Elucidation of a Novel Extracellular Calcium-binding Site on Metabotropic Glutamate Receptor 1α (mGluR1α) That Controls Receptor Activation

Abstract

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca²⁺ ([Ca²⁺]_o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca²⁺-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca²⁺-binding site in the mGluR1α ECD using a recently developed computational algorithm. This predicted site (comprising Asp-318, Glu-325, and Asp-322 and the carboxylate side chain of the receptor agonist, Glu) is situated in the hinge region in the ECD of mGluR1α adjacent to the reported Glu-binding site, with Asp-318 involved in both Glu and calcium binding. Mutagenesis studies indicated that binding of Glu and Ca²⁺ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca²⁺ ([Ca²⁺]_i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca²⁺-sensing capability largely intact. Mutating the predicted Ca²⁺-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca²⁺]_o and [Gd³⁺]_o but also, in some cases, to Glu. The dual activation of mGluR1α by [Ca²⁺]_o and Glu has important implications for the activation of other mGluR subtypes and related receptors. It also opens up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.

Introduction

Metabotropic glutamate receptors (mGluRs)² have key functions in a variety of different neurological processes, including memory, learning, pain, synaptic plasticity, and the control of the activity of various circuits throughout the brain (1). The mGluRs belong to family C of the large superfamily of G protein-coupled receptors (GPCRs). Family C GPCRs (also referred to as family 3 GPCRs, the nomenclature that will be utilized here) also include the Ca²⁺-sensing receptor (CaSR), GABA_B receptors, taste receptors, and putative pheromone receptors (2). All members of the family 3 GPCRs share similar domain architecture, including venus flytrap-like extracellular domains (ECD), heptahelical transmembrane domains, and intracellular C-terminal C-tails. The mGluRs fall into three groups and eight subtypes. Group I comprises mGluR1 and mGluR5 (3). mGluR1 is expressed mainly around a core of ionotropic glutamate receptors in the postsynaptic densities of neurons and functions as a disulfide-linked homodimer (4). Upon activation by its agonists, the intracellular domains of the group I mGluRs associate with the G protein G_q/11 to activate phospholipase C, which subsequently converts phosphatidylinositol bisphosphate (PIP₂) to diacylglycerol and inositol trisphosphate (IP₃), thereby releasing Ca²⁺ from the endoplasmic reticulum, as well as activating protein kinase C (PKC) and other downstream effectors (5).

The issues of whether mGluRs respond to extracellular calcium ([Ca²⁺]_o) and how calcium binding modulates the family 3 GPCRs have attracted extensive investigation. On the basis of sequence homology to CaSR, mGluRs were postulated to be capable of responding to [Ca²⁺]_o. [Ca²⁺]_o has been proposed to either activate mGluR1 directly or to act as a positive mGluR1 modulator (6, 7). Kubo et al. (6, 8) reported that [Ca²⁺]_o, as well as Glu, can trigger intracellular responses elicited by mGluR1, mGluR3, and mGluR5. [Ca²⁺]_o or Gd³⁺ further stimulate the activity of mGluR1α even after saturation of the Glu response and vice versa (6). In addition, mGluR1α responds to 5 mm [Ca²⁺]_o in Purkinje cells prepared from global mGluR1α knock-out mice in which the receptor has been specifically knocked into Purkinje cells, whereas the Purkinje cells from the mGluR1α global knock-out mice themselves cannot sense [Ca²⁺]_o (9, 10). On the basis of these studies, [Ca²⁺]_o is postulated to mediate postsynaptic efficacy through it action on mGluR1 (11). Moreover, Glu triggers [Ca²⁺]_i oscillations in a manner that is modulated by [Ca²⁺]_o (12), as R, 5-3, 5-Dihydroxylphenylglycine, an agonist of group I mGluRs, generated inward currents that were enhanced by [Ca²⁺]_o (10). In contrast, Nash et al. (13) concluded that mGluR1α is not a calcium-sensing receptor because its response to the agonist l-quisqualate is not sensitive to [Ca²⁺]_o. However, the effect of [Ca²⁺]_o on the EC₅₀ for quisqualate was not examined. Any putative Ca²⁺-binding sites capable of regulating mGluR signaling remain “invisible” in six crystal structures of the ECD of mGluR1α determined to date (14, 15), as well as the ECDs and cysteine-rich domains of mGluR3 and mGluR7 (15, 16). One Gd³⁺ ion binds to mGluR1 between the helices of lobe 2 (LB2) at the dimer interface of the ECD, far from the Glu-binding site (14, 17). Removing the Gd³⁺-binding residue, E238Q, eliminated sensitivity to Gd³⁺ but not sensitivity to [Ca²⁺]_o and Glu (17, 18). Two Gd³⁺ ions visible in the crystal structure were ignored by these authors, although one of them is located near the critical hinge region coordinated by Asp-322, Asp-324, and Asp-493 (14). This observation also suggests strongly that a Ca²⁺ ion could bind to this region of the protein. The invisibility of Ca²⁺-binding sites in the x-ray structures of the mGluRs represents a major challenge shared among other Ca²⁺-modulated proteins functioning at high Ca²⁺ concentrations, like those in the extracellular fluids, due to their low Ca²⁺-binding affinities (K_d, ∼0.1–1.5 mm) and irregular binding geometries (19). Our understanding of the role of Ca²⁺ as an extracellular signal acting via family 3 GPCRs beyond CaSR is severely hampered by the lack of adequate information about the location and properties of the Ca²⁺-binding sites of this class of proteins.

We report herein the identification of a novel Ca²⁺-binding site adjacent to the Glu-binding site in the hinge region of the ECD of mGluR1α (14, 15), which was found by using our recently developed the MUG (multiple geometries) algorithm (20–22). MUG is a graphic geometry-based Ca²⁺-binding site prediction software. It extracts oxygen clusters from Protein Data Bank (PDB) files and assumes a Ca²⁺ center for each cluster. The clusters then are verified by setting parameters for geometric filters that define the range of distance between oxygen atoms and Ca²⁺. The clusters satisfying the parameter setting were considered candidates for Ca²⁺-binding pockets. The putative Ca²⁺-binding pockets of lower quality were further modified by allowing rotation of the side chains of predicted liganding residues. To investigate a single Ca²⁺-binding site present within a short stretch of amino acids, normally less than 30 residues, we engineered the short loop into a scaffold protein, CD2. Intact CD2 does not bind Ca²⁺ and is tolerant of site-directed mutagenesis without undergoing changes in its overall structure. Then the metal-binding capabilities of the key predicted Ca²⁺-binding residues were further characterized using luminescence energy transfer (LRET) and site-directed mutagenesis (23, 24). Finally, we investigated the intracellular Ca²⁺ responses resulting from the binding of [Ca²⁺]_o, [Gd³⁺]_o, or Glu individually or of both [Ca²⁺]_o and Glu together by expressing wild type and mGluR1α variants with predicted ligand residues mutated. Our studies suggest that Asp-318, Asp-322, and Glu-325 at the predicted site are involved in Ca²⁺ binding and that mutation at Asp-318 and Glu-325 also abolishes responsiveness of the receptor to [Gd³⁺]_o. The ligands for Glu binding, in contrast, although also including Asp-318, are otherwise distinct (14, 15, 25). Notably, the carboxylate group of the side chain of Glu also contributes to the binding site for Ca²⁺. We propose a dual activation mechanism whereby the simultaneous binding of Glu and Ca²⁺, at their separate but partially overlapping binding sites, potentiates one another's actions to yield maximal activation of mGluR1α.

EXPERIMENTAL PROCEDURES

Computational Prediction of Ca²⁺-binding Sites in mGluR1α and Molecular Modeling

The three-dimensional coordinates of the crystal structures of the ECD of mGluR1α were obtained from the PDB (PDB entry codes: 1EWT, 1EWK (15), and 1ISR (14)). Hydrogen atoms were added using the Sybyl7.2 package (Tripos Inc., St. Louis, MO). The identification of putative Ca²⁺-binding sites in the ECD of mGluR1α was performed using MUG, a graph theory-based algorithm (21) developed by our laboratory. The Ca–O distance in the software was set to 1.6–3.1 Å with a set average cutoff of 2.4 Å (26, 27), and the O–O distance was set to 6.0 Å (21). Side chain atoms were rotated to accommodate Ca²⁺-induced local conformational changes (48). Furthermore, electrostatic surface potential maps were constructed using Delphi (28), and GRASP (29) was then used to render and modify the image. The linear, putative Ca²⁺-binding site was added into the scaffold protein CD2 between Ser-52 and Gly-53 with triple Gly linkers at both ends, and the combined grafting model was generated by Modeler 9v4 (30).

Plasmid, Expression, and Purification of Proteins

The predicted linear Ca²⁺-binding site, termed mGluR1-1, resides between Gly-316 and Gly-337 (GSDGWADRDEVIEGYEVEANGG). This sequence, grafted into CD2 between Ser-52 and Gly-53 in the plasmid pGEX-2T-CD2 (31), was named CD2.D1. The engineered protein was expressed as a GST fusion protein and purified using GS4B resin as described (32). Site-directed mutagenesis was performed using the multisite-directed mutagenesis kit (Stratagene, Cedar Creek, TX).

Tb³⁺ Titration and Ca²⁺ Competition

In Trp-sensitized Tb³⁺-LRET experiments, emission spectra from 500 to 580 nm were recorded with excitation set at 282 nm; slit widths were set at 8 nm for excitation and 12 nm for emission. A glass filter with a cutoff of 320 nm was utilized to circumvent secondary Rayleigh scattering. Tb³⁺ titration and metal competition assays were performed as described previously (24). 500 mm K⁺, 10 μm La³⁺, 10 μm Gd³⁺, 1 mm Mg²⁺, and 1 mm Ca²⁺, respectively, was used to selectively compete with Tb³⁺. Each experiment was carried out independently in triplicate.

Quantitatively Determined Membrane Expression of the mGluR1α Mutants Using Flow Cytometry

PcDNA-mGluR1α (donated by Dr. Randy Hall's laboratory) contained a FLAG tag at the N terminus of the receptor, and mCherry was genetically fused to the C terminus with a linker, GGNSGG. After 2 days of transient expression of mGluR1α and its mutants (D318I, D322I, E325I, and N335I) in HEK293 cells grown on polylysine-coated dishes, cells were incubated in 1× phosphate-buffered saline (PBS) supplemented with 1/1000 anti-FLAG and 1/100 fetal bovine serum (FBS) at 4 °C. The cells were then washed three times with 1× Tris-buffered saline (TBS) and fixed using 4% formaldehyde at room temperature for 15 min. After being washed three times with 1× TBS, the receptors on the cell surface were then labeled with Alexa Fluor® 488 goat anti-mouse IgG (Invitrogen) for 30 min at 37 °C. The cells were then collected in 1× PBS, and the intensity of green and red fluorescence was measured using LSRFortessa (BD Biosciences). The ratios of green and red fluorescence from mGluR1α and the mutated receptors were normalized to the amount of receptors expressing on the cell surface relative to total receptors (total cellular expression of receptor). Data were collected from three dishes.

Measurement of [Ca²⁺]_i Responses of mGluR1α and Its Mutants with or without [Ca²⁺]_o or Glu

Measurement of [Ca²⁺]_i was performed as described (24). In brief, wild type mGluR1α and its mutants (D318I, D322I, D324I, E325I, and E328I) were transiently transfected into HEK293 cells and cultured for 2 additional days. The cells on the coverslips were subsequently loaded using 4 μm Fura-2 AM in 2 ml of physiological saline buffer (10 mm HEPES, 140 mm NaCl, 5 mm KCl, 0.55 mm MgCl₂, and 1 mm CaCl₂, pH 7.4). The coverslips were mounted in a bathing chamber on the stage of a fluorescence microscope. Fura-2 emission signals from single cells excited at 340 or 380 nm were collected utilizing a Leica DM6000 fluorescence microscope in real time as the concentration of extracellular Ca²⁺ was increased in a stepwise manner. The ratio of emitted fluorescence at 510 nm resulting from excitation at 340 or 380 nm was further analyzed to obtain the intracellular Ca²⁺ response as a function of changes in [Ca²⁺]_o. Then, the sensitivity of mGluR1α and its mutants (D322I, D324I, E325I, and E328I) to extracellular glutamate was measured by increasing the extracellular glutamate concentration in the presence of 1.8 mm Ca²⁺. The glutamate concentrations at which the intracellular Ca²⁺ responses of mGluR1α and its mutants were first observed and, subsequently, saturated were determined. Moreover, to further characterize the influence of [Ca²⁺]_o to Glu-induced [Ca²⁺]_i release through wild type mGluR1α, an additional 5 or 10 mm Ca²⁺ was added to the perfusate. [Ca²⁺]_i was measured as described above during changes in [Ca²⁺]_o and/or Glu.

Measurement of Intracellular Ca²⁺ Release Mediated by mGluR1α and Its Mutants in the Presence of Extracellular Gd³⁺

Changes in [Ca²⁺]_i in response to the addition of Gd³⁺ were determined as just described. Specifically, cells were incubated in incubation buffer (140 mm NaCl, 4 mm KOH, 10 mm HEPES, 1.5 mm CaCl₂, 1 mm MgCl₂, 10 mm glucose, pH 7.4) for up to 1.5 h, and Gd³⁺ (made up in 140 mm NaCl, 4 mm KOH, 10 mm HEPES, and 0.3 mm MgCl₂, pH 7.4) was added at the concentrations described under “Results.” The [Ca²⁺]_i responses of mGluR1α after the introduction of mutations in the Glu-binding site were measured similarly.

Data Analysis and Curve Fitting

At each agonist concentration, all of the transfected cells in the microscopic field from three independent experiments were selected for analysis, and at least 60% of the cells displaying normal responses were analyzed. The cells that did not respond to the agonists or displayed a sigmoidal curve with a stable plateau after treatment with high [Ca²⁺]_o were excluded. These latter cells maintained a constant, high plateau of the intracellular Ca²⁺ concentration, perhaps because the plasma membrane was excessively permeable to Ca²⁺. To normalize the concentration response curves for the responses to [Ca²⁺]_o, the maximal response of wild type mGluR1α to extracellular Glu was set at 100% so that the maximal responses of mutant receptors to [Ca²⁺]_o or Glu were transformed into percentages relative to the response of WT mGluR1α to Glu. Data were fitted using the Hill equation as described previously (23).

RESULTS

Prediction of a Novel Ca²⁺-binding Site Adjacent to the Glu-binding Site in the ECD of mGluR1α

We recently developed the computational algorithm MUG, which predicts Ca²⁺-binding sites using graph theory by identifying all possible liganding oxygen clusters and finding maximal cliques. The positions of Ca²⁺ and its liganding groups in 144 calcium-binding proteins can be predicted with 0.22–0.49 Å accuracy by geometric filters established on the basis of an extensive survey of known Ca²⁺-binding sites in the Protein Data Bank (19). To accommodate Ca²⁺-induced conformational changes, the side chains of putative Ca²⁺-binding ligand residues were subjected to rotation using a rotamer library (MUG^SR) (48). Fig. 1 shows one predicted Ca²⁺-binding site identified here in the crystal structure of the mGluR1α ECD (PDB entry code: 1EWK) using the MUG algorithm. Two other predicted sites not included in this report were also revealed by MUG, one of them (site 2) residing in the Mg²⁺-binding pocket (Leu-86—Gly-102) inferred from the crystal structure and the other one located within a long loop (Asp-125—Lys-153) that was invisible in the crystal structure because of its high flexibility and was repaired using Modeler (30). The third predicted Ca²⁺-binding site (site 3), encompassing Ser-129 to Gly-144, was present within this missing loop.

FIGURE 1.

Predicted Ca²⁺-binding pocket in ECD of mGluR1α. A, location of the predicted Ca²⁺-binding site in the ECD (PDB entry code: 1EWK). The proposed key residues are indicated and highlighted in red (model generated by PyMOL). B, electrostatic potential map of ECD. The predicted Ca²⁺-binding site is in the hinge region and shares residue Asp-318 with the Glu-binding site. C, alignment of the grafted fragment in mGluRs and CaSR. Asp-318 is conserved in all of the receptors; Glu-325 is conserved in group I and II mGluRs; Asp-322 is conserved in group I mGluRs.

The predicted Ca²⁺-binding site studied in detail here comprises the carboxyl side chains of Asp-318 and Glu-325, the main chain carbonyl Asp-322 in a flexible loop of mGluR1α, and the carboxyl side chain of Glu-701 (a ligand for glutamate). This predicted Ca²⁺-binding site is located at the hinge region in the ECD adjacent to the reported Glu-binding site (Arg-74, Ser-165, Thr-188, Asp-208, Tyr-236, Asp-318, and Lys-409) (15, 25), with Asp-318 predicted to be involved in both Glu and Ca²⁺ binding. Thus, Ca²⁺ and Glu, when bound to the receptor, both bind to Asp-318. Asp-318 and Asp-322 can be identified in the Glu-free form (PDB entry code: 1EWT), whereas the direct binding of Ca²⁺ to the carboxyl side chain of the agonist Glu-701 is visualized only in the Glu-loaded form (PDB entry code: 1EWK). Thus, the agonist Glu provides an additional ligand for Ca²⁺ when the former is bound to the receptor, which is very different from intracellular Ca²⁺-binding trigger proteins such as calmodulin that lack any additional chelating groups from molecules other than the residues within the Ca²⁺-binding protein itself, except for water. Fig. 1B shows that the predicted Ca²⁺-binding pocket has a highly negatively charged surface as revealed by Delphi in the structures of three solved forms of the ECD within mGluR1α (Fig. 1B).

Obtaining Site-specific Ca²⁺/Ln³⁺-binding Affinities by a Grafting Approach

To probe the Ca²⁺-binding capability of the predicted Ca²⁺-binding site in mGluR1α, we utilized our grafting approach by inserting the protein sequence encompassing the putative mGluR1α Ca²⁺-binding site into the host protein, CD2.D1 (denoted as CD2-mGluR1-1). The inserted sequence contains all predicted Ca²⁺-binding residues except Glu-701. This approach had previously enabled us to obtain site-specific Ca²⁺ binding affinities of the EF-hand motifs from calmodulin and linear Ca²⁺ binding sequences, free from the limitations of working with membrane proteins (32, 33). The putative mGluR1α Ca²⁺-binding site was flanked by flexible triple-Gly linkers and inserted between Ser-52 and Gly-53 of CD2.D1 (Fig. 2A) to ensure a native-like conformation and close proximity (<15∼20 Å) to Trp-32 in order to enhance the Tb³⁺-LRET signal. Indeed, grafting the putative Ca²⁺-binding loop from mGluR1α did not significantly change the secondary and tertiary structures of CD2, as revealed by circular dichroism, Trp fluorescence, and NMR chemical shifts (supplemental Fig. S1). Fig. 2B shows that Tb³⁺ (which has the same coordination chemistry as Ca²⁺) elicits an increase in fluorescence of CD2.D1 at 550 nm when excited at 280 nm due to Tb³⁺- LRET. CD2.D1 had a Tb³⁺ binding affinity of 49 ± 9 μm (Fig. 2B and Table 1). Ca²⁺ displaced bound Tb³⁺ (Fig. 2B), thereby decreasing the Tb³⁺-LRET signal. CD2.D1 has a Ca²⁺ dissociation constant of 1.80 ± 0.12 mm determined in this manner (Table 1).

FIGURE 2.

Metal binding properties of the grafted Ca²⁺-binding site. A, three-dimensional illustration of the modeled structure of the engineered protein CD2.D1, based on the crystal structures of CD2 (PDB entry code: 1HNG (35)) and the mGluR1α ECD (PDB entry code: 1EWT (8)). B, Tb³⁺ titration and Ca²⁺ competition (shown as inset) of CD2.D1. The engineered protein bound Tb³⁺ and Ca²⁺ with dissociation constants of 49 ± 9 μm and 1.8 ± 0.1 mm, respectively. Substitution of putative metal-binding ligand residues with Ile decreases Tb³⁺ binding affinity. Tb³⁺ binding curves of a series of CD2-mGluR1 double mutants, E331I/E333I, D324I/E325I, D318I/D322I, and E328I/N335I. All measurements were carried out in a buffer containing 20 mm PIPES and 10 mm KCl, pH 6.8. C, Tb³⁺ binding curves of a series of CD2-mGluR1 double mutants: CD2.D1-1 (E331I/E333I), CD2.D1-2 (D324I/E325I), CD2.D1-3 (D318I/D322I), and CD2.D1-4 (E328I/N335I). All of the measurements were carried out in a buffer containing 20 mm PIPES and 10 mm KCl, pH 6.8. D318I/D322I obviously decreased Tb³⁺ binding affinity, whereas D324I/E325I displays two phases. The Tb³⁺ binding affinity of the engineered protein was clearly impaired by these two pair of mutations (n = 3). D, La³⁺ binding to the engineered protein CD2.D1 monitored by ¹D¹H NMR. 1 or 2 mm La³⁺ results in the peak split at the aromatic group region. E, metal selectivity of CD2.D1. The addition of 500 mm K⁺, 1 mm Ca²⁺, 1 mm Mg²⁺, 100 μm Gd³⁺, or 100 μm La³⁺ to the pre-equilibrated Tb³⁺ (30 μm) and protein (3 μm) solution was carried out independently. The resultant changes in the Tb³⁺ luminescence signal were monitored at 545 nm (*, p < 0.05; **, p < 0.01).

TABLE 1.

Tb³⁺ binding affinities of the engineered protein CD2.D1 and its double mutants (n = 3)

Proteins	Mutations	Dissociation constant (Kd)
CD2.D1	WT	49 ± 9 μm (Ca2+, 1.8 ± 0.1 mm)
CD2.D1-1	E331/E333I	85 ± 18 μm*
CD2.D1-2	D324/E325I	113 ± 5 μm
CD2.D1-3	D318/D322I	>4.8 mm*
CD2.D1-4	E328/N335I	299 ± 15 μm*

*, p < 0.05.

Because mGluR1α is modulated by various polyvalent cations, including Ca²⁺, Gd³⁺, Tb³⁺, La³⁺, Mn²⁺, and Mg²⁺ (6), we tested the metal binding selectivity of CD2.D1 by applying K⁺, Mg²⁺, La³⁺, or Gd³⁺ to compete with prebound Tb³⁺. Fig. 2C shows that the luminescence intensity of Tb³⁺ decreased significantly upon adding trivalent La³⁺ or Gd³⁺, indicating that Tb³⁺ bound to the pocket was replaced by these metal ions. Gd³⁺ had the strongest capacity to displace Tb³⁺. Similarly, adding La³⁺ to CD2.D1 produced a split in the resonance of CD2.D1 at 10 ppm (Fig. 2D). Ca²⁺ competed more effectively than Mg²⁺, whereas K⁺ failed to compete with Tb³⁺.

Next, we utilized mutagenesis studies to examine the contribution of proposed ligand-binding residues to metal-binding capability. Double substitutions of negatively charged residues by Ile to delete negative charges but preserve bulky side chains in the proposed binding pocket, as seen in the mutants D324I/E325I, D318I/D322I, and E328I/N335I, produced 2.3-, 6.1-, or 98-fold increases in the respective dissociation constant values (Fig. 2, B and E). However, removing the negative charges from the non-Ca²⁺-binding residues, Glu-331 and Glu-333 (E331I/E333I) (Fig. 2, B and E) produced a less than 2-fold change in the K_d, with a modest alteration in Tb³⁺ binding to the predicted binding pocket.

Membrane Expression of mGluR1α and Its Mutants

WT mGluR1α and its mutant forms (D318I, D322I, and E325I) were expressed heterogeneously in HEK293 cells. The receptors expressed on the cell membrane were visualized by confocal microscopy (supplemental Fig. S2) and flow cytometry (Fig. 3). Supplemental Fig. S2 shows that the mutations did not affect the distribution of the receptors on the membrane. We calculated the ratio of intensities of green and red fluorescence measured using flow cytometry (LSRFortessa, BD Biosciences); the mutant receptors displayed expression levels on the cell membrane comparable with that of the wild type receptor, although E325I displayed a somewhat lower membrane expression level (Fig. 3; n = 3). Thus, the mutations involving the predicted Ca²⁺-binding site had little effect on the surface expression of the respective mutant receptors.

FIGURE 3.

Surface expression of WT mGluR1α and its mutants. mGluR1α carries FLAG tag at its N terminus and mCherry at its C terminus. mGluR1α and its mutants were transiently expressed in HEK293 cells seeded on 50-mm dishes coated with polylysine. After incubation with anti-FLAG, the receptors on the membrane could be visualized using a secondary antibody, Alexa 488-anti-mouse IgG (Invitrogen). Emissions at 520 and 610 nm were collected by flow cytometry (LSRFortessa, BD Biosciences); these represent receptors present on the cell membrane and overall, respectively. Ratios of fluorescence at 520–610 nm indicate the membrane expression levels of WT mGluR1α and its mutants. Emission at 520 nm (green signal) reflects the membrane expressed receptors, whereas the red signal at 610 nm from mCherry is a measure of total expression of the receptor. NT indicates non-transfected cells, which display no fluorescence. Although E325I displays a relatively lower surface expression, the other mutants have membrane expression level comparable with that of WT mGluR1α (n = 3).

Extracellular Ca²⁺ Triggers mGluR1α-mediated Intracellular Responses

We next examined the mGluR-mediated intracellular Ca²⁺ responses in HEK293 cells transfected with mGluR1α-mCherry. The fluorescent protein mCherry was fused to mGluR1α to correlate cellular responses with the expression of mGluR1α. We chose HEK293 cells as a model because this cell line lacks endogenous mGluR1α (34). mGluR1α-mCherry was well expressed and correctly targeted to the cell membrane (Figs. 3 and 4A and supplemental Fig. S2), and Fura-2 was efficiently loaded (Fig. 4B). Single cell, real time imaging was performed using fluorescence microscopy. To minimize receptor desensitization by agonists, the responses to each concentration of added Ca²⁺ or Glu were examined using separate coverslips.

FIGURE 4.

Intracellular Ca²⁺ response of WT mGluR1α and its mutants. WT mGluR1α and D318I-mGluR1α were overexpressed in HEK293 cells. Fura-2 was then loaded into the cells, and the [Ca²⁺]_i level was measured by monitoring emission at 510 nm with excitation at 340 or 380 nm. A, red fluorescence of mCherry on the C terminus of mGluR1α indicates the presence of the receptor or its mutants. B, cells were loaded with Fura-2-AM to measure [Ca²⁺]_i level. C, [Ca²⁺]_i release triggered by [Ca²⁺]_o in WT mGluR1α and its mutants. D318I and E325I eliminate the [Ca²⁺]_i response, whereas D322I and S166A reduce it. NT, non-transfected cells.

In the absence of exogenous Glu, the addition of [Ca²⁺]_o at less than 1.8 mm did not induce any [Ca²⁺]_i response in HEK293 cells transfected with WT mGluR1α. Adding ≥3.0 mm Ca²⁺ to the medium elicited a transient [Ca²⁺]_i increase followed by a long lasting plateau (Fig. 4C); this response was maximal at ≥5.0 mm [Ca²⁺]_o, with an EC₅₀ of 3.0 mm. Furthermore, adding 0.5 or 30 μm Glu significantly enhanced the maximal response of mGluR1α to [Ca²⁺]_o by 1.3- and 1.7-fold, respectively (Fig. 5). The EC₅₀ value for [Ca²⁺]_o decreased from 3.0 to 2.8 and 0.1 mm, respectively (Fig. 5 and Table 2).

FIGURE 5.

Intracellular Ca²⁺ responses of WT mGluR1α and its mutants (D322I-mGluR1α, D324I-mGluR1α, E325I-mGluR1α, and E328I-mGluR1α) to [Ca²⁺]_o in the presence of Glu. Shown are the additional Glu-enhanced responses of WT mGluR1α and all of its mutants to [Ca²⁺]_o. The Glu concentrations that were added were determined by the responses of the wild type receptor or its mutants to Glu. That is, the Glu concentrations that evoked initial activation of or saturated the receptors were utilized for the [Ca²⁺]_o-induced responses of the wild type receptor and its mutants. [Ca²⁺]_i levels were measured by the same methods described above. Notably, E325I loses sensitivity to [Ca²⁺]_o but can still sense [Glu]_o. However, its responsiveness to [Ca²⁺]_o in the presence of high concentration [Glu]_o was not affected by increasing [Ca²⁺]_o (n = 3).

TABLE 2.

[Ca²⁺]_i responses of WT mGluR1α and its mutants to [Ca²⁺]_o and Glu (n = 3)

Variants	[Ca2+]o				Glu
	Glu concentration	EC50	nHill	Maximal responsea	EC50b	Maximal responsea
	μm	mm		%	μm	%
WT	0	3.0	1.7	75 ± 3	1.7	100 ± 3
	0.5	2.8	1.4	109 ± 7	0.9c	108 ± 1c
	30	0.1	0.5	128 ± 6	0.4d	130 ± 8d
D318I	0	None	None	No response	None	No response
D322I	0	4.3	2.0	63 ± 2	13.2	111 ± 2
	10	4.1	1.7	86 ± 5
	30	0.5	7.2	105 ± 3
E325I	0	None	None	No response	30.5	146 ± 2
	10	4.3	2.3	89 ± 12
	100	NDe	NDe	117 ± 3
D324I	0	3.9	4.1	104 ± 10	0.3	117 ± 7
	0.5	2.9	1.8	144 ± 4
	30	0.6	1.0	162 ± 6
E328I	0	2.5	2.7	102 ± 5	1.2	107 ± 12
	3	1.6	1.5	135 ± 13
	30	2.2	0.3	147 ± 3

^a The maximal responses are normalized to the maximal response of wild type mGluR1α to Glu.

^b Glu dose response was performed in 1.8 mm Ca²⁺.

^c Experiments performed in 5 mm Ca²⁺.

^d Experiments perfomed in 10 mm Ca²⁺.

^e ND, not determined.

The response of wild type mGluR1α to Glu was investigated in physiological saline buffer with 1.8 mm Ca²⁺. Wild type mGluR1α only responded at ≥0.5 μm Glu, and this response was saturated at 30 μm Glu. Fig. 6A shows that at 1.8 mm Ca²⁺, the addition of >30 μm Glu evoked large [Ca²⁺]_i responses. However, when [Ca²⁺]_o was reduced from 1.8 mm to close to zero (no Ca²⁺ added to the medium), 30 μm Glu still activated mGluR1α (Fig. 5). There was, however, a reduced maximal response (29%) (Fig. 5), possibly because of the depletion of intracellular calcium stores. This result suggests that Glu triggers the activation of mGluR1α and that this effect can be enhanced by [Ca²⁺]_o.

FIGURE 6.

Intracellular Ca²⁺ responses to extracellular Glu in HEK293 cells transfected with WT mGluR1α or its mutants. Three negatively charged residues in the predicted Ca²⁺-binding pocket (Asp-318, Asp-322, and Glu-325) were mutated into Ile. Along with WT mGluR1α, the mutants were transiently expressed in HEK293 cells. In the presence of 1.8 mm Ca²⁺, extracellular Glu-induced intracellular Ca²⁺ release was measured by recording emission intensities at 510 nm excited at 340 and 380 nm, respectively. A, responses to Glu of mutations on Ca²⁺-binding site. Except for mutant D318I, two other mutants, D322I, E325I, and WT mGluR1α display responsiveness to Glu. B, maximal response of WT mGluR1α and its mutants to Glu at a saturating concentration. Each single data point was performed in an individual dish, and the cells expressed mGluR1α showing responses to Glu were selected for analysis (n = 3).

To study the synergy of [Ca²⁺]_o and Glu binding to mGluR1α, the combination indices for quantitative evaluation of synergism were calculated using Calcusyn (35). With 0.5 or 30 μm Glu, the combination indices were <1 when [Ca²⁺]_o reached a physiological level (>0.5 mm) (supplemental Table S2), suggesting that Glu and Ca²⁺ synergistically modulate mGluR1α-mediated signaling.

Effects of [Ca²⁺]_o on Glu-induced [Ca²⁺]_i Release by Wild Type mGluR1α

To determine the role of [Ca²⁺]_o on Glu-mediated activation of wild type mGluR1α. 5 and 10 mm [Ca²⁺]_o were added to the perfusion system in addition to the 1.8 mm already present in the perfusate. As shown in Fig. 7, compared with the response in 1.8 mm [Ca²⁺]_o, both 5 and 10 mm [Ca²⁺]_o enhanced the responses of WT mGluR1α to Glu by reducing the EC₅₀ values from 1.7 to 0.9 and 0.4 μm, respectively (Table 2), although the maximal responses remained comparable with those at 1.8 mm [Ca²⁺]_o. This indicates that [Ca²⁺]_o potentiates the sensitivity of mGluR1α to Glu and that this effect increases at higher levels of [Ca²⁺]_o.

FIGURE 7.

Extracellular Ca²⁺ enhanced mGluR1α to sense extracellular Glu. Responses of WT mGluR1α to extracellular Glu were assessed in buffers containing additional [Ca²⁺]_o (1.8, 5, and 10 mm). The maximal responses of the receptor to [Glu]_o in 1.8, 5, and 10 mm [Ca²⁺]_o are comparable, but the EC₅₀ values for the responses in the presence of 5 and 10 mm [Ca²⁺]_o are significantly decreased. Clearly, higher [Ca²⁺]_o reduces the EC₅₀ of the receptor for [Glu]_o (n = 3).

Effects of Mutating Proposed Ca²⁺-binding Residues on [Ca²⁺]_o- or Glu-evoked [Ca²⁺]_i Responses

To further understand the potential physiological role of this proposed Ca²⁺-binding site, we compared the mGluR-mediated [Ca²⁺]_i responses in HEK293 cells transfected with the WT or mutated versions of mGluR1α. Fig. 4C shows that without Glu, substituting the predicted Ca²⁺-binding residues Asp-318 and Glu-325 with Ile eliminates the transient [Ca²⁺]_i response toward [Ca²⁺]_o. In addition, the mutation D322I also reduced the transient [Ca²⁺]_i response by 16–20% while increasing the EC₅₀ from 3.0 mm [Ca²⁺]_o for the wild type receptor to 4.2 mm for D322I (Fig. 5 and Table 2), although all mutants were expressed at levels comparable with the WT receptor, as assessed by immunofluorescence and flow cytometry (Fig. 3 and supplemental Fig. S2). These results suggest that the predicted Ca²⁺-binding residues, Asp-318, Glu-325, and Asp-322 (especially the first two), are important for the sensitivity of mGluR1α to modulation by [Ca²⁺]_o. However, S166A maintains Ca²⁺ sensitivity with a lower maximal response (Fig. 4C), although Ser-166 was previously reported to be a potential Ca²⁺-binding residue (6).

Effects of Mutating Predicted Ca²⁺-binding Residues on Glu-potentiated [Ca²⁺]_i Responses

The Ca²⁺-binding site identified here is adjacent to the previously defined Glu-binding site (Arg-78, Ser-165, Thr-188, Asp-208, Tyr-236, Asp-318, and Lys-409) (15, 25) (Fig. 1B). Asp-318 is the lone residue used in both the Glu- and Ca²⁺-binding sites. Figs. 4 and 6 show that the mutation D318I completely eliminates the [Ca²⁺]_i response of mGluR1α to both [Ca²⁺]_o and Glu, even at concentrations of the latter as high as 30 μm. In contrast, the mutant E325I completely abolishes [Ca²⁺]_o-mediated [Ca²⁺]_i responses without the agonist Glu (Figs. 4C and 5 and Table 2) but retains Glu-mediated [Ca²⁺]_i responses (Fig. 6A). However, its EC₅₀ value for Glu-mediated responses is increased by ∼18-fold (Fig. 6B and Table 2). These results further confirm that Glu-325 contributes to Ca²⁺ binding without directly liganding Glu (as shown by earlier studies of the binding site for Glu, which did not identify Glu-325 as a Glu ligand). However, the proximity of the Ca²⁺- and Glu-binding sites may produce indirect, conformational effects of mutating residue 325 on Glu binding. Furthermore, D322I exhibited a reduction in EC₅₀ for [Ca²⁺]_o by only 33%, consistent with it making a relatively minor contribution as a ligand for Ca²⁺ binding. In contrast to the marked impact of D318I and E325I on the EC₅₀ for [Ca²⁺]_o, removal of other charged residues, such as D324I and E328I, did not alter either the EC₅₀ (3 and 8% changes, respectively) or the magnitude of the response to [Ca²⁺]_o significantly in the absence of Glu (104 ± 10 and 102 ± 5, respectively, of the control level) (Fig. 5 and Table 2).

Effect of Mutating Glu-binding Site on [Ca²⁺]_i Responses to Glu and Ca²⁺

To further explore the synergistic interaction between the predicted Ca²⁺- and Glu-binding sites, four mutations at Glu ligand residues (S165A, T188A, D208I, and Y236F) were generated. Consistent with studies reported previously (25), T188A and D208I entirely eliminated Glu sensitivity, whereas S165A and Y236F could be activated only by high concentrations (100 μm) of Glu (Fig. 8A). Interestingly, all receptors with mutated Glu-binding ligand residues (exception for Asp-318) retained a sensitivity to [Ca²⁺]_o (Fig. 8B and Table 3), although their EC₅₀ values were increased compared with that of wild type mGluR1α (Table 3), again perhaps owing to local conformational effects of mutating the Glu-binding site on Ca²⁺ binding. S165A and D208I increased the EC₅₀ of the wild type receptor for [Ca²⁺]_o from 3.0 to 8.1 and 4.6 mm, respectively, although their maximal responses were comparable to that of the wild type receptor (Fig. 8B and Table 3). Conversely, T188A and D208I exhibited much reduced maximal responses (26 and 66%, respectively), whereas their EC₅₀ values were comparable with that of the wild type receptor (Table 3). Taken together, these data show that it is possible to generate mGluR1α variants responding to either Glu or to Ca²⁺ alone. Thus mGluR1α can function as a true [Ca²⁺]_o-sensing receptor, as certain mutants, such as S165A and D208I, do not respond to Glu but maintain their Ca²⁺-sensing capability with only a modest increase in the EC₅₀ for [Ca²⁺]_o.

FIGURE 8.

Receptors with mutations in the Glu-binding pocket can sense Ca²⁺ but not Glu in physiological buffer. Four residues from the reported Glu-binding pocket were selected to mutate into non-polar residues. Intracellular Ca²⁺ levels indicated by Fura-2 were recorded using fluorescence microscopy, which detected the ratio of the emission at 510 nm with excitation at 340 and 380 nm. A, T188A and D208I abolish sensitivity to Glu, but S165A and Y236F still respond to 100 μm Glu. B, mutants S165A, T188A, D208I, and Y236F remain capable of responding to [Ca²⁺]_o; the maximal responses of S165A, T188A, and D208I are comparable with that of WT mGluR1α, whereas Y236F decreases the sensitivity of the receptor to [Ca²⁺]_o (n = 3).

TABLE 3.

[Ca²⁺]_i response of mutants in Glu-binding site (n = 3)

Mutants	EC50	nHill	Maximal responsea
	mm		%
S165A	8.1	3.1	86 ± 3
T188A	3.4	2.2	74 ± 2
D208I	4.6	2.3	95 ± 3
Y236F	3.3	10.9	33 ± 3

^a Maximal responses are normalized to the maximal response of wild type mGluR1α to Glu.

Effects of Mutations in Predicted Ca²⁺-binding Site on Gd³⁺-induced [Ca²⁺]_i Responses

Gd³⁺ is also revealed at the hinge region in the Fourier map, where it shares residues Asp-322 and Asp-324 from the loop that contributes to Ca²⁺ binding (14). Because of the low resolution of this crystal structure (4 Å), the highly flexible loop that binds Gd³⁺ in the crystal structure, and the similarity of the binding geometries of Gd³⁺ and Ca²⁺, these two cations probably share, at least in part, the same residues. To address this possibility, the responses to [Gd³⁺]_o of D318I and E325I were compared with that of the wild type receptor. Consistent with results reported by Abe et al. (17, 36), the dose-response profiles of wild type mGluR1α display a bell-shaped curve. However, the introduction of the mutation D318I or E325I completely eliminated the receptor sensitivity to [Gd³⁺]_o (Fig. 9).

FIGURE 9.

Mutations D318I and E325I in the predicted Ca²⁺-binding site abolish [Ca²⁺]_i responses of the receptor to [Gd³⁺]_o. HEK293 cells expressing D318I, E325I, and WT mGluR1α were preincubated in 140 mm NaCl, 4 mm KOH, 10 mm HEPES, 1.5 mm Ca²⁺, 1 mm MgCl₂, and 10 mm glucose, pH 7.5, for up to 1.5 h before fluorescence microscopy was carried out. Gd³⁺ was added into the loading buffer (140 mm NaCl, 4 mm KOH, 10 mm HEPES, and 0.3 MgCl₂, pH 7.4) in the perfusion system. [Ca²⁺]_i levels indicated by Fura-2 are presented by the ratio of the fluorescence at 510 nm when excited at 340 and 380 nm as above. The [Ca²⁺]_i response of wild type mGluR1α displays a bell-shape curve, but D318I and E325I completely abolish [Ca²⁺]_i release to [Gd³⁺]_o (n = 3).

DISCUSSION

We utilized the computational algorithm MUG (22) to predict a novel Ca²⁺-binding site in mGluR1α adjacent to the Glu-binding site shown in Fig. 1. This predicted Ca²⁺-binding site (comprising Asp-318, Glu-325, Asp-322, and the carboxylate side chain of Glu-701) does not completely overlap the Glu-binding site (15, 25). However, both sites include Asp-318, which our data suggest is involved in both Glu and Ca²⁺ binding. The metal-binding capability of the predicted Ca²⁺-binding residues in mGluR1α was verified by a grafting approach. Like wild type mGluR1α, the predicted Ca²⁺-binding site grafted in a scaffold protein (CD2) exhibited metal selectivity for Ca²⁺ and its trivalent analogs, Gd³⁺, Tb³⁺, and La³⁺, in contrast to the physiological monovalent cation, K⁺, which exhibited no measurable affinity for the predicted site. The metal-binding capability of the predicted metal-binding ligand residues in mGluR1α was further verified by replacing negatively charged residues with Ile. The Ca²⁺ binding affinity of mGluR1α determined by the grafting approach (∼1.8 mm) is within the physiological concentration of [Ca²⁺]_o in the nervous system (0.8–1.5 mm) (37), although this Ca²⁺ binding constant may be changed slightly in vivo by the local microenvironment and/or the presence of Glu released by nearby cells.

We further demonstrated that mGluR1α could be activated by either Glu or [Ca²⁺]_o. Indeed, Glu and [Ca²⁺]_o act synergistically to elicit the maximal [Ca²⁺]_i responses observed here (Table 2). Mutating the Glu-binding site, such as T188A and D208I, in mGluR1α (Fig. 8A and Table 3), completely abolished the Glu-signaling capability of the receptor while leaving its Ca²⁺-sensing capability largely intact, with only modest increases in EC₅₀ for [Ca²⁺]_o. These results suggest that mGluR1α can function as a true [Ca²⁺]_o-sensing receptor, i.e. exhibiting robust activation by [Ca²⁺]_o, in the absence of added Glu. Although we cannot rule out release of low concentrations of Glu by the HEK293 cells, it should be noted that some mutants of the receptor retained responsiveness to [Ca²⁺]_o despite complete loss of responsiveness to Glu. Moreover, even the addition of high exogenous concentrations of Glu failed to activate these mutants.

We have also shown that mutating predicted Ca²⁺-binding residues abolishes or significantly not only reduces [Ca²⁺]_o sensitivity but also, in some cases, affects Glu-mediated responses. For example, E325I completely abolished [Ca²⁺]_o-mediated [Ca²⁺]_i responses in the absence of Glu (Fig. 3C and Table 2). At the same time, this mutant retained sensitivity to Glu, albeit with an ∼18-fold reduction in EC₅₀ (without any decrease in maximal response) (Fig. 5 and Table 2). Thus, despite its not being a Glu-binding residue, the presence of an intact Ca²⁺-binding ligand at Glu-325 considerably enhanced the affinity of mGluR1α for Glu. How could this take place? These results, in fact, are consistent with separate but overlapping Ca²⁺- and Glu-binding sites (Figs. 1 and 10). As noted, positively charged Arg-323 is also very close to Ca²⁺ in the model, but it might not affect Ca²⁺ binding because of the flexible backbone in the loop. Glu-325 is predicted to be a ligand for binding Ca²⁺ and not Glu. Glu-325 could be identified only in the x-ray structure of Glu-complexed mGluR1α (PDB entry code: 1EWK), indicating that 1) the binding of Glu could stabilize the side chain of Glu-325 in a conformation favorable for it to serve as a ligand for Ca²⁺ and/or 2) the bound Glu could then further enhance [Ca²⁺]_o binding by contributing an additional Ca²⁺-binding ligand. Conversely, binding of Ca²⁺ to mGluR1α could increase the affinity of the Glu-binding site for its ligand by providing Ca²⁺ as an additional Glu ligand (Glu-701). Overall, mutating residues involved only in binding [Ca²⁺]_o (with the exception of Asp-318) had less dramatic effects than mutations at the Glu-binding site. Most of these mutants can be fully activated upon binding Glu, because the addition of high levels of Glu restores their EC₅₀ and maximal responses to [Ca²⁺]_o to levels close to those of the wild type receptor. Additionally, a Gd³⁺ ion is also revealed in the crystal structure adjacent to the predicted Ca²⁺-binding site, and it shares the same loop that contributes to Ca²⁺ binding. Mutations in the Ca²⁺-binding residues in the loop, D318I and E325I, entirely eliminated Gd³⁺-induced [Ca²⁺]_i release (Fig. 9). Although Glu-238, located at the interface of the two protomers, has been reported as a functional Gd³⁺ site, I120A, another mutation located at the interface of the receptor loses sensitivity to [Ca²⁺]_o (25). This indicates that the mutations at the interface of the two monomers cause markedly reduced activation of the receptor by either [Gd³⁺]_o or [Ca²⁺]_o and that the site at the hinge region is a true Gd³⁺-binding site. This also highly suggests that our predicted Ca²⁺-binding site can likewise bind Gd³⁺.

FIGURE 10.

A dual activation model for mGluR1α involving both extracellular Ca²⁺ and Glu via the overlapping and interacting binding pockets for the two ligands at the hinge region of the ECD. Increasing either Glu or [Ca²⁺]_o partially activates mGluR1α. However, full activation of intracellular Ca²⁺ signaling by mGluR1α requires the simultaneous binding of both Glu and Ca²⁺; Asp-318 plays a key role in the synergy between the two agonists. In this sense, mGluR1α can be viewed as a coincidence detector, requiring the binding of both ligands for maximal intracellular signaling.

In view of these findings, we propose a working model of dual activation of mGluR1α by the two physiological activators, [Ca²⁺]_o and Glu, via their overlapping and interacting binding pockets at the hinge region of the ECD (Fig. 10). Increased concentrations of either Glu or [Ca²⁺]_o partially activate mGluR1α. However, full activation of mGluR1α with maximal sensitivity and maximal amplitude of the response to Glu requires simultaneous binding of both Glu and Ca²⁺, with Asp-318 playing a key role in the synergy between the two agonists. In this sense, mGluR1α can be viewed as a “coincidence detector,” requiring the binding of both ligands for maximal intracellular signaling.

Our proposed working model of mGluR1α is supported by previous studies from a number of groups working on both cultured cells and native brain tissue (6). Francesconi and Duvoisin (38) reported that mGluR1α in transfected cells is activated by [Ca²⁺]_o in the absence of Glu, indicating that mGluR1α is a Ca²⁺-sensing receptor. Kubo et al. (6) showed that [Ca²⁺]_o, as well as Glu, triggers intracellular responses in cultured cells and oocytes expressing mGluR1, mGluR3, and mGluR5. In terms of studies on endogenous mGluRs in native neurons, Tabata et al. (10) showed that [Ca²⁺]_o fails to induce [Ca²⁺]_i mobilization in the Purkinje cells from mGluR1 knock-out mice, whereas [Ca²⁺]_i is elevated by [Ca²⁺]_o in Purkinje cells from mGluR1α rescued mice. Furthermore, brain slice experiments from Hardingham et al. (11) demonstrated that [Ca²⁺]_o mediates postsynaptic efficacy via activation of group I mGluRs. Taken together, these studies represent strong evidence that [Ca²⁺]_o is a physiologically relevant modulator of mGluR1α activity in the central nervous system. Based on mutational analyses, the Ca²⁺- and Glu-binding sites were postulated to be close to one another but not completely overlapping (6). For example, S166D decreases [Ca²⁺]_i responses (6) and lowers sensitivity to Glu (39). Our data show that S166A maintains sensitivity to [Ca²⁺]_o albeit with reduced responsiveness (Fig. 4C). This residue is located at the ECD hinge joint but away from our predicted site in the flexible hinge region (Fig. 1B). The observed decrease in mGluR1α sensitivity observed with S166D could be a result of tuning the Ca²⁺ binding affinity and Ca²⁺-induced conformational change by altering electrostatic interactions around the predicted Ca²⁺-binding site (40).

As shown in Fig. 1C, the predicted Ca²⁺-binding residue Asp-322 is conserved in group I mGluRs. Glu-325 is highly conserved in group I (mGluR1 and mGluR5) and group II (mGluR2 and mGluR3) mGluRs (Fig. 1C). Interestingly, mGluR5 in group I and mGluR3 in group II sense [Ca²⁺]_o at physiological levels, whereas mGluR2 is activated only when [Ca²⁺]_o is more than 10 mm (6). On the basis of our observation that E325I abolished Ca²⁺-induced responses for mGluR1α but retained responsiveness to Glu, we concluded that Glu-325 might be very important for Ca²⁺ binding in the mGluR family generally. Analysis by the Contacts of Structural Units server indicates that Glu-325 interacts electrostatically with Arg-297 and Arg-323 in the Glu-bound structures of mGluR1α (41), suggesting that Glu-325 stabilizes the local structure through an electrostatic interaction. As revealed by the grafting approach (Fig. 2A), E325I significantly reduces the Ca²⁺-binding ability in mGluR1α, possibly by disturbing the favorable local charge environment.

Fig. 1C shows that Asp-318 in mGluR1α, located at the hinge region, is conserved in all members of the three GPCRs, corresponding to Asp-295 of mGluR2, Asp-301 of mGluR3, Asp-309 of mGluR8, and Glu-297 of CaSR (23, 24, 42). Figs. 4 and 6 clearly demonstrate that Asp-318 contributes not only to Ca²⁺- but also Glu-triggered [Ca²⁺]_i responses. This residue seems to play an essential role in the activation of mGluRs. Consistent with this finding, a D318A mutation was shown previously to reduce receptor expression on the membrane and abolish Glu-triggered [Ca²⁺]_i and inositol trisphosphate responses (25).

Our findings also appear to be applicable to other members of the three GPCRs, especially CaSR. The mutation E297I in CaSR, equivalent to D318I in mGluR1α, impairs receptor activation (23, 24, 42). Glu-297 is an important residue in our reported Ca²⁺-binding site in the CaSR hinge region (23, 24); the mutant E297I significantly impairs sensitivity to [Ca²⁺]_o with an EC₅₀ of 9.6 ± 0.2 mm [Ca²⁺]_o. Mutations at or around this Ca²⁺-binding site are also associated with clinical syndromes (autosomal dominant hypocalcemia and familial hypocalciuric hypercalcemia) because of either an increase or a decrease in the sensitivity of the respective receptors to [Ca²⁺]_o. Zhang et al. (43) and others (44, 45) have also reported that mutations around this site, Ser-147, Ser-170, Asp-190, Tyr-218, and E297K, impair the activation of human CaSR by [Ca²⁺]_o. Recently Silve et al. (42) have shown that the missense mutations E297K and Y218S significantly reduce the maximal response of the CaSR. Although E297K was considered a key factor in impairing protein folding, thus leading to lower expression on the cell surface and impaired responsiveness to [Ca²⁺]_o, our unpublished data³ show that E297I has a membrane expression level comparable with that of the wild type CaSR. Therefore, the low expression level of E297K could be, at least in part, the result of the substitution of an unfavorable positive charge, which modifies the local charge balance, leading to reduced folding efficiency. Furthermore, our assessment of the surface expression of D318I by flow cytometry showed that it was at the same level as wild type; this echoes the impact of mutating the equivalent residue in CaSR (e.g. Glu-297). It has been postulated that residues Ser-170, Asp-190, Gln-193, Ser-296, and Glu-297 are critical for Ca²⁺ binding to CaSR and functionality of the receptor (42), which is in excellent agreement with our prediction. In addition, CaSR functions primarily as a [Ca²⁺]_o-sensing receptor but can also integrate information about protein metabolism (i.e. amino acids) with that of divalent cations (e.g. calcium) (46). CaSR displays sensitivity to amino acids, especially phenylalanine and other aromatic amino acids, likely via three serine residues (Ser-169—Ser-171) at a site corresponding to the Glu-binding pocket in mGluR1α. The double mutation T145A/S170T specifically abolishes CaSR responsiveness to amino acids while leaving [Ca²⁺]_o sensing intact (47). Our unpublished data⁵ show that Ca²⁺ and phenylalanine also synergistically modulate the signaling functions of CaSR.

In summary, we have predicted and confirmed experimentally a calcium-binding site in the extracellular domain of mGluR1α. We have also shed new light on the co-activation of mGluR1α by Glu and [Ca²⁺]_o. These findings provide novel perspectives on mGluR1α, which may be viewed as capable of integrating information from two very different types of ligands (an amino acid neurotransmitter and a divalent cation). The levels of [Ca²⁺]_o in the brain are highly dynamic (37), and the affinity constants that we have determined in our studies on calcium binding to mGluR1α are well within the dynamic, physiological range of [Ca²⁺]_o in the brain. For family 3 GPCRs other than CaSR, the physiological importance of [Ca²⁺]_o binding has been uncertain; but the findings reported here may be useful in resolving this mystery by allowing for the development of knock-in mutations to mGluR1α, and resultant mouse models, that disrupt the ability of the receptor to bind [Ca²⁺]_o while leaving Glu binding intact. Moreover, because many of the key calcium-binding residues defined in our studies are conserved for other family 3 GPCRs, our findings may have relevance for a host of other receptors beyond just mGluR1α (6, 7). Family 3 GPCRs have tremendous potential as therapeutic targets, and therefore the advances described here may facilitate the development of novel family 3 GPCR-targeted drugs for use in the treatment of many different diseases.