Structural Basis for a Ca2+-Sensing Function of the Metabotropic Glutamate Receptors

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Science  13 Mar 1998:
Vol. 279, Issue 5357, pp. 1722-1725
DOI: 10.1126/science.279.5357.1722


The metabotropic glutamate receptors (mGluRs) are widely distributed in the brain and play important roles in synaptic plasticity. Here it is shown that some types of mGluRs are activated not only by glutamate but also by extracellular Ca2+(Ca2+ o). A single amino acid residue was found to determine the sensitivity of mGluRs to Ca2+ o. One of the receptors, mGluR1α, but not its point mutant with reduced sensitivity to Ca2+ o, caused morphological changes when transfected into mammalian cells. Thus, the sensing of Ca2+ o by mGluRs may be important in cells under physiological condition.

The mGluRs are a family of heterotrimeric guanosine triphosphate–binding protein (G protein)–coupled receptors (1, 2) that exist in various regions of the brain (3) and are known to play an important role in synaptic plasticity (4). Because of the structural similarity of mGluRs and the Ca2+ osensor of the parathyroid (2, 5, 6), we tested whether mGluRs function as Ca2+ sensors. We previously showed that a mGluR1 homolog isolated from the salmon brain can be activated not only by glutamate but also by Ca2+ o(7). Here we investigated whether rat mGluR1α and mGluR5 (which couple to Gq) and mGluR2 and mGluR3 (which couple to Gi) are activated by Ca2+ o.

The responses of mGluRs 1α and 5 to Ca2+ owere examined by monitoring the increase in Ca2+-activated Cl (Ca2+-Cl) current in aXenopus laevis oocyte expression system (8). The oocytes were bathed in Ca2+-free saline solution, and then the Ca2+ o concentration ([Ca2+]o) was increased while depolarizing pulses were applied. Oocytes expressing either mGluR1α or mGluR5 responded to 2 mM Ca2+, whereas noninjected oocytes did not (Fig. 1A). The response increased steeply in the physiological range of [Ca2+]o (Fig.1A), similarly to the response of the Ca2+ receptor (CaR) (5, 6, 9). When [Ca2+]o was raised, we also detected increases of the inositol trisphosphate (IP3) concentration in oocytes expressing mGluR1α (7) and of the intracellular concentration of Ca2+ ([Ca2+]i) in transfected CHO cells by Fluo-3 imaging (10). Because the CaR is activated not only by Ca2+ but also by various polyvalent cations (5), we examined the effects of polyvalent cations on mGluRs 1α and 5. Both were activated by polyvalent cations (Fig. 1B). Similar to the CaR (5), trivalent cations such as Gd3+ (Fig. 1B) were more effective than Ca2+ in activating mGluRs 1α and 5. Divalent cations other than Ca2+ (Fig. 1B) were less effective. Thus, mGluR1α and mGluR5 sense Ca2+ o and polyvalent cations as does the CaR.

Figure 1

Functional characterization of mGluRs 1α, 5, 2, and 3 as sensors for Ca2+ o. The activation of mGluRs 1α and 5 was monitored by an increase in Ca2+-Cl current (A and B) and that of mGluRs 2 and 3 was monitored by an increase in GIRK1/2 current (C) or by a decrease in cAMP concentration (D). (A) (Upper) Current traces of mGluR1α- or mGluR5-expressing oocytes or noninjected control oocytes before and after application of 2 mM Ca2+ o (8). (Lower) The time course of the response is shown by plotting the current amplitudes at the end of the depolarizing pulses against time. Arrows indicate 2 mM Ca2+ application. (Inset) The dose-response relation. Then values of each point were 3 to 7. Noninjected oocytes also showed a small, gradual increase in Ca2+-Cl current when high concentrations of Ca2+ o were applied, which could be due to the leak of Ca2+ into the oocytes. (B) Current traces before and after application of 1 mM Gd3+, Tb3+, or La3+ or 10 mM Mn2+, Mg2+, or Ba2+ to oocytes expressing mGluR1α or mGluR5. (C) Current traces recorded at –120 mV from oocytes expressing mGluR2 and GIRK1/2, mGluR3 and GIRK1/2, or GIRK1/2 alone. Either 40 μM glutamate (glu) or 5 mM Ca2+ o was applied at the times indicated by the bars. (D) Effect of glutamate or Ca2+ o to the cAMP concentration in CHO cell lines stably transfected with vector alone, mGluR2 cDNA, or mGluR3 cDNA (18). The cAMP concentrations were normalized by the concentration in the absence of glutamate or Ca2+ o. The error bars indicate standard deviation (n = 3).

The responses of the Gi-coupled mGluRs 2 and 3 were monitored by an increase in inward current of the coexpressed G protein–coupled, inwardly rectifying K+ channel, GIRK1/2 (11), in Xenopus oocytes (8). mGluR3 was activated by Ca2+ as well as glutamate, whereas mGluR2 showed no response to 5 mM Ca2+ o (Fig.1C). The response of mGluR3 to Ca2+ o varied between batches of oocytes. A response was detected in three out of nine batches, and in the three positive batches, the responses were observed in three out of five, two out of five, and two out of three oocytes. These results suggest that the link between Ca2+ o and activation of GIRK1/2 current by mGluR3 requires additional endogenous conditions. Even in the positive cells, an accurate dose-response analysis was not possible because the amplitude of the responses could not be determined accurately as a result of the decrease in the basal current level after the agonist was removed. The possible link of Ca2+ o stimulation with Gi was also confirmed as a decrease in the adenosine 3′,5′-monophosphate (cAMP) concentration by Ca2+ o in mGluR3-transfected CHO cells but not in mGluR2-transfected cells (Fig. 1D).

To make a quantitative comparison of the Ca2+ o-sensing properties of mGluRs 1α, 2, and 3, we assayed the activity of the same effector. Chimeric molecules were constructed in which the entire NH2-terminal extracellular domain of mGluR2 or mGluR3 was substituted for that of mGluR1α [R2(N)-R1 and R3(N)-R1, respectively] (12). R2(N)-R1 and R3(N)-R1 chimeras were expected to link to Gqproteins and activate Ca2+-Cl currents similar to mGluR1α. The sensitivity of these molecules to glutamate or Ca2+ o was compared (Fig.2) (8). The median effective concentration (EC50) values of the dose-response relation to glutamate did not differ significantly from each other. In contrast, the EC50 value of R2(N)-R1 to Ca2+ o (36.0 mM) was significantly higher than those of mGluR1α (4.7 mM) and R3(N)-R1 (2.2 mM) (Fig. 2B). Thus, mGluR1α and mGluR3 appear to be more sensitive to Ca2+ o than is mGluR2, and the structural basis of the Ca2+ o sensitivity would be predicted to lie in the NH2-terminal extracellular domain.

Figure 2

Comparison of the responses of R1(N)-R1, R2(N)-R1, and R3(N)-R1 chimeras to glutamate or Ca2+ o. (A) Examples of responses of R1(N)-R1, R2(N)-R1, and R3(N)-R1 to 10 μM glutamate (Glu) (left) or to 10 mM Ca2+ o (right). (B) Comparison of the dose-response relations of R1(N)-R1, R2(N)-R1, and R3(N)-R1 to glutamate (left) or Ca2+ o(right). The n values of each point are 3 to 7. The EC50values of the fittings were as follows: glutamate (R1, 4.6 μM; R2(N)-R1, 6.4 μM; R3(N)-R1, 7.2 μM) and Ca2+ o (R1, 4.7 mM; R2(N)-R1, 36.0 mM; R3(N)-R1, 2.2 mM). Three sets of data in each plot were obtained from the same batch of oocytes.

To identify the structural determinant of Ca2+ osensitivity within the NH2-terminal extracellular region, we tested whether the Ca2+ o-sensing site overlapped with the glutamate-binding site (8). After exposure to the saturating dose of glutamate (or Ca2+ o), mGluR1α still responded to Ca2+ o (or glutamate) (Fig.3A). Thus, there was no strong cross-desensitization, suggesting that Ca2+ oand glutamate are not recognized in an identical manner by mGluR1α. The response of mGluR1α to 5 mM Ca2+ o was partially blocked by 0.5 mM t-MCPG (S-α-methyl-4-carboxyphenylglycine), which inhibited the response to glutamate of the equivalent dose (5 μM) almost completely (Fig. 3B). Thus, the Ca2+ o-interacting site appears to be not identical to but to overlap with the glutamate-binding site.

Figure 3

Identification of the site responsible for the Ca2+ o-sensing function of mGluRs. (A) Effect of a saturating dose of glutamate (or Ca2+ o) to the response of mGluR1α to Ca2+ o (or glutamate). (B) Effect of t-MCPG on glutamate- or Ca2+ oinduced responses in oocytes expressing mGluR1α. The two traces on the left and right are from the same oocyte. A concentration of 0.5 mM t-MCPG, which almost completely blocked the response to 5 μM glutamate, partially blocked the response to an equivalent dose (5 mM) of Ca2+ o. (C) Alignments of the deduced amino acid sequences of mGluRs 2, 3, 1α, and 5 in the region adjacent to the glutamate-binding sites. The glutamate-binding sites, S165(R1) and T188(R1), are boxed, and the site that determines the sensitivity to Ca2+ o (S166 in R1) is shown by the arrow (19). (D) Comparison of the dose-response relations of the wild type (wt) (filled circles) and the point mutants (open triangles) of R1(N)-R1, R2(N)-R1, and R3(N)-R1. Sites shown by the arrow in (C) were mutated to D in R1 and R3, and to S in R2. The n value of each point was 3 to 7. The EC50 values of the fittings were as follows: glutamate (R1-wt, 22 μM; R1-mut, 32 μM; R2-wt, 9.6 μM; R2-mut, 6.9 μM; R3-wt, 9.1 μM; R3-mut, 8.4 μM) and Ca2+ o(R1-wt, 2.5 mM; R1-mut, 39 mM; R2-wt, 30 mM; R2-mut, 2.9 mM; R3-wt, 3.7 mM; R3-mut, 41 mM). Two sets of data in each plot are from the same batch of oocytes. The larger EC50 values for the glutamate response of wild-type mGluR1α and the mutant compared with the value in Fig. 2B was due to the difference in the batch of oocytes.

Structural and mutagenesis analyses previously revealed that the glutamate-binding sites of mGluR1α are located at Ser165and Thr188 (13). The amino acid sequences of mGluRs 1α, 2, 3, and 5 in the region adjacent to these sites were compared (Fig. 3C). Although mGluRs 2 and 3 exhibit high identity in this region, the residue next to the glutamate-binding site differs. The mGluRs 1α, 3, and 5 have a serine (S) at this position whereas mGluR2 has an aspartate (D). Thus, we hypothesized that this site determines the Ca2+ o sensitivity of mGluRs and examined the properties of the point mutants R1(N)-R1 (S166D), R2(N)-R1 (D146S), and R(3)-R1 (S152D) (8, 12). These mutations did not alter sensitivity to glutamate but did change the sensitivity to Ca2+ o (Fig. 3D). S166D of R1(N)-R1 and S152D of R3(N)-R1 showed much lower sensitivity to Ca2+ othan the wild type, and D146S of R2(N)-R1 had a high sensitivity to Ca2+ o (Fig. 3D). Thus, this site (S166 of mGluR1α) was critical for the determination of the sensitivity to Ca2+ o.

The above results were obtained by monitoring responses when [Ca2+]o was abruptly raised from 0 mM. It is important to know if the continuous stimulation by physiological concentrations of Ca2+ o through mGluR1α causes some intracellular events (that is, if the Ca2+ o-sensing function of mGluR1α plays a role under physiological conditions). We transfected CHO cells with wild-type mGluR1α or with the S166D mutant transiently (14) and compared the cell morphology (Fig.4A) and the alignment of actin filaments (Fig. 4B) (15). To analyze the cell morphology quantitatively, we classified the cells by a morphology index (the ratio of the long axis length to the short axis length). Control CHO cells mostly exhibited a square-shaped morphology, but the cells transfected with wild-type mGluR1α, identified by cotransfected green fluorescent protein (GFP), changed to a spindle or a bar shape (Fig. 4, A and C). The actin filaments of the transfected cells identified by antibody to mGluR1α (anti-mGluR1) were aligned in parallel (Fig. 4, B and C). In contrast, the morphological change of CHO cells transfected with the S166D mutant was much less (Fig. 4). When [Ca2+] in the culture medium was raised from 2 to 5 mM, the morphological change of the cells transfected with wild-type mGluR1α was more prominent, but control cells did not respond at all (10). Thus, the continuous stimulation of mGluR1α by 2 mM Ca2+in the medium caused the parallel alignments of the actin filaments and the morphological change of the transfected CHO cells, probably by means of an increase in second messenger activity. Indeed, the basal IP3 concentration in oocytes expressing mGluR1α in the frog saline solution was higher than in noninjected oocytes (7), and the cAMP concentration in CHO cells expressing mGluR1α was elevated not only by glutamate (16) but also by Ca2+ o (0 mM Ca2+ o, 100%; 2 mM, 148%; 10 mM, 180%), whereas cAMP in control cells was not elevated (0 mM Ca2+ o,100%; 2 mM, 77%, 10 mM, 96%).

Figure 4

Comparison of the morphology of control CHO cells and CHO cells transfected with wild-type mGluR1α (R1 wt) or with the S166D mutant (R1 S166D). (A) Observations of the living CHO cells transfected with pEGFP alone (left), wild type and pEGFP (middle), or S166D and pEGFP (right). The transfected cells were identified by the fluorescence of GFP (upper panels), and the morphology of the identified cells was observed with a phase-contrast microscope (lower panels). The scale bar in (B) indicates 100 μm for all photographs in (A) and (B). (B) Morphology of the actin filaments of CHO cells transfected with vector alone (left), wild-type mGluR1α (middle), or S166D (right). The cells transfected with mGluR1α were identified by staining with anti-mGluR1 (upper row, middle and right panels). The actin filaments were visualized by staining with FITC-phalloidin (lower panels). (C) Quantitative analysis of the cell morphology of the three groups in (B). The cells were classified into nine classes by the morphology index, and the number of cells of each class are shown.

What is the significance of the Ca2+-sensing function of mGluRs in the brain? As demonstrated above, it is possible that constant stimulation by Ca2+ o would cause an increase in the basal activity of the second messenger system such as IP3, protein kinase C, or cAMP in neurons expressing mGluR1α. This increase might play a role, for example, in the maintenance phase of long-term potentiation. Another possibility is that the dynamic fluctuation of [Ca2+]o in the synaptic cleft might stimulate mGluR1α effectively. The local [Ca2+]o in the narrow synaptic cleft is not constant but could fluctuate in the range between 0.8 and 1.5 mM (17). An increase of Ca2+ o from 0.5 mM (or 1 mM) to 1.5 mM caused responses in oocytes expressing mGluR1α (Fig. 5). It is possible that the activation of mGluR1α would be triggered by the fluctuation of local [Ca2+]o in the synaptic cleft.

Figure 5

Effect of [Ca2+ o] fluctuation on mGluR1α expressed in Xenopus oocytes. The [Ca2+ o] was changed in the range known to occur physiologically at the synaptic cleft (17).

Thus, mGluRs 1α, 5, and 3 but not mGluR2 are activated by Ca2+ o at physiological concentrations, and a single amino acid residue determines the sensitivity to Ca2+ o. Also, the Ca2+ o-sensing function of mGluR1α can play a role in generating morphological changes in transfected cells.

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