Receptor expression modulates calcium-sensing receptor mediated intracellular Ca2+ mobilization.

GENERAL

ENDOCRINOLOGY

Receptor Expression Modulates Calcium-Sensing Receptor Mediated Intracellular Ca2ⴙ Mobilization Sarah C. Brennan, Hee-Chang Mun, Katie Leach, Philip W. Kuchel, Arthur Christopoulos, and Arthur D. Conigrave School of Molecular Bioscience (S.C.B., H.-C.M., P.W.K., A.D.C.), University of Sydney, New South Wales 2006, Australia; and Monash Institute of Pharmaceutical Sciences and Department of Pharmacology (K.L., A.C.), Monash University, Parkville, Victoria 3052, Australia

Calcium-sensing receptors (CaSRs) are class C G protein-coupled receptors that respond to physiological activators, including extracellular Ca2⫹ (Ca2⫹ o ) and L-amino acids as well as the pharmaceutical calcimimetic, cinacalcet. Unlike Ca2⫹ o , which is an orthosteric agonist, L-amino acids and cinacalcet are positive allosteric modulators. CaSR expression levels vary considerably between tissues, but the physiological significance of these differences in expression for the effects of its activators is unknown. To investigate the impact of receptor expression on CaSR-mediated signaling we used a tetracycline-inducible expression system and focused on intracellular Ca2⫹ (Ca2⫹ i ) responses in single cells and considered both population and single-cell behavior. Increased receptor expression positively modulated CaSR-mediated Ca2⫹ mobilization in response to elevated i Ca2⫹ o , the amino acid L-phenylalanine, or the calcimimetic cinacalcet. It lowered threshold concentrations for the initiation of Ca2⫹ oscillations and for their transformation to sustained Ca2⫹ i i elevations, and it increased the proportions of responding cells. It also positively modulated the frequency of Ca2⫹ oscillations with the order of effectiveness: cinacalcet equal to or greater than i Ca2⫹ o greater than L-phenylalanine. The results indicate that receptor expression modulates key characteristics of the Ca2⫹ response at the single-cell level as well as the amplitude of whole-tissue i CaSR-mediated responses by recruiting quiescent cells into the active pool of responding cells. By lowering the threshold concentrations for Ca2⫹ o - and L-amino acid-induced responses below the physiological levels of these nutrients in plasma, mechanisms that up-regulate receptor expression can control tissue function in the absence of dynamic changes in ligand concentration. (Endocrinology 156: 1330 –1342, 2015)

he calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) that is widely expressed (1). It mediates feedback control of extracellular Ca2⫹ (Cao2⫹) by inhibiting parathyroid hormone secretion and renal Ca2⫹ reabsorption (2), and it modulates bone and cartilage development (3). It also plays distinct functional roles in tissues not involved in calcium metabolism (1). Although Cao2⫹ is its primary physiological agonist, the CaSR also responds to physiological and pharmacological modulators, including L-amino acids (4), ␥L-glutamyl peptides (5–7), and synthetic calcimimetics including cinacalcet (8, 9). L-amino acids and ␥L-glutamyl peptides

T

bind in the CaSR’s large extracellular Venus Fly-Trap domain (5, 10). In contrast, synthetic calcimimetics typically bind in the receptor’s heptahelical domain (11). CaSR expression levels vary between cell types (12–14), and changes in expression may influence agonist and modulator sensitivity under normal conditions. There may also be circumstances (eg, associated with the intrinsic control of cell differentiation or cell number) in which up-regulated receptor expression per se may drive cellular signaling responses in the presence of physiological levels of nutrient activators. For this to occur, high levels of CaSR expression should lower the threshold concentrations of

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received September 21, 2014. Accepted January 16, 2015. First Published Online January 21, 2015

Abbreviations: Cai2⫹, intracellular Ca2⫹; Cao2⫹, extracellular Ca2⫹; CaSR, calcium-sensing receptor; CHO, Chinese hamster ovary; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; HRP, horseradish peroxidase; IRU, integrated response unit; L-Phe, L-phenylalanine; mGlu-5, metabotropic glutamate receptor type 5; PSS, physiological saline solution; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline.

1330

endo.endojournals.org

Endocrinology, April 2015, 156(4):1330 –1342

doi: 10.1210/en.2014-1771

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 11:34 For personal use only. No other uses without permission. . All rights reserved.

doi: 10.1210/en.2014-1771


1331

Flp-In tetracycline-regulated expression HEK-293 cells (HEK-293-TREx cells), DMEM, trypsin [0.25% (w/v)]-EDTA, penicillin, and streptomycin were from Life Technologies. Tetracycline-reduced fetal bovine serum (FBS) was from Scientifix. Other reagents were from Sigma-Aldrich or Ajax Fine Chemicals. Cinacalcet HCl was synthesized in-house (18, 19). A list of antibodies and their suppliers is provided in an accompanying table.

immunoprecipitation assay buffer [25 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1.0% Nonidet P-40; 0.1% sodium dodecyl sulfate (SDS); 1.0% sodium deoxycholate; and 1⫻ protease inhibitor]. After centrifugation (12 000 ⫻ g, 30 min, 4°C), the supernatant was assayed for protein content (DC microplate protocol; Bio-Rad Laboratories). Protein equivalents (20 ␮g) were resuspended in a ratio of 4:1 in 5⫻ gel loading buffer (0.3 M Tris-HCl, pH 6.8; 10% SDS; 50% glycerol; 25% ␤-mercaptoethanol; 0.5% bromphenol blue) for 5 min and then loaded for SDS-PAGE. Samples, prestained standards (Life Technologies), and/or a biotinylated ladder (Cell Signaling Technologies) were passed through stacking (4.0% bis/acrylamide; 0.13 M Tris-HCl; 0.1% SDS, pH 6.8) and resolving (5.0% bis/acrylamide; 0.38 M Tris HCl; 0.1% SDS, pH 8.8) gels at room temperature. Gels were rinsed in transfer buffer [25 mM Tris; 193 mM glycine; 20% (vol/vol) methanol, pH 8.8] and then underwent electrotransfer to a nitrocellulose membrane (350 mA, 1 h). Membranes were then incubated in blocking buffer (5% (wt/ vol) skim milk powder in Tris buffered saline (TBS): 20 mM Tris; 500 mM NaCl, pH 7.4) for 1 h with agitation and washed 3⫻ for 5 min in TBS containing 0.2% (wt/vol) Tween 20. They were then divided into upper and lower sections between 80-kDa and 60-kDa bands. The upper membrane sections were incubated in TBS containing 5% (wt/vol) BSA and anti-CaSR ADD monoclonal antibody (1:500). The lower sections were incubated with TBS (5% BSA) containing an antitubulin monoclonal antibody (1:5000) to assess protein loading. Membranes were incubated overnight at 4°C with agitation and then washed with TBS containing 0.2% (wt/vol) Tween 20 and incubated for 1 h at room temperature with blocking buffer containing goat antimouse horseradish peroxidase (HRP)-conjugated IgG (diluted 1:20 000) and an antibiotin HRP-conjugated antibody (1:10 000). After washing, membranes were developed in the dark using ECL Plus Western blotting detection reagents (Amersham Biosciences). Protein bands were selected individually and pixel counts were obtained using GIMP software (http://www.gimp.org).

Generation of TREx293-CaSR cells and cell culture

Cell surface expression

TREx293-CaSR cells were generated by stably expressing a Myc-tagged full-length human CaSR in Flp-in HEK-293-TREx cells using hygromycin (200 ␮g mL⫺1) and blastocidin (5 ␮g mL⫺1) to select for colonies expressing the CaSR construct under the control of the tet repressor. TREx293-CaSR cells were routinely cultured using DMEM/10% tetracycline-depleted FBS 2–3 d prior to experiments. Cells were grown in six-well plates for expression studies or transferred onto 15-mm glass coverslips in 24-well plates for microfluorimetry experiments. Cells were cultured for 24 – 48 h and then induced by exposure to tetracycline (various concentrations). Pilot experiments demonstrated that a stable maximum level of expression was achieved from 24 to 36 h. For this reason, the time of exposure in the experiments described in Results was fixed at 24 h.

After removal of media, cells were incubated with rocking for 2 hours at 4°C with 1 ␮g mL⫺1 anti-cMyc antibody (9E10) in DMEM/10% FBS. Cell monolayers were detached by irrigation using 1 mL of ice-cold PBS and then pelleted (400 ⫻ g, 5 min, 4°C), resuspended in DMEM/10% FBS containing HRP-conjugated goat antimouse IgG antibody (1:5000), and incubated with shaking (1 h, 4°C) and then recentrifuged, washed twice with PBS (1 mL), and incubated with 3,3⬘, 5, 5⬘-tetramethyl benzidine (200 ␮L) for 20 min in the dark with gentle rocking. Reactions were stopped by the addition of equal volumes of 1.0 M HCl. After centrifugation, absorbances of supernatant samples were obtained (450 nm). Samples were analyzed in triplicate and median control values were normalized to 1.0.

nutrient activators below their physiological levels, ie, below 1.1 mM in the case of Cao2⫹ and below 3 mM in the case of the complement of L-amino acids (4). Receptor activation by submaximal Cao2⫹ induces low frequency (1– 4 min⫺1) intracellular Ca2⫹ (Ca2⫹ i ) oscillations in the context of protein kinase C-dependent phosphorylation of T888 in the C terminus (15–17). At high Cao2⫹, Ca2⫹ oscillations transform to sustained Ca2⫹ elei i vations accompanied by T888 dephosphorylation (17). However, the impact of receptor expression on CaSR-mediated Ca2⫹ signaling is unknown. i To investigate the effect of receptor expression level on CaSR-mediated Ca2⫹ signaling, we used human embryi onic kidney (HEK)-293 cells that stably expressed the CaSR under the control of a tetracycline-inducible promoter. We first evaluated the utility of the system for modulating total and surface receptor expression and then investigated the impacts of different levels of receptor expression on Cao2⫹- and modulator-induced Ca2⫹ mobii lization in single cells, focusing on the natural amino acid, L-phenylalanine (L-Phe) and the calcimimetic, cinacalcet.

Materials and Methods Reagents

Immunoblotting After the induction of CaSR expression, cells were washed twice with ice-cold PBS (137 mM NaCl; 2.7 mM KCl; 8.1 mM Na2HPO4; 1.76 mM KH2PO4, pH 7.4) and then lysed in radio-

Ca2ⴙ microfluorimetry i TREx293-CaSR cells were cultured on coverslips in 24-well plates and then loaded in the dark with fura2-AM (5 ␮M) in physiological saline solution (PSS): 125 mM NaCl, 4 mM KCl, 0.1% (wt/vol) D-glucose, 1 mM MgCl2, 20 mM HEPES-NaOH (pH 7.45) that contained 1 mM CaCl2, 0.8 mM NaH2PO4, and


1332

Brennan et al

Receptor Expression and CaSR-Mediated Signaling

1 mg mL⫺1 BSA (2 h, 37°C). The cells were washed and stored in loading solution (no fura2-AM) prior to experiments. Fura2loaded TREx293-CaSR cells were transferred into a perifusion chamber and then placed in the light path of a Zeiss Axiovert fluorescence microscope (⫻63 objective) and perifused with PSS containing various concentrations of Cao2⫹, L-Phe, and cinacalcet. Fura2-loaded TREx293-CaSR cells were excited using a Lambda DG-4 150 Watt xenon light source (Sutter Instruments), with alternating wavelengths of 340 and 380 nm at 0.5-s intervals, and imaged at 510 nm. For each data set, regions of interest of 10 individual cells were selected and digital images were captured using an AxioCam camera controlled by Stallion SB.4.1.0 PC software (Carl Zeiss).


was up-regulated by the addition of tetracycline to the culture medium. To investigate the effect of tetracycline on total CaSR expression, TREx293-CaSR cells were incubated with various concentrations of tetracycline for 24 h, and cell extracts were prepared for Western blotting. In all lanes, CaSR bands of approximately 160 and 140 kDa corresponded to the expected sizes of mature and immature, high-mannose subunit forms respectively (21, 22). Higher-molecular-mass bands (⬎ 200 kDa) were consistent with the expected mobilities of CaSR dimers.

Responding cells, Ca2ⴙ oscillation frequency, curve i fitting, and statistical analyses Single-cell Ca2⫹ mobilization data consisted of excitation i ratios (F340:F380) plotted against time (min). Ca2⫹ mobilizi ing responses arise from the release of Ca2⫹ from intracellular i stores and Cao2⫹ influx via plasma membrane channels. Where indicated, Ca2⫹ responses from single cells were integrated i according to the trapezoidal rule using the following equation:

冕

xn

f共 x兲dx ⬇ ⌬x

x0

冉

yn y0 ⫹ y1 ⫹ … ⫹ yn⫺1 ⫹ 2 2

冊

to provide results in the form of integrated response units (IRUs) and corrected for the baseline at a control Cao2⫹ concentration (0.5 mM Cao2⫹). Concentration-dependent response data were fitted to the following equation:

R ⫽ d ⫹ (a ⫺ d)Cb/(eb ⫹ Cb) in which a is the maximum response (EMAX); b is the Hill coefficient; e is the EC50; d is the minimum response; and C is the activator concentration. Estimates of curve-fitting parameters were obtained using GraphPad Prism version 5.0. Responding cells were identified by increases in baseline fluorescence greater than 2 SD above the mean for control after correction of baseline ratio data for small time-dependent shifts during the courses of experiments. The records from individual cells were processed for Ca2⫹ i oscillation frequency using wavelet analysis in Mathematica 7.0 (20). Frequency data from individual cells are expressed as means ⫾ SEM. Statistical analyses were performed using the ␹2 test for comparisons between the numbers and proportions of responding cells, Student’s unpaired t test for comparisons between log EC50 values, and one-way ANOVA with Dunnett/ Tukey’s posttest for other parameters using GraphPad Prism version 5.0.

Results Inducible expression of the CaSR in TREx293-CaSR cells CaSR expression in TREx293-CaSR cells was controlled via a cytomegalovirus promoter negatively modulated by Tet repressor function so that CaSR transcription

Figure 1. The effect of preincubation with tetracycline on total and surface expression of the CaSR. TREx293-CaSR cells were incubated with various concentrations of tetracycline for 24 h and then lysed for the determination of total and cell surface expression of the CaSR. A, Total immunoreactive CaSR expression was determined by Western blotting (representative blot from a total of four similar blots). B, Concentration-response curves for the effects of tetracycline on CaSR total and cell surface expression. Cell surface expression of the CaSR was determined by ELISA (fold changes relative to control uninduced cells; n ⫽ 4). Total expression was quantified by pixel counts of CaSR monomer bands (160 kDa plus 140 kDa) using GIMP imaging software (n ⫽ 4). The data are expressed as fold changes ⫾ SEM (n ⫽ 4). Similar results were obtained using pixel counts based on all bands detected from 120 kDa to greater than 200 kDa (not shown).


doi: 10.1210/en.2014-1771

Control TREx293-CaSR cells exhibited low but detectable levels of CaSR expression, despite the use of tetracycline-depleted FBS, consistent with the leaky promoter function (Figure 1A). Exposure to tetracycline (1–50 ng mL⫺1) for 24 h induced concentration-dependent increases in total expression, quantified using the 160-kDa and 140-kDa subunits, to a maximum 6.3- ⫾ 1.8-fold (n ⫽ 4; Figure 1B). Similar results were obtained quantifying all bands on the blots (not shown). Changes in surface expression were quantified using an ELISA based on a c-Myc epitope inserted between CaSR residues 371 and 372, in a loop tolerant of in-frame insertions (23). Surface expression was up-regulated in a concentration-dependent manner after preincubation with tetracycline to a maximum 8.3- ⫾ 0.6-fold (n ⫽ 4; Figure 1B). Effect of receptor expression on Ca2ⴙ o threshold concentration, Ca2ⴙ potency, and efficacy o TREx293-CaSR cells were exposed to various tetracycline concentrations (0 –50 ng mL⫺1) for 24 h and then loaded with fura2 and exposed to stepwise increments in


1333

Cao2⫹ (0.5–10 mM). Tetracycline (0.12 ␮M, ie, equivalent to 50 ng mL⫺1) had no acute effect on Ca2⫹ mobilization i in CaSR-expressing HEK-293 cells (not shown). Individual F340:F380 ratiometric traces were obtained (10 cells per experiment), and responding cells were identified by the presence of either repetitive Ca2⫹ oscillations or susi 2⫹ tained elevations in baseline Cai . In general, as Cao2⫹ increased, cellular Ca2⫹ responses were transformed from i Ca2⫹ oscillations to stable Ca2⫹ elevations. i i In uninduced TREx293-CaSR cells, even above a Cao2⫹ threshold of approximately 4 mM, the Ca2⫹ responses i mainly took the form of Ca2⫹ oscillations, and at the highi est Cao2⫹ tested (10 mM) less than 10% exhibited stable Ca2⫹ elevations (Figure 2A; n ⫽ 74 individual cells; eight i independent experiments). Increases in receptor expression increased the proportion of responding cells, especially at lower Cao2⫹ (⬃2.0 – 4.0 mM) as well as the proportion of cells that underwent transformation to sustained Ca2⫹ elevations (Figures 2 and 3). At 4.0 mM i Cao2⫹, tetracycline had highly significant positive effects on the proportions of cells that exhibited Ca2⫹ oscillations i at all three preincubation concentrations tested between 2 and 50 ng mL⫺1 (␹2 ⫽ 106 –123; df ⫽ 1; P ⬍ .001). Induction of CaSR expression by tetracycline (1–50 ng mL⫺1) for 24 h led to reductions in Cao2⫹ threshold at which Ca2⫹ mobilization was obi served (Figures 3 and 4) together with increases in Cao2⫹ potency and efficacy, as defined by decreases in EC50 and increases in EMAX respectively (Figure 4). Thus, exposure of TREx293-CaSR cells to tetracycline (50 ng mL⫺1) for 24 h lowered the Cao2⫹ threshold from approximately 2.0 mM to less than 1.0 mM and the EC50 for Cao2⫹ from 4.3 ⫾ 1.0 mM to 2.3 ⫾ 0.2 mM. The EMAX value increased 2-fold (Figure 4).

Figure 2. Impact of tetracycline-induced increases in CaSR expression on the proportions of cells that responded to Cao2⫹. TREx293-CaSR cells were preincubated with various concentrations of tetracycline for 24 h and then loaded with fura2-AM for microfluorimetry as described in Materials and Methods. Cells were perifused with PSS and exposed to stepwise increments in Cao2⫹. All individual cells from six to eight independent experiments were pooled to form separate populations for each tetracycline concentration. The panels display the percentages of total responding cells as well as cells exhibiting Cai2⫹ oscillations or sustained Cai2⫹ responses for each Cao2⫹ concentration tested. The preincubation tetracycline concentrations were as follows (in nanograms per milliliter⫺1): A, 0; B, 2; C, 10; and D, 50 ng mL⫺1. The data were obtained in 57–74 individual cells in six to eight independent experiments. The symbols denote the following: open circles, total responding cells; closed squares, cells exhibiting Cai2⫹ oscillations; and closed triangles, cells exhibiting sustained elevations in Cai2⫹.

Effect of receptor expression on Ca2ⴙ oscillation frequency i We next analyzed the effect of receptor expression on Ca2⫹ oscili lation frequency in responding cells. In uninduced cells, the frequency of Cao2⫹-induced Ca2⫹ osi cillations was 2–3 min⫺1, even at the highest Cao2⫹ tested, 10 mM, and, as noted above, less than 10%


1334

Brennan et al



mental Table 1). At Cao2⫹ greater than 4.0 mM, there was an increase in the proportion of cells in which Ca2⫹ oscillations were replaced by i stable elevations in Ca2⫹ and the i Ca2⫹ oscillation frequency of cells i that did not undergo this transformation fell. This apparently paradoxical finding suggests that these cells failed to achieve a threshold frequency required for the transformation to stable Ca2⫹ elevations. i Impact of receptor expression on L-Phe or cinacalcet-induced modulation of Ca2ⴙ responses i We next investigated the effects of the positive modulators L-Phe and cinacalcet on Cao2⫹-dependent Ca2⫹ i responses at low and high receptor 2⫹ 2⫹ Figure 3. Impact of receptor expression on Cao -induced Cai responses in single cells. expression. TRex293-CaSR cells TREx293-CaSR cells were preincubated with various tetracycline concentrations for 24 h and then were preincubated in the absence loaded with fura2-AM for microfluorimetry. The responses were obtained in representative cells preincubated with tetracycline at concentrations of 0 (A), 2 (B), 10 (C), or 50 (D) ng mL⫺1. The or presence of tetracycline (50 traces shown are representative of those obtained in six to eight independent experiments. ng mL⫺1) for 24 h and then loaded with fura2-AM. After equilibration of cells underwent transformation to stable elevations in PSS containing 0.5 mM Cao2⫹ for 3–5 min, the cells were 2⫹ in Cai (Figure 2A). (2 ␮M), Tetracycline-induced increases in receptor expression exposed to either L-Phe (10 mM) or cinacalcet 2⫹ led to increases in Ca2⫹ oscillation frequency that were followed by stepwise increments in Cao (0.5–10 mM). i 2⫹ most apparent at Cao approximately 4.0 mM (Supple- The concentrations selected for L-Phe and cinacalcet are considered maximally effective for CaSR-mediated Ca2⫹ i mobilization in HEK-293 cells (9, 24). In cells that were preincubated in the absence of tetracycline, 10 mM L-Phe lowered the Cao2⫹ threshold for Ca2⫹ oscillations from approximately 4.0 mM to 1.0 mM i (Figure 5A). In cells preincubated in the presence of tetracycline (50 ng mL⫺1), on the other hand, the Cao2⫹ threshold required for Ca2⫹ oscillations fell from around i 1.0 mM in the absence of L-Phe to approximately 0.7 mM in the presence of 10 mM L-Phe (Figure 5B). Thus, at levels of high receptor expression, the threshold for Cao2⫹ lay below its normal plasma level of 1.1–1.3 mM, and, in the presence of a maximally effective L-amino acid concentration, it fell further. In addition to lowering the Cao2⫹ threshold for Ca2⫹ i oscillations at low and high receptor expression, L-Phe Figure 4. The impact of receptor expression on the Cao2⫹ markedly increased the proportion of cells that exhibited concentration dependence of integrated Cai2⫹ responses. Batches of Ca2⫹ oscillations at submaximal Cao2⫹. The effect was i TREx293-CaSR cells were preincubated for 24 h in the absence (open most apparent at Cao2⫹ between 2.0 and 4.0 mM in cells circles) or presence of tetracycline at the following concentrations: 2 ⫺1 (closed circles); 10 (closed triangles); or 50 ng mL (closed squares). that had been preincubated in the absence of tetracycline Cells were then loaded with fura2-AM for microfluorimetry. Integrated and at Cao2⫹ between 0.5 and 2.0 mM, which encompasses responses were obtained for each activator concentration as described the physiological range in plasma (1.1–1.3 mM), in cells in Materials and Methods. The results were obtained in six to eight independent experiments in each case. preincubated in 50 ng mL⫺1 tetracycline (Figure 5B). At


doi: 10.1210/en.2014-1771


1335

and high receptor expression (Figure 5, C and D). At high receptor expression, cinacalcet also lowered the Cao2⫹ threshold at which sustained elevations in Ca2⫹ were observed i from approximately 4.0 mM to 2.0 mM (Figure 6D). Receptor expression also markedly increased the proportion of cells that responded to physiologically normal Cao2⫹ (1 mM) in the presence of 2 ␮M cinacalcet. Thus, in control cells the proportion of responding cells was less than 2%. However, in cells preincubated with 50 ng mL⫺1 tetracycline, it increased to 54% (␹2 ⫽ 203, P ⬍ .0001; compare Figure 5, C and D). Single-cell Ca2⫹ response data i were integrated to permit the analyFigure 5. Impacts of the positive modulators L-Phe or cinacalcet on the proportions of cells sis of the effects of the modulators on exhibiting Cao2⫹-induced Cai2⫹ oscillations at low and high receptor expression. TREx293-CaSR ⫺1 Cao2⫹ potency and efficacy. L-Phe cells were preincubated for 24 h with either 0 or 50 ng mL tetracycline and then loaded with and cinacalcet increased the recepfura2-AM for microfluorimetry as described in Materials and Methods. The cells were then exposed to stepwise increments in Cao2⫹ in either the absence or presence of L-Phe (10 mM) or tor’s sensitivity to Cao2⫹, as reported cinacalcet (2 ␮M), and the percentages of cells exhibiting Cai2⫹ oscillations were determined. The previously (Figure 6) (4, 9). In the effects of L-Phe (10 mM) are shown at low and high receptor expression in panel A and panel B, context of low receptor expression, respectively. The effects of cinacalcet (2 ␮M) are also shown at low and high receptor expression in panel C and panel D, respectively. The data from 26 –56 individual cells in three to eight 10 mM L-Phe and 2 ␮M cinacalcet independent experiments were combined to obtain the percentages shown. Open symbols, decreased the EC50 for Cao2⫹ from absence of modulator; closed symbols, presence of modulator; circles, preincubated in the 4.4 ⫾ 1.2 mM to 2.9 ⫾ 1.0 mM and absence of tetracycline; squares, preincubated in the presence of 50 ng mL⫺1 tetracycline. 2.8 ⫾ 1.0 mM, respectively, with maximum levels of receptor expression, 10 mM L-Phe also little or no effect on the maximum response (Figure 6, lowered the Cao2⫹ threshold at which sustained Ca2⫹ el- A and C). i evations were observed from approximately 4.0 –2.0 mM Preincubation with tetracycline to maximally increase (see Figure 6D). receptor expression decreased the EC50 for Cao2⫹ in the The impact of receptor expression on the proportion of absence of modulators to 2.9 ⫾ 0.2 mM Cao2⫹ (Figure 6, responding cells was particularly noticeable in the pres- C and D). In this setting, L-Phe (10 mM) and cinacalcet (2 ence of physiologically normal Cao2⫹ (1 mM) and high ␮M) further enhanced Cao2⫹ sensitivity, lowering the EC50 L-Phe concentration (10 mM). Thus, in cells preincubated values for Cao2⫹ to 1.9 ⫾ 0.4 mM and 1.7 ⫾ 0.3 mM, in the absence of tetracycline, the proportion of respond- respectively (P ⬍ .05; P ⬍ .05). However, cinacalcet had ing cells was 2.6%. However, after preincubation with no significant effect on EMAX and L-Phe reduced EMAX tetracycline (50 ng mL⫺1), the proportion of responding from 14.8 ⫾ 1.0 IRU to 11.5 ⫾ 0.9 IRU (P ⬍ .05, Figure cells increased to 35% (␹2 ⫽ 641, P ⬍ .0001; compare 6, B and D). Figure 5, A and B). An even more striking effect was obMaximum concentrations of the two modulators had 2⫹ served at submaximal Cao (2 mM) in the presence of 10 no apparent effects on Ca2⫹ oscillation frequency in cells i mM L-Phe. Thus, in control cells, the proportion of re- expressing the CaSR at low levels. However, they had sponding cells was 11%. In cells preincubated with tetra- differing effects on Ca2⫹ oscillation frequency in cells exi cycline (50 ng mL⫺1), however, the proportion of re- pressing the CaSR at high levels (Table 1). Thus, 10 mM sponding cells increased to 85% (␹2 ⫽ 250, P ⬍ .0001; L-Phe lowered the maximum Ca2⫹ oscillation frequency i compare Figure 5, A and B). observed across the range of Cao2⫹ concentrations tested Cinacalcet (2 ␮M) exhibited similar effects to L-Phe. from 4.3 ⫾ 0.3 min⫺1 to 3.5 ⫾ 0.2 min⫺1 (P ⫽ .03; Table Thus, it lowered the Cao2⫹ threshold for Ca2⫹ oscillations 1). In contrast, 2 ␮M cinacalcet raised the maximum obi and markedly increased the proportion of cells that ex- served Ca2⫹ oscillation frequency to 4.8 ⫾ 0.3 min⫺1 (P ⬍ i 2⫹ 2⫹ hibited Cai oscillations at submaximal Cao at both low .05; Table 1).


1336

Brennan et al



L-Phe

or cinacalcet at submaximal Cao2⫹ (2.5 mM). TRex293-CaSR cells were preincubated in the absence or presence of tetracycline (50 ng mL⫺1) for 24 h and then were loaded with fura2-AM. After equilibration in PSS containing 0.5 mM Cao2⫹ for 3–5 min, they were exposed to 2.5 mM Cao2⫹ followed by stepwise increments in L-Phe (0.1–30 mM) or cinacalcet (3 nM-3 ␮M). L-Phe and cinacalcet increased the proportion of cells that exhibited Ca2⫹ oscillations in response to 2.5 i mM Cao2⫹, and these effects were positively modulated by receptor expression. In uninduced cells exposed to 2.5 mM Cao2⫹, L-Phe (0 –30 mM) and cinacalcet (0 –3 ␮M) maximally increased the proportion of cells that exhibited Ca2⫹ oscillations from 2% i Figure 6. Impact of the positive modulators L-Phe or cinacalcet on Cao2⫹-induced Cai2⫹ responses at low and high receptor expression. TREx293-CaSR cells were preincubated for 24 h to 27% (Figure 7A) and from 2% to in either the absence or presence of tetracycline (50 ng mL⫺1) and then loaded with fura2-AM 77%, respectively (Figure 7B). Thus, for microfluorimetry as described in Materials and Methods. The cells were then exposed to at low receptor expression, cinacal2⫹ stepwise increments in Cao in either the absence or presence of L-Phe (10 mM) or cinacalcet (2 cet had a greater impact than L-Phe ␮M). The left-hand panels show representative Cai2⫹ traces obtained at low (A) or high (B) receptor expression in the absence of modulator (black) or presence of L-Phe (red) or cinacalcet on the proportion of responding (light blue). The impacts of the positive modulators on Cao2⫹ concentration response relationships cells. In cells that had been preincufor the integrated responses are shown at low and high receptor expression in panels C and D, bated for 24 h with 50 ng mL⫺1 tetrespectively. The symbols are as follows: open circles, control; closed squares, 10 mM L-Phe; closed triangles, 2 ␮M cinacalcet. The data were obtained in three to eight independent racycline, on the other hand, L-Phe experiments in each case. and cinacalcet both markedly increased the proportion of cells exhibImpact of receptor expression on the proportions 2⫹ iting Ca oscillations from approximately 40% to a maxof responding cells on exposure to various i imum level of 70%-80% in each case. Interestingly, at high modulator concentrations We next investigated the impact of receptor expression receptor expression, robust effects on the proportions of on Ca2⫹ responses induced by various concentrations of responding cells were observed, even at the lowest modi Table 1. Density

Impacts of Positive Modulators on Cao2⫹-Mediated Cai2⫹ Oscillation Frequency at Low and High Receptor Ca2ⴙ Oscillation Frequency, minⴚ1 i Tetracycline, 0 ng mLⴚ1

Cao2ⴙ, mM 1.0 2.0 4.0 6.0

Control NR NR 2.3 ⫾ 0.4 2.6 ⫾ 0.3

Tetracycline, 50 ng mLⴚ1

Cinacalcet

L-Phe

Control

NR 1.7 ⫾ 0.3 2.7 ⫾ 0.4 2.7 ⫾ 0.3

1.6 ⫾ 0.5 2.2 ⫾ 0.7 2.3 ⫾ 0.3 2.3 ⫾ 0.2

3.2 ⫾ 0.2 3.2 ⫾ 0.4 4.0 ⫾ 0.2 4.3 ⫾ 0.3

a

Cinacalcet

L-Phe

2.5 ⫾ 0.6 3.2 ⫾ 0.3 4.8 ⫾ 0.3b,c 3.5 ⫾ 1.0

2.8 ⫾ 0.4 2.8 ⫾ 0.2 3.5 ⫾ 0.2c 2.7 ⫾ 0.3b

Abbreviation: NR, no oscillatory responses. TREx293-CaSR cells were preincubated in the absence or presence of tetracycline (50 ng mL⫺1) for 24 hours and then loaded with fura2-AM and then analyzed for Cao2⫹-dependent Cai2⫹ responses in the absence or presence of L-Phe (10 mM) or cinacalcet (2 ␮M). Wavelet analysis was used to determine Cai2⫹ oscillation frequency. As seen in Supplemental Table 1, reductions in Cai2⫹ oscillation frequency were observed for Cao2⫹ of 6.0 mM or greater in cells that had not undergone the transformation to stable elevations in Cai2⫹. The results were obtained from 25–58 cells in three to nine independent experiments. a Observed in 5 of 10 cells in a single experiment. b P ⬍ .05 with respect to control at the same Cao2⫹. c P ⬍ .05 with respect to the maximum Cai2⫹ frequency observed in control cells.


doi: 10.1210/en.2014-1771


1337

concentrations of L-Phe induced only modest increases in Ca2⫹ frequency i at either low or high receptor expression (Figure 8A). Thus, in uninduced cells at 2.5 mM Cao2⫹, Ca2⫹ oscillai tion frequency was 1.0 ⫾ 0.1 min⫺1 at 0.1 mM L-Phe, and 1.4 ⫾ 0.4 min⫺1 at 30 mM L-Phe (P ⬍ .05; n ⫽ 10 cells in three experiments). In cells in which CaSR expression was maximally induced (tetracycline 50 Figure 7. Impact of receptor expression on modulator-induced increases in the proportions of ng mL⫺1; 24 h) and then exposed to responding cells. TREx293-CaSR cells were preincubated for 24 h in either the absence or presence of ⫺1 tetracycline (50 ng mL ) and then loaded with fura2-AM for single-cell microfluorimetry as described 2.5 mM Cao2⫹, Ca2⫹ oscillation frei in Materials and Methods. The cells were then exposed to a submaximal Cao2⫹ concentration (2.5 quency was 1.1 ⫾ 0.1 in the absence mM) in either the absence or presence of various concentrations of L-Phe (A) or cinacalcet (B), and 2⫹ of L-Phe and increased to 2.0 ⫾ 0.3 single-cell Cai responses were then analyzed for the proportions of responding cells. The data from 30 – 61 individual cells in three to seven independent experiments were combined to obtain the min⫺1 at 0.1 mM L-Phe and to 2.4 ⫾ percentages shown. Open circles, preincubated in the absence of tetracycline; closed circles, 0.2 min⫺1 at 30 mM L-Phe, respecpreincubated in the presence of tetracycline (50 ng mL⫺1). tively (33 cells in four experiments; Figure 8B). Although the difference ulator concentrations tested, ie, L-Phe (0.1 mM) and cinacalcet (3 nM), indicating that the threshold concentra- between control and L-Phe (30 mM) was statistically sigtions for activation by L-Phe and cinacalcet lay below 0.1 nificant (P ⬍ .001), no concentration-dependent increase 2⫹ mM and 3 nM, respectively (Figure 7). These experiments in Cai frequency was observed over the L-Phe concenidentify conditions, ie, high CaSR expression and sub- tration range (0.1–30 mM; P ⬎ .1), demonstrating that at 2⫹ maximal Cao2⫹, in which the normal fasting plasma total high CaSR expression and submaximal Cao , the effect of 2⫹ L-Phe on Cai oscillation frequency, like that on the proL-amino acid concentration (3 mM) is likely to robustly portion of responding cells, was independent of the conactivate the receptor. centrations tested encompassing the normal physiological range for total plasma amino acid concentration (⬃3– 6 Impact of receptor expression on Ca2ⴙ oscillation i mM). frequency in cells exposed to various modulator Cinacalcet positively modulated Ca2⫹ oscillation freconcentrations i 2⫹ 2⫹ quency at high but not low levels of receptor expression. Cells that exhibited Cai oscillations at 2.5 mM Cao 2⫹ were analyzed further for the impact of receptor expres- Thus, in uninduced cells exposed to 2.5 mM Cao , cina2⫹ 2⫹ sion on modulator-induced Cai oscillation frequency. calcet had no significant effect on Cai oscillation freOnce Ca2⫹ oscillations had been established, increasing quency as its concentration increased from 3 nM to 3 ␮M i (P ⬎ .1; Figure 8B). In cells preincubated for 24 h with tetracycline (50 ng mL⫺1), however, Ca2⫹ oscillation i frequency increased significantly from 1.1 ⫾ 0.2 to 3.7 ⫾ 0.3 min⫺1 as cinacalcet increased (P ⬍ .001; Figure 8B). Thus, the effect of cinacalcet on Ca2⫹ i oscillation frequency was dependent on receptor expression. Receptor expression had only modest impacts on the L-Phe or Figure 8. Impact of receptor expression on modulator-induced increases in Cai2⫹ oscillation cinacalcet concentration-response frequency. TREx293-CaSR cells were preincubated for 24 h in either the absence or presence of curves derived by integration of tetracycline (50 ng mL⫺1) and then loaded with fura2-AM for single-cell microfluorimetry as Ca2⫹ responses in active single cells described in Materials and Methods. The cells were then exposed to a submaximal Cao2⫹ i concentration (2.5 mM) in either the absence or presence of various concentrations of L-Phe (A) (data not shown), consistent with the or cinacalcet (B), and single-cell Cai2⫹ responses were then analyzed for Cai2⫹ oscillation low proportions of cells that underfrequency. The data from 30 – 61 individual cells in three to seven independent experiments were went transformation to stable Ca2⫹ i combined to obtain the percentages shown. Open circles, preincubated in the absence of ⫺1 tetracycline; closed circles, preincubated in the presence of tetracycline (50 ng mL ). elevations.


1338

Brennan et al


Discussion We have established the utility of TREx293-CaSR cells, HEK-293 cells that stably express the CaSR under the Tet repressor, for studies of receptor expression-dependent control of CaSR-mediated signaling and focused on single-cell Ca2⫹ responses, which permitted an analysis of the i behavior of cells within a population. The cells exhibited tetracycline concentration-dependent up-regulation of both total and plasma membrane CaSR expression (Figure 1). Although receptor expression was observed, even in the absence of tetracycline, indicating incomplete inhibitory control of the Tet repressor over transcription (19), total and cell surface receptor expression increased maximally by 6- to 8-fold after 24 hours of exposure to tetracycline, and the effects were concentration dependent in the range 0 –50 ng mL⫺1 (0 – 0.1 ␮M; Figure 1). Cell surface expression is a key determinant of plasma membrane receptor density, and similar approaches to that taken in the present study have been used to study the impact of cell surface expression on cell signaling for various receptors including the class C GPCR, metabotropic glutamate receptor type 5 (mGlu-5) (25). In this regard, it is notable that the impacts of tetracycline on total and cell surface expression were comparable at all concentrations tested and that the unglycosylated 120-kDa form was a minor component, even at the highest tetracycline concentrations used (see Figure 1). It was believed previously that receptor-dependent signaling occurred exclusively at the plasma membrane. It now seems clear, however, that receptor-dependent signaling also occurs intracellularly (26). For this reason, the term, receptor expression, referring to the intracellular as well as the extracellular pools of receptors, is preferred to the term, receptor surface expression or receptor density, in the present report. Receptor expression positively modulated Cao2⫹-induced single-cell Ca2⫹ responses in the absence, as well as i in the presence (see below), of chemical modulators, lowering the threshold Cao2⫹ at which Ca2⫹ oscillations api peared from approximately 2.0 mM to 1.0 mM, increasing the proportion of responding cells, increasing the frequency of Ca2⫹ oscillations, and lowering the threshold i Cao2⫹ at which Ca2⫹ oscillations transformed to stable i 2⫹ Cai elevations (Figures 1–3). Concentration-response analysis of the integrated data (Figure 4) indicated a positive impact of receptor expression on both Cao2⫹ potency and efficacy. Enhanced Cao2⫹ potency may have arisen as the number of activated receptors exceeded the capacity of the downstream signaling pathway. It is more difficult to conclude that the observed upper limit on EMAX depends on saturation of the available signaling apparatus by overexpressed CaSRs because both total and surface receptor


expression had reached apparent plateau levels at the highest tetracycline concentrations tested (Figure 1). The observed increases in efficacy as tetracycline concentration rose were dependent on a transformation of Ca2⫹ responses from oscillations to sustained elevations i and were presumably linked to dephosphorylation of CaSR residue T888 (17). A causal link between the effects might arise from Ca2⫹ frequency-dependent activation of i a protein phosphatase analogous to Ca2⫹ frequency-dei pendent control of calcineurin in the nuclear factor of activated T cells (NFAT) pathway (27). Physiological significance of the findings The findings have important implications for cell function under physiological conditions. In particular, the finding that at high receptor expression, the CaSR is active in the presence of physiological concentrations of Cao2⫹, and, indeed, normal fasting levels of L-amino acids, indicates that up-regulated receptor expression per se can drive changes in cell function and cell fate, independent of variations in activator concentrations. This explains how the CaSR can modulate the behavior of diverse tissues in the presence of tight regulation of Cao2⫹ (1.1–1.3 mM) and in a manner that may be insensitive to changes in the normal plasma total amino acid concentration [3–5 mM (28)]. Consistent with this concept, physiological Cao2⫹ levels efficiently modulate osteoblast differentiation and function (29). In tissues sensitive to modulation by upregulated CaSR expression, cell signaling responses would appear to be under the control of hormones and/or cytokines that modulate CaSR expression. Thus, the up-regulated CaSR, by responding to local nutrients present at near-constant physiological levels, may participate in a multireceptor signaling apparatus in which it couples activated hormone and cytokine receptors to downstream receptor targets, including the epithelial growth factor receptor, which is transactivated by the CaSR (30). Distinct from receptor expression-dependent modulation of function, it also seems likely that the high levels of CaSR expression reported in the parathyroid and renal cortical thick ascending limb (see the review in reference 2) support the responses to small deviations in Cao2⫹, both within and just outside the physiological range (1–1.5 mM) and that positive modulation by L-amino acids enhances the Cao2⫹ potency of Ca2⫹ mobilization, even at i fasting concentrations, by recruiting additional responding cells. In the gastrointestinal tract, in which the CaSR couples L-amino acid signals to the release of gut hormones (31–33), it seems plausible that receptor expression is adjusted to the Cao2⫹ and L-amino acid levels arising in the luminal fluid during the digestion of a protein-rich meal. It is notable, for example, that CaSR expression is lower


doi: 10.1210/en.2014-1771

in the luminal membrane than in the basolateral membrane of gastrin-secreting enteroendocrine G cells (34). Thus, the level of CaSR expression in the lumen-facing apical membrane may be calibrated downward to permit the detection of the higher levels of nutrient activators present in the gastric juice and intestinal fluid. Impact of receptor expression on the effects of CaSR modulators on single cell Ca2ⴙ responses i Both the natural modulator L-Phe and synthetic modulator cinacalcet lowered the Cao2⫹ threshold for cellular responses and markedly enhanced the proportions of responding cells at submaximal Cao2⫹ at both low and high expression (Figures 5 and 7). If these effects operate in vivo, the recruitment of quiescent cells into the active cell population (ie, cells responding to the local Cao2⫹ concentration) could have a major impact on endocrine tissues, such as the parathyroid and thyroid C cell, involved in hormone production and release. 2⫹ L-Phe and cinacalcet increased the frequencies of Cai 2⫹ oscillations at submaximal Cao (including 2.5 mM) and promoted the transformation of the Ca2⫹ responses from i oscillations to stable elevations. Similar results have been reported previously for L-Phe and phenylalkylamine calcimimetics [(35, 36); see review in reference 37]. The effects were observed at both low and high receptor expression but were more marked at high expression. In addition, whereas a maximally effective concentration of 2⫹ L-Phe increased the Cai oscillation frequency from the lowest frequencies observed, 0.5–1 min⫺1 to 2–3 min⫺1, a maximally effective concentration of cinacalcet increased Ca2⫹ frequency to 4 –5 min⫺1 (Figure 7 and Table 1). i The results indicate that compared with high Cao2⫹ or cinacalcet, L-Phe induces a characteristic intermediate- or submaximal-level Ca2⫹ oscillation frequency (Table 1), as i described previously (36, 38), as well as a submaximal Ca2⫹ plateau level (Figure 6). Intermediate Ca2⫹ frequeni i cies may be sufficient to control certain molecular responses, including the closure of plasma membrane K⫹ channels to induce depolarization of enteroendocrine cells (33) but may limit the ability of CaSR-binding L-amino acids to activate other signaling pathways (see review in reference 39). The adoption of intermediate rather than high Ca2⫹ oscillation frequencies upon exposure to high i 2⫹ L-Phe may also limit the magnitude of the Cai plateau observed via partial rather than full activation of the protein phosphatase that reverses T888 phosphorylation. Higher frequency Ca2⫹ oscillations appear to be rei quired for some CaSR-dependent signaling events. In human colonic epithelial cells, for example, CaSR-mediated high-frequency (3.5 min⫺1) but not low-frequency (1.5 min⫺1) Ca2⫹ oscillations, support the inhibition of cell i


1339

proliferation (40). Thus, the failure of L-Phe to increase Ca2⫹ frequency above 2.5 min⫺1, in the presence of a i supraphysiological Cao2⫹ level of 2.5 mM (Figure 7), and at high receptor expression, may limit its ability to drive signaling responses linked to changes in gene expression and proliferation. Receptor expression had differential effects on modulator-induced responses. In the case of L-Phe, high receptor expression induced a modest increase in Ca2⫹ oscillation i frequency that was apparently independent of concentration in the range 0.1–10 mM, encompassing the physiological range for total amino acids (3–5 mM). In the case of cinacalcet, on the other hand, high receptor expression induced a robust increase in Ca2⫹ oscillation frequency i with clearly enhanced activator sensitivity. Thus, receptor expression conferred enhanced sensitivity on both modulators, but the effect was greater, and more clearly concentration dependent, in the case of cinacalcet. The markedly enhanced sensitivity to the amino acid L-Phe at high levels of receptor expression suggests that even fasting plasma amino acid levels may maximally activate amino acid-sensitive signaling pathways in cell types such as the parathyroid and renal cortical thick ascending limb, which express the CaSR at the highest levels. Rey et al (38) have reported the existence of an amino acid-specific CaSR-mediated signaling pathway dependent upon G12, filamin, and Rho that may explain the differential sensitivities observed in the present study. As described herein for the L-Phe activated CaSR in HEK-293 cells, the frequency of Ca2⫹ oscillations in i metabotropic glutamate receptor type-5 (mGlu-5)-expressing Chinese hamster ovary (CHO) cells was insensitive to changes in the concentrations of either L-Glu or quisqualate but was highly sensitive to changes in receptor expression (25). It is possible that the response to enhanced receptor expression in mGlu-5-expressing CHO cells was dependent upon an unrecognized nutrient activator in the medium, eg, Cao2⫹, which, in addition to the CaSR, activates several other class C GPCRs including both type I metabotropic glutamate receptors mGlu-1 and mGlu-5 (41, 42). Thus, the impact of L-Glu, and the proposed impact of Cao2⫹, on cellular Ca2⫹ responses in i mGlu-5-expressing-CHO cells, might be analogous, respectively, to the effects of L-Phe and Cao2⫹ in CaSR-expressing HEK-293 cells. Clinical significance Changes in CaSR expression affect various pathophysiological processes. Thus, reduced CaSR expression is a feature of primary hyperparathyroidism, in which there is impaired Cao2⫹-dependent feedback control of PTH secretion (12), and in vivo assessment of Cao2⫹-regulated PTH


1340

Brennan et al


secretion correlates with CaSR expression levels in excised parathyroid tissue (43). Loss of receptor surface expression provides an explanation for the reductions in both Cao2⫹ and L-amino acid potency in adenomatous human parathyroid cells (12, 44, 45). In addition, down-regulated CaSR expression is associated with calcification in vascular smooth muscle cells (14) and with more aggressive, less differentiated forms of colon cancer (for review see reference 46). How impairments in CaSR signaling promote these outcomes is unclear. It seems likely, however, that reduced Cao2⫹ sensitivity arising from reduced surface expression plays an important role. Increased CaSR expression, on the other hand, has been observed in hippocampal neurons following forebrain ischemia acting to promote cell death (47) and in breast cancer cells that have metastasized to bone, whereby bone resorption promotes the local release of Cao2⫹ to stimulate local cell growth (48). Higher levels of CaSR expression may be required to support the higher Ca2⫹ oscillation frequeni cies required to drive these proliferative responses. The findings also have implications for new and existing applications of calcimimetics. In addition to acutely modulating function, cinacalcet promotes CaSR mRNA, protein and surface membrane expression (49 –51). Although the mechanisms that underlie these effects are not well understood, its effect on surface expression arises, in part, via pharmacochaperoning (51–53) and, in part, via a phenomenon known as agonist-directed insertional signaling (54). As demonstrated in the present study, increases in total and surface expression mediated by cinacalcet or other pharmacochaperones should lower the Cao2⫹ threshold, increase the number of responding cells, and increase the frequency of Ca2⫹ oscillations. Thus, i clinical studies demonstrating that cinacalcet is effective in normalizing plasma Ca2⫹ and PTH levels in patients with primary hyperparathyroidism (55, 56) might arise from up-regulated receptor expression as well as enhanced Cao2⫹ sensitivity at the cell surface. Conclusions Tetracycline-induced increases in CaSR expression in T-REx293-CaSR cells positively modulated CaSR-mediated Ca2⫹ mobilization in response to elevated Cao2⫹ and i the positive allosteric modulators, L-Phe and cinacalcet. Maximum levels of receptor expression lowered the threshold concentrations for Cao2⫹ and L-amino acid-induced Ca2⫹ responses to lie below their physiological levi els in plasma. Up-regulated receptor expression also positively modulated the proportion of responding cells, increased the frequency of CaSR-mediated Ca2⫹ oscillai 2⫹ tions, and lowered the Cao threshold for the transformation from Ca2⫹ oscillations to stable Ca2⫹ elevations. i i


Up-regulated expression of the CaSR per se may initiate CaSR-mediated signaling in the presence of physiological levels of its nutrient activators.

Acknowledgments S.C.B. thanks the European Union Marie Curie International Training Network for a postdoctoral fellowship. We thank Dr David Szekely for providing expert assistance with the wavelet analysis. Address all correspondence and requests for reprints to: Professor Arthur D. Conigrave, School of Molecular Bioscience, Charles Perkins Centre (D17), University of Sydney, NSW 2006, Australia. E-mail: [email protected]. Present address for S.C.B.: Cardiff School of Biosciences, Biomedical Sciences Building, Museum Avenue, Cardiff CF10 3AX, United Kingdom. A.C. and A.D.C. were supported by the National Health and Medical Research Council of Australia for project grant support, and P.W.K. was supported by a Discovery Project grant from the Australian Research Council. Disclosure Summary: The authors have nothing to disclose.

References 1. Riccardi D, Kemp PJ. The calcium-sensing receptor beyond extracellular calcium homeostasis: conception, development, adult physiology, and disease. Annu Rev Physiol. 2012;74:271–297. 2. Brown EM, MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev. 2001;81(1):239 –297. 3. Chang W, Tu C, Chen TH, Bikle D, Shoback D. The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development. Sci Signal. 2008;1(35):ra1. 4. Conigrave AD, Quinn SJ, Brown EM. L-amino acid sensing by the extracellular Ca2⫹-sensing receptor. Proc Natl Acad Sci USA. 2000; 97(9):4814 – 4819. 5. Wang M, Yao Y, Kuang D, Hampson DR. Activation of family C G-protein-coupled receptors by the tripeptide glutathione. J Biol Chem. 2006;281(13):8864 – 8870. 6. Ohsu T, Amino Y, Nagasaki H, et al. Involvement of the calciumsensing receptor in human taste perception. J Biol Chem. 2010; 285(2):1016 –1022. 7. Broadhead GK, Mun HC, Avlani VA, et al. Allosteric modulation of the calcium-sensing receptor by ␥-glutamyl peptides: inhibition of PTH secretion, suppression of intracellular cAMP levels, and a common mechanism of action with L-amino acids. J Biol Chem. 2011; 286(11):8786 – 8797. 8. Nemeth EF, Steffey ME, Hammerland LG, et al. Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA. 1998;95(7):4040 – 4045. 9. Nemeth EF, Heaton WH, Miller M, et al. Pharmacodynamics of the type II calcimimetic compound cinacalcet HCl. J Pharmacol Exp Therap. 2004;308(2):627– 635. 10. Mun HC, Franks AH, Culverston EL, Krapcho K, Nemeth EF, Conigrave AD. The Venus Fly Trap domain of the extracellular Ca2⫹sensing receptor is required for L-amino acid sensing. J Biol Chem. 2004;279(50):51739 –51744. 11. Petrel C, Kessler A, Dauban P, Dodd RH, Rognan D, Ruat M. Pos-


doi: 10.1210/en.2014-1771

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

itive and negative allosteric modulators of the Ca2⫹-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain. J Biol Chem. 2004;279(18):18990 – 18997. Kifor O, Moore FD Jr, Wang P, et al. Reduced immunostaining for the extracellular Ca2⫹-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab. 1996;81(4): 1598 –1606. Oda Y, Tu CL, Pillai S, Bikle DD. The calcium sensing receptor and its alternatively spliced form in keratinocyte differentiation. J Biol Chem. 1998;273(36):23344 –23352. Alam MU, Kirton JP, Wilkinson FL, et al. Calcification is associated with loss of functional calcium-sensing receptor in vascular smooth muscle cells. Cardiovasc Res. 2009;81(2):260 –268. Young SH, Wu SV, Rozengurt E. Ca2⫹-stimulated Ca2⫹ oscillations produced by the Ca2⫹-sensing receptor require negative feedback by protein kinase C. J Biol Chem. 2002;277(49):46871– 46876. Davies SL, Ozawa A, McCormick WD, Dvorak MM, Ward DT. Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity. J Biol Chem. 2007;282(20): 15048 –15056. McCormick WD, Atkinson-Dell R, Campion KL, Mun HC, Conigrave AD, Ward DT. Increased receptor stimulation elicits differential calcium-sensing receptor(T888) dephosphorylation. J Biol Chem. 2010;285(19):14170 –14177. Thiel OR, Bernard C, Tormos W, et al. Practical synthesis of the calcimimetic agent, cinacalcet. Tetrahedron Lett. 2008;49(1): 13–15. Davey AE, Leach K, Valant C, Conigrave AD, Sexton PM, Christopoulos A. Positive and negative allosteric modulators promote biased signaling at the calcium-sensing receptor. Endocrinology. 2012;153(3):1232–1241. Szekely D, Brennan SC, Mun HC, Conigrave AD, Kuchel PW. Effectors of the frequency of calcium oscillations in HEK-293 cells: wavelet analysis and a computer model. Eur Biophys J. 2009;39(1): 149 –165. Bai M, Quinn S, Trivedi S, et al. Expression and characterization of inactivating and activating mutations in the human Ca2⫹o-sensing receptor. J Biol Chem. 1996;271(32):19537–19545. Ray K, Clapp P, Goldsmith PK, Spiegel AM. Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction. J Biol Chem. 1998;273(51):34558 –34567. Bai M, Janicic N, Trivedi S, et al. Markedly reduced activity of mutant calcium-sensing receptor with an inserted Alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. J Clin Invest. 1997;99(8):1917–1925. Conigrave AD, Mun HC, Delbridge L, Quinn SJ, Wilkinson M, Brown EM. L-amino acids regulate parathyroid hormone secretion. J Biol Chem. 2004;279(37):38151–38159. Nash MS, Schell MJ, Atkinson PJ, Johnston NR, Nahorski SR, Challiss RA. Determinants of metabotropic glutamate receptor-5-mediated Ca2⫹ and inositol 1,4,5-trisphosphate oscillation frequency. Receptor density versus agonist concentration. J Biol Chem. 2002; 277(39):35947–35960. Irannejad R, Tomshine J, Tomshine J, et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature. 2013; 495(7442):534 –548. Colella M, Grisan F, Robert V, Turner J, Thomas A, Pozzan T. Ca2⫹ oscillation frequency decoding in cardiac cell hypertrophy: role of calcineurin/NFAT as Ca2⫹ signal integrators. Proc Natl Acad Sci USA. 2008;105(8):2859 –2864. Conigrave AD, Hampson DR. Broad-spectrum amino acid sensing by class 3 G-protein coupled receptors. Trends Endocrinol Metab. 2006;17(10):398 – 407. Dvorak M, Siddiqua A, Ward D, et al. Physiological changes in


30.

31.

32.

33.

34.

35.

36.

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

1341

extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones. Proc Natl Acad Sci USA. 2004;101(14):5140 –5145. MacLeod RJ, Yano S, Chattopadhyay N, Brown EM. Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism. Biochem Biophys Res Commun. 2004;320(2):455– 460. Feng J, Petersen CD, Coy DH, et al. Calcium-sensing receptor is a physiologic multimodal chemosensor regulating gastric G-cell growth and gastrin secretion. Proc Natl Acad Sci USA. 2010; 107(41):17791–17796. Liou AP, Sei Y, Zhao X, et al. The extracellular calcium sensing receptor is required for cholecystokinin secretion in response to Lphenylalanine in acutely isolated intestinal I cells. Am J Physiol Gastrointest Liver Physiol. 2011;300(4):G538 –G546. Wang Y, Chandra R, Samsa LA, et al. Amino acids stimulate cholecystokinin release through the calcium-sensing receptor. Am J Physiol Gastrointest Liver Physiol. 2011;300(4):G528 –G537. Ray J, Squires P, Curtis S, Meloche M, Buchan A. Expression of the calcium-sensing receptor on human antral gastrin cells in culture. J Clin Invest. 1997;99(10):2328 –2333. Miedlich S, Gama L, Breitwieser GE. Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations. J Biol Chem. 2002;277(51): 49691– 49699. Young SH, Rozengurt E. Amino acids and Ca2⫹ stimulate different patterns of Ca2⫹ oscillations through the Ca2⫹-sensing receptor. Am J Physiol Cell Physiol. 2002;282(6):C1414 –C1422. Breitwieser GE. Calcium sensing receptors and calcium oscillations: calcium as a first messenger. Curr Top Dev Biol. 2006;73:85–114. Rey O, Young SH, Yuan J, Slice L, Rozengurt E. Amino acid-stimulated Ca2⫹ oscillations produced by the Ca2⫹-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton. J Biol Chem. 2005;280(24):22875–22882. Conigrave A, Ward D. Calcium-sensing receptor (CaSR): pharmacological properties and signaling pathways. Best Pract Res Clin Endocrinol Metab. 2013;27(3):315–331. Rey O, Young SH, Jacamo R, Moyer MP, Rozengurt E. Extracellular calcium sensing receptor stimulation in human colonic epithelial cells induces intracellular calcium oscillations and proliferation inhibition. J Cell Physiol. 2010;225(1):73– 83. Soriano FX, Papadia S, Hofmann F, Hardingham NR, Bading H, Hardingham GE. Preconditioning doses of NMDA promote neuroprotection by enhancing neuronal excitability. J Neurosci. 2006; 26(17):4509 – 4518. Jiang Y, Huang Y, Wong HC, et al. Elucidation of a novel extracellular calcium-binding site on metabotropic glutamate receptor 1␣ (mGluR1␣) that controls receptor activation. J Biol Chem. 2010; 285(43):33463–33474. Cetani F, Picone A, Cerrai P, et al. Parathyroid expression of calcium-sensing receptor protein and in vivo parathyroid hormoneCa(2⫹) set-point in patients with primary hyperparathyroidism. J Clin Endocrinol Metab. 2000;85(12):4789 – 4794. Farnebo F, Enberg U, Grimelius L, et al. Tumor-specific decreased expression of calcium sensing receptor messenger ribonucleic acid in sporadic primary hyperparathyroidism. J Clin Endocrinol Metab. 1997;82(10):3481–3486. Mun HC, Brennan SC, Delbridge L, Wilkinson M, Brown EM, Conigrave AD. Adenomatous human parathyroid cells exhibit impaired sensitivity to L-amino acids. J Clin Endocrinol Metab. 2009; 94(9):3567–3574. Brennan SC, Thiem U, Roth S, et al. Calcium sensing receptor signalling in physiology and cancer. Biochim Biophys Acta. 2013; 1833(7):1732–1744. Kim JY, Kim N, Yenari MA, Chang W. Mild hypothermia suppresses calcium-sensing receptor (CaSR) Induction following fore-


1342

48.

49.

50.

51.

52.

Brennan et al


brain ischemia while increasing GABA-B Receptor 1 (GABA-B-R1) expression. Transl Stroke Res. 2011;2(2):195–201. Saidak Z, Boudot C, Abdoune R, et al. Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor. Exp Cell Res. 2009;315(12):2072– 2080. Mizobuchi M, Hatamura I, Ogata H, et al. Calcimimetic compound upregulates decreased calcium-sensing receptor expression level in parathyroid glands of rats with chronic renal insufficiency. J Am Soc Nephrol. 2004;15(10):2579 –2587. Mendoza FJ, Lopez I, Canalejo R, et al. Direct upregulation of parathyroid calcium-sensing receptor and vitamin D receptor by calcimimetics in uremic rats. Am J Physiol Renal Physiol. 2009;296(3): F605–F613. White E, McKenna J, Cavanaugh A, Breitwieser GE. Pharmacochaperone-mediated rescue of calcium-sensing receptor loss-offunction mutants. Mol Endocrinol. 2009;23(7):1115–1123. Cavanaugh A, McKenna J, Stepanchick A, Breitwieser GE. Calcium-sensing receptor biosynthesis includes a cotranslational con-

53.

54.

55.

56.


formational checkpoint and endoplasmic reticulum retention. J Biol Chem. 2010;285(26):19854 –19864. Leach K, Wen A, Cook A, Sexton P, Conigrave A, Christopoulos A. Impact of clinically relevant mutations on the pharmacoregulation and signaling bias of the calcium-sensing receptor by positive and negative allosteric modulators. Endocrinology. 2013;154(3):1105– 1116. Grant MP, Stepanchick A, Cavanaugh A, Breitwieser GE. Agonistdriven maturation and plasma membrane insertion of calcium-sensing receptors dynamically control signal amplitude. Sci Signal. 2011; 4(200):ra78. Shoback DM, Bilezikian JP, Turner SA, McCary LC, Guo MD, Peacock M. The calcimimetic cinacalcet normalizes serum calcium in subjects with primary hyperparathyroidism. J Clin Endocrinol Metab. 2003;88(12):5644 –5649. Peacock M, Bilezikian JP, Klassen PS, Guo MD, Turner SA, Shoback D. Cinacalcet hydrochloride maintains long-term normocalcemia in patients with primary hyperparathyroidism. J Clin Endocrinol Metab. 2005;90(1):135–141.


Blockade of NMDA receptor-mediated mobilization of intracellular Ca2+ prevents neurotoxicity.

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release.

Apparent noncompetitive antagonism of muscarinic receptor mediated Ca2+ mobilization by some muscarinic antagonists.

Heterologous supersensitization between serotonin2 and alpha 2-adrenergic receptor-mediated intracellular calcium mobilization in human platelets.

cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2+ mobilization.

Transient receptor potential-canonical 3 modulates sperm motility and capacitation-associated protein tyrosine phosphorylation via [Ca2+]i mobilization.

Differential regulation of thrombin- or ATP-induced mobilization of intracellular Ca2+ by prostacyclin receptor in mouse mastocytoma cells.

Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888.

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+.

Cholesterol modulates PAF-stimulated Ca2(+)-mobilization in monocytic U937 cells.

Intracellular aluminium inhibits acetylcholine- and caffeine-evoked Ca2+ mobilization.

Receptor-activated cytoplasmic Ca2+ spiking mediated by inositol trisphosphate is due to Ca2(+)-induced Ca2+ release.

3-iodothyronamine differentially modulates α-2A-adrenergic receptor-mediated signaling.

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Possible regulation of caffeine-induced intracellular Ca2+ mobilization by intracellular free Na+.

Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery.

The PICK1 Ca2+ sensor modulates N-methyl-d-aspartate (NMDA) receptor-dependent microRNA-mediated translational repression in neurons.

Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells.

Tetronothiodin, a novel CCKB receptor ligand, antagonizes cholecystokinin-induced Ca2+ mobilization in a pituitary cell line.

Glutamate-induced increases in intracellular Ca2+ in cultured frog tectal cells mediated by direct activation of NMDA receptor channels.

Muscarinic-receptor-mediated changes in intracellular Ca2+ and inositol 1,4,5-trisphosphate mass in a human neuroblastoma cell line, SH-SY5Y.

Epidermal growth factor selectively enhances NMDA receptor-mediated increase of intracellular Ca2+ concentration in rat hippocampal neurons.

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

Hemodialysis with cuprophane membrane modulates interleukin-2 receptor expression.