Biochemistry, physiology and pathophysiology of the extracellular calcium-sensing receptor

Calcium (Ca(2+)) has long been recognized as a physiologically indispensable ion owing to its numerous intra- and extracellular roles. More recently, it has become apparent that extracellular calcium (Ca(2+)(o)) also serves as an extracellular first messenger following the cloning of a Ca(2+)(o)-sensing receptor (CaR) that belongs to the superfamily of G protein-coupled receptors (GPCR). The CaR probably functions as a dimer in performing its central role of "sensing" minute alterations in Ca(2+)(o) and adjusting the secretion of parathyroid hormone (PTH) so as to normalize Ca(2+)(o) through the actions of PTH on the effector elements of the mineral ion homeostatic system (e.g., kidney, bone and intestine). Several inherited human conditions are caused by inactivating or activating mutations of this receptor, and mice have been generated with targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in patients with these conditions and in CaR-deficient mice have proven the physiological importance of the CaR in mineral ion homeostasis. An accumulating body of evidence, however, suggests that the CaR also plays numerous roles outside the realm of systemic mineral ion homeostasis. The receptor regulates processes such as cellular proliferation and differentiation, secretion, membrane polarization and apoptosis in a variety of tissues/cells. Finally, the availability of specific "calcimimetic", allosteric CaR activators - which are currently in clinical trials - will probably have therapeutic implications for diseases caused by malfunction of the CaR in tissues not only within, but also outside, the mineral ion homeostatic system.     

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes.     

Conditional knockout mouse for tissue-specific disruption of the cyclooxygenase-2 (Cox-2) gene

Cyclooxygenase-2 (Cox-2) modulates many normal functions, and appears to play a role in a wide variety of pathophysiologic conditions. Cox-2 gene expression is induced in many different cell types, in response to many distinct stimuli. We generated a conditional knockout mouse in which critical exons of the Cox-2 gene are flanked with loxP sites. Cox-2(flox/flox) mice appear normal and are fertile. Recombination at the loxP sites, loss of Cox-2 protein expression, and prevention of induced PGE2 accumulation are observed in Cox-2(flox/flox) mouse embryo fibroblasts following infection with an adenovirus expressing CRE recombinase. In vivo recombination at the Cox-2(flox) allele was demonstrated in the liver of Cox-2(flox/flox) mice following intravenous injection of adenovirus expressing CRE recombinase. Spatially and temporally restricted elimination of the Cox-2 gene in Cox-2(flox/flox) conditional knockout mice should provide a valuable tool to analyze the cell type-specific role of Cox-2 in many disease models.     

Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice

The aim of the present studies was to determine the effects of reduced or absent serotonin (5-HT) transporters (5-HTTs) on 5-HT2A and 5-HT2C receptors. The density of 5-HT2C receptors was significantly increased in the amygdala and choroid plexus of 5-HTT knockout mice. On the other hand, the density of 5-HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5-HTT knockout mice. However, 5-HT2A mRNA was not changed in any brain region measured. 5-HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5-HT2A receptors was evaluated by hormonal responses to (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5-HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5-HTTs changes the density of 5-HT2A and 5-HT2C receptors in a brain region-specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5-HTT knockout mice.     

The extracellular calcium-sensing receptor is expressed in rat microglia and modulates an outward K+ channel

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that "senses" extracellular calcium ions (Ca2+o) as an extracellular first messenger. In this report, we have shown that the CaR is expressed in primary cultures of microglial cells derived from rat brain as assessed by RT-PCR using four CaR-specific primer pairs followed by sequencing of the amplified products, by northern blot analysis using a CaR-specific probe, as well as by immunocytochemistry and western analysis utilizing a specific polyclonal anti-CaR antiserum. In addition, raising Ca2+o from 0.75 to 3.0 mM or addition of the polycationic CaR agonist neomycin or a "calcimimetic" CaR activator (R-467; NPS Pharmaceuticals) increased the open state probability (Po) of a Ca(+)-activated K+ channel having a unitary conductance of 84+/-4 pS, indicating that the channel is modulated by the CaR. Therefore, our data strongly suggest that a functional CaR is expressed in cultured rat microglia, similar to that in parathyroid gland and kidney, which could potentially play an important role(s) in regulating microglial function.     

Calcium receptor stimulates chemotaxis and secretion of MCP-1 in GnRH neurons in vitro: potential impact on reduced GnRH neuron population in CaR-null mice

The factors controlling the migration of mammalian gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus are not well understood. We studied whether the extracellular calcium-sensing receptor (CaR) promotes migration/chemotaxis of GnRH neurons. We demonstrated expression of CaR in GnRH neurons in the murine basal forebrain and in two GnRH neuronal cell lines: GT1-7 (hypothalamus derived) and GN11 (olfactory bulb derived). Elevated extracellular Ca(2+) concentrations promoted chemotaxis of both cell types, with a greater effect in GN11 cells. This effect was CaR mediated, as, in both cell types, overexpression of a dominant-negative CaR attenuated high Ca(2+)-stimulated chemotaxis. We also demonstrated expression of a beta-chemokine, monocyte chemoattractant protein-1 (MCP-1), and its receptor, CC motif receptor-2 (CCR2), in the hypothalamic GnRH neurons as well as in GT1-7 and GN11 cells. Exogenous MCP-1 stimulated chemotaxis of both cell lines in a dose-dependent fashion; the effect was greater in GN11 than in GT1-7 cells, consistent with the higher CCR2 mRNA levels in GN11 cells. Activating the CaR stimulated MCP-1 secretion in GT1-7 but not in GN11 cells. MCP-1 secreted in response to CaR stimulation is biologically active, as conditioned medium from GT1-7 cells treated with high Ca(2+) promoted chemotaxis of GN11 cells, and this effect was partially attenuated by a neutralizing antibody to MCP-1. Finally, in the preoptic area of anterior hypothalamus, the number of GnRH neurons was approximately 27% lower in CaR-null mice than in mice expressing the CaR gene. We conclude that the CaR may be a novel regulator of GnRH neuronal migration likely involving, in part, MCP-1.     

Normal hematopoiesis and inflammatory responses despite discrete signaling defects in Galpha15 knockout mice

Galpha15 activates phospholipase Cbeta in response to the greatest variety of agonist-stimulated heptahelical receptors among the four Gq class G-protein alpha subunits expressed in mammals. Galpha15 is primarily expressed in hematopoietic cells in fetal and adult mice. We disrupted the Galpha15 gene by homologous recombination in embryonic stem cells to identify its biological functions. Surprisingly, hematopoiesis was normal in Galpha15(-/-) mice, Galpha15(-/-) Galphaq(-/-) double-knockout mice (which express only Galpha11 in most hematopoietic cells), and Galpha11(-/-) mice, suggesting functional redundancy in Gq class signaling. Inflammatory challenges, including thioglycolate-induced peritonitis and infection with Trichinella spiralis, stimulated similar responses in Galpha15(-/-) adults and wild-type siblings. Agonist-stimulated Ca(2+) release from intracellular stores was assayed to identify signaling defects in primary cultures of thioglycolate-elicited macrophages isolated from Galpha15(-/-) mice. C5a-stimulated phosphoinositide accumulation and Ca(2+) release was significantly reduced in Galpha15(-/-) macrophages. Ca(2+) signaling was abolished only in mutant cells pretreated with pertussis toxin, suggesting that the C5a receptor couples to both Galpha15 and Galphai in vivo. Signaling evoked by other receptors coupled by Gq class alpha subunits appeared normal in Galpha15(-/-) macrophages. Despite discrete signaling defects, compensation by coexpressed Gq and/or Gi class alpha subunits may suppress abnormalities in Galpha15-deficient mice.     

Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice.

Heterotrimeric G proteins of the Gq class have been implicated in signaling pathways regulating cardiac growth under physiological and pathological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed G alpha q class genes, G alpha q and G alpha 11, demonstrate that at least two active alleles of these genes are required for extrauterine life. Mice carrying only one intact allele [G alpha q(-/+);G alpha 11(-/-) or G alpha q(-/-);G alpha 11(-/+)] died shortly after birth. These mutants showed a high incidence of cardiac malformation. In addition, G alpha q(-/-);G alpha 11(-/+) newborns suffered from craniofacial defects. Mice lacking both G alpha q and G alpha 11 [G alpha q(-/-);G alpha 11(-/-)] died at embryonic day 11 due to cardiomyocyte hypoplasia. These data demonstrate overlap in G alpha q and G alpha 11 gene functions and indicate that the Gq class of G proteins plays a crucial role in cardiac growth and development.     

Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.     

Extracellular Ca(2+) influx and NO generation are inhibited by small interference RNA targeting extracellular Ca(2+)-sensing receptor in human umbilical vein endothelial cells

To investigate the effect of Ca(2+)-sensing receptor (CaR) on Spermine-induced extracellular Ca(2+) influx and NO generation in human umbilical vein endothelial cells (HUVEC), the small interference RNA (siRNA) specifically targeting CaR gene was designed, synthesized and transfected into HUVEC according to the cDNA sequence of human CaR gene in GenBank. The transfection efficiency and the interference efficiency of CaR protein were determined by laser scanning confocal microscopy and Western blot, respectively. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by Fura-2/AM loading. The production of NO and the activity of endothelial nitric oxide synthase (eNOS) were determined by the DAF-FM diacetate (DAF-FM DA). Western blot results demonstrated that siRNA targeting the CaR specifically decreased the expression of CaR protein in CaR siRNA group 48 h after transfection (P < 0.05). At the same time, the Spermine-induced [Ca(2+)](i), eNOS activity and NO generation were also significantly reduced (P < 0.05) in CaR siRNA group compared with those in the untransfected or negative siRNA transfected group. In conclusion, the present study suggests that the CaR plays an important role in the Spermine-evoked process of extracellular Ca(2+) influx and NO generation in HUVEC.     

Role of ceramide in Ca2+-sensing receptor-induced apoptosis

Increased extracellular Ca(2+) ([Ca(2+)](o)) can damage tissues, but the molecular mechanisms by which this occurs are poorly defined. Using HEK 293 cell lines that stably overexpress the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, we demonstrate that activation of the CaR leads to apoptosis, which was determined by nuclear condensation, DNA fragmentation, caspase-3 activation, and increased cytosolic cytochrome c. This CaR-induced apoptotic pathway is initiated by CaR-induced accumulation of ceramide which plays an important role in inducing cell death signals by distinct G protein-independent signaling pathways. Pretreatment of wild-type CaR-expressing cells with pertussis toxin inhibited CaR-induced [(3)H]ceramide formation, c-Jun phosphorylation, and caspase-3 activation. The ceramide accumulation, c-Jun phosphorylation, and caspase-3 activation by the CaR can be abolished by sphingomyelinase and ceramide synthase inhibitors in different time frames. Cells that express a nonfunctional mutant CaR that were exposed to the same levels of [Ca(2+)](o) showed no evidence of activation of the apoptotic pathway. In conclusion, we report the involvement of the CaR in stimulating programmed cell death via a pathway involving GTP binding protein alpha subunit (Galpha(i))-dependent ceramide accumulation, activation of stress-activated protein kinase/c-Jun N-terminal kinase, c-Jun phosphorylation, caspase-3 activation, and DNA cleavage.     

Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton

The G protein-coupled Ca(2+)-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca(2+) and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here, we examined whether these differing patterns of [Ca(2+)](i) oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca(2+)](i) oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G(12) subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca(2+) or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.     

Heterotrimeric G alpha q/G alpha 11 proteins function upstream of vascular endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular permeability factor/VEGF signaling

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gbetagamma subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gbetagamma minigene, hbetaARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR.     

Expression and function of the extracellular calcium-sensing receptor in pancreatic beta-cells

The extracellular calcium-sensing receptor (CaR) was first identified in tissues involved in systemic Ca2+ homeostasis, where it acts to sense changes in circulating Ca2+. It has since been reported that the CaR is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. In the present study we have used an insulin-secreting pancreatic beta-cell line (MIN6) to investigate the expression and function of CaR, using the calcimimetic A568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca2+ ([Ca2+]o). Immunocytochemistry, Western blotting and RT-PCR confirmed the expression of CaR in MIN6 cells. CaR activation was associated with rapid and transient increases in [Ca2+]o, which were accompanied by the initiation of a marked but transient insulin secretory response. Stimulation of beta-cell secretory activity had no detectable effect on CaR mRNA levels, but CaR mRNA was markedly reduced by configuring MIN6 cells into islet- like structures. Our data are consistent with an important function for the beta-cell CaR in cell - cell communication within islets to co-ordinate insulin secretory responses.     

Direct activation of trpl cation channels by G alpha11 subunits.

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.     

Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity

The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr(888) contributing significantly to this effect. To determine whether CaR(T888) is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaR(pT888)). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaR(T888A), phorbol ester (PMA) treatment increased CaR(pT888) immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca(2+) concentration from 0.5 to 2.5 mM increased CaR(T888) phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca(2+) and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaR(T888A) did exhibit increased apparent agonist sensitivity, by converting intracellular Ca(2+) (Ca(2+)(i)) oscillations to sustained plateau responses in some cells, we still observed Ca(2+)(i) oscillations in a significant number of cells. This suggests that CaR(T888) contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca(2+)(i) oscillations. Finally, dephosphorylation of CaR(T888) was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca(2+)(i) oscillations. In addition, calyculin/PMA cotreatment increased CaR(T888) phosphorylation in bovine parathyroid cells. Therefore, CaR(T888) is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.     

Evidence for a functional calcium-sensing receptor that modulates myogenic tone in rat subcutaneous small arteries

Myogenic tone of small arteries is dependent on the presence of extracellular calcium (Ca(o)(2+)), and, recently, a receptor that senses changes in Ca(2+), the calcium-sensing receptor (CaR), has been detected in vascular tissue. We investigated whether the CaR is involved in the regulation of myogenic tone in rat subcutaneous small arteries. Immunoblot analysis using a monoclonal antibody against the CaR demonstrated its presence in rat subcutaneous arteries. To determine whether the CaR was functionally active, segments of artery (< 250 microm internal diameter) mounted in a pressure myograph with an intraluminal pressure of 70 mmHg were studied after the development of myogenic tone. Increasing Ca(o)(2+) concentration ([Ca(2+)](o)) cumulatively from 0.5 to 10 mM induced an initial constriction (0.5-2 mM) followed by dilation (42 +/- 5% loss of tone). The dose-dependent dilation was mimicked by other known CaR agonists including magnesium (1-10 mM) and the aminoglycosides neomycin (0.003-10 mM) and kanamycin (0.003-3 mM). PKC activation with the phorbol ester phorbol-12,13-dibutyrate (20nM) inhibited the dilation induced by high [Ca(2+)](o) or neomycin, whereas inhibition of PKC with GF109203X (10 microM) increased the responses to Ca(o)(2+) or neomycin, consistent with the role of PKC as a negative regulator of the CaR. We conclude that rat subcutaneous arteries express a functionally active CaR that may be involved in the modulation of myogenic tone and hence the regulation of peripheral vascular resistance.     

The role of the calcium-sensing receptor in cancer

The extracellular calcium-sensing receptor (CaR) is a versatile sensor of small, polycationic molecules ranging from Ca2+ and Mg2+ through polyarginine, spermine, and neomycin. The sensitivity of the CaR to changes in extracellular Ca2+ over the range of 0.05-5 mM positions the CaR as a key mediator of cellular responses to physiologically relevant changes in extracellular Ca2+. For many cell types, including intestinal epithelial cells, breast epithelial cells, keratinocytes, and ovarian surface epithelial cells, changes in extracellular Ca2+ concentration over this range can switch the cellular behaviour from proliferation to terminal differentiation or quiescence. As cancer is predominantly a disease of disordered balance between proliferation, differentiation, and apoptosis, disruptions in the function of the CaR could contribute to the progression of neoplastic disease. Loss of the growth suppressing effects of elevated extracellular Ca2+ have been demonstrated in parathyroid hyperplasias and in colon carcinoma, and have been correlated with changes in the level of CaR expression. Activation of the CaR has also been linked to increased expression and secretion of PTHrP (parathyroid hormone-related peptide), a primary causal factor in hypercalcemia of malignancy and a contributor to metastatic processes involving bone. Although mutation of the CaR does not appear to be an early event in carcinogenesis, loss or upregulation of normal CaR function can contribute to several aspects of neoplastic progression, so that therapeutic strategies directed at the CaR could potentially serve a supportive function in cancer management.     

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca(2+) signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca(2+) concentration ([Ca(2+)](o)) and modulation of cellular processes associated with acute or sustained changes in [Ca(2+)](o) are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca(2+)](o) signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca(2+)](o) activated PKC-alpha and PKC- in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca(2+)](o) required influx of Ca(2+)through Ni(2+)-sensitive Ca(2+)channels and phosphatidylinositol-dependent phospholipase C-beta activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-alpha or - with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca(2+)](o). Activation of ERK1/2 by high [Ca(2+)](o) was not necessary for the [Ca(2+)](o)-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca(2+)](o) signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.