Introduction

Summary Introduction Procedures Results Discussion References

The recent cloning of eight subtypes of mGluR has not only opened up new avenues for exploration of the central actions of this excitatory neurotransmitter but also expanded the potential to target drugs against specific receptor-mediated actions (1-3). Recently, novel synthetic glutamate analogues have been developed as ligands at different mGluRs, and of particular significance has been the development of competitive antagonists (4-7) and their use in identifying the involvement and roles of different mGluR subtypes in fundamental mechanisms such as long term potentiation and long term depression (8-12).

The mGluRs form a distinct branch of the G protein-coupled receptor superfamily, sharing topological organization but little sequence homology with other G protein-coupled receptors. Sequence homology and pharmacological profiling have allowed three subgroups of mGluRs, termed I, II, and III, to be described (2, 3). Group II mGluRs (types 2 and 3) and group III MGluRs (types 4, 6, 7 and 8) both couple to G proteins of the G_i/o family to inhibit adenylyl cyclase or modulate ion channel activities (2, 3); in contrast, group I mGluRs (types 1 and 5) activate PIC with the subsequent generation of the second messengers Ins(1,4,5)P₃ and diacylglycerol (2, 3, 13-17). The G protein or proteins responsible for coupling group I mGluRs to PIC have been the subject of some debate. Thus, although the phosphoinositide responses elicited by agonist stimulation of mGluR5 and the mGluR1 splice variant seem to be little affected by PTX treatment (14, 15), the response to mGluR1 activation is substantially attenuated by PTX when this splice variant is expressed in Chinese hamster ovary cells (13), BHK cells (15, 16), or Xenopus laevis oocytes (18). It has been concluded from such studies that mGluR1 can stimulate phosphoinositide hydrolysis via PTX-sensitive (G_i/o) and -insensitive (G_q/11) pathways, and it is possible that the distinct pathways may lead to the activation of distinct PIC isozymes.

In the current study, we examined the coupling of recombinant mGluR1 expressed in BHK cells using both [³H]InsP₁ and Ins(1,4,5)P₃ mass accumulations as indices of PIC activity. In contrast to previous reports (13, 15, 16), we do not observe a decrease in agonist-stimulated inositol (poly)phosphate accumulation after PTX pretreatment but rather find a profound increase in the ability of glutamate receptor agonists to increase PIC activity after toxin treatment. These data provide evidence consistent with a dual regulation of PIC by mGluR1 via G_q/11 and G_i/o.

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Cell culture. BHK cells stably expressing the T45A clone of the rat type 1 mGluR (15, 16) were cultured in Dulbecco's modified Eagle's (Glutamax-1) medium supplemented with 5% dialyzed fetal calf serum, 0.5 mg/ml G418, 50 µg/ml gentamicin, and 1 µM methotrexate. BHK-mGluR1 cells were maintained in a similar culture medium except that methotrexate was omitted (15). Vector control cells (BHK-570) (15) were cultured without G418 or methotrexate but in the presence of neomycin (0.1 mg/ml). Cells were maintained at 37° in a humidified atmosphere (95% air/5% CO₂) and were passaged every 4-5 days.

[³H]InsP₁ and Ins(1,4,5)P₃ determinations. BHK-mGluR cells were seeded onto 16-mm wells (24-well multidishes, Nunc, Naperville, CT) and, where indicated, labeled with 1 µCi/ml [³H]inositol for 48 hr. Treatment of monolayers with PTX was performed by the addition to the culture medium 22-24 hr before experimentation. Cells were washed four times with 1 ml of KHB (containing 118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 5 mM HEPES, and 10 mM D-glucose, pH 7.4, after equilibration with 95% O₂/5% CO₂) at 37°. Where GPT and pyruvate were added to decrease medium glutamate concentrations, these agents were present for 15 min before any other manipulations were performed and 30 min before agonist challenge. Where the effects of GPT per se were assessed, GPT/pyruvate- and pyruvate-only-treated cells were compared.

L-Glutamate assay. L-Glutamate in the cell monolayer incubation medium was determined after the addition of TCA (0.5 M final concentration) and extraction with diethylether as described above. A standard curve for known amounts of L-glutamate (0.1-100 µM) was also prepared in a diethylether-extracted-TCA `buffer-blank' solution. The spectrophotometric assay was performed according to manufacturer's instructions except that assay constituent volumes were adjusted to allow detection of L-glutamate at concentrations 0.3 µM.

Preparation of anti-mGluR1 antiserum. A 20-mer peptide (sequence: PNVTYASVILRDYKQSSSTC) corresponding to the final carboxyl-terminal 20 amino acids (residues 1180-1199; C-for-L substitution at position 1199) of the primary sequence of mGluR1 (20) was synthesized and coupled to the carrier protein keyhole limpet hemocyanin using glutaraldehyde. The peptide/keyhole limpet hemocyanin conjugate was purified by gel filtration and antisera against the conjugate raised in New Zealand White rabbits.

Immunoblotting. Membranes were prepared from control and PTX-treated (100 ng/ml, 24 hr) BHK-mGluR1 cells. Membrane proteins were resolved on 7.5% SDS-PAGE minigels and transferred onto nitrocellulose membranes. Blots were blocked in 5% nonfat dry milk/phosphate-buffered saline (blocking solution) overnight and then incubated for 2 hr with a 1:4000 dilution of the mGluR1 antiserum in the blocking solution. Blots were washed with three changes of phosphate-buffered saline for 30 min and incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2000 dilution in blocking solution) for 1 hr. After washing (30 min), immunoreactive proteins were detected with enhanced chemiluminescence (ECL; Amersham, Little Chalford, UK).

Materials. PTX, quisqualic acid, GPT (EC 2.6.1.2), and methotrexate were from Sigma Chemical (Poole, UK). L-Glutamate was obtained from BDH (Poole, UK), and the L-glutamate colorimetric assay kit was obtained from Boehringer-Mannheim (Mannheim, Germany). 4C3HPG and 1S,3R-ACPD were purchased from Tocris-Cookson (Bristol, UK). myo-[2-³H]inositol (70-120 Ci/mmol) and [³H]Ins(1,4,5)P₃ (30-60 Ci/mmol) were from Amersham. Dowex anion exchange resin AG1-X8 (200-400 mesh, formate form) was from BioRad (Watford, UK). All other chemicals were of analytical grade and were from Fisons (Loughborough, UK). Unless indicated, all cell culture media and reagents were obtained from Gibco Life Technologies (Paisley, UK).

	Results

Summary Introduction Procedures Results Discussion References

Time- and concentration-dependency of mGluR1 phosphoinositide responses. Receptor coupling to PIC in BHK-mGluR1 cells was investigated either by assessing [³H]InsP₁ accumulation in the presence of 10 mM LiCl in myo-[³H]inositol-prelabeled cells or measuring changes in endogenous Ins(1,4,5)P₃ levels. After the addition of 300 µM L-glutamate, [³H]InsP₁ accumulated linearly over the initial 2-30 min of agonist challenge but then plateaued (Fig. 1A). In contrast, Ins(1,4,5)P₃ levels increased to a peak at 30 sec and then declined toward a lower, but still elevated, plateau level (Fig. 1B). Fig. 1, C and D, shows concentration-dependent increases in [³H]InsP₁ (at 30 min) and Ins(1,4,5)P₃ (at 30 sec) accumulations, respectively, stimulated by L-glutamate, quisqualate, and 1S,3R-ACPD. L-Glutamate and quisqualate caused similar 8-9-fold increases in [³H]InsP₁ accumulation, but the latter agonist was ~10-fold more potent (Table 1). 1S,3R-ACPD seemed to be a partial agonist, eliciting a maximal response that was ~50% of that stimulated by L-glutamate or quisqualate. L-Glutamate and quisqualate caused similar maximal 4-5-fold increases in Ins(1,4,5)P₃ mass, with quisqualate again more potent that L-glutamate (Table 1). In contrast, 1S,3R-ACPD failed to increase significantly the steady state concentration of Ins(1,4,5)P₃ above basal levels, even at 1 mM.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Time courses and concentration-dependencies of agonist-stimulated [3H]InsP1 and Ins(1,4,5)P3 responses in BHK-mGluR1 cells. For subsequent measurement of [3H]InsP1 (A and C), BHK-mGluR1 cells were cultured in the presence of [3H]inositol (1 µCi/ml) for 48 hr, washed with KHB, and incubated in KHB plus 10 mM LiCl for 30 min before addition of the indicated concentrations of L-glutamate, quisqualate, or 1S,3R-ACPD for the times indicated (A) or 30 min (C). For [3H]InsP1, data are expressed as increases over basal labeling (644 ± 50 dpm/well; 16 experiments). For Ins(1,4,5)P3 mass determination (B and D), cells were washed with KHB and incubated for 30 min (no LiCl) before the addition of the indicated agonist concentrations for the times indicated (B) or 30 sec (D). All data are shown as mean ± standard error for at least four separate experiments performed in duplicate.

View this table: [in this window] [in a new window]   TABLE 1 EC50 values for L-glutamate-, quisqualate-, and 1S,3R-ACPD-stimulated [3H]InsP1 and Ins(1,4,5)P3 mass accumulations in BHK-mGluR1 cells Concentrations of L-glutamate, quisqualate, and 1S,3R-ACPD that stimulated half-maximal increases in [3H]InsP1 accumulation (assessed at 30 min in the presence of 10 mM LiCl) or Ins(1,4,5)P3 mass accumulation (assessed at 30 sec) are presented as log EC50 ± standard error values from analyses of at least three concentration-response curves, each performed in duplicate.

Agonist-independent activity of mGluR1. In agreement with the results obtained by Prézeau et al. (21) in LLC-PK1 and human embryonic kidney 293 cells transiently transfected to express mGluR1, direct comparison between the stably transfected BHK-mGluR1 cell line and vector-transfected BHK cells (labeled to equilibrium with myo-[³H]inositol) revealed that basal [³H]InsP₁ accumulation was increased in the mGluR1-expressing cells (BHK-570, 16,536 ± 245; BHK-mGluR1, 26,088 ± 1,030 dpm/mg of protein; four experiments; p < 0.001). This increase was not attributable to receptor activation by endogenous L-glutamate because pretreatment of cells with GPT and pyruvate had no effect on the different basal [³H]InsP₁ accumulations seen in vector- and mGluR1-transfected cells (data not shown).

Effects of PTX. The treatment of BHK-mGluR1 cells for 22-24 hr with 1, 10, or 100 ng/ml PTX had dramatic dose-related effects on basal and agonist-stimulated phosphoinositide turnover that were most marked at the highest concentration of PTX used (Fig. 2). Basal [³H]InsP₁ accumulation was dramatically elevated (12.8 ± 0.9-fold) after PTX pretreatment. Remarkably, this increase was greater than that seen in control cells stimulated with a maximally effective concentration of L-glutamate (Fig. 2A). However, in PTX-treated cells, L-glutamate still elicited a significant (p < 0.01) additional stimulatory effect, which was markedly diminished when expressed in relative terms (see Fig. 2C).

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Effects of PTX treatment on L-glutamate-stimulated [3H]InsP1 and Ins(1,4,5)P3 responses in BHK-mGluR1 cells. Cells were cultured in the absence (for Ins(1,4,5)P3 measurements) or presence (for [3H]InsP1 measurements) of [3H]inositol (1 µCi/ml) for 48 hr. Where indicated, PTX (100 ng/ml) was present for the final 24-hr period. Monolayers were washed with KHB and (A) incubated in KHB plus 10 mM LiCl for 30 min before the addition of vehicle or 300 µM L-glutamate for 30 min. B, Cells were incubated for 30 min (no LiCl) before the addition of vehicle or 300 µM L-glutamate for 30 sec. C, Concentration-effect curves for L-glutamate-stimulated [3H]InsP1 accumulations in control and PTX-treated cells expressed as fold-increases over respective basal values. All data are shown as mean ± standard error for at least four separate experiments performed in duplicate.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of PTX treatment on L-glutamate-stimulated [3H]InsP1 accumulation in BHK-mGluR1 cells. BHK-mGluR1 cells were cultured as described in Experimental Procedures. Cells were labeled with [3H]inositol (1 µCi/ml) for 48 hr, and where indicated, PTX (100 ng/ml) was present for the final 24-hr period. [3H]InsP1 accumulations were assessed after incubation in the presence of the indicated concentrations of L-glutamate plus 10 mM LiCl for 30 min. Basal [3H]InsP1 accumulations were control (Cont), 389 ± 34; +PTX, 449 ± 41 dpm/well (eight measurements). All data are shown as mean ± standard error for at least four separate experiments performed in duplicate.

Effects of PTX and enzymic removal of L-glutamate on agonist-stimulated phosphoinositide responses. Despite the lack of evidence that PTX may result in a change in L-glutamate "handling" in BHK cells, a number of studies have highlighted the problem of glutamate transport and the expression and signaling in cells transfected with cDNA for mGluRs (22, 23). Therefore, we examined the effects of incubation of control and PTX-treated BHK-mGluR1 cell monolayers with high activities of GPT and pyruvate to remove residual extracellular L-glutamate. Although this manipulation had no effect on basal [³H]InsP₁ accumulation in control cells, GPT/pyruvate (but not 5 mM pyruvate alone) caused a substantial decrease in PTX-treated cells, such that in the presence of 3 units/ml GPT plus pyruvate, there was a reduction in [³H]InsP₁ of >95% (Fig. 4).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of enzymic removal of extracellular L-glutamate on basal [3H]InsP1 accumulations in control and PTX-treated BHK-mGluR1 cells. Cells were cultured in the presence of [3H]inositol (1 µCi/ml) for 48 hr, and where indicated, PTX (100 ng/ml) was present for the final 24-hr period. Monolayers were washed with KHB and incubated in KHB in the absence or presence of GPT plus 5 mM pyruvate for 30 min. Then, 10 mM LiCl was then added for an additional 30-min period before acid termination. Data are expressed relative to [3H]InsP1 accumulation in control cells in the absence of GPT/pyruvate and presented as mean ± standard error for four separate experiments performed in duplicate.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent effects of quisqualate and 1S,3R-ACPD on [3H]InsP1 accumulations in control and PTX-treated BHK-mGluR1 cells in the presence of GPT/pyruvate. Cells were cultured in the presence of [3H]inositol (1 µCi/ml) for 48 hr, and where indicated, PTX (100 ng/ml) was present for the final 24-hr period. Cell monolayers were washed with KHB and incubated in KHB containing GPT (3 units/ml) plus 5 mM pyruvate for 30 min with 10 mM LiCl present for the last 15 min of this preincubation period. The indicated concentrations of quisqualate and 1S,3R-ACPD were then added to control or PTX-treated cells, and incubations were continued for an additional 30-min period before acid termination. All data are shown as mean ± standard error for at least four separate experiments performed in duplicate.

View this table: [in this window] [in a new window]   TABLE 2 Effects of PTX pretreatment on [3H]InsP1 accumulations stimulated by quisqualate, AlF4, or ionomycin in BHK-mGluR1 cells BHK-mGluR1 cell monolayers were labeled with myo-[3H]inositol (1 µCi/ml) for 48 hr and exposed to PTX (100 ng/ml) or vehicle for the last 24 hr of the labeling period. Incubations were performed in the presence of GPT (3 units/ml) plus pyruvate (5 mM) as described in Experimental Procedures. After stimulation with NaF (50 mM) plus AlCl3 (10 µM), ionomycin (5 µM), quisqualate (30 µM), or an appropriate vehicle for 30-min incubations was terminated, and [3H]InsP1 was extracted and separated as described in Experimental Procedures. Values are presented as mean ± standard error for three separate experiments performed in duplicate.

mGluR1 expression levels in control and PTX-treated BHK cells. Western blotting revealed that PTX pretreatment of BHK-mGluR1 cells had no discernible effect on levels of receptor expression (Fig. 6). Thus, the anti-mGluR1 antiserum identified similar levels of a protein of ~150 kDa in both control and PTX-treated BHK cells. In agreement with a previous report (24), additional immunoreactive material ran toward the top of the gel (Fig. 6). The identity of this band is unknown, although it has been proposed to represent an aggregated form of the mGluR1 receptor (24).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Relative expression of mGluR1 in control and PTX-treated BHK cells. Cells were cultured in the absence or presence of PTX (100 ng/ml) for 24 hr before the preparation of membranes as described in Experimental Procedures. Representative immunoblot for cell membranes prepared from BHK-mGluR1 (lanes 2-5) or untransfected (BHK570; lane 1) cells using an anti-mGluR1 antiserum. Lanes 3 and 5, from BHK-mGluR1 membranes prepared from cells exposed to PTX. Lanes 1, 2, and 4, from vehicle-treated control cells. Membranes were loaded with 10 µg (lanes 1-3) or 3 µg (lanes 4 and 5) of membrane protein/ml.

Lack of effect of PTX on antagonist action at mGluR1. The inhibitory effects of 4C3HPG (300 µM) on quisqualate-stimulated [³H]InsP₁ accumulations in control and PTX-treated BHK-mGluR1 cells incubated in the presence of GPT/pyruvate are shown in Fig. 7. The presence of the mGluR antagonist caused similar apparently parallel rightward shifts in the concentration-effect curves for quisqualate in both control cells [log EC₅₀: control, 5.44 ± 0.07; +4C3HPG, 4.46 ± 0.09; dose-ratio, 9.6 ± 1.0 (three experiments)] and PTX-treated cells [log EC₅₀: +PTX, 6.81 ± 0.16; +PTX + 4C3HPG, 5.73 ± 0.13; dose-ratio, 12.5 ± 1.9 (four experiments)] (Fig. 7). Using a rearrangement of the Gaddum equation (K_d = A/(DR  1), where K_d is the antagonist equilibrium dissociation constant, A is the concentration of antagonist used, and DR is the dose-ratio), we calculated K_d values for 4C3HPG of 35 and 26 µM in control and PTX-treated BHK-mGluR1 cells, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Antagonism of quisqualate-stimulated [3H]InsP1 accumulations in control and PTX-treated BHK-mGluR1 cells by 4C3HPG. Cells were cultured in the presence of [3H]inositol (1 µCi/ml) for 48 hr, and where indicated, PTX (100 ng/ml) was present for the final 24-hr period. Monolayers were washed with KHB and incubated in KHB containing GPT (3 units/ml) plus 5 mM pyruvate and 10 mM LiCl for 30 min; where indicated, 4C3HPG (300 µM) was present for the last 15 min of this preincubation and throughout the period of stimulation. The indicated concentrations of quisqualate were then added to control or PTX-treated cells, and incubations were continued for an additional 30-min period before acid termination. Data are shown as mean ± standard error for at three (control) or four (+PTX) separate experiments performed in duplicate.

	Discussion

Summary Introduction Procedures Results Discussion References

In agreement with reports in BHK (15, 16) and other (13, 22) cell types transfected to express mGluR1, we have shown that PIC activity is markedly stimulated by the addition of L-glutamate, quisqualate, and, to a lesser extent, 1S,3R-ACPD. Our data, which assess [³H]InsP₁ accumulation in the presence of Li⁺ as an index of PIC activation, also confirm the agonist potency ranking order of quisqualate > L-glutamate > 1S,3R-ACPD (with the latter agent a partial agonist with respect to this response) reported by others. Assessment of concentration-effect curves for agonist-stimulated increases in Ins(1,4,5)P₃ mass yielded EC₅₀ values for quisqualate- and L-glutamate-stimulated responses (assessed at 30 sec) that were similar to those obtained for [³H]InsP₁ accumulation (assessed at 30 min); however, the partial agonist 1S,3R-ACPD (even at 1 mM) failed to significantly increase Ins(1,4,5)P₃ accumulation. It should be emphasized that unlike [³H ]InsP₁ accumulation in the presence of Li⁺, Ins(1,4,5)P₃ mass accumulation is a more dynamic measurement that reflects the relative rates of synthesis and breakdown of this messenger; presumably the reduced ability of 1S,3R-ACPD to stimulate PIC activity via mGluR1 is insufficient to result in a detectable Ins(1,4,5)P₃ accumulation, despite an increased flux through this intermediate (25).

A recent report demonstrated apparent agonist-independent ("constitutive") activity of mGluR1 transiently expressed in either LLC-PK1 or HEK 293 cells (21). In agreement with these data, we have shown here that basal [³H]InsP₁ accumulation is increased (by ~50%) in BHK-mGluR1 cells compared with vector-transfected cells, and basal phosphoinositide hydrolysis rates are not significantly suppressed by the addition of an enzyme/cosubstrate (GPT/pyruvate) system for removing extracellular endogenous glutamate or an mGluR antagonist. To further explore the properties of this apparent agonist-independent activity, we choose to selectively manipulate the G protein populations present in BHK cells by using PTX.

The mGluR family members differ in a number of important respects from other members of the G protein-coupled receptor superfamily. Thus, the ligand binding domain (26, 27) and the intracellular domains of the receptor that control the specificity of linkage to G proteins (28, 29) of mGluRs have little in common with other G protein-coupled receptors. Similarly, in contrast to many receptors that link to -isozymes of PIC, the type 1 and 5 mGluRs and their splice variants exhibit varying susceptibilities to inhibition of effector coupling by PTX, suggesting that PIC activation is mediated by both G_q/11- and G_i/o-type G proteins (13-18). Thus, mGluR1-mediated activation of phosphoinositide hydrolysis has been reported to be inhibited by 40-80% after PTX pretreatment (13, 15, 16), suggesting that ADP-ribosylation and inactivation of G_i/o severely compromise the effector coupling of this splice variant. In the current study, we provided a radically contrasting picture of PTX effects on mGluR1-mediated phosphoinositide signaling by presenting evidence demonstrating that there is a marked increase in the ability of mGluR agonists to activate PIC after G_i/o inactivation in BHK-mGluR1 cells. One possible reason for the disparity between our data and those reported previously (13, 15, 16) may relate to the fact that the earlier studies reported only the effects of agonists as a "fold-over-basal" response, and therefore the major action of PTX on "basal" values may have been overlooked in these studies.

Initial experiments demonstrated that 24-hr pretreatment with 1-100 ng/ml PTX caused a dramatic dose-related increase in [³H]InsP₁ accumulation in the absence of added agonist and at a maximally effective dose of toxin was 12-16-fold greater than in control cells. The facts that similar, very low levels of glutamate (1 µM) were observed in incubation medium taken from control and PTX-treated cell monolayers and that exogenous L-glutamate stimulated a further increase in [³H]InsP₁ accumulation initially suggested that PTX was causing a dramatic unmasking of agonist-independent (constitutive) mGluR1 activity. Despite such evidence, previous work by other groups (22), including studies performed in BHK cells (23), has highlighted the problems that can be associated with systems in which endogenous L-glutamate can contribute to, or account for, receptor activation. Thus, Desai et al. (22) reported that mGluR agonists stimulate a much more dramatic increase in phosphoinositide hydrolysis in AV12 cells transfected to express the human mGluR1 in the presence compared with the absence of the cotransfected Na⁺-dependent glutamate transporter GLAST (30). Under the assay conditions described by Desai et al. (22), extracellular L-glutamate concentration was much greater (30 µM) if cells were not engineered to express GLAST, leading to tonic receptor activation and desensitization. However, in the current study, much lower levels of L-glutamate (1 µM) were detected, suggesting that BHK cells may differ from AV12 cells in their handling of transplasmalemmal glutamate movements or may already possess a glutamate transporter favoring accumulation from the extracellular medium (31). However, more subtle effects can also occur. Thomsen et al. (23) reported that agents that do not directly interact with mGluR1 can, nevertheless, activate phosphoinositide hydrolysis by stimulating heteroexchange mediated by the endogenous glutamate transporter and having the net effect of causing L-glutamate release into the extracellular space sufficient to activate mGluR1.

In view of this evidence, we carried out further experiments using the enzyme/cosubstrate addition (GPT + 5 mM pyruvate) used by others to reduce extracellular glutamate concentrations (32). Although GPT/pyruvate had no effect on [³H]InsP₁ responses in control cells, the elevated basal phosphoinositide hydrolysis in PTX-treated cells was dramatically and fully attenuated under these conditions. In addition, further analysis of the concentration-dependencies of quisqualate- and 1S,3R-ACPD-stimulated [³H]InsP₁ accumulation revealed that in the presence of GPT/pyruvate, PTX pretreatment increased the maximal response elicited by the full agonist quisqualate and dramatically reduced the EC₅₀ value (~65-fold) compared with control cells. The partial agonist 1S,3R-ACPD stimulated a [³H]InsP₁ response that was similarly affected with a 2-3-fold increase in the maximal response and a 10-fold leftward shift in EC₅₀ value. These dramatic changes in the stimulation of phosphoinositide responses seem to occur without any detectable PTX-induced changes in BHK cell mGluR1 expression assessed by immunoblotting.

The sensitization and increased responsiveness of the phosphoinositide response to mGluR agonists after PTX treatment fully account for the original observations of dramatic elevations in basal [³H]InsP₁ accumulation. Thus, although a medium L-glutamate concentration of ~1 µM may have no significant stimulatory effect in control cells, it is sufficient to substantially stimulate phosphoinositide turnover after "sensitization" by PTX treatment. The effect of PTX seems to be much more dramatic in BHK cells expressing the mGluR1 splice variant; although the maximal agonist-stimulated response seen in mGluR1-expressing BHK cells is increased, a sensitization of the signaling pathway to agonist is not evident. Although further studies are required, these data implicate the carboxyl terminus of mGluR1, in which differential splicing of this receptor occurs (3), as an important domain in the interaction with PTX-sensitive G protein or proteins. Our attempts to examine the effects of PTX on receptor-independent activations of PIC revealed a small enhancement of enzyme activity by AlF₄-mediated G protein activation in PTX-treated cells, which would be consistent with a dual regulation of this effector by G_q/11 and G_i/o proteins. However, the small responses obtained with AlF₄ and the lack of knowledge of the relative activation of the different G proteins by this agent preclude confident interpretation.

Our observations strongly suggest that the ability of activated mGluR1 to link to PIC activation (probably via G_q/11) is negatively modulated by G_i/o proteins and that ADP-ribosylation by PTX neutralizes this inhibitory influence (Fig. 8). Consistent with this model is very recent work from this laboratory (33) that provides evidence that glutamate-stimulated [³⁵S]GTPS binding in BHK-mGluR1 cell membranes involves both PTX-sensitive and -insensitive G proteins. Furthermore, it is interesting to note that the maximum extent of mGluR1-mediated G protein activation by glutamate is modest (300-400 fmol/mg of protein), suggesting that cell-surface mGluR1 capable of interacting with G proteins are not dramatically overexpressed in this model cell system (33).

View larger version (11K): [in this window] [in a new window]   Fig. 8.   Possible G protein-mediated dual regulation of PIC activity by mGluR1 in BHK cells and the effect of PTX treatment.

Inhibitory effects of PTX-sensitive G proteins on PIC activity have been suspected for some time (34), although whether such modulations occur via direct inhibition of PIC, through activation of a distinct signal transduction pathway, or at a noneffector site in the receptor-effector coupling pathway has often been difficult to establish (34-36). However, a number of studies have reported direct inhibitory G_i/o effects on PIC activity in intact cells (37, 38) and permeabilized cell and membrane preparations (37, 39). Of particular interest with respect to the current study is the finding of Watkins et al. (40) that G_i2 expression can negatively modulate agonist-stimulated PIC activity. They reported that in mouse F9 tetracarcinoma and rat osteosarcoma cells, down-regulation of G_i2 by transfection with RNA antisense to this protein or overexpression of a constitutively active Q205L/G_i2 caused increases and decreases, respectively, in basal phosphoinositide turnover and potentiated or abolished the Ins(1,4,5)P₃ response to thrombin and an ₁-adrenoceptor agonist (40). Contrary to initial indications, our data provide no evidence for an enhancement of basal phosphoinositide hydrolysis by PTX per se, but the current data are consistent with those of Watkins et al. (40) with respect to the effect of PTX to enhance agonist-stimulated phosphoinositide responses.

In conclusion ,the present data provide strong evidence that ADP-ribosylation and inactivation of G_i/o proteins by PTX result in a dramatic enhancement of mGluR1-PIC signaling via G_q/11. It will be important to establish whether this phenomenon occurs for mGluR1 in other cell types, whether it depends on receptor expression levels, and whether it is specific to this subtype. Overall, however, these observations are consistent with a dual regulation of receptor-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.