55 [0.45-0.65] and 0.63 [0.57-0.7], respectively. For positive patients with imaging, sestamibi was inconclusive or nonlocalizing for 13 of 23 and showed multiglandular disease for localizing scans in 7 of 10. Ultrasound was nonlocalizing or equivocal for 11 of 13. In total, 212 of 401 patients (53%) underwent surgery; 100 of 212 were tested preoperatively (0/100 positive, 5/100 variants of unknown significance) and 112 of 212 patients were tested postoperatively (15/112 positive, 10/112 variants of unknown significance)., Conclusion: Familial hypocalciuric hypercalcemia demonstrates non-negligible biochemical overlap with primary hyperparathyroidism, with many patients having a urine calcium value greater than the American Association of Endocrine Surgeons suggested threshold for considering genetic evaluation. More frequent genetic testing beyond urine measurements may help identify more patients with familial hypocalciuric hypercalcemia., Competing Interests: Conflict of Interest/Disclosure The authors have no related conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Objective: The exact underlying mechanism for the differential clinical profiles of symptomatic and asymptomatic primary hyperparathyroidism (PHPT) patients has not been fully elucidated, and efforts to define the molecular mechanisms underlying the phenotypic heterogeneity of PHPT have been limited. The aim of this study was to explore the underlying molecular mechanisms involved in the pathogenesis of symptomatic and asymptomatic sporadic PHPT in Asian Indians., Methods: A prospective cohort study was conducted at a tertiary care hospital in North India. PHPT patients who underwent parathyroidectomy were included. The main outcome was the comparison of vitamin D receptor (VDR), calcium-sensing receptor (CaSR), cyclin D 1 (CD1), and parathyroid hormone (PTH) mRNA levels between symptomatic and asymptomatic PHPT patients and controls determined via quantitative real-time polymerase chain reaction (qRT-PCR)., Results: Forty-two PHPT patients were studied. The mean (SD) age was 49.7 (12.8) years. Twenty patients were asymptomatic. The median PTH levels were significantly greater in the symptomatic group than in the asymptomatic group (878 vs 653 pg/mL). CaSR and VDR mRNAs were significantly lower in both symptomatic and asymptomatic patients than in controls. CD1 and PTH mRNAs were significantly increased in symptomatic patients, but not in asymptomatic PHPT patients compared with controls. Symptomatic PHPT patients had significantly greater CD1 mRNA expression and reduced CaSR expression than asymptomatic patients., Conclusion: Symptomatic PHPT patients had significantly greater CD1 mRNA expression and lower CaSR expression than asymptomatic patients, underscoring the importance of the molecular mechanisms underlying the phenotypic heterogeneity of PHPT., Competing Interests: Conflict of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Familial hypocalciuric hypercalcemia (FHH) is typically a benign condition characterized by elevated serum calcium, low urinary calcium excretion, and non-suppressed circulating levels of parathyroid hormone (PTH), usually requiring no intervention. FHH is inherited in an autosomal-dominant manner. Three subtypes are described, representing variants in genes with critical roles in extracellular calcium-sensing. FHH1, due to heterozygous inactivating variants in the calcium-sensing receptor gene (CASR), accounts for the majority of cases. FHH2, due to variants in GNA11, encoding the α-subunit of the downstream signaling protein, G11, is the rarest form of FHH. FHH3, resulting from variants in AP2S1, may present with a more pronounced phenotype than FHH1 or FHH2. We describe herein a newborn girl presenting with in utero femoral fractures, hypercalcemia, hypophosphatemia, and elevated circulating PTH. She was diagnosed with mild hyperparathyroidism and provided supplemental phosphate upon hospital discharge. However, serum calcium and PTH remained elevated at 5 mo of age. The combination of low-calcium formula and cinacalcet improved the biochemical profile. No pathogenic variants in the coding region of CASR were identified; subsequent whole exome sequencing revealed a G- > T transition at c.44 (p.R15L) in AP2S1. Family studies identified this variant in the father and an affected brother. The mother was unexpectedly found to be hypocalcemic and was diagnosed with idiopathic hypoparathyroidism. This case demonstrates successful treatment of FHH3 using a low-calcium formula to limit dietary calcium availability and cinacalcet to modify PTH levels., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Female mice are more resistant to obesogenic effects of a high-fat diet (HFD), compared to male mice. Although the underlying mechanisms are poorly understood, sex hormones seem to play an important role. Interestingly, the activity of the oestrogen receptor-α (ERα) is affected by the calcium-sensing-receptor (CaSR). Therefore, we investigated sex-differences upon diet-induced obesity and the role of adipocyte-specific CaSR herein., Methods: Adipocyte-specific Casr deficient mice (AdipoqCre + Casr flox ) and control mice (Casr flox ) were injected with AAV8-PCSK9 to make them prone to develop atherosclerosis and fed an obesity-inducing diet for 12 weeks., Findings: Female mice have lower visceral white adipose tissue (vWAT) mass compared to male mice, while this sex-difference is abolished upon adipocyte-specific Casr deficiency. Furthermore, while females showed elevated levels of inflammatory cytokines and CD3 + CD8 + T cell accumulation in vWAT, compared to males, adipocyte-specific Casr deficiency abrogated this sex-phenotype and demonstrated an inhibition of inflammatory signalling pathways. The expression of Erα, as well as associated genes involved in adipocyte differentiation, was increased in female mice in a mostly adipocyte-specific Casr dependent manner. Interestingly, circulating lipid levels were reduced in female compared to male mice, which correlated with decreased atherosclerotic plaque formation. These systemic effects were abrogated upon adipocyte-specific Casr deficiency., Interpretation: Our findings indicate that female mice show a more pronounced vWAT dysfunction compared to males upon obesity. This sex effect is abolished upon adipocyte-specific Casr deficiency. In contrast, females show diminished atherosclerotic plaque formation compared to males, an effect that was abrogated by adipocyte-specific Casr deficiency., Funding: This work was supported by a grant from the Interdisciplinary Center for Clinical Research within the faculty of Medicine at the RWTH Aachen University, by the Corona Foundation, by the Deutsche Forschungsgemeinschaft (DFG), the BMBF and Free State of Bavaria and the DZHK., Competing Interests: Declaration of interests None., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Familial Hypocalciuria Hypercalcemia (FHH) is an inherited disease with autosomal dominant transmission characterized by the presence of usually mild-to-moderate hypercalcemia, hypophosphatemia, hypocalciuria, and normal or moderately increased PTH values. Generally, FFH is asymptomatic although symptoms related to elevated plasma calcium values such as asthenia, intense thirst, polyuria, polydipsia or confusional state may occur. Three types of FHH, which differ in the genetic alterations underlying the condition, are described. The majority of FHH cases are classified as type 1 (about 65 percent of cases), due to mutation in the gene for the calcium-sensitive receptor CASR, expressed on chromosome (Chr) 3q13.3-21, which encodes for a calcium-sensitive receptor G-protein-coupled protein of the plasma membrane. FHH types 2 and 3 are due to GNA11 and AP2S1 mutations, respectively, and other genes involved in the pathogenesis of the disease have likely yet to be identified. Rarely, familial hypocalciuric hypercalcemia may not recognize a genetic cause but be caused by autoantibodies directed against CASR. The frequency of the disease is not known and is estimated, probably by default, because of paucisymptomatic presentation of the disease, to be around 1:80000 cases. Recognition of FHH is especially important for differential diagnosis with primary hyperparathyroidism, which has a much higher incidence, about 1:1000 cases. This allows for the identification of patients at risk for chondrocalcinosis and/or pancreatitis. Clinical suspicion must be raised in cases of hypercalcaemia associated with hypocalciuria, and genetic analysis is fundamental in the differential diagnosis toward forms of primary hyperparathyroidism that might result in unnecessary surgical interventions. We describe a clinical case in which a novel inactivating mutation of CASR leading to FHH type 1 was found., (Copyright by Società Italiana di Nefrologia SIN, Rome,Italy.)

Cisplatin (DDP) is commonly used in the treatment of non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD), and the primary cause for its clinical inefficacy is chemoresistance. Here, we aimed to investigate a novel mechanism of chemoresistance in LUAD cells, focusing on the calcium-sensing receptor (CaSR). In this study, high CaSR expression was detected in DDP-resistant LUAD cells, and elevated CaSR expression is strongly correlated with poor prognosis in LUAD patients receiving chemotherapy. LUAD cells with high CaSR expression exhibited decreased sensitivity to cisplatin, and the growth of DDP-resistant LUAD cells was inhibited by cisplatin treatment in combination with CaSR suppression, accompanied by changes in BRCA1 and cyclin B1 protein expression both in vitro and in vivo . Additionally, an interaction between CaSR and KIF11 was identified. Importantly, suppressing KIF11 resulted in decreased protein levels of BRCA1 and cyclin B1, enhancing the sensitivity of DDP-resistant LUAD cells to cisplatin with no obvious decrease in CaSR. Here, our findings established the critical role of CaSR in promoting cisplatin resistance in LUAD cells by modulating cyclin B1 and BRCA1 and identified KIF11 as a mediator, highlighting the potential therapeutic value of targeting CaSR to overcome chemoresistance in LUAD., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose of Review: Parathyroid hormone (PTH) is the major peptide hormone regulator of blood calcium homeostasis. Abnormal PTH levels can be observed in patients with various congenital and acquired disorders, including chronic kidney disease (CKD). This review will focus on rare human diseases caused by PTH mutations that have provided insights into the regulation of PTH synthesis and secretion as well as the diagnostic utility of different PTH assays., Recent Findings: Over the past years, numerous diseases affecting calcium and phosphate homeostasis have been defined at the molecular level that are responsible for reduced or increased serum PTH levels. The underlying genetic mutations impair parathyroid gland development, involve the PTH gene itself, or alter function of the calcium-sensing receptor (CaSR) or its downstream signaling partners that contribute to regulation of PTH synthesis or secretion. Mutations in the pre sequence of the mature PTH peptide can, for instance, impair hormone synthesis or intracellular processing, while amino acid substitutions affecting the secreted PTH(1-84) impair PTH receptor (PTH1R) activation, or cause defective cleavage of the pro-sequence and thus secretion of a pro- PTH with much reduced biological activity. Mutations affecting the secreted hormone can alter detection by different PTH assays, thus requiring detailed knowledge of the utilized diagnostic test., Summary: Rare diseases affecting PTH synthesis and secretion have offered helpful insights into parathyroid biology and the diagnostic utility of commonly used PTH assays, which may have implications for the interpretation of PTH measurements in more common disorders such as CKD., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Kidney aging accelerates the progression of various acute and chronic kidney diseases and can also induce pathological changes in other organs throughout the body. Circular RNAs (circRNAs) have been demonstrated to play a vital role in aging and age-related diseases. However, biological functions and the underlying molecular mechanism of circRNAs in kidney aging remain largely unclear. Uncovering the functions of circRNAs in kidney aging and their underlying regulatory mechanisms may shed new light on the development of novel diagnostic and therapeutic strategies for human aging. Here, we report the important role of circVmn2r1 in the progression of kidney aging. We found that circVmn2r1 was one of the top expressed circRNAs in mouse kidney by RNA sequencing and was significantly upregulated in 24-month-old mouse kidney compared to 3-month-old. More importantly, we demonstrated that overexpression of circVmn2r1 promoted kidney aging in senescence-accelerated mouse prone 8 mice. Cellular assays with mouse kidney tubular epithelium (TCMK-1) cells under both gain-of-function and loss-of-function conditions demonstrated that circVmn2r1 inhibited proliferation and promoted senescence, whereas miR-223-3p counteracted these effects. Mechanistic analysis demonstrated that circVmn2r1 acted as a miR-223-3p sponge to relieve the repressive effect of miR-223-3p on its target NLRP3, which we proved could inhibit proliferation and promote senescence of TCMK-1 cells. Our results indicate that circVmn2r1 promotes kidney aging through acting as a miR-223-3p sponge, consequently upregulating NLRP3 expression, and can be a valuable diagnostic marker and an important therapeutic target for kidney aging., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purpose of Review: Activation of the calcium-sensing receptor (CASR) in the parathyroid gland suppresses the release of parathyroid hormone (PTH). Furthermore, activation of the renal CASR directly increases the urinary excretion of calcium, by inhibiting transepithelial calcium transport in the nephron. Gain-of-function mutations in the CASR gene lead to autosomal dominant hypocalcemia 1 (ADH1), with inappropriately low PTH levels and hypocalcemia, indicative of excessive activation of the parathyroid CASR. However, hypercalciuria is not always observed. The reason why the manifestation of hypercalciuria is not uniform among ADH1 patients is not well understood., Recent Findings: Direct activation of the CASR in the kidney has been cumbersome to study, and an indirect measure to effectively estimate the degree of CASR activation following chronic hypercalcemia or genetic gain-of-function CASR activation has been lacking. Studies have shown that expression of the pore-blocking claudin-14 is strongly stimulated by the CASR in a dose-dependent manner. This stimulatory effect is abolished after renal Casr ablation in hypercalcemic mice, suggesting that claudin-14 abundance may gauge renal CASR activation. Using this marker has led to unexpected discoveries regarding renal CASR activation., Summary: These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium-Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). In line with these findings, we provide evidence that, with respect to wild-type mice, CaSR knock-in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2-targeting miRNA137 consistent with a post-transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT-305, a CaSR antagonist, increased AQP2 expression and reduced AQP2-targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short-term vasopressin response by increasing AQP2-pS261, as well as AQP2 abundance, via the p38MAPK-ATF1-miR137 pathway. KEY POINTS: Calcium-Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis. In vivo treatment with calcilytic JTT-305/MK-5442 ameliorates most of the ADH phenotypes of the CaSR knock-in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin-sensitive aquaporin-2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response. The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone-independent action through vasopressin-dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct. The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2-mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Purpose: Calcium-sensing receptor (CASR) influences the expression pattern of multiple genes in renal tubular epithelial cells. The objective of this inquiry was to explore the molecular mechanisms of CASR in renal tubular epithelial cells and nephrolithiasis., Methods: HK-2 cells were transfected with lentiviruses carrying either CASR (named CASR) or an empty vector negative control (named NC), as well as shRNA intended to target CASR (named shCASR) or its corresponding negative control (named shNC). CCK-8 assay was used to detect the effect of CASR on the proliferation of HK-2 cells. RNA-Sequencing was applied to explore potential pathways regulated by CASR in HK-2 cells., Results: PCR and western blot results showed that CASR expression was significantly increased in CASR cells and was decreased in shCASR cells when compared to their corresponding negative control, respectively. CCK-8 assay revealed that CASR inhibited the proliferation of HK-2 cells. RNA-Sequencing results suggested that the shCASR HK-2 cells exhibited a significant up-regulation of 345 genes and a down-regulation of 366 genes. These differentially expressed genes (DEGs) were related to cell apoptosis and cell development. In CASR HK-2 cells, 1103 DEGs primarily functioned in mitochondrial energy metabolism, and amino acid metabolism. With the Venn diagram, 4 DEGs (Clorf116, ENPP3, IL20RB, and CLDN2) were selected as the hub genes regulated by CASR. Enrichment analysis revealed that these hub genes were involved in cell-cell junction, and epithelial cell development., Conclusions: In summary, our investigation has the potential to offer novel perspectives on CASR regulating cell-cell junction in HK-2 cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Understanding the molecular factors involved in the development of uterine myomas may result in the use of pharmacological drugs instead of aggressive surgical treatment. ANG1, CaSR, and FAK were examined in myoma and peripheral tissue samples taken from women after myoma surgery and in normal uterine muscle tissue samples taken from the control group. Tests were performed using tissue microarray immunohistochemistry. No statistically significant differences in ANG1 expression between the tissue of the myoma, the periphery, and the normal uterine muscle tissue of the control group were recorded. The CaSR value was reduced in the myoma and peripheral tissue and normal in the group of women without myomas. FAK expression was also lower in the myoma and periphery compared to the healthy uterine myometrium. Calcium supplementation could have an effect on stopping the growth of myomas.

The calcium-sensing receptor (CaSR) is a critical mediator of calcium homeostasis in various tissues. Its role in human reproduction, especially in sperm function and male fertility, remains not fully elucidated. This study investigates the expression patterns of CaSR in normal and abnormal sperm and spermatogenic cells and evaluates its potential effect on sperm motility and morphology. Using immunohistochemistry (IHC), quantitative PCR (qPCR), we assessed the expression levels of CaSR in normal sperm, spermatogonia, and cases of asthenozoospermia, oligozoospermia, and teratozoospermia. In vitro functional assays were performed to analyze the effects of CaSR modulation on sperm motility under varying conditions, including the presence of specific CaSR agonists and antagonists. Our study revealed distinct patterns of CaSR expression in normal sperm and spermatogonia compared with those in abnormal sperm samples, particularly in cases of asthenozoospermia, oligozoospermia, and teratozoospermia. A marked decrease in CaSR expression was evident in these abnormal samples, highlighting its significance in normal sperm functionality. Functional assays further elucidated the role of CaSR in sperm motility. Activation of CaSR through specific agonists enhanced sperm motility, while inhibition by antagonists led to reduced motility. Our findings suggest that CaSR plays a significant role in maintaining sperm functionality and that changes in its expression may be associated with male infertility. These insights into the molecular underpinnings of sperm physiology highlight CaSR as a potential therapeutic target for treating certain forms of male infertility.

Calcium-sensing receptors (CaSRs) are G protein-coupled receptors that help maintain Ca 2+ concentrations, modulating calciotropic hormone release (parathyroid hormone (PTH), calcitonin and 1,25-dihydroxyvitamin D) by direct actions in the kidneys, gastrointestinal tract and bone. Variability in population calcium levels has been attributed to single nucleotide polymorphisms in CaSR genes, and several conditions affecting calcium and phosphate homeostasis have been attributed to gain- or loss-of-function mutations. An example is autosomal dominant hypercalciuric hypocalcaemia, because of a missense mutation at codon 128 of chromosome 3, as reported in our specific case and her family. As a consequence of treating symptomatic hypocalcaemia as a child, this female subject slowly developed progressive end-stage kidney failure because of nephrocalcinosis and nephrolithiasis. After kidney transplantation, she remains asymptomatic, with decreased vitamin D and elemental calcium requirements, stable fluid and electrolyte homeostasis during intercurrent illnesses and has normalised urinary calcium and phosphate excretion, reducing the likelihood of hypercalciuria-induced graft impairment. We review the actions of the CaSR, its role in regulating renal Ca 2+ homeostasis along with the impact of a proven gain-of-function mutation in the CaSR gene resulting in autosomal dominant hypercalciuric hypocalcaemia before and after kidney transplantation., (© 2024 The Authors. Internal Medicine Journal published by John Wiley & Sons Australia, Ltd on behalf of Royal Australasian College of Physicians.)

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr ( Casr BCH002 ), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double Casr BCH002 /PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion. NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (Casr BCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed Casr BCH002 mice with PTH deficient mice. Heterozygous Casr BCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in Casr BCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, Casr BCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH., (© 2024. The Author(s).)

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors., (© 2024. The Author(s).)

The calcium-sensing receptor (CaSR), a G protein-coupled receptor, regulates Ca 2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, it is reported to play roles in cellular differentiation and migration and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knockout (KO) ( Eso CaSR -/- ) model showed a significant reduction in the levels of adherens and tight junction proteins and had a marked buildup of bacteria on the luminal esophageal surface. To further examine the role of CaSR, we used RNA sequencing to determine gene expression profiles in esophageal epithelia of control and Eso CaSR -/- mice RNA Seq data indicated upregulation of gene sets involved in DNA replication and cell cycle in Eso CaSR -/- . This is accompanied by the downregulation of gene sets involved in the innate immune response and protein homeostasis including peptide elongation and protein trafficking. Ingenuity pathway analysis (IPA) demonstrated that these genes are mapped to important biological networks including calcium and Ras homologus A (RhoA) signaling pathways. To further explore the bacterial buildup in Eso CaSR -/- esophageal tissue, 16S sequencing of the mucosal-associated bacterial microbiome was performed. Three bacterial species, g&#95;Rodentibacter , s &#95;Rodentibacter&#95;unclassified , and s&#95;Lactobacillus&#95;hilgardi were significantly increased in Eso CaSR -/- . Furthermore, metagenomic analysis of 16S sequences indicated that pathways related to oxidative phosphorylation and metabolism were downregulated in Eso CaSR -/- tissues. These data demonstrate that CaSR impacts major pathways of cell proliferation, differentiation, cell cycle, and innate immune response in esophageal epithelium. The disruption of these pathways causes inflammation and significant modifications of the microbiome. NEW & NOTEWORTHY Calcium-sensing receptor (CaSR) plays a significant role in maintaining the barrier function of esophageal epithelium. Using RNA sequencing, we show that conditional deletion of CaSR from mouse esophagus causes upregulation of genes involved in DNA replication and cell cycle and downregulation of genes involved in the innate immune response, protein translation, and cellular protein synthesis. Pathway analysis shows disruption of signaling pathways of calcium and actin cytoskeleton. These changes caused inflammation and esophageal dysbiosis.

Objective: The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897&#95;2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants., Design and Methods: To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs., Results: Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT., Conclusions: This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

We report a 45-year-old woman with persistent post-parathyroidectomy PTH-dependent asymptomatic hypercalcemia who, after evaluation in our bone clinic, was diagnosed as having familial hypocalciuric hypercalcemia based on concomitant hypocalciuria and a family history of asymptomatic hypercalcemia. Genetic study shows a variant of uncertain significance in the CASR gene. To our knowledge, this is the first report on a Chilean family with HHF.

Context: Although a monoallelic mutation in the calcium-sensing receptor ( CASR ) gene causes familial hypocalciuric hypercalcemia (FHH), the functional characterization of the identified CASR mutation linked to the clinical response to calcimimetics therapy is still limited., Objective: A 45-year-old male presenting with moderate hypercalcemia, hypocalciuria, and inappropriately high parathyroid hormone (PTH) had a good response to cinacalcet (total serum calcium (Ca 2+ ) from 12.5 to 10.1 mg/dl). We identified the genetic mutation and characterized the functional and pathophysiological mechanisms, and then linked the mutation to calcimimetics treatment in vitro ., Design: Sanger sequencing of the CASR , GNA11 , and AP2S1 genes was performed in his family. The simulation model was used to predict the function of the identified mutant. In vitro studies, including immunoblotting, immunofluorescence, a cycloheximide chase study, Calbryte™ 520 Ca 2+ detection, and half-maximal effective concentration (EC 50 ), were examined., Results: This proband was found to carry a de novo heterozygous missense I554N in the cysteine-rich domain of CASR , which was pathogenic based on the different software prediction models and ACGME criteria. The simulation model showed that CASR I554N mutation decreased its binding energy with Ca 2+ . Human CASR I554N mutation attenuated the stability of CASR protein, reduced the expression of p-ERK 1/2, and blunted the intracellular Ca 2+ response to gradient extracellular Ca 2+ (eCa 2+ ) concentration. The EC 50 study also demonstrated the correctable effect of calcimimetics on the function of the CASR I554N mutation., Conclusion: This novel CASR I554N mutation causing FHH attenuates CASR stability, its binding affinity with Ca 2+ , and the response to eCa 2+ corrected by therapeutic calcimimetics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lin, Ding, Huang, Chen, Lee, Sung and Lin.)

Rainforest hunter-gatherers from Southeast Asia are characterized by specific morphological features including a particularly dark skin color (D), short stature (S), woolly hair (W), and the presence of steatopygia (S)-fat accumulation localized in the hips (DSWS phenotype). Based on previous evidence in the Andamanese population, we first characterized signatures of adaptive natural selection around the calcium-sensing receptor gene in Southeast Asian rainforest groups presenting the DSWS phenotype and identified the R990G substitution (rs1042636) as a putative adaptive variant for experimental follow-up. Although the calcium-sensing receptor has a critical role in calcium homeostasis by directly regulating the parathyroid hormone secretion, it is expressed in different tissues and has been described to be involved in many biological functions. Previous works have also characterized the R990G substitution as an activating polymorphism of the calcium-sensing receptor associated with hypocalcemia. Therefore, we generated a knock-in mouse for this substitution and investigated organismal phenotypes that could have become adaptive in rainforest hunter-gatherers from Southeast Asia. Interestingly, we found that mouse homozygous for the derived allele show not only lower serum calcium concentration but also greater body weight and fat accumulation, probably because of enhanced preadipocyte differentiation and lipolysis impairment resulting from the calcium-sensing receptor activation mediated by R990G. We speculate that such differential features in humans could have facilitated the survival of hunter-gatherer groups during periods of nutritional stress in the challenging conditions of the Southeast Asian tropical rainforests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Background: Enterotoxins produce diarrhea through direct epithelial action and indirectly by activating the enteric nervous system. Calcium-sensing receptor (CaSR) inhibits both actions. The latter has been well documented in vitro but not in vivo . The hypothesis to be tested was that activating CaSR inhibits diarrhea in vivo ., Aim: To determine whether CaSR agonists ameliorate secretory diarrhea evoked by cholera toxin (CTX) in mice., Methods: CTX was given orally to C57BL/6 mice to induce diarrhea. Calcium and calcimimetic R568 were used to activate CaSR. To maximize their local intestinal actions, calcium was administered luminally via oral rehydration solution (ORS), whereas R568 was applied serosally using an intraperitoneal route. To verify that their actions resulted from the intestine, effects were also examined on Cre-lox intestine-specific CaSR knockouts. Diarrhea outcome was measured biochemically by monitoring changes in fecal Cl - or clinically by assessing stool consistency and weight loss., Results: CTX induced secretory diarrhea, as evidenced by increases in fecal Cl - , stool consistency, and weight loss following CTX exposure, but did not alter CaSR, neither in content nor in function. Accordingly, calcium and R568 were each able to ameliorate diarrhea when applied to diseased intestines. Intestinal CaSR involvement is suggested by gene knockout experiments where the anti-diarrheal actions of R568 were lost in intestinal epithelial CaSR knockouts ( villin Cre/Casr flox/flox ) and neuronal CaSR knockouts ( nestin Cre/Casr flox/flox )., Conclusion: Treatment of acute secretory diarrheas remains a global challenge. Despite advances in diarrhea research, few have been made in the realm of diarrhea therapeutics. ORS therapy has remained the standard of care, although it does not halt the losses of intestinal fluid and ions caused by pathogens. There is no cost-effective therapeutic for diarrhea. This and other studies suggest that adding calcium to ORS or using calcimimetics to activate intestinal CaSR might represent a novel approach for treating secretory diarrheal diseases., Competing Interests: Conflict-of-interest statement: All the authors report having no relevant conflicts of interest for this article., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Context: Familial hypocalciuric hypercalcemia type 1 (FHH-1) defines an autosomal dominant disease, related to mutations in the CASR gene, with mild hypercalcemia in most cases. Cases of FHH-1 with a short QT interval have not been reported to date., Objective: Three family members presented with FHH-1 and short QT interval (<360 ms), a condition that could lead to cardiac arrhythmias, and the effects of cinacalcet, an allosteric modulator of the CaSR, in rectifying the abnormal sensitivity of the mutant CaSR and in correcting the short QT interval were determined., Methods: CASR mutational analysis was performed by next-generation sequencing and functional consequences of the identified CaSR variant (p.Ile555Thr), and effects of cinacalcet were assessed in HEK293 cells expressing wild-type and variant CaSRs. A cinacalcet test consisting of administration of 30 mg cinacalcet (8 Am) followed by hourly measurement of serum calcium, phosphate, and parathyroid hormone during 8 hours and an electrocardiogram was performed., Results: The CaSR variant (p.Ile555Thr) was confirmed in all 3 FHH-1 patients and was shown to be associated with a loss of function that was ameliorated by cinacalcet. Cinacalcet decreased parathyroid hormone by >50% within two hours, and decreases in serum calcium and increases in serum phosphate occurred within 8 hours, with rectification of the QT interval, which remained normal after 3 months of cinacalcet treatment., Conclusion: Our results indicate that FHH-1 patients should be assessed for a short QT interval and a cinacalcet test used to select patients who are likely to benefit from this treatment., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Introduction: Breast cancer (BC) is a common cancer characterized by a high molecular heterogeneity. Therefore, understanding its biological properties and developing effective treatments for patients with different molecular features is imperative. Calcium-sensing receptor (CaSR) has been implicated in several regulatory functions in various types of human cancers. However, its underlying pathological mechanism in BC progression remains elusive., Methods: We utilized The Cancer Genome Atlas and Gene Expression Omnibus databases to explore the function of CaSR in the metastasis of BC. Gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Gene Set Enrichment Analysis of biological processes and cell signaling pathways revealed that CaSR could be activated or inhibited. Importantly, quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to verify the gene expression of the CaSR. Wound healing and transwell assays were conducted to assess the effect of CaSR on the migration of BC cells., Results: We demonstrated that CaSR expression in metastatic BC was higher than that in non-metastatic BC. It is the first time that database information has been used to reveal the biological process and molecular mechanism of CaSR in BC. Moreover, the CaSR expression in normal breast epithelial cells was notably less compared to that in BC cells. The activation of CaSR by Cinacalcet (a CaSR agonist) significantly enhanced the migration of BC cells, whereas NPS-2143 (a CaSR antagonist) treatment dramatically inhibited these effects., Conclusion and Future Perspective: Bioinformatics techniques and experiments demonstrated the involvement of CaSR in BC metastasis. Our findings shed new light on the receptor therapy and molecular pathogenesis of BC, and emphasize the crucial function of CaSR, facilitating the metastasis of BC., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Calcium-sensing receptor (CASR), primarily found in the parathyroid gland and other tissues, plays a crucial role in sensing and regulating extracellular calcium, which was also aberrantly expressed in human tumors. Nevertheless, a comprehensive analysis of CASR in pan-cancer has yet to be conducted. To gain a better understanding of CASR in pan-cancer, data profiles on CASR cancers were collected from TCGA database. The expression level, clinical significance, prognostic value, and potential mechanisms of CASR in pan-cancer were analyzed via multiple public databases. The functional assays were conducted using human kidney renal clear cell carcinoma (KIRC) cell lines, clinical samples, and nude mice. Our research revealed that the abnormal expression of CASR was found in a variety of tumors. The expression and mutation of CASR were significantly associated with tumor prognosis and stage. Pathway analyses suggested that CASR was involved in the epithelial-mesenchymal transition (EMT) progress. Besides, CASR expression was correlated with immune inhibitory genes and immunotherapy in cancers. Particularly in KIRC, we established that CASR mRNA and protein levels were downregulated in clinical samples and cell lines. Moreover, a Cox regression analysis revealed that CASR was an independent prognostic factor in both TCGA-KIRC samples and clinical samples from our center. In vitro and in vivo experiments revealed that blocking CASR with lentivirus could suppress tumor growth and invasion, and EMT progress in KIRC cells. In summary, our study provides a comprehensive bioinformatic analysis of CASR in pan-cancer, offering deeper insights into its function and the EMT mechanism in KIRC, warranting further investigation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

This study aimed to test the hypothesis that the calcium-sensing receptor (CaSR) can protect intestinal epithelial barrier integrity and decrease inflammatory response mediated by the Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway. IPEC-J2 monolayers were treated without or with TNF-α in the absence or presence of CaSR antagonist (NPS 2143), CaSR overexpression, and Rac1 silencing, PLCγ1 silencing or spermine. Results showed that spermine increased transepithelial electrical resistance (TER), tight junction protein levels, the protein concentration of Rac1/PLC-γ1 signaling pathway, and decreased paracellular permeability in the presence of TNF-α. NPS2143 inhibited spermine-induced change in above-mentioned parameters. CaSR overexpression increased TER, the levels of tight junction proteins and the protein concentration of CaSR, phosphorylated PLCγ1, Rac1, and IP3, and decreased paracellular permeability and contents of interleukin-8 (IL-8) and TNF-α after TNF-α challenge. Rac1 and PLCγ1 silencing inhibited CaSR-induced increase in barrier function and the protein concentration of phosphorylated PLCγ1, Rac1, and IP3, and decrease in contents of IL-8 and TNF-α after TNF-α challenge. These results suggest that CaSR activation protects intestinal integrity and alleviates the inflammatory response by activating Rac1 and PLCγ1 signaling after TNF-α challenge, and spermine can maintain barrier function via CaSR/Rac1/PLC-γ1 pathway.

The parathyroid gland plays an essential role in mineral and bone metabolism. Cultivation of physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. In this study, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. Parathyroid cell differentiation occurred in accordance with embryologic development. Differentiated cells, which expressed the parathyroid hormone, adopted unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

Haematopoietic stem cells (HSC) reside in the bone marrow microenvironment (BMM), where they respond to extracellular calcium [eCa 2+ ] via the G-protein coupled calcium-sensing receptor (CaSR). Here we show that a calcium gradient exists in this BMM, and that [eCa 2+ ] and response to [eCa 2+ ] differ between leukaemias. CaSR influences the location of MLL-AF9 + acute myeloid leukaemia (AML) cells within this niche and differentially impacts MLL-AF9 + AML versus BCR-ABL1 + leukaemias. Deficiency of CaSR reduces AML leukaemic stem cells (LSC) 6.5-fold. CaSR interacts with filamin A, a crosslinker of actin filaments, affects stemness-associated factors and modulates pERK, β-catenin and c-MYC signaling and intracellular levels of [Ca 2+ ] in MLL-AF9 + AML cells. Combination treatment of cytarabine plus CaSR-inhibition in various models may be superior to cytarabine alone. Our studies suggest CaSR to be a differential and targetable factor in leukaemia progression influencing self-renewal of AML LSC via [eCa 2+ ] cues from the BMM., (© 2023. Springer Nature Limited.)

Background and Aims: Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway., Methods: In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated., Results: In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, β-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins G i , G q, and G βγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/G i /mTORC1 and CaSR/G q /mTORC1 pathways., Conclusion: We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/G i /mTORC1 and CaSR/G q /mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Objective: To explore the miRNAs that down- regulate calcium-sensing receptor (CaSR) in secondary hyperparathyroidism (SHPT) and their effects on parathyroid hormone (PTH) secretion., Methods: Whole transcriptome sequencing was performed for 6 normal parathyroid tissue samples and 11 SHPT parathyroid tissue samples. Based on bioinformatic prediction, we screened out 7 candidate miRNAs that regulate CaSR, among which the most likely miRNA for CaSR regulation was identified by double luciferase test. We detected the differential expression of miR-301a-5p and CaSR mRNA in SHPT and normal parathyroid tissue using qRT-PCR, and analyzed the correlation between their expressions and serum PTH levels of the patients. Western blotting was used to detect the expression of CaSR protein in primary SHPT parathyroid cells transfected with miR-301a-5p mimics or inhibitors, and the level of PTH in the supernatant of the cell culture was determined., Results: Among the preliminarily selected 7 miRNAs that potentially regulate CaSR (miR-15a-5p, miR-15b-5p, miR- 16- 5p, miR- 221- 3p, miR- 222- 3p, miR- 301a- 5p and miR- 503- 5p), miR- 301a-5p was significantly upregulated in SHPT compared with normal parathyroid tissue ( P < 0.05), and its expression appeared to be positively correlated with PTH level, but this correlation was not statistically significant ( P > 0.05); The expression of CaSR mRNA was significantly downregulated in SHPT ( P < 0.05), and its expression tended to inversely correlate with the patient's PTH level, but the correlation was not statistically significant ( P > 0.05). In primary culture of SHPT parathyroid cells, miR-301a-5p overexpression caused a significant decrease of CaSR protein expression ( P < 0.05), and conversely, inhibition of miR-301a-5p expression increased the expression of CaSR protein ( P < 0.05). Although miR-301a-5p overexpression did not significantly affect PTH secretion of the cells ( P > 0.05), inhibition of iR-301a-5p expression strongly increased the secretion of PTH ( P < 0.05)., Conclusion: MiR-301a-5p affects PTH secretion in SHPT possibly by regulating the expression of CaSR.

Although ethanol treatment is widely used to activate oocytes, the underlying mechanisms are largely unclear. Roles of intracellular calcium stores and extracellular calcium in ethanol-induced activation (EIA) of oocytes remain to be verified, and whether calcium-sensing receptor (CaSR) is involved in EIA is unknown. This study showed that calcium-free ageing (CFA) in vitro significantly decreased intracellular stored calcium (sCa) and CaSR expression, and impaired EIA, spindle/chromosome morphology and developmental potential of mouse oocytes. Although EIA in oocytes with full sCa after ageing with calcium does not require calcium influx, calcium influx is essential for EIA of oocytes with reduced sCa after CFA. Furthermore, the extremely low EIA rate in oocytes with CFA-downregulated CaSR expression and the fact that inhibiting CaSR significantly decreased the EIA of oocytes with a full complement of CaSR suggest that CaSR played a significant role in the EIA of ageing oocytes. In conclusion, CFA impaired EIA and the developmental potential of mouse oocytes by decreasing sCa and downregulating CaSR expression. Because mouse oocytes routinely treated for activation (18 h post hCG) are equipped with a full sCa complement and CaSR, the present results suggest that, while calcium influx is not essential, CaSR is required for the EIA of oocytes.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP 87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP 87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-Cre ERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP 87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP 87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The calcium-sensing receptor (CaSR) plays a crucial role in maintaining the balance of calcium in the body. Altered signaling through the CaSR has been linked to the development of various tumors, such as colorectal and breast tumors. This retrospective study enrolled 79 patients who underwent surgical removal of invasive breast carcinoma of no special type (NST) to explore the expression of the CaSR in breast cancer. The patients were categorized based on age, tumor size, hormone receptor status, HER2 status, Ki-67 proliferation index, tumor grade, and TNM staging. Immunohistochemistry was conducted on core needle biopsy samples to assess CaSR expression. The results revealed a positive correlation between CaSR expression and tumor size, regardless of the tumor surrogate subtype ( p = 0.001). The expression of ER exhibited a negative correlation with CaSR expression ( p = 0.033). In contrast, a positive correlation was observed between CaSR expression and the presence of HER2 receptors ( p = 0.002). Increased CaSR expression was significantly associated with lymph node involvement and the presence of distant metastasis ( p = 0.001 and p = 0.038, respectively). CaSR values were significantly higher in the patients with increased Ki-67 ( p = 0.042). Collectively, higher CaSR expression in breast cancer could suggest a poor prognosis and treatment outcome regardless of the breast cancer subtype.

Fibroblast growth factor (FGF)-23 and calcium-sensing receptor (CaSR) have previously been postulated to be parts of a negative feedback regulation of the intestinal calcium absorption to prevent excessive calcium uptake and its toxicity. However, the underlying mechanism of this feedback regulation remained elusive, especially whether it required transcription of FGF-23. Herein, we induced calcium hyperabsorptive state (CHS) by exposing intestinal epithelium-like Caco-2 monolayer to 30 mM CaCl 2 and 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] after which FGF-23 mRNA levels and transepithelial calcium flux were determined. We found that CHS upregulated FGF-23 transcription, which was reverted by CaSR inhibitors (Calhex-231 and NPS2143) but without effect on CaSR transcription. Although 10 nM 1,25(OH) 2 D 3 was capable of enhancing transepithelial calcium flux, the higher-than-normal calcium inundation as in CHS led to a decrease in calcium flux, consistent with an increase in FGF-23 protein expression. Administration of inhibitors (≤10 μM CN585 and cyclosporin A) of calcineurin, a mediator of CaSR action to control transcription and production of its target proteins, was found to partially prevent FGF-23 protein production and the negative effect of CHS on calcium transport, while having no effect on FGF-23 mRNA expression. Direct exposure to FGF-23, but not FGF-23 + PD173074 (FGFR1/3 inhibitor), also completely abolished the 1,25(OH) 2 D 3 -enhanced calcium transport in Caco-2 monolayer. Nevertheless, CHS and CaSR inhibitors had no effect on the mRNA levels of calcineurin (PPP3CB) or its targets (i.e., NFATc1-4). In conclusion, exposure to CHS induced by high apical calcium and 1,25(OH) 2 D 3 triggered a negative feedback mechanism to prevent further calcium uptake. CaSR and its downstream mediator, calcineurin, possibly contributed to the regulatory process, in part by enhancing FGF-23 production to inhibit calcium transport. Our study, therefore, corroborated the physiological significance of CaSR-autocrine FGF-23 axis as a local feedback loop for prevention of excessive calcium uptake., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Narattaphol Charoenphandhu reports financial support was provided by National Research Council of Thailand. Kannikar Wongdee reports financial support was provided by Thailand Science Research and Innovation., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Familial hypocalciuric hypercalcemia type 2 (FHH2) and autosomal dominant hypocalcemia type 2 (ADH2) are due to loss- and gain-of-function mutations, respectively, of the GNA11 gene that encodes the G protein subunit Gα11, a signaling partner of the calcium-sensing receptor (CaSR). To date, four probands with FHH2-associated Gα11 mutations and eight probands with ADH2-associated Gα11 mutations have been reported. In a 10-year period, we identified 37 different germline GNA11 variants in >1200 probands referred for investigation of genetic causes for hypercalcemia or hypocalcemia, comprising 14 synonymous, 12 noncoding, and 11 nonsynonymous variants. The synonymous and noncoding variants were predicted to be benign or likely benign by in silico analysis, with 5 and 3, respectively, occurring in both hypercalcemic and hypocalcemic individuals. Nine of the nonsynonymous variants (Thr54Met, Arg60His, Arg60Leu, Gly66Ser, Arg149His, Arg181Gln, Phe220Ser, Val340Met, Phe341Leu) identified in 13 probands have been reported to be FHH2- or ADH2-causing. Of the remaining nonsynonymous variants, Ala65Thr was predicted to be benign, and Met87Val, identified in a hypercalcemic individual, was predicted to be of uncertain significance. Three-dimensional homology modeling of the Val87 variant suggested it was likely benign, and expression of Val87 variant and wild-type Met87 Gα11 in CaSR-expressing HEK293 cells revealed no differences in intracellular calcium responses to alterations in extracellular calcium concentrations, consistent with Val87 being a benign polymorphism. Two noncoding region variants, a 40bp-5'UTR deletion and a 15bp-intronic deletion, identified only in hypercalcemic individuals, were associated with decreased luciferase expression in vitro but no alterations in GNA11 mRNA or Gα11 protein levels in cells from the patient and no abnormality in splicing of the GNA11 mRNA, respectively, confirming them to be benign polymorphisms. Thus, this study identified likely disease-causing GNA11 variants in <1% of probands with hypercalcemia or hypocalcemia and highlights the occurrence of GNA11 rare variants that are benign polymorphisms. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)