Positive Allosteric Modulation of the Calcium-sensing Receptor by Physiological Concentrations of Glucose*

The calcium-sensing receptor (CaSR) is activated by various cations, cationic compounds, and amino acids. In the present study we investigated the effect of glucose on CaSR in HEK293 cells stably expressing human CaSR (HEK-CaSR cells). When glucose concentration in the buffer was raised from 3 to 25 mm, a rapid elevation of cytoplasmic Ca2+ concentration ([Ca2+]c) was observed. This elevation was immediate and transient and was followed by a sustained decrease in [Ca2+]c. The effect of glucose was detected at a concentration of 4 mm and reached its maximum at 5 mm. 3-O-Methylglucose, a non-metabolizable analogue of glucose, reproduced the effect of glucose. Sucrose also induced an elevation of [Ca2+]c in HEK-CaSR cells. Similarly, sucralose was nearly as effective as glucose in inducing elevation of [Ca2+]c. Glucose was not able to increase [Ca2+]c in the absence of extracellular Ca2+. The effect of glucose on [Ca2+]c was inhibited by NPS-2143, an allosteric inhibitor of CaSR. In addition, NPS-2143 also inhibited the [Ca2+]c responses to sucralose and sucrose. Glucose as well as sucralose decreased cytoplasmic cAMP concentration in HEK-CaSR cells. The reduction of cAMP induced by glucose was blocked by pertussis toxin. Likewise, sucralose reduced [cAMP]c. Finally, glucose increased [Ca2+]c in PT-r parathyroid cells and in Madin-Darby canine kidney cells, both of which express endogenous CaSR. These results indicate that glucose acts as a positive allosteric modulator of CaSR.

The calcium-sensing receptor (CaSR) 2 was originally identified in parathyroid cells as a receptor that regulates secretion of parathyroid hormone by sensing changes in the concentration of extracellular calcium (1). Elevation of extracellular Ca 2ϩ is detected by this receptor, and phospholipase C is activated leading to elevation of cytoplasmic Ca 2ϩ concentration ([Ca 2ϩ ] c ) (1). The molecular structure of CaSR was revealed by the expression cloning of CaSR from the bovine parathyroid gland (2). CaSR is a glycosylated protein with a molecular mass of ϳ120 KDa and belongs to the family C of G protein-coupled receptors (GPCRs). It has a large extracellular domain, namely the Venus flytrap domain (VFTD), and like other members of the family C GPCR, CaSR forms a dimer to exert its function. In addition to parathyroid glands, CaSR is expressed in tissues involved in the regulation of Ca 2ϩ homeostasis including kidney, intestine, and bone. This receptor is also expressed in various other tissues and organs including neurons and glial cells (3) and may modulate the function of these cells (3)(4)(5). Although CaSR was identified as a receptor sensing extracellular Ca 2ϩ , many studies revealed that CaSR is in fact activated by a variety of ligands. Those include various cations such as Mg 2ϩ , Mn 2ϩ , Sr 2ϩ , Ni 2ϩ , and Gd 3ϩ , amino acids, polypeptides, aminoglycosides, and polyamines (4,5). Among them, a principal agonist for CaSR is Ca 2ϩ , which binds to a pocket in the VFTD (6). Binding Ca 2ϩ to the binding pocket in the VFTD may cause a conformational change in the receptor molecule, which eventually activates trimeric G proteins, leading to activation of the effector molecules. Amino acids bind to an adjacent site in the VFTD and act as allosteric activators of CaSR (4).
An interesting feature of CaSR is that various synthetic allosteric modulators bind to domains other than VFTD, especially the transmembrane domain, and modulate the function of CaSR in positive and negative manners. For example, calcimimetics bind to the transmembrane domain and stabilize the receptor in the active form (5). Conversely, NPS-2143, an allosteric inhibitor of CaSR, binds to the transmembrane domain of CaSR and may stabilize the inactive conformation of CaSR (7). In addition to the above-mentioned agonists, CaSR is also activated by the bitter compound named denatonium (8).
We have been studying the cell-surface receptor activated by glucose in pancreatic ␤-cells and enteroendocrine cells (9 -11). During the course of study, we found that physiological concentrations of glucose activate CaSR expressed in HEK293 cells. In the present study we characterized the effect of glucose and other compounds presenting sweet taste on CaSR in HEK293 cells stably expressing human CaSR (HEK-CaSR cells). The results indicate that glucose acts as an allosteric modulator of CaSR.

Results
Effect of Ca 2ϩ on [Ca 2ϩ ] c in HEK-CaSR Cells-We established HEK293 cells stably expressing human CaSR (hCaSR). As shown in Fig. 1A, HEK-CaSR cells expressed a considerably high amount of mRNA for hCaSR compared with that in parental HEK293 cells. Fig. 1B shows the protein expression of CaSR in HEK-CaSR cells. As can been seen, CaSR was detected in HEK-CaSR but not in HEK293 cells. Immunocytochemistry revealed that a considerable amount of CaSR was expressed on the cell surface of HEK-CaSR cells (Fig. 1C). We then monitored changes in [Ca 2ϩ ] c in HEK-CaSR cells using fluo-8. In the presence of 2.5 mM extracellular calcium, oscillation of [Ca 2ϩ ] c was observed in 34.0 Ϯ 2.0% (mean Ϯ S.E., n ϭ 3) of the cells examined (Fig. 1D). In the presence of 1.3 mM extracellular calcium, oscillation of [Ca 2ϩ ] c was observed in 16.2 Ϯ 1.0% (n ϭ 3) of the cells (Fig. 1E). The amplitude and frequency of the peaks of [Ca 2ϩ ] c in the presence of 1.3 mM extracellular calcium were considerably lower compared with those in the presence of 2.5 mM extracellular calcium. In the absence of extracellular calcium, no oscillation of [Ca 2ϩ ] c was observed (Fig. 1F). Note that oscillation of [Ca 2ϩ ] c was not observed in naïve HEK293 cells in the presence of 1.3 and 2.5 mM Ca 2ϩ (Fig. 1G). The addition of cinacalcet, an allosteric activator of CaSR (12), induced a large transient elevation of [Ca 2ϩ ] c in the presence of 1.3 mM extracellular calcium in HEK-CaSR cells (Fig. 1H). In HEK293 cells, cinacalcet did not affect [Ca 2ϩ ] c (Fig. 1I).
Effect of Glucose on [Ca 2ϩ ] c in HEK-CaSR Cells-In the presence of 1.3 mM extracellular calcium, oscillation of [Ca 2ϩ ] c was observed in some cells. When ambient glucose concentration was raised from 3 mM to 25 mM, an application of the high concentration of glucose induced an immediate transient elevation of [Ca 2ϩ ] c in 64.1 Ϯ 9.9% (n ϭ 8) of the cells examined ( Fig. 2A) ] c , ambient glucose should be kept at a low concentration, for example 3 mM. The effect of glucose on [Ca 2ϩ ] c was not observed when the ambient glucose was kept at 5.5 mM. This is probably because 5.5 mM glucose induces nearly maximal allosteric stimulation. Glucose-induced changes in [Ca 2ϩ ] c were inhibited by U73122, a nonspecific inhibitor of phospholipase C, and [Ca 2ϩ ] c response to 25 mM glucose was observed in only 25% of the cells in the presence of 10 M U73122 (13). An inactive analogue of U73122 was not effective. In addition, YM-254890, an inhibitor of G q (14), completely inhibited glucose-evoked changes in [Ca 2ϩ ] c (Fig. 2E). Note that in the presence of YM254890, basal oscillation of [Ca 2ϩ ] c was not observed. We also examined the effect of glucose on the activation of protein kinase C (PKC). To this end, we monitored the translocation of MARCKS from the plasma membrane to cytosol. As shown in Fig. 2F, the addition of glucose induced an increase in MARCKS in the cytosol. We next determined whether or not glucose modulated the sensitivity of CaSR to Ca 2ϩ . To this end we measured the response of CaSR to Ca 2ϩ in different ambient glucose concentrations. As shown in Fig. 2G, glucose did not affect the sensitivity of CaSR to Ca 2ϩ .
Elevation of [Ca 2ϩ ] c induced by glucose was dependent on extracellular calcium; glucose did not induce a peak of [Ca 2ϩ ] c in the absence of extracellular calcium (Fig. 3A). The effect of glucose was independent of glucose metabolism as 3-O-methylglucose, a non-metabolizable glucose analogue, increased [Ca 2ϩ ] c , as did glucose (Fig. 3B). We then examined the effect of NPS-2143, an allosteric inhibitor of CaSR that blocks [Ca 2ϩ ] c responses to Ca 2ϩ and other allosteric agonists (7). In the presence of NPS-2143, [Ca 2ϩ ] c response to 25 mM glucose was observed in Ͻ10% of the cells (Fig. 3C), and [Ca 2ϩ ] c response was blunted in these responsive cells (Fig. 3D). When cells were stimulated by 8 mM glucose in the presence of NPS-2143, [Ca 2ϩ ] c response was blunted and was detected in Ͻ2% of the cells (Fig. 3, C and E). NPS-2143 also inhibited translocation of MARCKS induced by glucose (Fig. 3, F and G). The effect of glucose on [Ca 2ϩ ] c was not inhibited by lactisole, an inhibitor of the sweet taste receptor subunit T1R3 (15) (Fig. 3H). In fact, the peak of [Ca 2ϩ ] c induced by glucose was higher, and [Ca 2ϩ ] c response was observed in nearly 90% of the cells. Therefore, the [Ca 2ϩ ] c response to glucose was rather enhanced by lactisole.
We then examined the effect of Ca 2ϩ on [Ca 2ϩ ] c in the presence and absence of glucose. In the presence of 3 mM glucose, elevation of extracellular calcium from 1.3 mM to 2.2 mM resulted in a rapid elevation of [Ca 2ϩ ] c (Fig. 4A). After the peak of [Ca 2ϩ ] c , [Ca 2ϩ ] c decreased gradually, and oscillatory elevation of [Ca 2ϩ ] c was observed in some cells. In the absence of glucose, the addition of 2.2 mM calcium induced a rapid peak of [Ca 2ϩ ] c , which was followed by oscillatory decay of [Ca 2ϩ ] c (Fig. 4B). It is possible that removal of glucose affected the cellular functions because of the lack of fuels. To rule this out we performed the same experiments by adding glyceraldehyde   (Fig. 5D). Likewise, glycyrrhizin, a natural sweet compound, induced biphasic changes in [Ca 2ϩ ] c in HEK-CaSR cells (Fig. 5E). The effect of sucralose on [Ca 2ϩ ] c was inhibited by NPS-2143. In the presence of NPS-2143, only 2% of the cells responded to sucralose (Fig. 5F). In those responsive cells, elevation of [Ca 2ϩ ] c was blunted (Fig. 5G). Similarly, the effect of sucrose was also inhibited by NPS-2143 (data not shown).
Effect of Glucose on [cAMP] c in HEK-CaSR Cells-We then measured changes in [cAMP] c by using Flamindo2 (16). As shown in Fig. 6A, an elevation of [cAMP] c by forskolin induced a decrease in the fluorescence ratio of Flamindo2, indicating that forskolin elevated [cAMP] c . As shown in Fig. 6B, the addition of 5 mM glucose induced an immediate and sustained increase in the fluorescence, which indicates that glucose induced a rapid and sustained decrease in [cAMP] c . The effect of glucose was observed in almost all of the cells examined. Fig.  6C demonstrates the effect of cinacalcet, an allosteric activator   OCTOBER (Fig. 6D). The effect of glucose on [Ca 2ϩ ] c was inhibited by NPS-2143 (Fig. 6, E and F). Note that, in HEK-CaSR cells pretreated with pertussis toxin (PTX), the effect of glucose on [cAMP] c was inhibited (Fig. 6G).   was observed. This type of [Ca 2ϩ ] c response was observed in almost all of the cells examined. The effect of glucose was inhibited by NPS-2143 (Fig. 7G). Note that higher concentration of NPS-2143 was needed to inhibit rat CaSR (Fig. 7H). We then stimulated PT-r cells with an artificial sweetener, sucralose. As shown in Fig. 8A, 10 8B). This type of [Ca 2ϩ ] c response was observed in 38.0 Ϯ 4.6% (n ϭ 3) of the cells. We then knocked down CaSR by using siRNA in PT-r cells. In CaSR knockdown cells, mRNA level for CaSR was 30% that in control cells. In these cells, the effect of glucose was not observed (Fig. 8C).

Effect of Glucose on [Ca 2ϩ ] c in Cells Expressing Endogenous
We next examined the effect of glucose on [Ca 2ϩ ] c in Madin-Darby canine kidney (MDCK) cells (18), a cell line derived from renal tubular epithelial cells expressing CaSR. In these cells the elevation of extracellular Ca 2ϩ from 1.3 mM to 2.2 mM induced an oscillatory elevation of [Ca 2ϩ ] c (Fig. 9A). This type of response was observed in 48.0 Ϯ 5.0% (n ϭ 3) of the cells. The addition of cinacalcet induced an immediate and sustained elevation of [Ca 2ϩ ] c in nearly all of the cells (Fig. 9B). As in PT-r cells, elevation of glucose from 3 mM to 5 mM induced an oscillatory elevation of [Ca 2ϩ ] c in 49% (mean of two) of MDCK cells (Fig. 9C). When ambient glucose concentration was further increased to 12 mM, glucose induced an immediate elevation of [Ca 2ϩ ] c , which was followed by an oscillatory elevation of [Ca 2ϩ ] c in 72.2 Ϯ 4.2 (n ϭ 3) of the cells (Fig. 9D). NPS-2143 inhibited the effect of glucose in MDCK cells (Fig. 9, E and F). As shown in Fig. 9G, 10 mM sucralose induced a similar oscillatory elevation of [Ca 2ϩ ] c as glucose. This type of elevation of [Ca 2ϩ ] c was observed in 44% (mean of two) of the cells. We then knocked down CaSR in MDCK cells using siRNA. In knocked down cells, the mRNA level of CaSR was 30% that of control cells. Glucose-induced elevation of [Ca 2ϩ ] c was inhibited in CaSR knockdown cells (Fig. 9H).

Discussion
In the present study we showed for the first time that glucose allosterically modulated CaSR and increased [Ca 2ϩ ] c in HEK-CaSR cells. The elevation of [Ca 2ϩ ] c was due to the allosteric stimulation of CaSR by glucose as glucose did not affect [Ca 2ϩ ] c in naive HEK293 cells. The effects of glucose on CaSR are unique in several respects.
First, glucose exerts its effect at relatively low and physiologically relevant concentrations. In HEK-CaSR cells, an elevation of [Ca 2ϩ ] c was observed when ambient glucose was raised from 3 to 4 mM, and surprisingly 5 mM glucose elicited the maximal effect. This implies that in physiological settings, CaSR is already stimulated by the plasma glucose at least to some extent. We have to mention that most of the previous studies to  characterize the function of CaSR in vitro were performed in the buffer containing physiological concentration of glucose, for example 5.5 mM glucose. This means that CaSR was already stimulated significantly by the ambient glucose. In our experiments, we characterized the function of CaSR in the presence of 3.0 mM ambient glucose. Therefore, CaSR is less active in our experimental conditions. Most of the natural agonists for CaSR known to date are inorganic cations and organic cationic compounds (5). In addition, aromatic amino acids such as Phe, Tyr, His, and Trp act as positive allosteric modulators (19). Sugars including glucose and sucrose as well as artificial sweeteners should be considered as allosteric modulators of CaSR. Our results are apparently different from those by Quinn et al. (20) and Rogachevskaja (8). They showed that sucrose and sucrose octaacetate did not affect [Ca 2ϩ ] c in HEK-CaSR cells. The absence of the effect of these sugars may have been due to the higher ambient glucose concentration. Although they did not mention the exact concentration of glucose in the buffer used in their measurements, it is likely that they used the buffer containing 5.5 mM glucose. If so, CaSR was already activated by glucose, and the effect of sucrose was difficult to detect.
Second, although glucose increased [Ca 2ϩ ] c in HEK-CaSR cells in physiological conditions, glucose is not capable of activating CaSR in the absence of extracellular Ca 2ϩ . In this regard glucose is not a genuine agonist for CaSR, and instead, it acts as an allosteric modulator of this receptor. In accordance with this notion, an allosteric inhibitor of CaSR, NPS-2143, inhibited the effect of glucose on CaSR. This is in line with the observation that NPS-2143 is able to inhibit the effect of Ca 2ϩ and calcimimetics such as NPS R-467 (21). Glucose and other sweet compounds should be listed as allosteric modulators of CaSR.
Third, CaSR is activated not only by glucose but also by sucrose and other natural and synthetic sweeteners, all of which activate the sweet taste receptor (22). The effects of sucrose and other sweeteners were due to activation of CaSR but not endogenous T1R3. This is deduced because these compounds were ineffective in HEK293 cells. Furthermore, the expression of T1R3 in HEK293 and HEK-CaSR was detectable by RT-PCR, but the expression level was very low. Also, the effects of these compounds were not inhibited by lactisole, an inhibitor of T1R3 (12). In activating CaSR, however, these sweet taste compounds are slightly less potent compared with glucose. This is quite different from what we know about the activation of the sweet taste receptor by these sweet compounds; sucralose and sucrose are much more potent than glucose in activating the sweet taste receptor (22). Although the rank of potency is different, these compounds resemble glucose in some respects in activating CaSR. For example, these sweet molecules induced a biphasic response of [Ca 2ϩ ] c ; that is, an initial transient elevation of [Ca 2ϩ ] c followed by a sustained decrease in [Ca 2ϩ ] c . In addition, as in the case of glucose, the effects of sucralose and sucrose were inhibited by NPS-2143 (Fig. 4F). At present, the exact binding site for glucose in CaSR is not certain. In this regard, all these sweet taste molecules activate the sweet taste receptor, a heterodimer of T1R2 and T1R3 (23), both of which possess the VFTD. All these sweet compounds except glycyrrhizin bind to the VFTD of the sweet taste receptor subunit (22). Given the similarity of the stereostructure of the VFTD among members of the family C GPCR (24), it is possible that glucose and sucrose interact with a site in VFTD of CaSR. This possibility should be assessed experimentally in the near future.
Fourth, glucose-induced biphasic changes in [Ca 2ϩ ] c ; a transient elevation was followed by a sustained decrease in [Ca 2ϩ ] c ( Fig. 2A). This is a rather unusual [Ca 2ϩ ] c response to Ca 2ϩ mobilizing agonists. At present, the mechanism for the sustained reduction of [Ca 2ϩ ] c in response to glucose is not clear. The sustained decrease in [Ca 2ϩ ] c may be explained by the extrusion of Ca 2ϩ to endoplasmic reticulum or other organelles together with suppression of Ca 2ϩ entry. The exact mechanism for the unique action of glucose remains to be determined.
Fifth, glucose decreased [cAMP] c in HEK-CaSR cells (Fig.  5A). This response is mediated by the activation of G i , as it was blocked by the pretreatment with pertussis toxin. In HEK-CaSR cells, cinacalcet also decreased [cAMP] c (Fig. 5C). As in parathyroid cells (25,26), CaSR is coupled to G i in HEK-CaSR cells.
The present results clearly show that CaSR is allosterically stimulated by glucose at relatively low concentrations. The physiological significance of this glucose effect, however, is not certain at present. In this regard, the glucose sensitivity of CaSR is quite high, and CaSR is already activated by physiological concentrations of glucose in the plasma. Therefore, most of the previous studies on CaSR performed in vitro and in vivo were carried out under the conditions where CaSR is activated at least to some extent by glucose. Recently, CaSR is shown to function as the kokumi receptor in taste buds of the tongue (27). CaSR is expressed in some population of the type 2 taste cells in the taste buds (28). When stimulated by an agonist, for example ␥-glutamylcysteinylglycine (GSH), GSH activates the kokumi pathway, which enhances the sweet, salty, and umami tastes (27). When sweet substances such as sucrose are ingested, both CaSR and the sweet taste receptor may be activated, and the sweet sensation is enhanced by the activation of the kokumi pathway.
We have been studying the cell-surface receptor activated by glucose in pancreatic ␤-cells (9,11). In these cells, glucose activates cell-surface receptor, which is dependent on T1R3 (11). Presumably, a homodimer of T1R3 may function as a glucosesensing receptor. Compared with the cell-surface receptor in ␤-cells, glucose sensitivity of CaSR is quite high. CaSR, which is expressed considerably in ␤-cells (29,30), may be already activated in the presence of physiological concentrations of glucose. There is a possibility that CaSR and T1R3 form a heterodimer. If this is the case, what is the glucose sensitivity of the putative heterodimer? Does the heterodimer also function as the glucose-sensing receptor? Further studies are clearly necessary to address these issues.
In summary, CaSR is allosterically stimulated by physiological concentrations of glucose in HEK-CaSR cells. This sugar acts as an allosteric modulator of CaSR and elicits unique patterns of intracellular signals.

Experimental Procedures
Materials-Sucralose, 3-O-methyl-D-glucose, and lactisole were obtained from Sigma. Glucose, sucrose, acesulfame-K, and glycyrrhizin were from Wako Pure Chemicals (Osaka, Japan), and cinacalcet was from Chemscene LLC (Monmouth Junction, NJ). NPS-2143 was kindly provided by Dr. Yuzuru Eto (Ajinomoto Co., Kawasaki, Japan). These chemicals were dissolved in a solution composed of 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1.3 mM CaCl 2 , 10 mM HEPES/NaOH (pH 7.3), and 3 mM glucose unless otherwise mentioned. This solution was named extracellular solution (ECS) for descriptive purposes applied to the experimental conditions. All of the chemical reagents used to prepare the ECS were obtained from Wako Pure Chemicals except HEPES (Dojindo Molecular Technologies, Kumamoto, Japan). U73122, an inhibitor of phospholipase C (PLC) (13), and its inactive analogue U73343 were obtained from EDM Millipore (Billerica, MA). YM25489, an inhibitor of G q (14), was a generous gift from Dr. Jun Takasaki (Yamanouchi Pharmaceutical, Ibaraki, Japan).
Cell Culture-HEK293 cells obtained from the Riken Cell Bank (Riken Bioresource Center, Ibaraki, Japan) were grown in Dulbecco's modified Eagle's medium (DMEM) containing a high concentration of glucose (Invitrogen), 10% fetal bovine serum (FBS) (Sigma), MEM non-essential amino acids solution (Invitrogen), and penicillin-streptomycin solution (Wako Pure Chemicals). HEK293 cells stably expressing hCaSR were continuously selected by adding G418 sulfate (Wako Pure Chemicals) to the growth medium. PT-r cells (a rat parathyroid cell line) (17) were grown in Epilife medium (Invitrogen) and antibiotics. MDCK cells (18) obtained from the American Type Culture Collection were maintained in a "complete medium" composed of ␣-MEM (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine (Invitrogen), and penicillin-streptomycin solution. MDCK cells were grown to confluence in 35-mm glass bottom culture dishes in ␣-MEM. Cells were then washed twice with minimum essential medium for suspension culture (S-MEM, Invitrogen) and incubated in S-MEM supplemented with 5% dialyzed fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin solution (low calcium medium). Cells were incubated in low calcium medium for 16 h and then prepared for the corresponding measurement. All of the aforementioned cells were cultured in a humidified incubator with 95% air and 5% CO 2 at 37°C and kept in a warmed chamber that maintained the extracellular solution in a temperature range that oscillated between 35°C and 38°C during the experiment.
Establishment of HEK-CaSR Cells-Transfection was carried out using an expression vector encoding hCaSR kindly provided by Dr. Edward Brown (Brigham and Women's Hospital, Boston, MA). Transfection was achieved using the Attractene Transfection Reagent (Qiagen, Hilden, Germany) according to the manufacturer's protocol. hCaSR-transfected HEK293 cells were selected using the functional expression of the neomycin resistance gene included in the plasmid sequence for 14 days. Sixty resistant colonies were expanded, and their hCaSR mRNA expression was evaluated by using real-time quantitative PCR (31). Primers used in this study are shown in Table 1 . For the measurement, cells were placed inside a heated chamber MI-IBC attached to the microscope stage and connected to a microscope objective lens heater through a temperature controller (Olympus, Tokyo, Japan). The temperature of the aqueous extracellular environment was tightly controlled between 35°C and 38°C. Visualization was performed using an UPlanApo 10ϫ/0.40 objective lens (Olympus). To detect the fluorescence images, we used the AQUACOSMOS/ASHURA imaging system (Hamamatsu Photonics, Hamamatsu, Japan) (32). The values (F) were normalized to each initial value (F 0 ), and the relative fluorescence change was referred to as F/F 0 (32). The stimulation was performed by slowly applying the stimulus using a perfusion system inserted through a small opening in the top of the warm chamber. For the analyses of the effects of agonists and inhibitors, representative traces (each one corresponding to one cell) were selected. Selected patterns were considered representative when they showed a similar reaction in Ͼ80% of all the examined cells in the observed microscopic field. Four to seven representative traces are presented. For the determination of the dose-response relationship, the sensitivity degree to a certain stimulus was estimated by calculating the percentage of reactive cells and averaging the results obtained from at least 10 samples corresponding to each experimental protocol. Results are expressed as the mean Ϯ S.E. of accumulated data obtained in multiple experiments. Statistical analysis was performed using Student's t test. To monitor activation of PKC, we monitored translocation of a PKC substrate myristoylated alanine-rich protein kinase C substrate (MARCKS) (18). MARCKS is located in the plasma membrane in a unphosphorylated state but is translocated to the cytosol upon phosphorylation. Using cells transfected with EGFP-N 2 -MARCKS, we monitored translocation of MARCKS from the plasma membrane to the cytosol as described previously (33).

Measurement of Cytoplasmic [cAMP] c in HEK-CaSR Cells-
Changes in cytoplasmic cAMP ([cAMP] c ) were monitored by using Flamindo2 (16). HEK-CaSR cells were transiently transfected with 1.2 g of plasmid encoding Flamindo2 using 4.5 l of Attractene transfection reagent (Qiagen) per dish. Transfected cells were cultured for 3 days at 30°C. For imaging exper-  Human GAPDH  5Ј-GATCATCAGCAATGCCTCCT-3Ј  5Ј-TGTGGTCATGAGTCCTTCCA-3Ј  Human CaSR  5Ј-GCTCCTGTGCAGACATCAAG-3Ј  5Ј-AGGTGCCAGTTGATGATGGA-3Ј  Rat GAPDH  5Ј-TGCCACTCAGAAGACTGTGG-3Ј  5Ј-TTCAGCTCTGGGATGACCTT-3Ј  Rat CaSR  5Ј-GCCTCAGAAGAATGCCATGG-3Ј  5Ј-TCTGCACACTGTAGGGGAAG-3Ј Activation of Calcium-sensing Receptor OCTOBER 28, 2016 • VOLUME 291 • NUMBER 44 iments, transfected cells were incubated in ECS, and after extracellular solution temperature reached the predetermined values, cells were visualized using an UPlanApo 10ϫ/0.40 objective lens (Olympus). A 485-nm dichroic mirror (U-MGF-PHQ fluorescence filter cube, Olympus) and an ORCA-3CCD digital camera (Hamamatsu Photonics) containing an RGB prism were used for the measurement of a single wavelength of Flamindo2. Neutral density filters were not needed. For detection of the yellow fluorescence protein variant (citrine), we used AQUACOSMOS/ASHURA imaging system (Hamamatsu Photonics). The values (F) were normalized to each initial value (F 0 ), and the relative fluorescence change was referred to as F/F 0 . Stimulus was applied as described above. The analyses of the reaction to each particular stimulus were processed, and data were presented in the same way as described for the measurement of [Ca 2ϩ ] c . Immunoblotting-HEK293 and HEK-CaSR cells were washed with phosphate-buffered saline (PBS) and lysed with PBS containing complete Mini EDTA-free (Roche Diagnostics). Cell lysates were cleared by centrifugation. Supernatants were mixed with sample buffer (8 M urea, 5% SDS, 50 mM Tris/HCl (pH 6.8), 0.343 M DTT, and 0.005% bromophenol blue) and separated on 4 -15% Mini-PROTEAN TGX Gel (Bio-Rad) before transfer to Immobilon-P Membrane (Merck Millipore). Membranes were blocked for 1 h at room temperature using blocking buffer containing 5% skim milk and 0.05% Polysorbate 20 (MP Biomedicals, LLC; Santa Ana, CA) in Tris-buffered saline (TBS). Membranes were incubated with primary antibodies, anti-calcium sensing receptor antibody (ab19347; Abcam; Cambridge, UK) and anti-␤-tubulin antibody (ab11308; Abcam), at a 1:1000 dilution at room temperature for 2 h. Membranes were incubated with secondary antibody coupled to horseradish peroxidase, anti-mouse IgG, HRP-linked whole Ab sheep (NA931V; GE Healthcare) at a 1:1000 dilution at room temperature for 1 h. Protein was visualized by luminate (Merck Millipore).
Immunocytochemistry-HEK293 and HEK-CaSR cells were washed with PBS, fixed with 3% paraformaldehyde at room temperature for 40 min, washed with PBS, and blocked with Block Ace (DS Pharma Biomedical Co., Ltd.; Osaka, Japan) at room temperature for 40 min. Cells were incubated with primary antibodies and DYKDDDDK tag antibody (#2368; Cell Signaling Technology, Inc; Danvers, MA) at a 1:1000 dilution at room temperature for 1 h and washed with PBS. Cells were incubated with secondary antibodies, goat anti-rabbit IgG (HϩL) secondary antibody and Alexa Fluor 568 conjugate (A-11036; Thermo Fisher; Waltham, MA), at a 1:1000 dilution at room temperature for 1 h, after which the samples were washed with PBS for 5 min twice and incubated with DAPI (46190; Thermo Fisher) at a 1:1000 dilution at room temperature for 5 min before they were mounted on the slide glass. The cells were observed under a confocal laser scanning microscope (FV1000; Olympus).
Knockdown of CaSR Using siRNA-MDCK and PT-r cells were grown in 35-mm dishes until reaching ϳ80% confluence. Then the cells were transfected with 1.2 g of CaSR siRNA duplex for the dog gene (Sigma) or ON-TARGT plus SMART pool siRNA for the rat gene (GE Healthcare). Transfection was performed using Attractene reagent according to the manufacturer's instruction. After 24 h, the cells were tested in their response to stimulants or prepared for measurement of mRNA expression.
Author Contributions-J. M. performed, analyzed, and interpreted the biochemical experiments. Y. N., M. N., and A. F. performed and analyzed the experiments. K. S. provided a cell line and interpreted the results. T. K. provided the cAMP indicator and interpreted the results. I. K. designed and drafted the article. All authors reviewed the results and approved the final version of the manuscript.