Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α*

Background: Extracellular Ca2+ alters mGluR1α activity but by an unknown mechanism. Results: Mutations in predicted Ca2+-binding sites modulated the potency of both orthosteric and allosteric modulators. Conclusion: Ca2+ binding exerts multiple types of effects on mGluR1α. Significance: Improved knowledge of the mechanisms underlying the actions of Ca2+ on mGluR1α activity could facilitate development of isoform-selective drugs and/or suggest ways to tune the actions of available drugs. Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca2+ enhanced mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca2+ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.

Metabotropic glutamate receptor 1␣ (mGluR1␣), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1␣ is also modulated by extracellular Ca 2؉ . However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca 2؉ -binding site(s) on mGluR1␣ may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca 2؉ -binding site in the hinge region of mGluR1␣ is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca 2؉ enhanced mGluR1␣-mediated intracellular Ca 2؉ responses evoked by the orthosteric agonist L-quisqualate. Conversely, extracellular Ca 2؉ diminished the inhibitory effect of the mGluR1␣ orthosteric antagonist (S)-␣-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1acarboxylate ethyl ester) allosteric modulators of mGluR1␣ potentiated and inhibited responses to extracellular Ca 2؉ , respectively, in a manner similar to their effects on the response of mGluR1␣ to glutamate. Mutations at residues predicted to be involved in Ca 2؉ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist L-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca 2؉ . These studies reveal that binding of extracellular Ca 2؉ to the predicted Ca 2؉binding site in the extracellular domain of mGluR1␣ modu-lates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1␣.
The eight subtypes of metabotropic glutamate receptors (mGluRs) 2 belong to family C of the G protein-coupled receptors (GPCRs) and possess a large extracellular domain (ECD), a transmembrane domain (TMD), and a cytosolic C-terminal tail. The mGluRs are widely expressed in the central nervous system and play critical roles in regulating neuronal excitability and synaptic plasticity at both excitatory and inhibitory synapses (1). Extensive structural studies have revealed that the endogenous agonist L-glutamate (L-Glu), the major excitatory neurotransmitter in the central nervous system, binds at the hinge region of the ECD within the Venus fly trap motif of the receptor to activate the protein. This subsequently stimulates phospholipase C and leads to accumulation of inositol trisphosphate and an increase of intracellular calcium concentration ([Ca 2ϩ ] i ) (2)(3)(4).
In recent years, mGluRs have received increasing interest as potential drug targets for the treatment of a range of psychiatric and neurological diseases (5) (see Fig. 1). The ligands targeting mGluRs can be classified as orthosteric agonists and antagonists as well as allosteric modulators. Orthosteric agonists and antagonists induce and attenuate, respectively, the activity of the receptor by competitively binding to the L-Glu-binding pocket. L-Quisqualate (L-Quis), the most potent agonist of mGluR1 reported to date (6,7), has been speculated to share nearly the same binding pocket as L-Glu (8,9). In contrast, (S)-MCPG is an analog of L-Glu and is a non-selective competitive antagonist that has been shown to occupy the L-Glu-binding pocket, thereby blocking the function of group I/II members in the mGluR family (10). On the other hand, allosteric modulators bind to sites other than the orthosteric center to affect the activity of the receptor. Ro 67-4853 is a positive allosteric modulator (PAM) of mGluR1 that enhances the potency of L-Glu by interacting with the TMD of the receptor. CPCCOEt is a negative allosteric modulator (NAM) that inhibits the activation of mGluR1 by L-Glu by specifically binding to a site that involves the third extracellular loop of mGluR1␣ (11).
Like other members of the family C GPCRs, such as the calcium-sensing receptor, mGluR1␣ senses [Ca 2ϩ ] o using the extracellular domain (12,13). By transient expression of mGluR1␣ in oocytes, Kubo et al. (4) demonstrated that mGluR1-mediated activation of Ca 2ϩ -activated Cl Ϫ channels is modulated by [Ca 2ϩ ] o in addition to L-Glu. Purkinje cells from mGluR1 knock-out mice lose sensitivity to [Ca 2ϩ ] o , and this sensitivity to [Ca 2ϩ ] o was restored after mGluR1 was genetically reintroduced into the mice (14). There are sparse reports of [Ca 2ϩ ] o affecting the action of various classes of compounds acting on mGluRs (15). However, it is not clear how [Ca 2ϩ ] o is able to modulate the activity of mGluR1 or the actions of various mGluR1 ligands, and no Ca 2ϩ -binding sites have been identified in the 15 structures solved by x-ray crystallography to date (Protein Data Bank).
Using our recently developed computational algorithm, we identified a novel potential [Ca 2ϩ ] o -binding site within the hinge region of the ECD of mGluR1␣ adjacent to the reported L-Glu-binding site (16,17). It comprises Asp-318, Glu-325, Asp-322, and the carboxylate side chain of the natural agonist L-Glu. The carboxylate side chains of both L-Glu and Asp-318 are involved in both L-Glu and [Ca 2ϩ ] o binding. Our previous mutagenesis study indicated that binding of L-Glu and Ca 2ϩ to their distinct but partially overlapping binding sites synergistically modulates mGluR1␣-mediated activation of [Ca 2ϩ ] i signaling. Mutating the L-Glu-binding site completely abolished L-Glu signaling but left its Ca 2ϩ -sensing capability largely intact. Mutating predicted Ca 2ϩ -binding residues not only abolished or significantly reduced the sensitivity of mGluR1␣ to [Ca 2ϩ ] o but also in some cases to L-Glu (18).
In the present study, we first demonstrated that our predicted Ca 2ϩ -binding site is adjacent to the orthosteric agonist and antagonist interaction sites. We then examined the role of [Ca 2ϩ ] o in modulating the actions of different orthosteric ligands acting on mGluR1␣, including L-Quis and (S)-MCPG as well as reciprocal interactions between Ca 2ϩ and the mGluR1 allosteric modulators Ro 67-4853 and CPCCOEt. Our results suggest that [Ca 2ϩ ] o modulates the sensitivity of mGluR1␣ to not only orthosteric agonists and antagonists but also to allosteric modulators likely by interacting with the predicted [Ca 2ϩ ] o -binding site in the ECD of the receptor.

EXPERIMENTAL PROCEDURES
Docking L-Quis to ECD-mGluR1␣ Using AutoDock Vina and Hinge Motion Analysis-To elucidate binding of L-Quis to the ECD of mGluR1␣, L-Quis was docked into the crystal structure (Protein Data Bank 1EWK). After removing the coordinates of the bound endogenous ligand, L-Glu, the Protein Data Bank file was loaded into AutoDock tools to add polar hydrogen atoms and choose the docking center and grid box. The docking work was carried out by the AutoDock tool Vina (Scripps). The binding residues were analyzed by measuring the atoms within 6 Å of L-Quis. The L-Glu-and the (S)-MCPG-binding sites within the hinge region were analyzed using Dymdon.
Molecular Dynamics Simulation and Correlation Analysis Using AMBER-The initial coordinates for all the simulations were taken from a 2.20-Å resolution x-ray crystal structure (Protein Data Bank code 1EWK; Ref. 19). The AMBER 10 suite of programs (20) was used to carry out all of the simulations in an explicit TIP3P (transferable intermolecular potential 3P) water model (21) using the modified version of the all-atom Cornell et al. (22) force field and the reoptimized dihedral parameters for the peptide -bond (23). The crystal structure contains only Glu substrate. Ca 2ϩ ion was placed at the suggested Ca 2ϩ -binding site that is defined by residues Asp-318, Asp-322, and Glu-325. An initial 2-ns simulation was performed using NOE restraint during the equilibration to reorient the side chain residues in the Ca 2ϩ -binding site, but no restraints were used during the actual simulation. A total of four molecular dynamics simulations were carried out for 50 ns each on wild type and three mutant mGluRs. The mutations were D318I, D322I, and E325I. First, our structures were minimized to achieve the lowest energy conformation in each complex. The structures were then equilibrated for 2 ns, starting the molecular dynamics simulations from the equilibrated structures. During the simulations, an integration time step of 0.002 ps was used to solve Newton's equation of motion. The long range electrostatic interactions were calculated using the particle mesh Ewald method (24), and a cutoff of 9.0 Å was applied for non-bonded interactions. All bonds involving hydrogen atoms were restrained using the SHAKE algorithm (25). The simulations were carried out at a temperature of 300 K and a pressure of 1 bar. A Langevin thermostat was used to regulate the temperature with a collision frequency of 1.0 ps Ϫ1 . The trajectories were saved every 500 steps (1 ps). The trajectories were then analyzed using the ptraj module in AMBER 10.
Constructs, Site-directed Mutagenesis, and Expression of mGluR1␣ Variants-The red fluorescent protein mCherry was genetically tagged to the C terminus of mGluR1␣ by a flexible linker, GGNSGG (18). Point mutations were introduced using a site-directed mutagenesis kit (Stratagene). HEK293 cells were seeded and cultured on glass coverslips. mGluR1␣ and its mutants were transfected into cells utilizing Lipofectamine 2000 (Invitrogen). The cells were then incubated for an additional 2 days so that mGluR1␣ and its mutants were expressed at sufficient levels for study. Cells were fixed on the coverslips with 4% formaldehyde, and nuclei were stained with DAPI. The expression of mGluR1␣ and its variants was detected by measuring red fluorescence using confocal microscopy at 587 nm.
Determining the Effect of [Ca 2ϩ ] o on Activation of mGluR1␣ and Its Mutants by L-Quis-Measurement of [Ca 2ϩ ] i was performed as described (13). In brief, wild type mGluR1␣ was transiently transfected into the cells and cultured for an additional 2 days. The cells on the coverslips were subsequently loaded using 4 M Fura-2 AM in 2 ml of physiological saline buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 0.55 mM MgCl 2 , and 1 mM CaCl 2 , pH 7.4) for 30 min. The coverslips were then mounted in a bathing chamber on the stage of a fluorescence microscope at room temperature. Fura-2 emission signals at 510 nm from single cells excited at 340 or 380 nm were collected utilizing a Leica DM6000 fluorescence microscope in real time as the concentration of L-Quis was progressively increased in the presence or absence of [Ca 2ϩ ] o . The ratio of fluorescence emitted at 510 nm resulting from excitation at 340 or 380 nm was further analyzed to obtain the [Ca 2ϩ ] i response as a function of changes in L-Quis. Only the individual cells expressing mCherry were selected for analysis. Determining the Effects of [Ca 2ϩ ] o on the Potency of Ro 67-4853 on mGluR1␣-Fura-2 AM was used for monitoring [Ca 2ϩ ] i in real time as described above. Ro 67-4853 did not potentiate mGluR1␣ in the absence of L-Glu (26,27). To obtain the [Ca 2ϩ ] i readout, HEK293 cells expressing mGluR1␣ were preincubated with 0.5 mM Ca 2ϩ and 5 nM Ro 67-4853 for at least 10 min. Cells loaded with Fura-2 AM were mounted onto a chamber perfused with saline buffer. The concentration of Ro 67-4853 was increased stepwise in the presence of 0. 5  Binding to mGluR1␣ and Its Mutants-HEK293 cells transiently transfected with wild type mGluR1␣ or its mutants were maintained in a 5% CO 2 37°C incubator for an additional 48 h as before. Cells were then collected in ice-cold hypotonic buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 5 mM KCl, 0.5 mM EDTA, and 1% protease inhibitors at pH 7.0 -7.5). The cell pellet was washed twice more using hypotonic buffer to remove the L-Glu in the cellular debris. The crude membrane protein (100 g) was mixed with 30 nM L-[ 3 H]Quis in 100 l of hypotonic buffer. The nonspecific binding was determined by measuring bound

Predicted [Ca 2ϩ ] o -binding Site Is Adjacent to Orthosteric
Agonist and Antagonist-binding Sites-Using our recently developed computational algorithms, we have identified a novel potential Ca 2ϩ -binding site at the hinge region of the ECD of mGluR1␣ (18). Fig. 1 shows that the predicted Ca 2ϩbinding site comprises Asp-318, Glu-325, Asp-322, and the carboxylate side chain of the natural agonist L-Glu in the hinge region in the ECD of mGluR1␣ adjacent to the reported L-Glubinding site. Asp-318 is involved in both L-Glu and Ca 2ϩ binding (18).
Using the crystal structure (Protein Data Bank code 1EWK; closed-open form) of the ECD of the receptor and the AutoDock Vina program, we modeled the binding site for the orthosteric agonist L-Quis. As shown in Fig. 1B, the docked binding site of the agonist L-Quis corresponds well with the L-Glu-binding residues previously suggested by the crystal structure. Our predicted Ca 2ϩ -binding site is also adjacent to the L-Quis pocket and interacts with L-Quis similarly to L-Glu (Fig. 1B). In the reported crystal structure of mGluR1 complexed with an orthosteric antagonist, (S)-MCPG (Protein Data Bank code 1ISS), (S)-MCPG interacts with Tyr-74, Trp-110, Ser-165, Thr-188, and Lys-409 in lobe 1 and Asp-208, Tyr-236, and Asp-318 in lobe 2 ( Fig. 1B) (10). It shares with L-Glu most of the residues of the L-Glu-binding pocket (10) and is also adjacent to our predicted Ca 2ϩ -binding site.
We next performed molecular dynamics simulations to reveal any possible interaction between our predicted [Ca 2ϩ ] obinding site and the orthosteric ligand-binding site. Residues involved in the [Ca 2ϩ ] o -binding pocket, such as Asp-318, Asp-322, and Glu-325, have strong correlated motions as expected given their roles as [Ca 2ϩ ] o -binding ligands. In addition, residues Asp-318 and Arg-323 residing within the same loop as the predicted Ca 2ϩ -binding site are also concurrently correlated. As shown in Fig. 2, most of the critical L-Glu-binding residues, including Trp-110, Ser-165, Thr-188, Asp-208, Tyr-236, Asp-318, and Arg-323, are well correlated to the [Ca 2ϩ ] o -binding site (Asp-318, Asp-322, and Glu-325). However, mutations at the charged residues involved in [Ca 2ϩ ] o binding, such as D318I and E325I, markedly attenuated the correlation of the Ca 2ϩbinding site with the L-Glu-binding pocket. The Ca 2ϩ -binding site in mutant D318I only correlates with Gly-293 and Asp-208, and mutant D325I only correlates with Tyr-236 and Gly-293. The mutant D322I also exhibited impaired correlation between the [Ca 2ϩ ] o -binding site and L-Glu-binding site but to a lesser degree. As shown in Table 1, Asp-318 in the [Ca 2ϩ ] o -binding site still correlates with four residues in the L-Glu-binding pocket (Fig. 2). Similarly, residues that are involved in binding L-Quis and (S)-MCPG also correlate well with residues involved in the predicted [Ca 2ϩ ] o -binding site. Results from these analyses and our previous studies on the effect of binding of [Ca 2ϩ ] o to its site on L-Glu-mediated activation of mGluR1 led us to hypothesize that [Ca 2ϩ ] o regulates the effects of orthosteric ligands on mGluR1␣.

Ca 2ϩ Enhances Sensitivity of Activation of mGluR1␣ by L-Quis by Increasing L-[ 3 H]Quis Binding via Interaction with the [Ca 2ϩ ] o -binding Site of the Receptor-To test the effect of [Ca 2ϩ
] o on the activation of mGluR1␣ by the orthosteric agonist L-Quis, we performed a single cell fluorescence imaging assay by measuring changes in [Ca 2ϩ ] i using HEK293 cells tran-siently transfected with mGluR1␣ and loaded with Fura-2. To eliminate any potential effect of trace L-Glu secreted from cells, experiments were conducted using continuous superfusion of cells with an L-Glu-free buffer. Fig. 3, A-D, show that L-Quis induced intracellular calcium responses mediated by mGluR1 in a manner similar to the activation of the receptor by L-Glu. [Ca 2ϩ ] o behaved as a PAM of the L-Quis response and induced a leftward shift in the L-Quis concentration-response curve for activation of mGluR1a (Fig. 3, A-D). In the absence of [Ca 2ϩ ] o (Ca 2ϩ -free buffer with less than 2 M calcium), the EC 50 for the activation of mGluR1a by L-Quis is 12.8 nM. The addition of 1.8  Table 2). Importantly, this mutation significantly reduced the [Ca 2ϩ ] o -mediated enhancement in potency for L-Quis from 4.6-to 1.6-fold in 1.8 mM [Ca 2ϩ ] o , although both the potency and efficacy of L-Quis-mediated activation of the E325I mutant were still enhanced rela-tive to WT mGluR1 (Fig. 3, A-D). As L-Glu could potentially serve as a ligand for binding of Ca 2ϩ to its pocket, L-Glu or L-Quis binding could rescue the mutated Ca 2ϩ -binding pocket, thus enhancing the Ca 2ϩ sensitivity of the mutant. On the other hand, mutant D322I exhibited WT-like behavior in its response to L-Quis both in the absence and presence of [Ca 2ϩ ] o (Fig. 3, A-D, and Table 2), consistent with Asp-322 contributing to [Ca 2ϩ ] o binding to a lesser degree with only its main chain oxygen serving as a ligand atom. We also observed WT-like modulation of the L-Glu response of D332I by Ca 2ϩ (18). These        Table  4). The maximal response was also significantly decreased by 40 M CPCCOEt, although the maximal response with 5 M CPCCOEt was still comparable. This indicates that 30 mM [Ca 2ϩ ] o cannot completely reverse the antagonism induced by CPCCOEt, and thus the inhibitory effects of CPCCOEt on the response of mGluR1␣ to [Ca 2ϩ ] o appear to be non-competitive ( Fig. 5B and Table 4).

The mGluR1␣ PAM Ro 67-4853 Potentiates Activation of mGluR1 by [Ca 2ϩ ] o -The finding that CPCCOEt inhibited activation of mGluR1 by [Ca 2ϩ ] o suggests that the CPCCOEt site in the transmembrane-spanning domain of mGluR1 and the [Ca 2ϩ
] o -binding site in the ECD of the receptor interact in a manner similar to the interactions between the orthosteric L-Glu-binding site and the allosteric CPCCOEt site. We performed analogous experiments to determine whether the mGluR1 PAM Ro 67-4853, which binds to the extracellular loops of the TMDs of mGluR1␣ (2, 29) (Fig. 1B), can also potentiate responses to [Ca 2ϩ ] o . Fig. 6A shows that L-Glu-induced activation of WT mGluR1␣ was enhanced by the addition of 10 or 100 nM Ro 67-4853 using single cell [Ca 2ϩ ] i imaging. We then examined the effects of Ro 67-4853 on the [Ca 2ϩ ] o sensitivity of wild type mGluR1␣ in the absence of L-Glu. Fig. 6B shows that both 30

TABLE 3 Addition of 0.5 mM (S)-MCPG decreases the responses of mGluR1␣ to [Ca 2؉ ] o and L-Glu
The [Ca 2ϩ ] i response to [Ca 2ϩ ] o and L-Glu in the absence or presence of 0.5 mM (S)-MCPG were obtained by measuring the ratiometric change of Fura-2 AM fluorescence.

Response to [Ca 2؉ ] o
Response to L-Glu  6B and Table 5).
To further evaluate the effect of Ro 67-4853 on mGluR1␣, HEK293 cells transiently expressing mGluR1␣ were preincubated with 0.5 mM Ca 2ϩ and 5 nM Ro 67-4853 for up to 10 min, and then the responses to multiple concentrations of Ro 67-4853 were tested. In the presence of 0.5 mM [Ca 2ϩ ] o , Ro 67-4853 enhanced L-Glu-induced mGluR1␣ activity in a concentration-dependent manner. Increasing [Ca 2ϩ ] o to 1.8 mM significantly increased the potency of a low dosage of Ro 67-4853 for mGluR1␣ (p Ͻ 0.05) (Fig. 6C). At the same time, the EC 50 value decreased from 20.7 to 10.0 nM (Fig. 6C and Table 5). Interestingly, [Ca 2ϩ ] i oscillations were observed when the cells were treated with Ro 67-4853 (data not shown). Similar to the Ca 2ϩ -sensing receptor, three different patterns of response were noted (30) Fig. 1), we then performed studies using an mGluR variant with a key [Ca 2ϩ ] o -binding ligand residue mutated, E325I. Fig. 1B shows that Glu-325 is not directly involved in L-Glu binding, and variant E325I is able to sense L-Glu in a manner similar to WT (18). Fig. 7A shows that addition of 30 M L-Glu enhanced the responsiveness of E325I to Ro 67-4853. Of note, Fig. 7B shows that E325I responded to 10 M Ro 67-4853 in the absence of L-Glu in [Ca 2ϩ ] o -free saline. Increasing [Ca 2ϩ ] o from 0.5 to 1.8 mM did not affect the sensitivity of E325I to Ro 67-4853, but elevating [Ca 2ϩ ] o increased the responses of WT mGluR1␣ to 300 nM Ro 67-4853 (Fig. 7B). This suggests that mutating the Ca 2ϩ -binding site (E325I) eliminates the effect of Ca 2ϩ on Ro 67-4853 but not on WT mGluR1␣. To determine whether the receptors were saturated by Ro 67-4853, higher concentrations of the PAM were applied to both WT mGluR1 and E325I. As shown in Fig. 7B, higher concentrations of Ro 67-4853 increased the responses of both WT mGluR1 and E325I. This result suggests that [Ca 2ϩ ] o binding at its predicted site in the hinge region is essential for the positive allosteric action of this modulator.

DISCUSSION
In this study, we demonstrated that [Ca 2ϩ ] o had significant modulating effects on the actions of various orthosteric and allosteric ligands on mGuR1a as assessed using a functional readout (i.e. [Ca 2ϩ ] i responses) in receptor-transfected HEK293 cells.
[Ca 2ϩ ] o exerted several different effects on the compounds studied here, including the orthosteric agonist L-Quis, the orthosteric antagonist (S)-MCPG, and allosteric modulators, e.g. the PAM Ro 67-4853 and the NAM CPCCOEt.
As shown in Fig. 1, the predicted [Ca 2ϩ ] o -binding site partially overlaps the predicted orthosteric binding site for the agonist L-Quis and the antagonist (S)-MCPG. We have previously    (18). However, activation of GPCRs is also known to induce Ca 2ϩ influx through store-operated Ca 2ϩ entry channels (31,32). By utilizing Gd 3ϩ , an inhibitor of these Ca 2ϩ channels, we noted that mGluR1a still could induce an increase in [Ca 2ϩ ] i (18 (Fig. 3). ] o -binding site with the adjacent binding site for orthosteric agonists and antagonists. We first showed that the L-Quis-binding pocket predicted here using AutoDock Vina overlaps extensively with the L-Glu-binding pocket in the reported crystal structure ( Table 6). The side chain of Asp-318 is involved in both [Ca 2ϩ ] o and agonist binding. In our earlier study, the   (18). In this study, it also completely eliminated L-Quis-mediated activation of mGluR1 (Fig. 3E). This finding is supported by a previous report that the mutants T188A, D208A, Y236A, and D318A abolished the sensitivity of the receptor to both L-Quis and L-Glu, whereas the mutants R78E and R78L exhibited clearly impaired L-Quis binding (8,9). The key residue Glu-325 is involved in [Ca 2ϩ ] o binding, and the mutant E325I indeed significantly impaired both the [Ca 2ϩ ] o and L-Glu sensitivity of the receptor (Fig. 3). On the other hand, variant D322I produced less reduction of the modulatory effects of [Ca 2ϩ ] o on both L-Quis and L-Glu agonist action, which is consistent with its lesser role in [Ca 2ϩ ] o binding with a contribution of only a main chain ligand atom (Fig. 1).   Our observed effect of [Ca 2ϩ ] o on responses to orthosteric agonists and antagonists of mGluR1 is consistent with molecular dynamics simulation studies performed here on the correlated motions of the hinge region in the ECD of mGluR ( Fig. 2 and Table 1). We observed a strong correlation among residues in the predicted [Ca 2ϩ ] o -binding site and residues involved in the orthosteric binding sites shared by L-Glu, L-Quis, and (S)-MCPG. Interestingly, mutation of the [Ca 2ϩ ] o -binding site largely removed this correlation. Fig. 1A shows that the predicted [Ca 2ϩ ] o -binding site at the hinge region is conserved in the group I mGluRs, e.g. mGluR1 and mGluR5 (18) (12,13,38,39). In recent years, increasing numbers of family C GPCRs have been found to exhibit synergistic modulation of the primary orthosteric agonist by allosteric modulators. Sweet enhancers binding to the hinge region of the human taste receptor are known to stabilize the active form of the receptor, thus leading to altered perception of sweet taste, whereas IMP and L-Glu also synergistically activate the umami taste receptor (40,41). It is also interesting to note that an allosteric ligand suggested to act at the ECD domain of mGluR is located at the hinge region (42,43). Thus, our work has strong implications for the role of the hinge region of the ECD in modulating action of small molecule ligands on family C GPCRs.
As for allosteric modulators targeting the TMDs, the binding sites of positive and negative modulators of mGluR1␣ are distinct (44). These allosteric modulators effectively modulate receptor activation by L-Glu, but little is known about the effects of the endogenous mineral ion Ca 2ϩ on these modulators. In this study, the effects of [Ca 2ϩ ] o on CPCCOEt (NAM) and Ro 67-4853 (PAM) were further assessed.
The non-competitive NAM CPCCOEt is known to inhibit the L-Glu response by binding to Thr-815 and Ala-818 on the seventh transmembrane helix (45,46). Our data shown in Fig. 5 support the contention that CPCCOEt, acting as a non-competitive inhibitor, also can diminish the [Ca 2ϩ ] i response of mGluR1␣. Interestingly, increasing [Ca 2ϩ ] o restored the [Ca 2ϩ ] o sensitivity of the receptor. CPCCOEt not only inhibits proliferation of melanoma cells but also reverses morphine tolerance (47,48). Thus, the findings in this study indicate that a novel drug targeting the [Ca 2ϩ ] o -binding site in mGluR1 has the potential to tune the therapeutic effect of CPCCOEt on melanoma or addiction. Val-757 in the TMD was revealed to be critical to the activation of mGluR1 by the PAMs (27,44 (Fig. 7). PAMs binding to the TMDs have been shown to enhance L-Quis binding to mGluR1␣ (27). It is possible that the incomplete reduction in the inhibitory effect of MCPG by [Ca 2ϩ ] o is due to an additional synergistic effect involving the TMD region of the receptor. By tagging the FRET pair YFP/cyan fluorescent protein to the two intracellular loops 2 (i2) of the dimeric mGluR1␣, Tateyama et al. (49) observed that the rearrangement of the TMD induced by L-Glu was reversed by (S)-MCPG. Such an integrated effect of the TMD with the ECD region is further supported by studies of mGluRs with deletions of the Venus fly trap. It was found that PAMs not only potentiate the action of agonists on the full-length receptors but sometimes can display strong agonist activity on Venus fly trap-truncated receptors (50,51). The Venus fly traps of the ECDs are not only responsible for agonist-induced activation but also prevent PAMs from activating the full-length receptor (50,51). Taken together, our study reveals that [Ca 2ϩ ] o binding at the hinge region is likely to be responsible for its capacity to modulate action of other allosteric modulators.  Tables 4 and  5). Over the past decade, many new PAMs and NAMs for various receptors have been developed, and the potential exists for developing allosteric modulators with greater subtype specificity than is possible for orthosteric agonists (52). The co-activation induced by endogenous agonists and PAMs binding to the hinge regions of receptors could be a common feature of family C GPCRs. These data provide further insight into the modulation of mGluR1␣ by [Ca 2ϩ ] o and suggest that [Ca 2ϩ ] o has the potential to modulate the profile of a variety of agents acting on mGluR1␣, including agonists, antagonists, and allosteric modulators.
In conclusion, we investigated the effects of [Ca 2ϩ ] o on the modulation of mGluR1␣ by orthosteric agonists and an orthosteric antagonist as well as by a PAM and NAM and found that [Ca 2ϩ ] o enhanced the actions of agonists and PAMs but attenuated the actions of antagonists and NAMs. These findings provide new insights into the targeting of mGluR1␣ by different classes of ligands. In addition to the specific relevance of these findings for understanding the nature of allosteric modulation of mGluR1␣, they may also have general relevance for understanding the modulation of family C GPCRs by extracellular ions, such as Ca 2ϩ .