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1.  Impaired Osteoblast Function in GPRC6A Null Mice 
Journal of Bone and Mineral Research  2009;25(5):1092-1102.
GPRC6A is a widely expressed orphan G protein–coupled receptor that senses extracellular amino acids, osteocalcin, and divalent cations in vitro. GPRC6A null (GPRC6A−/−) mice exhibit multiple metabolic abnormalities including osteopenia. To investigate whether the osseous abnormalities are a direct function of GPRC6A in osteoblasts, we examined the function of primary osteoblasts and bone marrow stromal cell cultures (BMSCs) in GPRC6A−/− mice. We confirmed that GPRC6A−/− mice exhibited a decrease in bone mineral density (BMD) associated with reduced expression of osteocalcin, ALP, osteoprotegerin, and Runx2-II transcripts in bone. Osteoblasts and BMSCs derived from GPRC6A−/− mice exhibited an attenuated response to extracellular calcium-stimulated extracellular signal-related kinase (ERK) activation, diminished alkaline phosphatase (ALP) expression, and impaired mineralization ex vivo. In addition, siRNA-mediated knockdown of GPRC6A in MC3T3 osteoblasts also resulted in a reduction in extracellular calcium-stimulated ERK activity. To explore the potential relevance of GPRC6A function in humans, we looked for an association between GPRC6A gene polymorphisms and BMD in a sample of 1000 unrelated American Caucasians. We found that GPRC6A gene polymorphisms were significantly associated with human spine BMD. These data indicate that GRPC6A directly participates in the regulation of osteoblast-mediated bone mineralization and may mediate the anabolic effects of extracellular amino acids, osteocalcin, and divalent cations in bone. © 2010 American Society for Bone and Mineral Research.
PMCID: PMC3153369  PMID: 19874200
GPRC6A; G protein–coupled receptor (GPCR); osteoblast; bone mineral density; gene polymorphisms
2.  GPRC6A Null Mice Exhibit Osteopenia, Feminization and Metabolic Syndrome 
PLoS ONE  2008;3(12):e3858.
GPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown.
Methods/Principal Findings
In this study, we created and characterized the phenotype of GPRC6A−/− mice. We observed complex metabolic abnormalities in GPRC6A−/− mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A−/− mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A−/− mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A−/− mice exhibited increments in urine Ca/Cr and PO4/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A−/− mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone.
GPRC6A−/− mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.
PMCID: PMC2585477  PMID: 19050760
3.  Osteoblast Calcium-Sensing Receptor Has Characteristics of ANF/7TM Receptors 
Journal of cellular biochemistry  2005;95(6):1081-1092.
There is evidence for a functionally important extracelluar calcium-sensing receptor in osteoblasts, but there is disagreement regarding its identity. Candidates are CASR and a putative novel calcium-sensing receptor, called Ob.CASR. To further characterize Ob.CASR and to distinguish it from CASR, we examined the extracellular cation-sensing response in MC3T3-E1 osteoblasts and in osteoblasts derived from CASR null mice. We found that extracellular cations activate ERK and serum response element (SRE)-luciferase reporter activity in osteoblasts lacking CASR. Amino acids, but not the calcimimetic NPS-R568, an allosteric modulator of CASR, also stimulate Ob.CASR-dependent SRE-luciferase activation in MC3T3-E1 osteoblasts. In addition, we found that the dominant negative Gαq(305–359) construct inhibited cation-stimulated ERK activation, consistent with Ob.CASR coupling to Gαq-dependent pathways. Ob.CASR is also a target for classical GPCR desensitization mechanisms, since β-arrestins, which bind to and uncouple GRK phosphorylated GPCRs, attenuated cation-stimulated SRE-luciferase activity in CASR deficient osteoblasts. Finally, we found that Ob.CASR and CASR couple to SRE through distinct signaling pathways. Ob.CASR does not activate RhoA and C3 toxin fails to block Ob.CASR-induced SRE-luciferase activity. Mutational analysis of the serum response factor (SRF) and ternary complex factor (TCF) elements in SRE demonstrates that Ob.CASR predominantly activates TCF-dependent mechanisms, whereas CASR activates SRE-luciferase mainly through a RhoA and SRF-dependent mechanism. The ability of Ob.CASR to sense cations and amino acids and function like a G-protein coupled receptor suggests that it may belong to the family of receptors characterized by an evolutionarily conserved amino acid sensing motif (ANF) linked to an intramembranous 7 transmembrane loop region (7TM).
PMCID: PMC1360183  PMID: 15962313
G-protein coupled receptors; calcium-sensing; osteoblasts; β-arrestin; Gαq; ERK; SRE; CASR, calcium-sensing receptor; Ob.CASR, osteoblastic calcium-sensing receptor; GPCR, G-protein coupled receptor; ERK, extracellular signal-regulated kinase; SRE, serum response element; SRF, serum response factor; TCF, ternary complex factor
4.  GPRC6A regulates prostate cancer progression 
The Prostate  2011;72(4):399-409.
GPRC6A is a nutrient sensing GPCR that is activated in vitro by a variety of ligands, including amino acids, calcium, zinc, osteocalcin (OC) and testosterone. The association between nutritional factors and risk of prostate cancer, the finding of increased expression of OC in prostate cancer cells and the association between GPRC6A and risk of prostate cancer in Japanese men implicates a role of GPRC6A in prostate cancer.
We examined if GPRC6A is expressed in human prostate cancer cell lines and used siRNA-mediated knockdown GPRC6A expression in prostate cancer cells to explore the function of GPRC6A in vitro. To assess the role GPRC6A in prostate cancer progression in vivo we intercrossed Gprc6a−/− mice onto the TRAMP mouse prostate cancer model.
GPRC6A transcripts were markedly increased in prostate cancer cell lines 22Rv1, PC-3 and LNCaP, compared to the normal prostate RWPE-1 cell line. In addition, a panel of GPRC6A ligands, including calcium, OC, and arginine, exhibited in prostate cancer cell lines a dose-dependent stimulation of ERK activity, cell proliferation, chemotaxis, and prostate specific antigen and Runx 2 gene expression. These responses were inhibited by siRNA-mediated knockdown of GPRC6A. Finally, transfer of Gprc6a deficiency onto a TRAMP mouse model of prostate cancer significantly retarded prostate cancer progression and improved survival of compound Gprc6a−/−/TRAMP mice.
GPRC6A is a novel molecular target for regulating prostate growth and cancer progression. Increments in GPRC6A may augment the ability of prostate cancer cells to proliferate in response to dietary and bone derived ligands.
PMCID: PMC3183291  PMID: 21681779
GPRC6A; GPCR; calcium; osteocalcin; siRNA; prostate cancer; cell proliferation; metastases
5.  Gprc5a deletion enhances the transformed phenotype in normal and malignant lung epithelial cells by eliciting persistent Stat3 signaling induced by autocrine Lif 
Cancer research  2010;70(21):8917-8926.
Signal transduction and activator of transcription 3 (Stat3) is activated by cytokines and growth factors in lung cancers and regulates expression of genes implicated in cell growth, survival, and transformation. Previously, we found that mice with a deletion of the G protein-coupled receptor, family C, group 5, member a (Gprc5a) gene develop lung tumors indicating that Gprc5a is a tumor suppressor. Herein, we show that epithelial cells from Gprc5a knockout mouse lung (Gprc5a−/− cells) survive better in vitro in medium deprived of exogenous growth factors and form more colonies in semi-solid medium than their counterparts from wildtype mice (Gprc5a+/+ cells). Stat3 Tyrosine 705 phosphorylation and expression of several Stat3-regulated anti-apoptotic genes were higher in Gprc5a−/− than in Gprc5a+/+ cells. Both cell types secreted Leukemia inhibitory factor (Lif), however, whereas Stat3 activation was persistent in Gprc5a−/− cells it was transient in Gprc5a+/+ cells. Lung adenocarcinoma cells isolated from Gprc5a−/− mice also exhibited autocrine Lif-mediated Stat3 activation. The level of Socs3, the endogenous Stat3 inhibitory protein, was higher in Gprc5a+/+ than in Gprc5a−/− cells and expression of the tumor suppressor stabilized Socs3. Inhibition of Stat3 signaling in Gprc5a−/− normal and cancer cells by the Jak2 inhibitor AG490 or by a dominant negative Stat3(Y705F) increased starvation-induced apoptosis and inhibited colony formation. These results demonstrate that persistent Stat3 activation is important for the survival and transformation of Gprc5a−/− lung cells and suggest that the tumor suppressive effects of Gprc5a are mediated, at least in part, by inhibition of Stat3 signaling via Socs3 stabilization.
PMCID: PMC2970717  PMID: 20959490
Stat3; Gprc5a; lung cancer; Lif; apoptosis
6.  Pharmacology of the calcium sensing receptor 
Calcium sensing receptor (CASR) is a G-protein couple receptor which plays a key role in calcium homeostasis in vertebrates. Its extracellular domain is sensitive to divalent cations, aminoacids and polyamines. In parathyroid glands, CASR activation causes parathyroid hormone (PTH) reduction and subsequently a decrease in blood calcium concentration. In PTH-dependent disorders, e.g. primary and secondary hyperparathyroidism (HPT), the need for therapeutic options other than surgery led to the synthesis of various allosteric CASR agonists (calcimimetics), such as cinacalcet. Cinacalcet is the only calcimimetic approved for HPT secondary to chronic kidney disease (CDK), parathyroid carcinoma, and, in some countries, primary HPT. Clinical trials showed that cinacalcet reduced PTH and calcemia both in CDK and primary HPT, lowering the risk of bone fractures, surgery, and cardiovascular complications in the former patients. Long-term safety and pharmacoeconomics have to be fully tested yet. Few both in vitro and in vivo studies showed an association between Arg990Gly-CASR polymorphism and cinacalcet sensitivity, though in patients with severe CASR inactivating mutations the drug substantially retained its positive clinical effects.
Recently, a new class of allosteric antagonists of CASR, i.e. calcilytics, has been synthesized. Calcilytics are structurally similar to calcimimetics, but exert their effects acting on a different allosteric site. Infusion of calcilytics was followed by transient rise in PTH and calcium. One of these compounds, ronacaleret, was able to increase femur BMD in post menopausal women, but with induction of mild hyperparathyroidism. In the future, calcilytics may contribute to the osteoporosis treatment choice.
PMCID: PMC3917576  PMID: 24554924
CASR; calcium sensing receptor; cinacalcet; calcium metabolism; ronacaleret
7.  A Flanking Gene Problem Leads to the Discovery of a Gprc5b Splice Variant Predominantly Expressed in C57Bl/6J Mouse Brain and in Maturing Neurons 
PLoS ONE  2010;5(4):e10351.
Gprc5b, a retinoic acid-inducible orphan G protein–coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins possibly involved in non-canonical Wnt signaling. Many GPCR transcripts are alternatively spliced, which diversifies this class of proteins in their cell- and tissue-specific signaling, regulatory and/or pharmacological properties. We previously generated p97FE65 isoform-specific knockout mice that showed learning/memory deficits. In this study, we further characterized the 97FE65 null mice using cDNA microarray and RT-PCR analyses.
Methodology/Principal Findings
We discovered a novel brain-specific C-terminal splice variant of Gprc5b, Gprc5b_v2, which was differentially expressed in p97FE65 wild type and null mouse brains. The null mice were generated in 129/Sv ES cells, and backcrossed to C57Bl/6J for ten generations. We found that expression of Gprc5b_v2 mRNA in the brains of p97FE65 null mice was dramatically down-regulated (more than 20 fold) compared to their wild type littermates. However, expression profiles of Gprc5b variants and SNP analysis surrounding the FE65 locus suggest that the down-regulation is unlikely due to the altered FE65 function, but rather is caused by gene retention from the 129/Sv ES cells. Consistently, in contrast to ubiquitously expressed Gprc5b_v1, Gprc5b_v2 was predominantly expressed in the brain tissues of C57Bl/6J mice. The alternative splicing of the 3′ terminal exon also altered the protein coding sequences, giving rise to the characteristic C-termini. Levels of Gprc5b_v2 mRNA were increased during neuronal maturation, paralleling the expression of synaptic proteins. Overexpression of both Gprc5b variants stimulated neurite-like outgrowth in a neuroblastoma cell line.
Our results suggest that Gprc5b-v2 may play a role during brain maturation and in matured brain, possibly through the regulation of neuronal morphology and protein-protein interaction. This study also highlights the fact that unexpected gene retention following repeated backcrosses can lead to important biological consequences.
PMCID: PMC2859937  PMID: 20436672
8.  Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors 
Nature Communications  2012;3:1329-.
Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca2+ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.
Levels of extracellular calcium can increase at sites of infection and inflammation; however, the physiological significance of this has been unclear. This work shows that extracellular calcium acts as a danger signal, triggering the NLRP3 inflammasome via two G protein-coupled receptors.
PMCID: PMC3535422  PMID: 23271661
9.  Calcium Sensing Receptor Expression in Ovine Amniotic Fluid Mesenchymal Stem Cells and the Potential Role of R-568 during Osteogenic Differentiation 
PLoS ONE  2013;8(9):e73816.
Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.
PMCID: PMC3767786  PMID: 24040082
10.  Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells 
PLoS ONE  2014;9(6):e98777.
Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.
PMCID: PMC4048219  PMID: 24905090
11.  The calcium-sensing receptor changes cell shape via a β-arrestin-1–ARNO–ARF6–ELMO protein network 
Journal of cell science  2007;120(Pt 15):2489-2497.
G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves β-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative β-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, β-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter β-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, β-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of β-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation.
PMCID: PMC3324777  PMID: 17623778
Calcium-sensing receptor; Cytoskeleton; β-arrestin 1; ARF6; ARNO; ELMO
12.  Calcium-Sensing Receptor-Mediated Osteogenic and Early-Stage Neurogenic Differentiation in Umbilical Cord Matrix Mesenchymal Stem Cells from a Large Animal Model 
PLoS ONE  2014;9(11):e111533.
Umbilical cord matrix mesenchymal stem cells (UCM-MSCs) present a wide range of potential therapeutical applications. The extracellular calcium-sensing receptor (CaSR) regulates physiological and pathological processes. We investigated, in a large animal model, the involvement of CaSR in triggering osteogenic and neurogenic differentiation of two size-sieved UCM-MSC lines, by using AMG641, a novel potent research calcimimetic acting as CaSR agonist.
Methodology/Principal Findings
Large (>8µm in diameter) and small (<8µm) equine UCM-MSC lines were cultured in medium with high calcium (Ca2+) concentration ([Ca2+]o; 2.87 mM) and dose-response effects of AMG641 (0.01 to 3µM) on cell proliferation were evaluated. Both cell lines were then cultured in osteogenic or neurogenic differentiation medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) AMG641 (0.05, 0.1 or 1 µM) with high [Ca2+]o and 4) the CaSR antagonist NPS2390 (10 mM for 30 min) followed by incubation with AMG641 in high [Ca2+]o. Expression of osteogenic or neurogenic differentiation biomarkers was compared among groups. In both cell lines, AMG641 dose-dependently increased cell proliferation (up to P<0.001). Osteogenic molecular markers expression was differentially regulated by AMG641, with stimulatory (OPN up-regulation) in large or inhibitory (RUNX2 and OPN down-regulation) effects in small cells, respectively. AMG641 significantly increased alkaline phosphatase activity and calcium phosphate deposition in both cell lines. Following treatment with AMG641 during osteogenic differentiation, in both cell lines CaSR expression was inversely related to that of osteogenic markers and inhibition of CaSR by NPS2390 blocked AMG641-dependent responses. Early-stage neurogenic differentiation was promoted/triggered by AMG641 in both cell lines, as Nestin and CaSR mRNA transcription up-regulation were observed.
Calcium- and AMG641-induced CaSR stimulation promoted in vitro proliferation and osteogenic and early-stage neurogenic differentiation of UCM-MSCs. CaSR activation may play a fundamental role in selecting specific differentiation checkpoints of these two differentiation routes, as related to cell commitment status.
PMCID: PMC4224416  PMID: 25379789
13.  Calcium Nutrition and Extracellular Calcium Sensing: Relevance for the Pathogenesis of Osteoporosis, Cancer and Cardiovascular Diseases 
Nutrients  2013;5(1):302-327.
Through a systematic search in Pubmed for literature, on links between calcium malnutrition and risk of chronic diseases, we found the highest degree of evidence for osteoporosis, colorectal and breast cancer, as well as for hypertension, as the only major cardiovascular risk factor. Low calcium intake apparently has some impact also on cardiovascular events and disease outcome. Calcium malnutrition can causally be related to low activity of the extracellular calcium-sensing receptor (CaSR). This member of the family of 7-TM G-protein coupled receptors allows extracellular Ca2+ to function as a “first messenger” for various intracellular signaling cascades. Evidence demonstrates that Ca2+/CaSR signaling in functional linkage with vitamin D receptor (VDR)-activated pathways (i) promotes osteoblast differentiation and formation of mineralized bone; (ii) targets downstream effectors of the canonical and non-canonical Wnt pathway to inhibit proliferation and induce differentiation of colorectal cancer cells; (iii) evokes Ca2+ influx into breast cancer cells, thereby activating pro-apoptotic intracellular signaling. Furthermore, Ca2+/CaSR signaling opens Ca2+-sensitive K+ conductance channels in vascular endothelial cells, and also participates in IP3-dependent regulation of cytoplasmic Ca2+, the key intermediate of cardiomyocyte functions. Consequently, impairment of Ca2+/CaSR signaling may contribute to inadequate bone formation, tumor progression, hypertension, vascular calcification and, probably, cardiovascular disease.
PMCID: PMC3571650  PMID: 23340319
Calcium intake; calcium-sensing receptor; Wnt signaling; vitamin D; bone formation; colorectal cancer; breast cancer; hypertension; vasculature; cardiac functions
14.  Calcimimetic R-568 and Its Enantiomer S-568 Increase Nitric Oxide Release in Human Endothelial Cells 
PLoS ONE  2012;7(1):e30682.
Calcimimetics, such as R-568, are thought to activate G protein-linked Ca2+-sensing receptor (CaSR) by allosterically increasing the affinity of the receptor for Ca2+ allowing for efficient control of uremic hyperparathyroidism. Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects.
Methodology/Principal Findings
Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular Ca2+ levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay (both in HUVECs and in Human Aortic Endothelial Cells) and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca2+ levels by mobilization of Ca2+ from intracellular stores, which in turn augmented NO release by a time- and Ca2+-dependent increase in eNOS-ser1177 phosphorylation levels.
Taken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents.
PMCID: PMC3266284  PMID: 22295103
15.  Elevation of Extracellular Ca2+ Induces Store-Operated Calcium Entry via Calcium-Sensing Receptors: A Pathway Contributes to the Proliferation of Osteoblasts 
PLoS ONE  2014;9(9):e107217.
The local concentration of extracellular Ca2+ ([Ca2+]o) in bone microenvironment is accumulated during bone remodeling. In the present study we investigated whether elevating [Ca2+]o induced store-operated calcium entry (SOCE) in primary rat calvarial osteoblasts and further examined the contribution of elevating [Ca2+]o to osteoblastic proliferation.
Cytosolic Ca2+ concentration ([Ca2+]c) of primary cultured rat osteoblasts was detected by fluorescence imaging using calcium-sensitive probe fura-2/AM. Osteoblastic proliferation was estimated by cell counting, MTS assay and ATP assay. Agonists and antagonists of calcium-sensing receptors (CaSR) as well as inhibitors of phospholipase C (PLC), SOCE and voltage-gated calcium (Cav) channels were applied to study the mechanism in detail.
Our data showed that elevating [Ca2+]o evoked a sustained increase of [Ca2+]c in a dose-dependent manner. This [Ca2+]c increase was blocked by TMB-8 (Ca2+ release inhibitor), 2-APB and BTP-2 (both SOCE blockers), respectively, whereas not affected by Cav channels blockers nifedipine and verapamil. Furthermore, NPS2143 (a CaSR antagonist) or U73122 (a PLC inhibitor) strongly reduced the [Ca2+]o-induced [Ca2+]c increase. The similar responses were observed when cells were stimulated with CaSR agonist spermine. These data indicated that elevating [Ca2+]o resulted in SOCE depending on the activation of CaSR and PLC in osteoblasts. In addition, high [Ca2+]o significantly promoted osteoblastic proliferation, which was notably reversed by BAPTA-AM (an intracellular calcium chelator), 2-APB, BTP-2, TMB-8, NPS2143 and U73122, respectively, but not affected by Cav channels antagonists.
Elevating [Ca2+]o induced SOCE by triggering the activation of CaSR and PLC. This process was involved in osteoblastic proliferation induced by high level of extracellular Ca2+ concentration.
PMCID: PMC4177836  PMID: 25254954
16.  Comparison of human chromosome 19q13 and syntenic region on mouse chromosome 7 reveals absence, in man, of 11.6 Mb containing four mouse calcium-sensing receptor-related sequences: relevance to familial benign hypocalciuric hypercalcaemia type 3 
Familial benign hypocalciuric hypercalcaemia (FBHH) is a genetically heterogeneous disorder that consists of three designated types, FBHH1, FBHH2 and FBHH3, whose chromosomal locations are 3q21.1, 19p and 19q13, respectively. FBHH1 is caused by mutations of a calcium-sensing receptor (CaSR), but the abnormalities underlying FBHH2 and FBHH3 are unknown. FBHH3, also referred to as the Oklahoma variant (FBHHOk), has been mapped to a 12cM interval, flanked by D19S908 and D19S866. To refine the location of FBHH3, we pursued linkage studies using 24 polymorphic loci. Our results establish a linkage between FBHH3 and 17 of these loci, and indicate that FBHH3 is located in a 4.1 Mb region flanked centromerically by D19S112 and telomerically by rs245111, which in the syntenic region on mouse chromosome 7 contains four Casr-related sequences (Gprc2a-rss). However, human homologues of these Gprc2a-rss were not found and a comparative analysis of the 22.0 Mb human and 39.3 Mb mouse syntenic regions showed evolutionary conservation of two segments that were inverted with loss from the human genome of 11.6 Mb that contained the four Gprc2a-rss. Thus, FBHH3 cannot be attributed to Gprc2a-rss abnormalities. DNA sequence analysis of 12 other genes from the interval that were expressed in the parathyroids and/or kidneys did not detect any abnormalities, thereby indicating that these genes are unlikely to be the cause of FBHH3. The results of this study have refined the map location of FBHH3, which will facilitate the identification of another CaSR or a mediator of calcium homeostasis.
PMCID: PMC2842244  PMID: 19809483
calcium; linkage; rearrangement
17.  Comparison of human chromosome 19q13 and syntenic region on mouse chromosome 7 reveals absence, in man, of 11.6Mb containing 4 mouse calcium sensing receptor related sequences: relevance to familial benign hypocalciuric hypercalcaemia type 3 
Familial benign hypocalciuric hypercalcaemia (FBHH) is a genetically heterogenous disorder that consists of three types designated, FBHH1, FBHH2 and FBHH3 whose chromosomal locations are 3q21.1, 19p and 19q13, respectively. FBHH1 is caused by mutations of a calcium sensing receptor (CaSR), but the abnormalities underlying FBHH2 and FBHH3 are unknown. FBHH3, also referred to as the Oklahoma variant (FBHHOk), has been mapped to a 12cM interval, flanked by D19S908 and D19S866. To refine the location of FBHH3, we pursued linkage studies using 24 polymorphic loci. Our results establish linkage between FBHH3 and 17 of these loci, and indicate that FBHH3 is located in a 4.1Mb region flanked centromerically by D19S112 and telomerically by rs245111, which in the syntenic region on mouse chromosome 7 contains 4 Casr related sequences (Gprc2a-rss). However, human homologues of these Gprc2a-rss were not found and a comparative analysis of the 22.0Mb human and 39.3Mb mouse syntenic regions showed evolutionary conservation of 2 segments that were inverted with loss from the human genome of 11.6Mb that contained the 4 Gprc2a-rss. Thus, FBHH3 cannot be due to Gprc2a-rss abnormalities. DNA sequence analysis of 12 other genes from the interval that were expressed in the parathyroids and/or kidneys did not detect any abnormalities, thereby indicating that these genes are unlikely to be the cause of FBHH3. The results of this study have refined the map location of FBHH3, which will facilitate the identification of another CaSR or a mediator of calcium homeostasis.
PMCID: PMC2842244  PMID: 19809483
calcium; linkage; rearrangement
18.  Epithelial calcium-sensing receptor activation by eosinophil granule protein analog stimulates collagen matrix contraction 
Pediatric research  2012;73(4 0 1):414-419.
Eosinophils reside in normal gastrointestinal tracts and increase in disease. Receptors for eosinophil derived granule proteins (EDGPs) have not been identified but highly cationic molecules, similar to eosinophil proteins, bind extracellular calcium-sensing receptors (CaSR). We hypothesized that stimulation of CaSR by eosinophil proteins activates epithelial cells.
Caco2 intestinal epithelial cells, AML14.3D10 eosinophils, wild type human embryonic kidney 293 (HEK293) cells not expressing CaSR (HEK-WT) or CaSR transfected HEK293 cells (HEK-CaSR) were stimulated with an eosinophil protein analog poly-L-arginine (PA) and phosphorylated extracellular signal-regulated kinases 1/2 (pERK) was measured. Functional activation was measured with collagen lattice contraction assays.
Co-culture of Caco2 cells with AML14.3D10 eosinophils augmented lattice contraction compared to lattices containing Caco2 cells alone. PA stimulation of Caco2 lattices augmented contraction. HEK-CaSR stimulation with PA or Ca2+ resulted in greater pERK activation than stimulated HEK-WT cells. PA stimulated greater HEK-CaSR lattice contraction than unstimulated lattices. Contraction of PA stimulated and unstimulated HEK-WT lattices did not differ.
Exposure of intestinal epithelia to the EDGP analog, PA, stimulates CaSR dependent ERK phosphorylation and epithelial mediated collagen lattice contraction. We speculate that EDGP release within the epithelial layers activates the CaSR receptor leading to matrix contraction and tissue fibrosis.
PMCID: PMC4321999  PMID: 23269116
19.  Molecular evolution of a chordate specific family of G protein-coupled receptors 
Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success.
We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling.
GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become chordates.
PMCID: PMC3238225  PMID: 21827690
GPRC5; chordate development; retinoic acid; phylogeny; GPCR
20.  Spontaneous glutamate release is independent of calcium influx and tonically activated by the calcium-sensing receptor 
Spontaneous release of glutamate is important for maintaining synaptic strength and controlling spike timing in the brain. Mechanisms regulating spontaneous exocytosis remain poorly understood. Extracellular calcium concentration ([Ca2+]o) regulates Ca2+ entry through voltage-activated calcium channels (VACCs) and consequently is a pivotal determinant of action potential-evoked vesicle fusion. Extracellular Ca2+ also enhances spontaneous release, but via unknown mechanisms. Here we report that external Ca2+ triggers spontaneous glutamate release more weakly than evoked release in mouse neocortical neurons. Blockade of VACCs has no effect on the spontaneous release rate or its dependence on [Ca2+]o. Intracellular [Ca2+] slowly increases in a minority of neurons following increases in [Ca2+]o. Furthermore, the enhancement of spontaneous release by extracellular calcium is insensitive to chelation of intracellular calcium by BAPTA. Activation of the calcium-sensing receptor (CaSR), a G-protein coupled receptor present in nerve terminals, by several specific agonists increased spontaneous glutamate release. The frequency of spontaneous synaptic transmission was decreased in CaSR mutant neurons. The concentration effect relationship for extracellular calcium regulation of spontaneous release was well described by a combination of CaSR-dependent and CaSR-independent mechanisms. Overall these results indicate that extracellular Ca2+ does not trigger spontaneous glutamate release by simply increasing calcium influx but stimulates CaSR and thereby promotes resting spontaneous glutamate release.
PMCID: PMC3097128  PMID: 21430159
21.  G protein-coupled receptor, family C, group 5 (GPRC5B) downregulation in spinal cord neurons is involved in neuropathic pain 
Korean Journal of Anesthesiology  2014;66(3):230-236.
G protein-coupled receptor, family C, group 5 (GPRC5B), a retinoic acid-inducible orphan G-protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins presumably related in non-canonical Wnt signaling. In this study, we investigated altered GPRC5B expression in the dorsal horn of the spinal cord after spinal nerve injury and its involvement in the development of neuropathic pain.
After induction of anesthesia by intraperitoneal injection of pentobarbital (35 mg /kg), the left L5 spinal nerve at the level of 2 mm distal to the L5 DRG was tightly ligated with silk and cut just distal to the ligature. Seven days after nerve injury, animals were perfused with 4% paraformaldehyde, and the spinal cords were extracted and post-fixed at 4℃ overnight. To identify the expression of GPRC5B and analyze the involvement of GPRC5B in neuropathic pain, immunofluorescence was performed using several markers for neurons and glial cells in spinal cord tissue.
After L5 spinal nerve ligation (SNL), the expression of GPRC5B was decreased in the ipsilateral part, as compared to the contralateral part, of the spinal dorsal horn. SNL induced the downregulation of GPRC5B in NeuN-positive neurons in the spinal dorsal horn. However, CNPase-positive oligodendrocytes, OX42-positive microglia, and GFAP-positive astrocytes were not immunolabeled with GPRC5B antibody in the spinal dorsal horn.
These results imply that L5 SNL-induced GPRC5B downregulation may affect microglial activation in the spinal dorsal horn and be involved in neuropathic pain.
PMCID: PMC3983420  PMID: 24729846
GPCR5B; Microglial activation; Neuroglial cell; Neuropathic pain; Spinal nerve injury
22.  Receptor-activity-modifying proteins are required for forward trafficking of the calcium-sensing receptor to the plasma membrane 
Journal of cell science  2005;118(Pt 20):4709-4720.
The calcium-sensing receptor (CaSR) is a class III G-protein-coupled receptor (GPCR) that responds to changes in extracellular calcium concentration and plays a crucial role in calcium homeostasis. The mechanisms controlling CaSR trafficking and surface expression are largely unknown. Using a CaSR tagged with the pH-sensitive GFP super-ecliptic pHluorin (SEP-CaSR), we show that delivery of the GPCR to the cell surface is dependent on receptor-activity-modifying proteins (RAMPs). We demonstrate that SEP-CaSRs are retained in the endoplasmic reticulum (ER) in COS7 cells that do not contain endogenous RAMPs whereas they are delivered to the plasma membrane in HEK 293 cells that do express RAMP1. Coexpression of RAMP1 or RAMP3, but not RAMP2, in COS7 cells was sufficient to target the CaSR to the cell surface. RAMP1 and RAMP3 colocalised and coimmunoprecipitated with the CaSR suggesting that these proteins associate within the cell. Our results indicate that RAMP expression promotes the forward trafficking of the GPCR from the ER to the Golgi apparatus and results in mature CaSR glycosylation, which is not observed in RAMP-deficient cells. Finally, silencing of RAMP1 in the endogenously expressing HEK293 cells using siRNA resulted in altered CaSR traffic. Taken together, our results show that the association with RAMPs is necessary and sufficient to transfer the immature CaSR retained in the ER towards the Golgi where it becomes fully glycosylated prior to delivery to the plasma membrane and demonstrate a role for RAMPs in the trafficking of a class III GPCR.
PMCID: PMC3311923  PMID: 16188935
Calcium-sensing receptor; Receptor-activity-modifying protein; siRNA; pHluorin; GPCR
23.  Functional Expression of the Extracellular Calcium Sensing Receptor (CaSR) in Equine Umbilical Cord Matrix Size-Sieved Stem Cells 
PLoS ONE  2011;6(3):e17714.
The present study investigates the effects of high external calcium concentration ([Ca2+]o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.
Methodology/Principal Findings
A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca2+]o and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca2+]o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca2+]o was not effective in this cell line. In small cells, both higher [Ca2+]o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca2+]o and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.
In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.
PMCID: PMC3060090  PMID: 21437284
24.  Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. 
Journal of Clinical Investigation  1997;99(8):1917-1925.
Missense mutations have been identified in the coding region of the extracellular calcium-sensing receptor (CASR) gene and cause human autosomal dominant hypo- and hypercalcemic disorders. The functional effects of several of these mutations have been characterized in either Xenopus laevis oocytes or in human embryonic kidney (HEK293) cells. All of the mutations that have been examined to date, however, cause single putative amino acid substitutions. In this report, we studied a mutant CASR with an Alu-repetitive element inserted at codon 876, which was identified in affected members of families with the hypercalcemic disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), to understand how this insertion affects CASR function. After cloning of the Alu-repetitive element into the wild-type CASR cDNA, we transiently expressed the mutant receptor in HEK293 cells. Expression of mutant and wild-type receptors was assessed by Western analysis, and the effects of the mutation on extracellular calcium (Ca2+(o)) and gadolinium (Gd3+(o)) elicited increases in the cytosolic calcium concentration (Ca2+(i)) were examined in fura-2-loaded cells using dual wavelength fluorimetry. The insertion resulted in truncated receptor species that had molecular masses some 30 kD less than that of the wild-type CASR and exhibited no Ca2+(i) responses to either Ca2+(o) or Gd3+(o). A similar result was observed with a mutated CASR truncated at residue 876. However, the Alu mutant receptor had no impact on the function of the coexpressed wild-type receptor. Interestingly, the Alu mutant receptor demonstrated decreased cell surface expression relative to the wild-type receptor, whereas the CASR (A877stop) mutant exhibited increased cell surface expression. Thus, like the missense mutations that have been characterized to date in families with FHH, the Alu insertion in this family is a loss-of-function mutation that produces hypercalcemia by reducing the number of normally functional CASRs on the surface of parathyroid and kidney cells. In vitro transcription of exon 7 of the CASR containing the Alu sequence yielded the full-length mutant product and an additional shorter product that was truncated due to stalling of the polymerase at the poly(T) tract. In vitro translation of the mutant transcript yielded three truncated protein products representing termination in all three reading frames at stop codons within the Alu insertion. Thus sequences within the Alu contribute to slippage or frameshift mutagenesis during transcription and/or translation.
PMCID: PMC508016  PMID: 9109436
25.  Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release in vivo 
BMC Physiology  2004;4:5.
The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium.
To demonstrate regulatory effects of CaSR on calcitonin release, we studied calcitonin responsiveness to calcium in normal and CaSR heterozygous-ablated (Casr+/-) mice. Casr+/- mice have hypercalcemia and hypocalciuria, and live normal life spans. Each mouse received either 500 μl of normal saline or one of two doses of elemental calcium (500 μmol/kg or 5 mmol/kg) by intraperitoneal injection. Ionized calcium was measured at baseline and 10 minutes, and serum calcitonin was measured on the 10 minute sample.
At baseline, Casr+/- mice had a higher blood calcium, and in response to the two doses of elemental calcium, had greater increments and peak levels of ionized calcium than their wild type littermates. Despite significantly higher ionized calcium levels, the calcitonin levels of Casr+/- mice were consistently lower than wild type at any ionized calcium level, indicating that the dose-response curve of calcitonin to increases in ionized calcium had been significantly blunted or shifted to the right in Casr+/- mice.
These results confirm that the CaSR is a physiological regulator of calcitonin; therefore, in response to increases in ionized calcium, the CaSR inhibits parathyroid hormone secretion and stimulates calcitonin secretion.
PMCID: PMC419359  PMID: 15099400
Animal models/rodent; calcitonin; familial hypocalciuric hypocalcemia; calcium receptor; gene knock-out

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