Human Calcium-sensing Receptor Research Articles

Heterogenous Origins of Calcium Homeostasis Disorders Arising from Five Heterozygous Calcium-Sensing Receptor Variants.

The human calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis, and most identified CASR variants are associated with hypercalcemic and hypocalcemic disorders. Here we characterized the pharmacological implications of five heterozygous CASR variants from individuals with familial hypocalciuric hypercalcemia 1 [FHH1: Y63C, I81T, Q459R, W818stop] or autosomal dominant hypocalcemia 1 [ADH1: R955stop]. Total and cell surface expression levels of wild-type (WT) and variant CaSRs expressed in human embryonic kidney 293T (HEK293T) cells were determined using ELISA, and the pharmacological properties of the receptors were delineated in two functional assays. The Y63C and I81T variations in the extracellular domain (ECD) of CaSR yielded markedly reduced cell surface expression and Ca2+ responsiveness, while Q459R displayed WT-like expression and functional properties. Truncation of the 7-transmembrane domain (7TMD) in W818stop eliminated cell surface expression, whereas R955stop in the intracellular carboxy-terminal yielded modestly increased surface expression and Ca2+ potency compared with WT CaSR. Interestingly, the effectiveness of positive allosteric modulators (PAMs) at the variants varied. Ca2+-mediated signaling through Y63C and I81T was significantly augmented by 7TMD-binding PAMs (NPS R-568 and Evocalcet) but not by ECD-binding PAMs (Etelcalcetide and Nb4), whereas signaling through Q459R and R955stop were robustly potentiated by all four PAMs. While the molecular phenotypes exhibited by the five CaSR variants concord with the clinical phenotypes in individuals harboring them, CASR variant-induced calcium homeostasis disorders clearly arise from diverse molecular origins, and the effectiveness of calcimimetics in these disorders could differ depending on the specific variants.

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

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

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

Exploring Adaptive Phenotypes for the Human Calcium-Sensing Receptor Polymorphism R990G.

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

Evaluating the anti-urolithiasis potential of Ficus religiosa seed GC MS evaluated phytoconstituents based on their in-vitro antioxidant properties and in-silico ADMET and molecular docking studies

BackgroundUrolithiasis, the deposit of stones in the urinary tract is a pertinent clinical issue in daily practice that imposes a burden on the human health system. Ficus religiosa plant has historically been useful in preventing urolithiasis. There is currently no information on phytochemical profiling that specifies the precise phytochemicals in the seed that are active against urolithiasis.MethodsF. religiosa seeds were extracted with different solvents in increasing order of their polarity by Soxhlet extraction. All the extracts were evaluated for their antioxidant potential. GC–MS profiling of the most potent antioxidant F. religiosa seed extract was done to evaluate the phytoconstituents. To evaluate the pharmacokinetics and drug-likeness properties of these compounds in silico ADMET analysis was done. To comprehend the binding potential of the best ADMET evaluated phytochemicals contained in the F. religiosa seed extract against the several protein targets (matrix metalloproteinases (MMP-2, MMP-9), and Human calcium-sensing receptor (CaSR)) and antioxidant enzymes (Glutathione S-transferase (GST), glutathione-disulfide reductase (GR), glutathione peroxidase (GPX), and superoxide dismutase (SOD)) involved in urolithiasis, multi targets based virtual screening tests were done using Autodock Vina tool.ResultsGC–MS profiling revealed the presence of 53 different compounds. Of all these compounds, based on ADMET analysis 2-Methoxy-4-vinylphenol; 3,5-Di-tert-butylphenol; diethyl benzene-1,2-dicarboxylate; 4-hydroxy-3,5-dimethoxybenzaldehyde; 2-methoxy-4-prop-2-enylphenol; and bis (2-methyl propyl) benzene-1,2-dicarboxylate were found to have best pharmacokinetics and drug-likeness properties. In the autodocking studies, 3,5-Di-tert-butyl phenol is proved to be the best of all in terms of binding energies with the selected targets.ConclusionThe findings of this study suggest a framework for employing F. religiosa seed ethyl acetate extract as a potent herbal treatment for urolithiasis.

Pharmacological profile of upacicalcet, a novel positive allosteric modulator of calcium-sensing receptor, in vitro and in vivo

Upacicalcet (formerly SK-1403/AJT240) is a novel non-peptide calcimimetic agent that acts as a calcium-sensing receptor (CaSR) agonist for the treatment of secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). We compared upacicalcet with other calcimimetics (etelcalcetide or cinacalcet) and examined its in vitro and in vivo characteristics in terms of its human CaSR agonistic activity, its efficacy in normal and CKD rats after a single administration, and its effect on gastric emptying in rats. Upacicalcet activated human CaSR depending on the extracellular calcium (Ca2+) concentration without exhibiting an agonistic action when the extracellular Ca2+ level was below the physiological level. On the other hand, etelcalcetide had an agonistic activity even in the absence of physiological levels of extracellular Ca2+. The intravenous administration of upacicalcet to normal and double-nephrectomized rats dose-dependently (0.03–3mg/kg and 0.3–30mg/kg, respectively) decreased the serum intact parathyroid hormone (iPTH) and serum Ca2+ levels; however, the effect of upacicalcet on the reduction in serum Ca2+ disappeared at extracellular Ca2+ levels below the physiologically range, even when administered at a dose higher (100-fold) than the effective dose. Furthermore, upacicalcet did not affect gastric emptying in normal rats when administered up to a dose of 10mg/kg (300-fold higher than the dose affecting serum iPTH levels), while the administration of cinacalcet significantly slowed gastric emptying by approximately 50%. These findings suggest that upacicalcet has potential as an alternative calcimimetic agent with good pharmacological properties and a lower risk of hypocalcemia and gastrointestinal complications.

Oral expressions and functional analyses of the extracellular calcium-sensing receptor (CaSR) in chicken

In vertebrates, the extracellular calcium-sensing receptor (CaSR) plays a key role in calcium homeostasis by sensing slight changes in extracellular Ca2+. CaSR is also expressed in mammals including rodent taste cells and is involved in sensing kokumi, a rich, savory quality that enhances the intensities of salty, sweet, and umami tastes. In this study, we focused on chicken CaSR (cCaSR) since calcium is an essential nutrient that is necessary for making eggshell and for the extremely rapid initial growth of bones. First we confirmed that cCaSR is expressed in taste cells. Next we cloned the cCaSR gene from kidney and transiently transfected human embryonic kidney 293 T (HEK293T) cells with the recombinant cCaSR, or empty vector and looked for the agonists and allosteric modulators (including kokumi substances) of cCaSR by Ca2+ imaging. We found that cCaSR was activated by extracellular Ca2+ and Mg2+ in a dose dependent manner. Several L-amino acids and kokumi substances such as glutathione enhanced the response of cCaSR. In addition, NPS2143 as a negative allosteric modulator of human CaSR negatively modulated the response of cCaSR. These results suggest that cCaSR can sense extracellular Ca2+ and Mg2+ as well as positive and negative allosteric modulators. Taken together, the results imply that CaSR might be a multifunctional receptor for calcium, amino acids, and kokumi substances in chicken. The present finding that functional CaSR is expressed in the chicken oral tissues will allow us to further elucidate the physiological role of CaSR in the chickens' taste sense, and to create new feeds that will contribute to the poultry industry.

Extracellular domain of human calcium sensing receptor immobilized to silica beads as biomaterial: A rapid chromatographic method for recognizing ligands from complex matrix ‘Shuangdan’

Human calcium-sensing receptor (CaSR), a member of the G-protein-coupled receptor superfamily (GPCR), has been a therapeutic target for developing new drugs against calciotropic disorders and non-calciotropic diseases. The highly efficient methodologies for pursuing novel ligands/drugs remained a challenge due to the redundant purification processes of membrane protein in some widely-used methods including NMR, X-ray crystallography, Fluorescence Titration Spectroscopy, and Circular Dichroism. Herein, extracellular domain (ECD) of CaSR as its functional fragment was used to develop a rapid chromatographic method, which involved the synthesis of stationary phase material based on the site-specific covalent reaction of Halogenated alkane dehalogenase (Halo)-tagged ECD of CaSR in cell lysate with 6-chlorocaproic acid modified silica beads, the use of the immobilized CaSR column for revealing the interaction of three known agonists with CaSR and further screening ligands from complex matrix like Chinese herb medicine ‘Shuangdan’. The immobilized CaSR column was prepared rapidly without the protein purification and retained a good stability and specificity for at least 35 days. It was revealed that one type of binding sites occurred on CaSR with the binding affinity of neomycin > gentamicin-C / kanamycin, presumably which related to the number of structural amino groups attached. This method allowed for recognizing specifically novel ligands from ’Shuangdan’, demonstrating one type of binding sites on CaSR with the binding affinity of gallic acid > caffeic acid > paeonol. These results indicated that, the immobilization of a representative extracellular domain of CaSR to silica beads as biomaterial is feasible to develop a new rapid method, which can be successfully applied in screening novel ligands efficiently from complex matrices.

Upacicalcet Is a Novel Secondary Hyperparathyroidism Drug that Targets the Amino Acid Binding Site of Calcium-Sensing Receptor

The human calcium-sensing receptor (CaSR) is a G protein-coupled receptor that maintains extracellular Ca2+ homeostasis by regulating the secretion of parathyroid hormone. Upacicalcet is a novel positive allosteric modulator of CaSR that is used for the treatment of secondary hyperparathyroidism. In the present study, to clarify the binding site of upacicalcet to CaSR, we conducted binding studies and agonistic activity studies in HEK-293T cells expressing human CaSR (intact and mutant) and an in silico docking-simulation analysis. As a result, upacicalcet competed with L-tryptophan and was thought to affect the amino acid binding site. In addition, the effects of substitutions at the amino acid binding site on the binding abilities to upacicalcet as well as the effects on receptor function as measured using inositol-1 monophosphate accumulation were examined. Upacicalcet interacted with several CaSR residues that constitute the amino acid binding site. Based on these results, we performed an in silico analysis and obtained a binding mode, consistent with the in vitro study results. Our study revealed that upacicalcet is a novel secondary hyperparathyroidism drug that targets the amino acid binding site of CaSR. Upacicalcet is expected to become a new treatment option for secondary hyperparathyroidism because the binding site differs from that of conventional drugs; consequently, it may be effective for patients who are not sensitive to conventional drugs, and it may have a superior safety profile. SIGNIFICANCE STATEMENT: Upacicalcet interacts with several residues that constitute the amino acid binding site of the calcium-sensing receptor (CaSR) and shows a potent positive allosteric activity. This mechanism differs from those of conventional drugs. Therefore, upacicalcet can be regarded as a novel secondary hyperparathyroidism drug that acts on the amino acid binding site of CaSR.

Symmetric activation and modulation of the human calcium-sensing receptor

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

159 In vitro Characterization of Potential Pig Calcium-sensing Receptor (CaSR) Ligands and Cell Signaling Pathways Related to CaSR Activation by a Dual-luciferase Reporter Assay

Abstract The calcium-sensing receptor (CaSR) is a pivotal regulator of calcium homeostasis. Our previous study has found that pig CaSR (pCaSR) is widely expressed in intestinal segments in weaned piglets. To characterize the activation of pCaSR by potential ligands and related cell signaling pathways, a dual-luciferase reporter assay was employed for the ligands screening and molecular docking was utilized to predict the binding mode of identified ligands. Our results showed that the dual-luciferase reporter assay system was well suited for pCaSR research and its ligand screening. The extracellular calcium activated pCaSR in a concentration-dependent manner with a half-maximal effective concentration (EC50) = 4.74 mM through the Gq/11 signaling pathway, EC50 = 2.85 mM through extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation signaling pathway, and EC50 = 2.26 mM through the Ras homolog family member A (RhoA) activation signaling pathway. Moreover, the activation of pCaSR stimulated by extracellular calcium showed biased agonism through three main signaling pathways: ERK1/2 phosphorylation signaling, Gq/11 signaling, and G12/13 signaling. Both L-Tryptophan and α-casein (90–95) could activate the pCaSR in the presence of extracellular calcium. Furthermore, we characterized the L-tryptophan binding pocket formed by pCaSR residues TRP 70, SER 147, ALA168, SER 169, SER 170, ASP 190, GLU 297, ALA 298, and ILE 416, as well as the α-casein (90–95) binding pocket formed by pCaSR residues PRO188, ASN189, GLU191, HIS192, LYS225, LEU242, ASP480, VAL486, GLY487, VAL513, and TYR514. In conclusion, similar to the human CaSR, the pCaSR also shows biased agonism through three main signaling pathways and both α-casein (90–95) and L-tryptophan are agonists for pCaSR. Furthermore, the binding sites of α-casein (90–95) and L-tryptophan are mainly located within the extracellular domain of pCaSR.

Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells.

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes.

Structural insights into the activation of human calcium-sensing receptor.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and agonist+PAM bound states. Complemented with previously reported structures of CaSR, we show that in addition to the full inactive and active states, there are multiple intermediate states during the activation of CaSR. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data show that agonist binding leads to compaction of the dimer, proximity of the cysteine-rich domains, large-scale transitions of seven-transmembrane domains, and inter- and intrasubunit conformational changes of seven-transmembrane domains to accommodate downstream transducers. Our results reveal the structural basis for activation mechanisms of CaSR and clarify the mode of action of Ca2+ ions and L-amino acid leading to the activation of the receptor.

Asymmetric activation of the calcium-sensing receptor homodimer.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor.

Kokumi taste perception is functional in a model carnivore, the domestic cat (Felis catus)

Kokumi taste is a well-accepted and characterised taste modality and is described as a sensation of enhancement of sweet, salty, and umami tastes. The Calcium Sensing Receptor (CaSR) has been designated as the putative kokumi taste receptor for humans, and a number of kokumi-active ligands of CaSR have been discovered recently with activity confirmed both in vivo and in vitro. Domestic cats (Felis catus) are obligate carnivores and accordingly, their diet is abundant in proteins, peptides, and amino acids. We hypothesised that CaSR is a key taste receptor for carnivores, due to its role in the detection of different peptides and amino acids in other species. Using in silico, in vitro and in vivo approaches, here we compare human CaSR to that of a model carnivore, the domestic cat. We found broad similarities in ligand specificity, but differences in taste sensitivity between the two species. Indeed our in vivo data shows that cats are sensitive to CaCl2 as a kokumi compound, but don’t show this same activity with Glutathione, whereas for humans the reverse is true. Collectively, our data suggest that kokumi is an important taste modality for carnivores that drives the palatability of meat-derived compounds such as amino acids and peptides, and that there are differences in the perception of kokumi taste between carnivores and omnivores.

Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca2+ homeostasis in the blood. The general consensus is that extracellular Ca2+ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca2+ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca2+ and L-Trp binding sites and conformational changes of the ECD upon Ca2+/L-Trp binding. However, it remains to be understood at the structural level how Ca2+/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca2+- or L-Trp-bound states, and Ca2+/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca2+ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca2+ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca2+. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.

Effects of pharmacological calcimimetics on colorectal cancer cells over-expressing the human calcium-sensing receptor

The calcium-sensing receptor (CaSR) is a ubiquitously expressed multifunctional G protein-coupled receptor. Several studies reported that the CaSR plays an anti-inflammatory and anti-tumorigenic role in the intestine, and that it is down-regulated during colorectal carcinogenesis. We hypothesized that positive allosteric CaSR modulators (type II calcimimetics) selectively targeting the intestinal cells could be used for the treatment of intestinal pathologies. Therefore, the aim of this study was to determine the effect of pharmacological stimulation of CaSR on gene expression in vitro and on tumor growth in vivo.We stably transduced two colon cancer cell lines (HT29 and Caco2) with lentiviral vectors containing either the CaSR fused to GFP or GFP only. Using RNA sequencing, RT-qPCR experiments and ELISA, we determined that CaSR over-expression itself had generally little effect on gene expression in these cells. However, treatment with 1 μM of the calcimimetic NPS R-568 increased the expression of pro-inflammatory factors such as IL-23α and IL-8 and reduced the transcription of various differentiation markers in the cells over-expressing the CaSR. In vivo, neither the presence of the CaSR nor p.o. treatment of the animals with the calcimimetic cinacalcet affected tumor growth, tumor cell proliferation or tumor vascularization of murine HT29 xenografts.In summary, CaSR stimulation in CaSR over-expressing cells enhanced the expression of inflammatory markers in vitro, but was not able to repress colorectal cancer tumorigenicity in vivo. These findings suggest potential pro-inflammatory effects of the CaSR and type II calcimimetics in the intestine.

N-terminal [Glu]3 moiety of γ-glutamyl peptides contributes largely to the activation of human calcium-sensing receptor, a kokumi receptor.

γ-glutamyl peptides have been suggested to impart kokumi properties to foods by activating human calcium-sensing receptor (hCaSR). In this study, the relationship between γ-glutamyl peptide structure and hCaSR activity was systematically analyzed using γ-[Glu](n=0-4)-α-[Glu](n=0-3)-Tyr. Our results suggest that N-terminal [Glu]3 moiety is very important for hCaSR activities of γ-glutamyl peptides.

Negative allosteric modulators of the human calcium-sensing receptor bind to overlapping and distinct sites within the 7-transmembrane domain.

Negative allosteric modulators (NAMs) that target the calcium-sensing receptor (CaS receptor) were originally developed for the treatment of osteoporosis by stimulating the release of endogenous parathyroid hormone, but failed in human clinical trials. Several chemically and structurally distinct NAM scaffolds have been described, but it is not known how these different scaffolds interact with the CaS receptor to inhibit receptor signalling in response to agonists. In the present study, we used a mutagenesis approach combined with analytical pharmacology and computational modelling to probe the binding sites of four distinct NAM scaffolds. Although all four scaffolds bind to the 7-transmembrane and/or extracellular or intracellular loops, they occupy distinct regions, as previously shown for positive allosteric modulators of the CaS receptor. Furthermore, different NAM scaffolds mediate negative allosteric modulation via distinct amino acid networks. These findings aid our understanding of how different NAMs bind to and inhibit the CaS receptor. Elucidation of allosteric binding sites in the CaS receptor has implications for the discovery of novel allosteric modulators.