Homology modeling of the transmembrane domain of the human calcium sensing receptor and localization of an allosteric binding site

A homology model for the human calcium sensing receptor (hCaR) transmembrane domain utilizing bovine rhodopsin (bRho) structural information was derived and tested by docking the allosteric antagonist, NPS 2143, followed by mutagenesis of predicted contact sites. Mutation of residues Phe-668 (helix II), Arg-680, or Phe-684 (helix III) to Ala (or Val or Leu) and Glu-837 (helix VII) to Ile (or Gln) reduced the inhibitory effects of NPS 2143 on [Ca2+]i responses. The calcimimetic NPS R-568 increases the potency of Ca2+ in functional assays of CaR. Mutations at Phe-668, Phe-684, or Glu-837 attenuated the effects of this compound, but mutations at Arg-680 had no effect. In all cases, mutant CaRs responded normally to Ca2+ or phenylalanine, which act at distinct site(s). Discrimination by the Arg-680 mutant is consistent with the structural differences between NPS 2143, which contains an alkyl bridge hydroxyl group, and NPS R-568, which does not. The homology model of the CaR transmembrane domain robustly accounts for binding of both an allosteric antagonist and agonist, which share a common site, and provides a basis for the development of more specific and/or potent allosteric modulators of CaR. These studies suggest that the bRho backbone can be used as a starting point for homology modeling of even distantly related G protein-coupled receptors and provide a rational framework for investigation of the contributions of the transmembrane domain to CaR function.     

Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations

Activation of the calcium sensing receptor (CaR) by small increments in extracellular calcium (Ca(2+)(e)) induces intracellular calcium (Ca(2+)(i)) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca(2+)(i) oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca(2+)(e) at eliciting Ca(2+)(i) oscillations, particularly at low [Ca(2+)](e) (as low as 0.1 mm). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60-70 to 30-45 s, depending upon the concentrations of both Ca(2+)(e) and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca(2+)(i) oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca(2+)(i) oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca(2+) or NPS R-568 to induce Ca(2+)(i) oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca(2+)-dependent activation and regular Ca(2+)(i) oscillations. Alanine scanning within the amino acid sequence from Arg(873) to His(879) reveals a linkage between the cooperativity for Ca(2+)-dependent activation and establishment and maintenance of intracellular Ca(2+) oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.     

L-phenylalanine and NPS R-467 synergistically potentiate the function of the extracellular calcium-sensing receptor through distinct sites

The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) can be potentiated by allosteric activators including calcimimetics and l-amino acids. In this study, we found that many mutations had differential effects on the functional modulation of the CaR by these two allosteric activators, supporting the idea that these modulators act through distinct sites. 10 mm l-phenylalanine and 1 microm NPS R-467, submaximal doses of the two agents, each elicited similar modulation of R185Q. However, there are different relative potencies for these two modulators with some receptors being more responsive to l-phenylalanine and others being more responsive to NPS R-467. The responsiveness of the CaR to Ca(2+)(o) appears to be essential to observe the potentiating action of l-phenylalanine but not of NPS R-467 on the receptor. NPS R-467 reduces the Hill coefficients of the wild-type as well as mutant receptors, suggesting that engagement of all Ca(2+) binding sites is not required when the receptor is activated by NPS R-467. In contrast, l-phenylalanine has little effect on the Hill coefficients of mutant receptors. The two-site model is further supported by the observation that these two classes of modulators exert a synergistic effect on CaRs with inactivating mutations that are responsive to both modulators.     

Cellular "sensing" of extracellular calcium (Ca(2+)(o)): emerging roles in regulating diverse physiological functions

The extracellular Ca(2+) (Ca(2+)(o))-sensing receptor (CaR) critically influences Ca(2+)(o) homeostasis by regulating parathyroid hormone (PTH) secretion and renal Ca(2+) handling. Moreover, its expression in intestinal and bone cells suggests roles in all of the organs involved in maintaining systemic Ca(2+)(o) homeostasis. This G-protein coupled receptor is also expressed in a wide variety of additional cells throughout the body. While our understanding of its role(s) outside of the system governing Ca(2+)(o) metabolism remains rudimentary, the CaR will probably emerge as a versatile regulator of diverse cellular functions, including proliferation, differentiation, apoptosis, gene expression and maintenance of membrane potential. Finally, the recently developed, "calcimimetic" CaR activators, exemplified by a NPS R-467 and NPS R-568, provide novel approaches to treating diseases that previously had no effective medical therapies: topic likewise covered in this review.     

Identification of acidic residues in the extracellular loops of the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+ and a positive allosteric modulator

We investigated the role of the eight acidic residues in the extracellular loops (exo-loops) of the seven-transmembrane domain of the human Ca(2+) receptor (hCaR) in receptor activation by Ca(2+) and in response to a positive allosteric modulator, NPS R-568. Both in the context of the full-length receptor and of a truncated receptor lacking the extracellular domain (Rho-C-hCaR), we mutated each acidic residue to alanine, singly and in combination, and tested the effect on expression of the receptor, on activation by Ca(2+), and on NPS R-568 augmentation of sensitivity to Ca(2+). Of the eight acidic residues, mutation of any of three in exo-loop 2, Asp(758), Glu(759), and Glu(767), increased the sensitivity of both the full-length hCaR and of Rho-C-hCaR to activation by Ca(2+). Mutation of all five acidic residues in exo-loop 2, whether in the full-length receptor or in Rho-C-hCaR, impaired cell surface expression of the mutant receptor and thereby largely abolished response to Ca(2+). Mutation of Glu(837) in exo-loop 3 to alanine did not alter Ca(2+) sensitivity of the full-length receptor, but in both the latter context and in Rho-C-hCaR, alanine substitution of Glu(837) drastically reduced sensitivity to NPS R-568. Our data point to a key role of three specific acidic residues in exo-loop 2 in hCaR activation and to Glu(837) at the junction between exo-loop 3 and transmembrane helix seven in response to NPS R-568. We speculate on the basis of these results that the three acidic residues we identified in exo-loop 2 help maintain an inactive conformation of the seven-transmembrane domain of the hCaR.     

Calcium-sensing receptor-mediated ERK1/2 activation requires Galphai2 coupling and dynamin-independent receptor internalization

The calcium-sensing receptor (CaR) recently has been shown to activate MAP kinase (ERK1/2) in various cell types as well as in heterologous expression systems. In this study we show that the CaR agonist NPS R-467 (1 microm), which does not activate the CaR by itself, robustly activates ERK1/2 in the presence of a low concentration of Ca(2+) (0.5 mm CaCl(2)) in human embryonic kidney (HEK) cells permanently expressing the human CaR (HEK-hCaR). Ca(2+) (4 mm) also activates ERK1/2 but with differing kinetics. CaR-dependent ERK1/2 activation begins to desensitize to 4 mm Ca(2+) after 10 min, whereas there is no desensitization to NPS R-467/CaCl(2) as late as 4 h. Moreover, recovery from desensitization occurs as rapidly as 30 min with 4 mm CaCl(2). Pretreatment of HEK-hCaR cells with concanavalin A (250 microg/ml) to block CaR internalization completely eliminated the NPS R-467/CaCl(2)-mediated ERK1/2 activation but did not block the 2-min time point of 4 mm Ca(2+)-mediated ERK1/2 activation. Neither dominant-negative dynamin (K44A) nor dominant-negative beta-arrestin inhibited ERK1/2 activation by either CaR agonist treatment, suggesting that CaR-elicited ERK1/2 signaling occurs via a dynamin-independent pathway. Pertussis toxin pretreatment partially attenuated the 4 mm Ca(2+)-ERK1/2 activation; this attenuated activity was completely restored by co-expression of the Galpha(i2) (C351I) but not Galpha(i1) (C351I) or Galpha(i3) (C351I) G proteins, PTX-insensitive G protein mutants. Taken together, these data suggest that both 4 mm Ca(2+) and NPS R-467/CaCl(2) activate ERK1/2 via distinguishable pathways in HEK-hCaR cells and may represent a nexus to differentially regulate differentiation versus proliferation via CaR activation.     

The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.     

Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.     

Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin

Calcium-sensing receptors (CaR) contribute to regulation of systemic calcium homeostasis by activation of G(q)- and G(i)-linked signaling pathways in the parathyroids, kidney, and intestine. Little is known about the mechanisms regulating CaR synthesis and degradation. Screening of a human kidney yeast two-hybrid library identified the E3 ubiquitin ligase dorfin as a binding partner for the intracellular carboxyl terminus of CaR. Interaction between CaR and dorfin was confirmed by coimmunoprecipitation from HEK293 cells. Ubiquitination of CaR was observed in the presence of the proteasomal inhibitor MG132; mutation of all putative intracellular loop and carboxyl-terminal lysine residues abolished ubiquitination of CaR. Coexpression with dorfin decreased the amount of total CaR protein and increased CaR ubiquitination, whereas a dominant negative fragment of dorfin had opposite effects. The AAA-ATPase p97/valosin-containing protein associates with both CaR and dorfin in HEK293 cells. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, induced the appearance of the unglycosylated 115-kDa CaR form, which was further increased by exposure to MG132, or upon transfection with a dorfin dominant negative construct, suggesting that dorfin-mediated proteasomal degradation of immature CaR occurs from the endoplasmic reticulum. Because endogenous CaR in Madin-Darby canine kidney cells is also subject to degradation from the endoplasmic reticulum, dorfin-mediated ubiquitination may contribute to a general mechanism for CaR quality control during biosynthesis.     

Calcium-sensing receptor modulates cell adhesion and migration via integrins

The calcium-sensing receptor (CaSR) is a family C G protein-coupled receptor that is activated by elevated levels of extracellular divalent cations. The CaSR couples to members of the G(q) family of G proteins, and in the endocrine system this receptor is instrumental in regulating the release of parathyroid hormone from the parathyroid gland and calcitonin from thyroid cells. Here, we demonstrate that in medullary thyroid carcinoma cells, the CaSR promotes cellular adhesion and migration via coupling to members of the integrin family of extracellular matrix-binding proteins. Immunopurification and mass spectrometry, co-immunoprecipitation, and co-localization studies showed that the CaSR and β1-containing integrins are components of a macromolecular protein complex. In fibronectin-based cell adhesion and migration assays, the CaSR-positive allosteric modulator NPS R-568 induced a concentration-dependent increase in cell adhesion and migration; both of these effects were blocked by a specific CaSR-negative allosteric modulator. These effects were mediated by integrins because they were blocked by a peptide inhibitor of integrin binding to fibronectin and β1 knockdown experiments. An analysis of intracellular signaling pathways revealed a key role for CaSR-induced phospholipase C activation and the release of intracellular calcium. These results demonstrate for the first time that an ion-sensing G protein-coupled receptor functionally couples to the integrins and, in conjunction with intracellular calcium release, promotes cellular adhesion and migration in tumor cells. The significance of this interaction is further highlighted by studies implicating the CaSR in cancer metastasis, axonal growth, and stem cell attachment, functions that rely on integrin-mediated cell adhesion.     

Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators

The human extracellular Ca(2+)-sensing receptor (CaR), a member of the G protein-coupled receptor family 3, plays a key role in the regulation of extracellular calcium homeostasis. It is one of just a few G protein-coupled receptors with a large number of naturally occurring mutations identified in patients. In contrast to the small sizes of its agonists, this large dimeric receptor consists of domains with topologically distinctive orthosteric and allosteric sites. Information derived from studies of naturally occurring mutations, engineered mutations, allosteric modulators and crystal structures of the agonist-binding domain of homologous type 1 metabotropic glutamate receptor and G protein-coupled rhodopsin offers new insights into the structure and function of the CaR.     

A region in the seven-transmembrane domain of the human Ca2+ receptor critical for response to Ca2+

Of 12 naturally occurring, activating mutations in the seven-transmembrane (7TM) domain of the human Ca2+ receptor (CaR) identified previously in subjects with autosomal dominant hypocalcemia (ADH), five appear at the junction of TM helices 6 and 7 between residue Ile819 and Glu837. After identifying a sixth activating mutation in this region, V836L, in an ADH patient, we studied the remaining residues in this region to determine whether they are potential sites for activating mutations. Alanine-scanning mutagenesis revealed five additional residues in this region that when substituted by alanine led to CaR activation. We also found that, whereas E837A did not activate the receptor, E837D and E837K mutations did. Thus, region Ile819-Glu837 of the 7TM domain represents a "hot spot" for naturally occurring, activating mutations of the receptor, and most of the residues in this region apparently maintain the 7TM domain in its inactive configuration. Unique among the residues in this region, Pro823, which is highly conserved in family 3 of the G protein-coupled receptors, when mutated to either alanine or glycine, despite good expression severely impaired CaR activation by Ca2+. Both the P823A mutation and NPS 2143, a negative allosteric modulator that acts on the 7TM through a critical interaction with Glu837, blocked activation of the CaR by various ADH mutations. These results suggest that the 7TM domain region Ile819-Glu837 plays a key role in CaR activation by Ca2+. The implications of our finding that NPS 2143 corrects the molecular defect of ADH mutations for treatment of this disease are also discussed.     

Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells

Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10(-4)M), or 10% cyclodextrin, for 6h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro.     

AMSH regulates calcium-sensing receptor signaling through direct interactions

Calcium-sensing receptor (CaR) activates intracellular pathways controlling calcium homeostasis. CaR carboxyl-terminal mutants associated with metabolic diseases suggest that unidentified proteins interact with the carboxyl-terminal region of this receptor. To address this possibility, we screened for CaR-interacting proteins using the carboxyl terminus of CaR (CaRDelta895-1075 deletion mutant). We identified AMSH, an ubiquitin isopeptidase, as a CaR-interacting partner. AMSH caused a decrease on the signaling properties of wild-type and mutant CaR. Our results indicate that AMSH, which has been recently characterized as a regulator of the endosomal sorting of epidermal growth factor receptor, represents a novel modulator of CaR signaling.     

Positive and negative allosteric modulators of the Ca2+-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain

A three-dimensional model of the human extracellular Ca(2+)-sensing receptor (CaSR) has been used to identify specific residues implicated in the recognition of two negative allosteric CaSR modulators of different chemical structure, NPS 2143 and Calhex 231. To demonstrate the involvement of these residues, we have analyzed dose-inhibition response curves for the effect of these calcilytics on Ca(2+)-induced [(3)H]inositol phosphate accumulation for the selected CaSR mutants transiently expressed in HEK293 cells. These mutants were further used for investigating the binding pocket of two chemically unrelated positive allosteric CaSR modulators, NPS R-568 and (R)-2-[1-(1-naphthyl)ethylaminomethyl]-1H-indole (Calindol), a novel potent calcimimetic that stimulates (EC(50) = 0.31 microM) increases in [(3)H]inositol phosphate levels elicited by activating the wild-type CaSR by 2 mM Ca(2+). Our data validate the involvement of Trp-818(6.48), Phe-821(6.51), Glu-837(7.39), and Ile-841(7.43) located in transmembranes (TM) 6 and TM7, in the binding pocket for both calcimimetics and calcilytics, despite important differences observed between each family of compounds. The TMs involved in the recognition of both calcilytics include residues located in TM3 (Arg-680(3.28), Phe-684(3.32), and Phe-688(3.36)). However, our study indicates subtle differences between the binding of these two compounds. Importantly, the observation that some mutations that have no effect on calcimimetics recognition but which affect the binding of calcilytics in TM3 and TM5, suggests that the binding pocket of positive and negative allosteric modulators is partially overlapping but not identical. Our CaSR model should facilitate the development of novel drugs of this important therapeutic target and the identification of the molecular determinants involved in the binding of allosteric modulators of class 3 G-protein-coupled receptors.     

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.     

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.     

The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.     

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids

The Ca²⁺-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca²⁺ or Mg²⁺ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca²⁺ (Ca_i²⁺) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G_q/11 or G_i/o signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca_i²⁺ mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists. cytoskeleton; signaling     

Calcium sensing receptor in developing human airway smooth muscle

Airway smooth muscle (ASM) regulation of airway structure and contractility is critical in fetal/neonatal physiology in health and disease. Fetal lungs experience higher Ca²⁺ environment that may impact extracellular Ca²⁺ ([Ca²⁺]_o) sensing receptor (CaSR). Well-known in the parathyroid gland, CaSR is also expressed in late embryonic lung mesenchyme. Using cells from 18-22 week human fetal lungs, we tested the hypothesis that CaSR regulates intracellular Ca²⁺ ([Ca²⁺]_i) in fetal ASM (fASM). Compared with adult ASM, CaSR expression was higher in fASM, while fluorescence Ca²⁺ imaging showed that [Ca²⁺]_i was more sensitive to altered [Ca²⁺]_o. The fASM [Ca²⁺]_i responses to histamine were also more sensitive to [Ca²⁺]_o (0–2 mM) compared with an adult, enhanced by calcimimetic R568 but blunted by calcilytic NPS2143. [Ca²⁺]_i was enhanced by endogenous CaSR agonist spermine (again higher sensitivity compared with adult). Inhibition of phospholipase C (U73122; siRNA) or inositol 1,4,5-triphosphate receptor (Xestospongin C) blunted [Ca²⁺]_o sensitivity and R568 effects. NPS2143 potentiated U73122 effects. Store-operated Ca²⁺ entry was potentiated by R568. Traction force microscopy showed responsiveness of fASM cellular contractility to [Ca²⁺]_o and NPS2143. Separately, fASM proliferation showed sensitivity to [Ca²⁺]_o and NPS2143. These results demonstrate functional CaSR in developing ASM that modulates airway contractility and proliferation.