Intracellular Ca²⁺ oscillations generated via the extracellular Ca²⁺-sensing receptor (CaSR) in response to extracellular Ca²⁺ or L-phenylalanine: impact of the highly conservative mutation Ser170Thr

Abstract

The extracellular Ca²⁺-sensing receptor (CaSR) is an allosteric protein that responds to changes in the extracellular concentration of Ca²⁺ ([Ca²⁺]_e) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca²⁺ concentration ([Ca²⁺]_i). An increase in [Ca²⁺]_e stimulates sinusoidal oscillations in [Ca²⁺]_i whereas aromatic amino acid-induced CaR activation in the presence of a threshold [Ca²⁺]_e promotes transient oscillations in [Ca²⁺]_i. Here, we examined spontaneous and ligand-evoked [Ca²⁺]_i oscillations in single HEK-293 cells transfected with the wild type CaSR or with a mutant CaSR in which Ser170 was converted to Thr (CaSR_S170T). Our analysis demonstrates that cells expressing CaSR_S170T display [Ca²⁺]_i oscillations in the presence of low concentrations of extracellular Ca²⁺ and respond to L-Phe with robust transient [Ca²⁺]_i oscillations. Our results indicate that the S170T mutation induces a marked increase in CaSR sensitivity to [Ca²⁺]_e and imply that the allosteric regulation of the CaSR by aromatic amino acids is not only mediated by an heterotropic positive effect on Ca²⁺ binding cooperativity but, as biased agonists, aromatic amino acids stabilize a CaSR conformation that couples to a different signaling pathway leading to transient [Ca²⁺]_i oscillations.

1. Introduction

The extracellular Ca²⁺-sensing receptor (CaSR), a member of the C family of heptahelical G protein-coupled receptors (GPCRs), is an allosteric protein that plays a major physiological role in correcting small changes in extracellular concentration of Ca²⁺ ([Ca²⁺]_e) by inhibiting parathyroid hormone secretion and renal Ca²⁺ absorption [1]. Interestingly, subsequent studies demonstrated that the CaSR recognizes other ligands and is expressed in many other tissues and organs, including the gastrointestinal tract, brain, pituitary, thyroid, skin, breast, pancreas, lung, bone, and heart [2], suggesting that this receptor plays additional, yet less well defined, physiological roles in the regulation of normal and abnormal cell function [3,4,5,6]. Indeed, recent evidence indicates that the CaSR is implicated in the negative control of colon cell proliferation [7] and cancer [8], nutrient sensing [4], epithelial transport [9], inflammation [10], bone turnover [11] and stem cell differentiation [6]. Thus, the signaling mechanisms triggered via CaSR activation are attracting intense attention.

A number of studies of CaSR activation in individual living cells have shown that intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillates upon stimulation of CaSR by an elevation in [Ca²⁺]_e within a physiological range [12,13,14,15,16,17,18]. Oscillatory changes in [Ca²⁺]_i in response to receptor stimulation is a fundamental mechanism of cell signaling implicated in the regulation of Ca²⁺- and calmodulin-dependent protein kinase II [19], conventional protein kinase C (PKC) isoforms [15,20], mitochondrial function [21,22], and nuclear transcriptional activity leading to differential gene expression [23,24]. We proposed that [Ca²⁺]_i oscillations induced by activation of the CaSR in response to an increase in extracellular Ca²⁺ results from negative feedback involving PKC-mediated phosphorylation of the CaSR at the inhibitory residue Thr⁸⁸⁸ [14,25]. In addition to its role as sensor of [Ca²⁺]_e, the CaSR is also stimulated by aromatic amino acids [26] which, like [Ca²⁺]_e, induce striking and lasting CaSR-mediated [Ca²⁺]_i oscillations [13,15,16]. However, the patterns of [Ca²⁺]_i oscillations induced by these agonists are different. Aromatic amino acid stimulation of the CaSR in the presence of a threshold [Ca²⁺]_e induces repetitive, low frequency [Ca²⁺]_i spikes that return to the base-line level, a pattern known as transient oscillations [13,15,16]. The amplitude, frequency, and duration of [Ca²⁺]_i oscillations are increasingly recognized to encode important information for a variety of biological processes, including metabolism and gene expression.

The most striking structural feature of the family C GPCR is its large extracellular domain, which consists of ~600 amino acid residues arranged in two predicted lobes referred as the Venus Flytrap Domain (VFTD), the site of numerous disease-causing mutations in this receptor [27,28]. The VFTD contains multiple binding sites for extracellular Ca²⁺ [29] and the allosteric binding site for amino acids. The residues Ser-147, Ser-170, Asp-190, Tyr-218 and Glu-297 of the VFTD comprise the first Ca²⁺ binding site which is considered the most important and operational under physiological conditions [27,30]. A number of studies identified Ser-170 as playing a critical role in Ca²⁺ binding and signal generation since mutation of Ser-170 to Ala completely abolished the response of the mutant CaSR to even very high [Ca²⁺]_e [31] without affecting its membrane expression [30,31]. In contrast, a conservative mutation of Ser-170 to Thr (S170T) appeared to have very little effect on CaSR responsiveness to an increase in [Ca²⁺]_e but impaired L-phenylalanine (L-Phe) sensing [29,31] though the ability of this CaSR mutant to mediate transient oscillations in single cells was not examined. The detailed study of the impact of mutations in the N-terminal VFTD is of importance to advance understanding of the molecular mechanisms of CaSR regulation.

Here, we examined spontaneous and ligand-evoked [Ca²⁺]_i oscillations in single HEK-293 cells transfected with the wild type CaSR or with CaSR in which Ser170 was mutated to Thr (CaSR_S170T). Our analysis demonstrates that cells expressing CaSR_S170T display [Ca²⁺]_i oscillations in the presence of low concentrations of extracellular Ca²⁺ and respond to L-Phe with robust transient [Ca²⁺]_i oscillations. We propose that the S170T mutation increases CaSR sensitivity to [Ca²⁺]_e and separates the positive heterotropic effect that enhances the apparent affinity of the CaSR for extracellular Ca²⁺ from the stabilization of a receptor conformation that promotes transient oscillations in the presence of a threshold [Ca²⁺]_e.

2. Materials and Methods

2.1 Materials

Fura 2-AM, Dulbecco’s Modified Eagle’s Medium (DMEM), and Hanks Balanced Salt Solution (HBSS) were obtained from Invitrogen (Carlsbad, CA).

2.2 Cell culture and transfection

Human Embryonic Kidney (HEK-293) cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in a humidified incubator under 10% CO₂ and 90% air at 37°C, as described previously [13,25,32]. For experimental purposes, cells were plated onto 18-mm diameter glass coverslips inside 35-mm plastic dishes, where they could be dually transiently transfected with a plasmid encoding the human CaSR or a mutant CaSR receptor (CaSR_S170T). Site-directed mutagenesis to convert Ser170 of the CaSR into Thr was performed as previously described [33]. Identification of cells transiently transfected with pCR3.1-CaSRwt (0.5 µg/dish) or pCR3.1-CaSR_S170T (0.5 µg/ dish) was achieved by co-transfection with pDsRed-Express (BD Biosciences) (0.5 µg/dish), a vector that encodes a red fluorescent protein. After 16 h, the cultures were loaded with the Ca²⁺ indicator Fura-2 as described below.

2.3 Measurement of [Ca²⁺]_i

[Ca²⁺]_i was measured in single cells loaded with the calcium indicator fura-2 as previously described [13]. Briefly, cells were incubated in saline solution containing 138 mM NaCl, 4 mM NaHCO₃, 0.3 mM Na₂HPO₄, 5 mM KCl, 0.3 mM KH₂PO₄, 1.5 mM CaCl₂, 0.5 mM MgCl₂, 0.4 mM MgSO₄, 5.6 mM D-glucose, 20 mM HEPES, pH: 7.4 which was supplemented with 5 µM fura-2 AM for 45–60 min at 37°C before [Ca²⁺]_i imaging. The cells were then washed and placed in an experimental chamber that was perfused with saline solution at 1.5 ml/min at 37°C. The chamber in turn was placed on the stage of an inverted microscope connected to a digital imaging system. Ratios of images (340 nm excitation/ 380 nm excitation, emission filter 520 nm) were obtained at 1.5 sec intervals. A region of interest covering 15 µm × 15 µm was defined over each cell, and the average ratio intensity over the region was converted to [Ca²⁺]_i using an standard curve constructed with a series of calibrated buffered calcium solutions (Calcium Calibration Buffer Kit #2, Invitrogen Corp).

2.4 Western blots and indirect immunofluorescence

HEK-293 cells were transfected with vectors encoding wild type human CaSR or CaSR_S170T at 0.5 µg/ dish and analyzed by Western blot 16 h post-transfection using a murine monoclonal antibody against the CaSR (Affinity BioReagents). The images were captured using a luminescent image analyzer LAS-4000 mini (Fujifilm Life Sciences). For indirect immunofluorescence, the transfected cells were fixed 16 h post-transfection in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 7 min at 24°C to only reveal surface expression of the receptor. The fixed cells were first incubated during 2 h at 24°C in blocking buffer (PBS-1% gelatin) and then 4 h with an anti-CaSR murine monoclonal antibody (Affinity BioReagents) diluted in blocking buffer. The cells were then washed with PBS at 25°C and incubated during 2 h at 24°C with Alexa 488-conjugated rabbit-anti mouse (Invitrogen) diluted in blocking buffer. After extensive washes at 24°C with PBS, the samples mounted with a gelvatol-glycerol solution containing 2.5% 1,4-diazobicyclo-[2.2.2]octane. The samples were examined with a epifluorescence microscope (Zeiss Axioskop) as previously described [33]. The selected cells displayed in the appropriate figures were representative of 80% of the population of positive cells.

4. Results

In order to examine the impact of mutations in the 1^st Ca²⁺ binding site of the VFTD, we analyzed the pattern of [Ca²⁺]_i response in individual HEK-293 cells transiently co-transfected with a plasmid encoding the human wild type CaSR or with a CaSR containing a highly conservative substitution in Ser-170 to Thr (CaSR_S170T) and a plasmid encoding a red fluorescent protein (pDsRed-Express) to facilitate the identification of the transfected cells. After 16 h, the cells were loaded with the fluorescent Ca²⁺ indicator fura 2-AM and incubated in the presence of medium containing 1.5 mM [Ca²⁺]_e. Single cell imaging of fura-2 loaded HEK-293 cells expressing the CaSR or CaSR_S170T revealed that a rise in the [Ca²⁺]_e from 1.5 mM to 5 mM produced a rapid elevation in [Ca²⁺]_i followed by a sustained plateau in cells transfected with either the wild type CaSR (Fig. 1 A) or mutant CaSR_S170T (Fig. 1 B, C). The maximal response of CaSR_S170T to the increase in [Ca²⁺]_e was similar to that of the wild type receptor (Fig. 1 A, B). Most transfected cells with CaSR or CaSR_S170T responded to the increase in [Ca²⁺]_e (Fig. 1 D). Accordingly, equivalent levels of CaSR and CaSR_S170T were expressed on the cell membrane of HEK-293 cells, as judged by indirect immunofluorescence (Fig. 1 F, G) and total cellular expression of CaSR and CaSR_S170T was also equal, as shown by immunoblotting (Fig. 1 H).

Figure 1.

Changes in [Ca²⁺]_i in HEK-293 cells transfected with CaSR (WT) A, or CaSR_S170T (S170T) B,C, in response to an increase in [Ca²⁺]_e from 1.5 mM to 5 mM. The addition of Ca²⁺ is indicated by upward arrows whereas the return of [Ca²⁺]_e to 1.5 mM is indicated by the downward arrows. D, Bar chart: Percentage of HEK-293 cells transfected with CaSR (WT) or CaSR_S170T which responded to an increase in [Ca²⁺]_e from 1.5 mM to 5 mM. E, F, G. Fixed cells were labeled with an anti-CaSR murine monoclonal antibody to detect surface expressed receptor. E. Control, no transfection. F. Transfected with CaSR(WT). G. Transfected with CaR_S170T. Scale Bar 30 µM. H. Western Blot analysis of control, no transfection (−), CaSR (WT), and CaSR_S170T (S170T) cell lysates.

Although the results in Fig. 1 indicated that the responses of wild type and mutant CaSR to an increase in the [Ca²⁺]_e were virtually identical, analysis of [Ca²⁺]_i prior to simulation revealed an important difference between CaSR and CaSR_S170T in the dynamics of basal [Ca²⁺]_i. In agreement with our previous results, most cells transfected with wild type CaSR and incubated in the presence of medium containing 1.5 mM [Ca²⁺]_e exhibited a stable [Ca²⁺]_i with only a small proportion of cells (~10%) exhibiting spontaneous oscillatory activity.

In striking contrast, a substantial proportion of cells transfected with CaSR_S170T display spontaneous oscillatory fluctuations in [Ca²⁺]_i. The difference in spontaneous [Ca²⁺]_i oscillations is illustrated in Fig. 2 A, showing a pseudo-colored images of clusters of cells transfected with wild type CaSR or CaSR_S170T incubated for various times in medium containing 1.5 mM [Ca²⁺]_e. Cells labeled O show a spontaneous increase in [Ca²⁺]_i which declines, and then increases again (oscillatory response) while other cells exhibited a stable [Ca²⁺]_i. Examples of tracings corresponding to HEK-293 cells transfected with CaSR_S170T displaying spontaneous [Ca²⁺]_i oscillations are illustrated in Fig. 2 B. The examples also illustrate that the oscillatory frequency is markedly higher in cells expressing CaSR_S170T. The difference in basal [Ca²⁺]_i oscillations between CaSR and CaSR_S170T was substantiated by analyzing 714 individual cells. We found a marked increase in the proportion of cells that display spontaneous [Ca²⁺]_i oscillations when transfected with CaSR_S170T (Fig. 2 C, bars). These results demonstrate, for the first time, that a sub-population of HEK-293 cells expressing CaSR_S170T (~27%) displays spontaneous [Ca²⁺]_i oscillations while the majority of cells transfected with wild type CaSR exhibited a stable [Ca²⁺]_i. In agreement with our previous results [13], we did not detect any [Ca²⁺]_i oscillations in non-transfected HEK-293 cells or in cells transfected with vector.

Figure 2.

Spontaneous [Ca²⁺]_i oscillations of HEK-293 cells transfected with wild type CaSR (WT) or CaSR_S170T (S170T) and incubated in medium containing 1.5 mM [Ca²⁺]_e. A. Pseudo-colored images of clusters of cells showing changes in [Ca²⁺]_i with time. Shown is one cycle of rest to peak response. Spontaneously oscillating cells (O) are marked with an arrow. B. Typical traces of [Ca²⁺]_i as a function of time (s) for a single HEK-293 cell transfected with the wild type CaSR or a single HEK-293 cells transfected with CaSR_S170T. C. Percentage of spontaneously oscillating HEK-293 cells from a total of 544 transfected with the wild type CaSR and from 170 cells transfected with CaSR_S170T.

The results shown in Fig. 2 prompted us to hypothesize that HEK-293 cells transfected with CaSR_S170T have increased sensitivity to extracellular Ca²⁺. To examine this possibility, we monitored basal [Ca²⁺]_i oscillations in HEK-293 cells transfected with wild type CaSR or CaSR_S170T incubated in medium containing different [Ca²⁺]_e. Cells incubated in medium containing 0.8 mM Ca²⁺ show striking differences, as shown in Fig. 3. We found that ~20% of HEK-293 cells expressing CaSR_S170T exhibit spontaneous [Ca²⁺]_i oscillations with a frequency of 3 spikes/min. In sharp contrast, only ~3% of cells transfected with wild type CaSR exhibited oscillatory activity of lower frequency (2 spikes/min) whereas 97% of transfected cells showed stable [Ca²⁺]_i. A further decrease in [Ca²⁺]_e to 0.4 mM completely eliminated any spontaneous [Ca²⁺]_i oscillations in HEK 293 cells transfected with wild type CaSR while 10% of HEK-293 cells expressing CaSR_S170T exhibited spontaneous [Ca²⁺]_i oscillations with a frequency of 2.7 spikes/min (Fig. 3). A small subpopulation of HEK 293 cells expressing CaSR_S170T show spontaneous [Ca²⁺]_i oscillations at a [Ca²⁺]_e as low as 0.2 mM. The threshold [Ca²⁺]_e required for spontaneous [Ca²⁺]_i oscillations in individual HEK-293 cells transfected with either CaSR or CaSR_S170T was 0.6mM and 0.2 mM, respectively, as extrapolated from the data in Fig. 3. These results support the hypothesis that a highly conserved mutation of the CaSR at Ser170 increases the sensitivity of the CaSR for extracellular Ca²⁺.

Figure 3.

Percentage of spontaneously oscillating HEK-293 cells transfected with wild type CaSR (WT) or CaSR_S170T (S170T), (left panels) and their oscillating frequencies (right panels) as a function of varying [Ca²⁺]_e. The data is a summary derived from a total of 1,289 HEK-293 cells transfected with wild type CaSR and 1,386 HEK-293 cells transfected with CaSR_S170T. Numbers show number of cells measured at each [Ca²⁺]_e.

In addition to its role as sensor of [Ca²⁺]_e, the CaSR is also stimulated by aromatic amino acids [26] that induce striking and lasting transient [Ca²⁺]_i oscillations characterized by low frequency [Ca²⁺]_i spikes that return to the base-line level [13]. These oscillations are mediated by a phospholipase C-independent pathway that involves Rho, filamin and TRPC1 [13,15,16,18]. In contrast, CaSR activation in response to modest increase in [Ca²⁺]_e (e.g. from 1.5 to 3 mM) produces high frequency sinusoidal oscillations upon a raised plateau level of [Ca²⁺]_i [13,15,16]. Consequently, we next determined whether CaSR_S170T mediates different oscillatory patterns, i.e. sinusoidal and transient oscillations in response to different agonists in cells initially incubated in low [Ca²⁺]_e. To examine this possibility, HEK-293 cells transfected with CaSR_S170T were incubated in medium containing 0.8 mM [Ca²⁺]_e to diminish spontaneous [Ca²⁺]_i oscillations and then stimulated by an increase of [Ca²⁺]_e to 3 mM or by addition of 5 mM L-Phe. As shown in Fig. 4, HEK-293 cells expressing CaSR_S170T displayed sinusoidal oscillations in response to an increase in [Ca²⁺]_e to 3 mM and striking transient oscillations in response to 5 mM L-Phe in the presence of 0.8 mM [Ca²⁺]_e (Fig. 4). The responses were comparable to those showed by HEK-293 cells expressing wild type CaSR but incubated initially in medium containing 1.5 mM [Ca²⁺]_e and thus substantiating that the Ser170 to Thr mutation decreases the [Ca²⁺]_e required for detecting transient oscillations in response to aromatic amino acids.

Figure 4.

Oscillatory responses in [Ca²⁺]_i in HEK-293 expressing CaSR_S170T (S170T) cells when challenged with increases in [Ca²⁺]_e or L-phenylalanine (L-Phe). The cells were initially incubated in medium containing [Ca²⁺]_e at 0.8 mM. After recording basal [Ca²⁺]_i the cells were challenged by either increasing the [Ca²⁺]_e to 3mM or 5 mM L-Phe (upward arrows). Downward arrow marks return to medium containing [Ca²⁺]_e at 1.5 mM.

5. Discussion

Oscillatory changes in [Ca²⁺]_i in response to receptor stimulation is a fundamental mechanism of cell signaling implicated in the regulation of signal transduction [15,19,20], metabolism [21,22], and differential gene expression [23,24]. Previous studies identified Ser-170 in the N-terminal region of the VFTD as a critical residue in Ca²⁺ binding and signal generation since mutation of Ser-170 to Ala completely abolished the response to even very high concentrations of extracellular Ca2+ [31] without affecting its membrane expression [30,31]. In the current study we analyzed [Ca2+]_i in single HEK-293 cells expressing wild type CaSR or a receptor with a highly conservative mutation of Ser-170 to Thr. Our results demonstrated that cells expressing CaSRS170T exhibit spontaneous oscillatory activity when incubated in medium containing 1.5 mM [Ca2+]_e. Analysis of spontaneous [Ca²⁺]_i oscillations as a function of [Ca²⁺]_e revealed that HEK-293 cells expressing CaSR_S170T display [Ca²⁺]_i oscillations of higher frequency at markedly lower [Ca²⁺]_e as compared with HEK-293 cells expressing wild type CaSR. Furthermore, HEK-293 cells expressing CaSR_S170T display sinusoidal [Ca²⁺]_i oscillations in response to an increase in [Ca²⁺]_e or transient oscillations when challenged by L-Phe, provided that cells are initially incubated in medium containing a low [Ca²⁺]_e. We conclude that a small increase in the size of the side chain of Ser170 (Ser-to-Thr substitution in the VTFD) is sufficient to induce a marked increase in sensitivity to [Ca²⁺]_e of the CaSR.

Previous studies concluded that mutation of Ser-170 to Thr impairs amino acid sensing [30,31], as judged by experiments in which the EC₅₀ value for [Ca²⁺]_e remains unchanged in the presence of L-Phe. In contrast, we demonstrated that L-Phe induced transient oscillations in cells expressing CaSR_S170T incubated in medium containing a low [Ca²⁺]_e. We propose that the S170T mutation separates two different allosteric effects of aromatic amino acids on the CaSR. Specifically, (1) the stabilization of a conformation that promotes transient oscillations in the presence of a threshold level of [Ca²⁺]_e via a multi-protein complex that includes Rho, filamin-A, and TRPC1 from (2) the positive heterotropic effect that causes an increase in the affinity of the CaSR for extracellular Ca²⁺. We obtained similar results using a different CaSR mutant in which Glu127 was replaced by Ala, an activating mutation. As with CaSR_S170T, cells expressing the CaSR_E127A mutation displayed [Ca²⁺]_i transient oscillations in response to L-Phe. We conclude that the allosteric regulation of the CaSR by aromatic amino acids is not only mediated by an heterotropic positive effect on Ca²⁺ binding cooperativity but, as a biased agonist, aromatic amino acids stabilize a CaSR conformation that couples to a different signaling pathway leading to transient [Ca²⁺]_i oscillations.

HIGHLIGHTS.

• [Ca²⁺]_i oscillations were analyzed in HEK-293 cells expressing CaSR and CaSR_S170T

• CaSR_S170T mediated basal [Ca²⁺]_i oscillations even in medium with low [Ca²⁺]_e

• CaSR_S170T produced transient [Ca²⁺]_i oscillations in response to L-phenylalanine.

• CaSR_S170T separates enhanced sensitivity to [Ca²⁺]_e from transient oscillations