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Characterization of [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors
Neuropharmacology 39 (2000) 1700–1706 www.elsevier.com/locate/neuropharm
Characterization of [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors Christophe Schweitzer, Claudia Kratzeisen, Geo Adam, Kenneth Lundstrom, Pari Malherbe, Serge Ohresser, Heinz Stadler, Ju¨rgen Wichmann, Thomas Woltering, Vincent Mutel * Pharmaceutical Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland Accepted 30 November 1999
Abstract The binding properties of [3H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave KD values of 20±5 and 53±8 nM and Bmax values of 474±161 and 667±89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl2, DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [3H]-LY354740 binding with similar potencies on both receptors. l-CCG I, l-AP4, l-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [3H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and l-AP3 had a 6-fold and DTT a 28fold preference for mGlu2 over mGlu3. ZnCl2, at 10 mM, inhibited more than 70% of [3H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [3H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPγS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [3H]-LY354740 binding on mGlu3 receptors. 2000 Elsevier Science Ltd. All rights reserved. Keywords: [3H]-LY354740; Binding; mGlu2; mGlu3; Pharmacology
1. Introduction Eight G-protein-coupled metabotropic glutamate (mGlu) receptors have been cloned so far (for review, see Conn and Pin, 1997). Based on their sequence similarities, signal transduction and agonist rank order of potencies, these receptors have been subdivided into three groups. Group I mGlu receptors, which couple to phosphoinositide (PI) hydrolysis, are activated by the weak group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) and the potent non-selective agonist quisqualate (Aramori and Nakanishi, 1992; Ito et al., 1992). Group I comprises mGlu1, which is preferentially
* Corresponding author. Tel.: +41-61-688-7584; fax: +41-61-6884484. E-mail address: [email protected] (V. Mutel).
blocked by (+)-2-methyl-4-carboxyphenylglycine (Salt and Turner, 1998) and selectively blocked by 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (Annoura et al., 1996; Litschig et al., 1999), and mGlu5, which is preferentially activated by the weak agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) (Doherty et al., 1997) and selectively blocked with high potency by methylphenylethynylpyridine (MPEP) (Gasparini et al., 1999). Group II mGlu receptors, which are negatively coupled to cyclic AMP production, are activated by the group II specific and potent agonist (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740) (Schoepp et al., 1997) and blocked by the potent although not group II selective antagonist 2-amino2- (2-carboxycyclopropan-1-yl) -3- (dibenzopyran-4-yl) propanoic acid (LY341495) (Kingston et al., 1998). This group comprises mGlu2 and mGlu3 (Tanabe et al. 1992,
0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 6 5 - 8
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1993), for which there are so far no subtype selective agonists or antagonists, although mGlu3 is preferentially activated by N-acetyl-l-aspartyl-l-glutamic acid (NAAG) (Wroblewska et al., 1997). Finally, group III mGlu receptors, which are also negatively coupled to cyclic AMP production, are activated by the group III preferential agonist l(+)-2-amino4-phosphonobutyric acid (l-AP4) and potently blocked by the group II/III antagonist (R,S)-α-cyclopropyl-4phosphonophenylglycine (CPPG) (Toms et al., 1996). This is the largest group and comprises mGlu4, mGlu6, mGlu7 and mGlu8 for which there are so far no subtype selective agonists or antagonists. The binding of the new metabotropic receptor ligand [3H]-LY354740 has been characterized to rat brain membranes and sections as well as in human brain sections (Richards et al., 1998; Schaffhauser et al., 1998). As [3H]-LY354740 labeled multiple populations of binding sites in rat brain homogenates it appeared useful to report its binding characteristics on the cloned rat mGlu2 and 3 receptors. Here we have expressed the recombinant receptors by infection of suspension cultures of Chinese hamster ovary (CHO) cells with Semliki Forest virus (SFV) particles.
2. Methods 2.1. Materials 1,4-Dithiothreitol (DTT) was obtained from Boehringer Mannheim (Mannheim, Germany). l-Aspartate, lcysteic acid (l-CA), l-homocysteic acid (l-HCA), Nmethyl-d-aspartic acid (NMDA) and guanosine-5⬘-O-(3thiotriphosphate) (GTPγS) were obtained from Sigma (Buchs, Switzerland). l-Glutamate and quisqualate were obtained from RBI (Zu¨rich, Switzerland). l-Cysteinesulfinic acid (l-CSA), l-homocysteinesulfinic acid (lHCSA), ibotenic acid, N-acetyl-l-aspartyl-l-glutamic acid (NAAG), (S)-3,5-dihydroxyphenylglycine (DHPG), l(+)-2-amino-3-phosphonopropionic acid (l-AP3), l(+)2-amino-4-phosphonobutyric acid (l-AP4), l(+)-2amino-5-phosphonopentanoic acid (l-AP5), (1S,3R)-1aminocyclopentane-1,3-dicarboxylic acid (1S,3RACPD), 2(S)-α-ethylglutamic acid (EGLU), 2S,1⬘S,2⬘S2-(2⬘-carboxycyclopropyl)glycine (l-CCGI) and (R,S)α-methyl-4-phosphonophenyl glycine (MPPG) were purchased from Tocris Cookson (Bristol, UK). (2S,2⬘R,3⬘R)-2-(2⬘,3⬘-dicarboxycyclopropyl) glycine (DCG IV), (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY354740) and 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl)propanoic acid (LY341495, mixture of four diastereoisomers) were synthesized at Hoffmann-La Roche Ltd by Dr J. Wichman, G. Adam and T. Woltering respectively. [3H]-LY354740 (s.a. 35 Ci/mmol) was synthesized by Dr P. Huguenin
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at the Roche Chemical and Isotope Laboratories following a synthetic route devised by Dr H. Stadler. 2.2. Subcloning of the rat mGlu2 and mGlu3 receptors cDNAs clones for the rat mGlu2 and mGlu3 receptor genes in the pBluescript II vector were obtained from Professor S. Nakanishi (Kyoto, Japan). Using the PCR techniques, the coding regions of mGlu2 and mGlu3 genes were amplified with a Kosak consensus sequence (CCACCATG) preceded by a BamH I site (in the case of mGlu3) or by a Bgl II site (in the case of mGlu2). Similarly, a Not I site was introduced at the 3⬘ end. The 3.2-kb mGlu2 (Bgl II/Not I) and 3.1-kb mGlu3 (BamH I/Not I) fragments were then subcloned into the BamH I/Not I sites of pSFV2gen vector. 2.3. Virus production and recombinant receptor expression Recombinant Semliki Forest virus particles containing the mGlu2 and 3 receptor genes were generated as described previously (Lundstrom et al., 1994). Briefly, RNAs were transcribed in vitro from linearized pSFV2gen-mGlu2, mGlu3 and pSFV-Helper2 (Berglund et al., 1993) plasmids in 40 mM HEPES/KOH, pH 7.4, 6 mM MgOAc, 10 mM spermidine/HCl in the presence of 1 mM CAP, 10 mM DTT, 1 mM each of ATP, UTP and CTP, 0.5 mM GTP, 50 U RNAse inhibitor and 60 U SP6 RNA polymerase at 37°C for 1 h. The transcripts were co-electroporated into baby hamster kidney (BHK21) cells at 1500 V and 25 µF. The virus stocks were collected 24 h later, and activated by α-chymotrypsin digestion. Confluent BHK cells were infected with the recombinant SFV particles and receptor expression was assayed by [35S]-methionine labeling 16–18 h post-infection. Labeled cell lysates were subjected to 10% SDS– PAGE and the protein expression verified by autoradiography. For large-scale membrane preparations, CHO cells adapted to grow in serum-free suspension cultures (1–4 liters) were infected with recombinant SFV-mGlu particles as described previously (Schlaeger and Lundstrom, 1999). 2.4. Membrane preparation Cells, cultured as above, were harvested and washed three times with cold PBS and frozen at ⫺80°C. The pellet was resuspended in cold 20 mM HEPES-NaOH buffer containing 10 mM ethylene diamine tetraacetic acid (EDTA) (pH 7.4), and homogenized with a polytron (Kinematica, AG, Littau, Switzerland) for 10 s at 10 000 rpm. After centrifugation for 30 min at 4°C, the pellet was washed once with the 20 mM HEPES–NaOH buffer containing 0.1 mM EDTA (pH 7.4), centrifuged and
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resuspended in a smaller volume of a cold Tris–HCl 50 mM, MgCl2 2 mM, pH 7.4 binding buffer. The membrane suspension was frozen at ⫺80°C before use. Protein content was measured by the Pierce method (Socochim, Lausanne, Switzerland) using bovine serum albumin as standard. 2.5. [3H]-LY354740 binding After thawing, the membranes were diluted in binding buffer. Their final concentration in the assay was 25 µg protein/ml. Saturation isotherms were determined by incubating various [3H]-LY354740 concentrations (1– 300 nM) for 1 h at room temperature. For inhibition experiments, membranes were incubated with 10 and 20 nM of [3H]-LY354740 for mGlu2 and mGlu3 receptors, respectively, for 1 h at room temperature in the presence of various concentration of inhibitors. At the end of the incubation, the membranes were filtered onto Whatmann GF/C glass fiber filters and washed five times with cold binding buffer. Non-specific binding was measured in the presence of 10 µM DCG IV. The radioactivity was measured by liquid scintillation after transfer of the filter in plastic vials containing 10 ml of Ultima-gold (Packard, Zu¨rich, Switzerland) in a Tri-Carb 2500 TR counter (Packard). 2.6. Data analysis The inhibition curves were fitted with a four-parameter logistic equation giving IC50 values, and Hill coefficients using the iterative non-linear curve-fitting software Origin (Microcal Software Inc., Northampton, MA, USA). Saturation experiments were analysed with the same program using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, B=(Bmax×[F])/(KD+[F]), where B is the amount of ligand bound at equilibrium, Bmax is the maximum number of binding sites, [F] is the concentration of free ligand and KD is the ligand dissociation constant. Ki values were calculated using the Cheng and Prusoff equation (Cheng and Prusoff, 1973). Each experiment was performed at least three times in duplicate.
3. Results There was no significant [3H]-LY354740 binding on control untransfected CHO cell membranes. Using SFVinfected cell membranes, the saturation analysis provided dissociation constant (KD) values of 20±5 and 53±8 nM and maximal number of binding sites (Bmax values) of 474±161 and 667±89 fmol/mg protein for
mGlu2 and mGlu3 receptors, respectively (Fig. 1). Although the affinity values were statistically significantly different (Student’s t-test, p⬍0.0001), the maximal number of binding sites did not differ between the two receptors. CaCl2 up to 10 mM and NMDA, kainate and DHPG up to 1 mM had no significant effect on [3H]-LY354740 binding to both mGlu2 and mGlu3 receptors. As shown in Fig. 2A and B for selected compounds and Table 1 for Ki values, the most potent compounds for both receptors were the agonist and antagonist from Eli Lilly, LY354740 and LY341495. Glutamate showed Ki values close to 1 and 0.4 µM at mGlu2 and 3 receptors, respectively. DCG IV, LY341495 and ibotenate inhibited the binding with similar potencies on both receptors. l-CCG I, l-AP4, l-AP5, LY354740 and 1S,3R-ACPD were 2to 4-fold more potent inhibitors of [3H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and l-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. All these compounds inhibited the binding to the same maximum level. Finally, the effects of endogenous ligands have been tested and apart from l-CA which inhibited [3H]LY354740 binding to mGlu2 and mGlu3 receptors with Ki values of 21 and 80 µM, respectively, the other compounds, l-aspartate, l-CSA, l-HCSA and l-HCA, were weak inhibitors with poor selectivity (see Table 1). ZnCl2, at 10 mM, inhibited more than 70% of [3H]LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [3H]-LY354740 binding to mGlu3 receptors. The effect of ZnCl2 on [3H]LY354740 binding to both receptors was complex. With mGlu2, the inhibition curve was better fitted with a twosite model (Fig. 3) with a high-affinity site having an IC50 of 60±20 µM which represents almost 60% of the total and a second low-affinity site with an IC50 of 19±8 mM. With mGlu3, this ion gave a first phase of almost 40% of inhibition of [3H]-LY354740 binding at up to 30 µM, followed by a reversal of the inhibition back to the control value with further increase of ZnCl2 concentration (Fig. 3). Finally, GTPγS partially inhibited concentrationdependently [3H]-LY354740 binding to mGlu2 (maximum 50%) with an IC50 of 6.4±0.5 nM without affecting the binding to mGlu3 receptors (data not shown). For LY354740, LY341495, DCG IV, l-CCG I, glutamate, 1S,3R-ACPD, MPPG, l-AP3, l-AP4, l-AP5, EGLU, quisqualate, NAAG and ibotenate we have examined the correlation between the affinities found using mGlu2 and mGlu3 receptors. As shown in Fig. 4 there was an overall good correlation between the Ki values obtained with mGlu2 or mGlu3 receptors. As expected, the outliers were compounds which showed
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Fig. 1. Saturation analysis of [3H]-LY354740 binding to rat mGlu2 (䊏) and mGlu3 (쐌) receptors from SFV-infected CHO cells membranes isolated and incubated at room temperature for 1 h with various concentrations of [3H]-LY354740. Data are expressed as fmoles of specific bound [3H]-LY354740 per mg of protein and are the mean±standard deviation (bars) of at least three individual experiments performed in duplicate. Inset: Scatchard transform of the data (total [3H]-LY354740/bound [3H]-LY354740 is expressed as a function of the [3H]-LY354740 bound).
some preference for one receptor (preferential 2 under and 3 above the line). The correlation coefficient R2 was equal to 0.83.
4. Discussion We have already reported the binding characteristics of [3H]-LY354740 to rat brain membranes and rat and human brain sections (Richards et al., 1998; Schaffhauser et al., 1998). Using rat brain cortex homogenate this radioactive ligand was found to bind to two sites with Kd values of 5 and 60 nM, respectively (Schaffhauser et al., 1998). In the present study, using transiently transfected cells we found Kd values of 20 and 53 nM for mGlu2 and 3 receptors respectively. The Kd value for the mGlu3 receptor is very close to the value found for the low-affinity site in rat brain cortex membranes, whereas the Kd value of the ligand for the mGlu2 receptor is 4-fold higher than the one found for the high-affinity site in the rat brain membranes. We have observed that the binding of [3H]-LY354740 to mGlu2 receptor is sensitive to GTPγS. This might indicate, as has been described for several agonist ligands of G-protein-coupled receptors (Branchek et al., 1990;
Mazzoni et al., 1993), that the coupling state of the receptor could influence their affinities. We have also measured a higher affinity (Kd value of 10 nM) of this ligand using membranes of CHO cells stably transfected with the mGlu2 receptor and this might indicate again that the level of receptor and the coupling state could influence the affinity for agonist ligands. Interestingly, the [3H]-LY354740 binding to mGlu3 receptors was not sensitive to GTPγS confirming the results of Laurie et al. (1995) who reported the lack of sensitivity to GTPγS of the [3H]-glutamate binding to mGlu3 receptor. Owing to the differential properties of those receptors, our results suggest that the low-affinity site identified in the rat brain cortex membranes would correspond to mGlu3 receptors and that the high-affinity site might correspond to mGlu2 receptors. Moreover, some compounds such as NAAG, EGLU or MPPG, which appeared to be more than 5-fold selective for one or the other receptor, inhibited [3H]-LY354740 binding in rat brain homogenate in a biphasic manner with high and low affinities (Schaffhauser et al., 1998). Furthermore, using functional assays, LY354740 had EC50 values of 5 and 24 nM at mGlu2 and 3 receptors respectively, but showed no effect at mGlu1, 5, 4 and 7 receptors up to 300 µM and displayed only low micromolar affinities for mGlu8a
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Fig. 2. Inhibition of [3H]-LY354740 binding to rat mGlu2 (A) or mGlu3 (B) receptors from SFV-infected cell by: LY341495, DCG IV, glutamate, 1S,3R-ACPD, MPPG, quisqualate and NAAG. Results are expressed as a percentage of [3H]-LY354740 specific bound and are the mean±standard deviation (bars) of three individual experiments performed in duplicate.
and b receptors (Schoepp et al., 1997; Wu et al., 1998; Malherbe et al., 1999). This lends further support to the probability that, in rat brain membranes, [3H]-LY354740 binds only to the group II mGlu receptors. ZnCl2 is a well-known modulator of protein–protein interaction (Berg and Shi, 1996) and the zinc ion has been reported to be stored in neuronal synaptic vesicles and released upon neuron depolarization to reach a concentration of around 300 µM in the synaptic cleft (Assaf and Chung, 1984; Perez-Clausell and Danscher, 1985). In our experiments, this ion had a pronounced differential effect on the two receptors, inhibiting almost 60% of [3H]-LY354740 binding to mGlu2 receptor at 300 µM without any effect on mGlu3 at this concentration. At 10 mM, it inhibited almost 70% of the [3H]-LY354740 binding to mGlu2 without affecting the binding to mGlu3 receptors. ZnCl2 is potentially a very useful tool to discriminate between mGlu2 and 3 receptors using [3H]-LY354740 and its effect will be studied on the binding of this ligand in human and rat brain sections.
MGlu3 has been shown to be stimulated by Ca2+, whereas mGlu2 receptors are insensitive to this ion and the critical site for the determination of the Ca2+ sensitivity was attributed to a serine residue at position 152 of mGlu3 which correspond to an aspartic acid in the mGlu2 receptor (Kubo et al., 1998). Although we have not seen any significant effect of Ca2+ on [3H]LY354740 binding to mGlu2 and 3 receptors, we cannot rule out that part of the differential effect of ZnCl2 is not mediated via this site. The biphasic effect of ZnCl2 on [3H]-LY354740 binding to both receptors strongly suggests that this ion interacts with multiple sites which seem to have different properties on each receptor. Using functional assays in intact cells, potencies of 4– 20 and 4–5 µM have been reported for glutamate at mGlu2 and 3 receptors, respectively (Conn and Pin, 1997). Our results showed that glutamate is slightly more active on mGlu3 than 2 with an affinity close to 400 nM for the former receptor. Interestingly, apart from what was found with glutamate, numerous discrepancies were found when comparing the pharmacology reported using functional assays in intact cells and the results we obtained with binding assays. Quisqualate was reported to have an EC50 value greater than 1 mM on mGlu2, whereas we found a Ki value close to 100 µM using both [3H]-LY354740 and [3H]-DCG IV binding (this study; Cartmell et al., 1998). Using cAMP measurement in intact cells, l-AP4 was reported to be devoid of functional activity at both mGlu2 and 3 receptors (Conn and Pin, 1997). However, this compound was able to inhibit concentration-dependently [3H]-LY354740 binding to mGlu2 receptor with a Ki value of 129 µM. We have previously shown that l-AP3, l-AP5 and l-AP4 were able to antagonize 1S,3R-ACPD-stimulated GTPγ35S binding to mGlu2 receptor-transfected cell membranes with Ki values of 11, 22 and 143 µM, respectively (Cartmell et al., 1998) and the potency of l-AP4 correlates well with its Ki value determined in binding experiment. Interestingly, Laurie et al. (1995) have shown that l-AP4 inhibited [3H]-glutamate binding to mGlu3 receptor with a Ki value of 282 µM which is very close to the Ki value of 216 µM we found using [3H]-LY354740 binding, and our result for l-AP3 (114 µM) is also in agreement with the Ki value of 125 µM reported by this group with the same receptor. It appeared, thus, that the exclusive use of functional assays with intact cells to define receptor pharmacology and compound specificity can be misleading, particularly when uptake phenomena are known to take place. Furthermore, the lack of knowledge of receptor expression and occupancy has to be avoided in order to perform meaningful pharmacological comparisons. In this regard, the use of radiolabeled ligands will become essential and should be more extensively used in the course of mGlu receptors characterization. Finally, the mGlu2 receptor expressed using the SFV vector had characteristics very
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Table 1 Ki and Hill number values±standard deviation for various compounds to inhibit [3H]-LY354740 binding to cloned rat mGlu2 and 3 receptors Compound
mGlu2 Ki (µM)
mGlu2 Hill
mGlu3 Ki (µM)
mGlu3 Hill
LY341495 LY354740 DCG IV l-CCG I Glutamate 1S,3R-ACPD MPPG l-AP3 l-CA EGLU l-AP5 l-HCA l-HCSA Quisqualate l-AP4 Ibotenate NAAG l-CSA l-Aspartate DTT
0.009±0.001 0.013±0.002 0.11±0.02 0.14±0.02 1.2±0.2 4.5±0.5 18±5 19±3 21±5 27±2 51±8 83±27 99±28 113±30 129±25 139±48 236±113 250±48 320±130 453±200
0.96±0.09 0.72±0.05 0.83±0.1 0.78±0.05 1.055±0.07 1.072±0.08 1±0.1 1.15±0.3 0.9±0.1 1.021±0.1 0.8±0.1 1.2±0.4 1±0.2 0.84±0.3 0.99±0.2 0.9±0.2 0.91±0.3 1.2±0.4 1.1±0.5 0.5±0.1
0.01±0.002 0.051±0.003 0.15±0.05 0.3±0.07 0.44±0.05 16±3.6 104±28 114±24 80±19 4±0.6 136±22 51±21 208±37 22±5 216±79 123±50 19±1 320±50 420±40 12850±684
0.82±0.1 0.94±0.04 0.77±0.2 0.86±0.2 1.19±0.1 0.77±0.1 0.73±0.3 0.61±0.1 0.95±0.2 0.6±0.1 1.6±0.4 0.7±0.1 1.2±0.2 0.75±0.1 1.24±0.2 0.91±0.2 0.96±0.05 0.99±0.1 0.9±0.4 0.75±0.3
Fig. 3. Effect of ZnCl2 on [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptor-transfected cell membranes. Results are expressed as a percentage of [3H]-LY354740 specific bound and are the mean±standard deviation (bars) of four individual experiments performed in duplicate.
similar to that of the stably expressed mGlu2 receptor. This SFV-vector system of expression offered several advantages, particularly as their was no need to block the receptor to maintain the expression as was the case for the stable line. In this regard, it seems particularly appropriate to express receptors for which the natural ligand is secreted by the cells.
Fig. 4. Correlation between Ki values for LY354740, LY341495, DCG IV, l-CCG I, glutamate, 1S,3R-ACPD, MPPG, l-AP3, l-AP4, l-AP5, EGLU, quisqualate, NAAG and ibotenate obtained using [3H]LY354740 binding to rat mGlu2 and to rat mGlu3 receptors transiently expressed in CHO cells using SFV vectors (this study). The values have been converted to their negative logarithms.
Acknowledgements We thank Dr E.-J. Schlaeger and M. Fogetta for providing the CHO suspension cultures and A. Nilly and S. Chaboz for their skillful technical assistance.
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receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding. Molecular Pharmacology 55, 453–461. Lundstrom, K., Mills, A., Buell, G., Allet, E., Adami, N., Liljestro¨m, P., 1994. High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. European Journal of Biochemistry 224, 917–921. Malherbe, P., Kratzeisen, C., Lundstrom, K., Richards, J.G., Mutel, V., 1999. Cloning and functional expression of alternative spliced variants of the human metabotropic glutamate receptor 8. Molecular Brain Research 67, 201–210. Mazzoni, M.R., Martini, C., Lucacchini, A., 1993. Regulation of agonist binding to A2a adenosine receptors: effects of guanine nucleotides (GDP[S] and GTP[S]) and Mg2+ ion. Biochimica et Biophysica Acta 1220, 76–84. Perez-Clausell, J., Danscher, G., 1985. Intravesicular localization of zinc in rat telencephalic boutons: a histochemical study. Brain Research 337, 91–98. Richards, J.G., Messer, J., Bleuel, Z., Malherbe, P., Faull, R.L.M., Mutel, V., 1998. Cellular sites of synthesis and protein expression of mGlu2/3 glutamate receptors in human postmortem brain revealed by hybridization histochemistry and [3H]LY354740 radioautography. Society for Neuroscience Meeting, Los Angeles, USA. Salt, T.E., Turner, J.P., 1998. Reduction of sensory and metabotropic glutamate receptor responses in the thalamus by the novel metabotropic glutamate receptor-1-selective antagonist S-2-methyl-4carboxy-phenylglycine. Neuroscience 85, 655–658. Schaffhauser, H., Richards, J.G., Cartmell, J., Chaboz, S., Kemp, J.A., Klingelschmidt, A. et al., 1998. In vitro binding characteristics of a new selective group II metabotropic glutamate receptor radioligand, [3H]LY354740, in rat brain. Molecular Pharmacology 53, 228–233. Schlaeger, E.-J., Lundstrom, K., 1999. Effect of temperature on recombinant protein expression in Semliki Forest virus infected mammalian cell lines grown in serum-free suspension cultures. Cytotechnology 28, 205–211. Schoepp, D.D., Johnson, B.G., Wright, R.A., Salhoff, C.R., Mayne, N.G., Wu, S. et al., 1997. LY354740 is a potent and highly selective group II metabotropic glutamate receptor agonist in cells expressing human glutamate receptors. Neuropharmacology 36, 1–11. Tanabe, Y., Masu, M., Ishii, T., Shigemoto, R., Nakanishi, S., 1992. A family of metabotropic glutamate receptors. Neuron 8, 169–179. Tanabe, Y., Nomura, A., Masu, N., Shigemoto, R., Nakanishi, S., 1993. Signal transduction, pharmacological properties, and expression of two rat metabotropic glutamate receptors, mGluR3 and mGluR4a. Journal of Neuroscience 13, 1372–1378. Toms, N.J., Jane, D.E., Kemp, M.C., Bedingfield, J.S., Roberts, P.J., 1996. The effects of (RS)-alpha-cyclopropyl-4-phosphonophenylglycine ((RS)-CPPG), a potent and selective metabotropic glutamate receptor antagonist. British Journal of Pharmacology 119, 851–854. Wroblewska, B., Wroblewski, J.T., Pshenichkin, S., Surin, A., Sullivan, S.E., Neale, J.H., 1997. N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells. Journal of Neurochemistry 69, 174–181. Wu, S., Wright, R.A., Rockey, P.K., Burgett, S.G., Arnold, J.S., Rosteck, P.R., 1998. Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression and comparison of pharmacological properties in RGT cells. Molecular Brain Research 53, 88–97.
Characterization of [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors Christophe Schweitzer, Claudia Kratzeisen, Geo Adam, Kenneth Lundstrom, Pari Malherbe, Serge Ohresser, Heinz Stadler, Ju¨rgen Wichmann, Thomas Woltering, Vincent Mutel * Pharmaceutical Division, Preclinical CNS Research, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland Accepted 30 November 1999
Abstract The binding properties of [3H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave KD values of 20±5 and 53±8 nM and Bmax values of 474±161 and 667±89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl2, DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [3H]-LY354740 binding with similar potencies on both receptors. l-CCG I, l-AP4, l-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [3H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and l-AP3 had a 6-fold and DTT a 28fold preference for mGlu2 over mGlu3. ZnCl2, at 10 mM, inhibited more than 70% of [3H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [3H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPγS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [3H]-LY354740 binding on mGlu3 receptors. 2000 Elsevier Science Ltd. All rights reserved. Keywords: [3H]-LY354740; Binding; mGlu2; mGlu3; Pharmacology
1. Introduction Eight G-protein-coupled metabotropic glutamate (mGlu) receptors have been cloned so far (for review, see Conn and Pin, 1997). Based on their sequence similarities, signal transduction and agonist rank order of potencies, these receptors have been subdivided into three groups. Group I mGlu receptors, which couple to phosphoinositide (PI) hydrolysis, are activated by the weak group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) and the potent non-selective agonist quisqualate (Aramori and Nakanishi, 1992; Ito et al., 1992). Group I comprises mGlu1, which is preferentially
* Corresponding author. Tel.: +41-61-688-7584; fax: +41-61-6884484. E-mail address: [email protected] (V. Mutel).
blocked by (+)-2-methyl-4-carboxyphenylglycine (Salt and Turner, 1998) and selectively blocked by 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (Annoura et al., 1996; Litschig et al., 1999), and mGlu5, which is preferentially activated by the weak agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) (Doherty et al., 1997) and selectively blocked with high potency by methylphenylethynylpyridine (MPEP) (Gasparini et al., 1999). Group II mGlu receptors, which are negatively coupled to cyclic AMP production, are activated by the group II specific and potent agonist (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740) (Schoepp et al., 1997) and blocked by the potent although not group II selective antagonist 2-amino2- (2-carboxycyclopropan-1-yl) -3- (dibenzopyran-4-yl) propanoic acid (LY341495) (Kingston et al., 1998). This group comprises mGlu2 and mGlu3 (Tanabe et al. 1992,
0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 6 5 - 8
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1993), for which there are so far no subtype selective agonists or antagonists, although mGlu3 is preferentially activated by N-acetyl-l-aspartyl-l-glutamic acid (NAAG) (Wroblewska et al., 1997). Finally, group III mGlu receptors, which are also negatively coupled to cyclic AMP production, are activated by the group III preferential agonist l(+)-2-amino4-phosphonobutyric acid (l-AP4) and potently blocked by the group II/III antagonist (R,S)-α-cyclopropyl-4phosphonophenylglycine (CPPG) (Toms et al., 1996). This is the largest group and comprises mGlu4, mGlu6, mGlu7 and mGlu8 for which there are so far no subtype selective agonists or antagonists. The binding of the new metabotropic receptor ligand [3H]-LY354740 has been characterized to rat brain membranes and sections as well as in human brain sections (Richards et al., 1998; Schaffhauser et al., 1998). As [3H]-LY354740 labeled multiple populations of binding sites in rat brain homogenates it appeared useful to report its binding characteristics on the cloned rat mGlu2 and 3 receptors. Here we have expressed the recombinant receptors by infection of suspension cultures of Chinese hamster ovary (CHO) cells with Semliki Forest virus (SFV) particles.
2. Methods 2.1. Materials 1,4-Dithiothreitol (DTT) was obtained from Boehringer Mannheim (Mannheim, Germany). l-Aspartate, lcysteic acid (l-CA), l-homocysteic acid (l-HCA), Nmethyl-d-aspartic acid (NMDA) and guanosine-5⬘-O-(3thiotriphosphate) (GTPγS) were obtained from Sigma (Buchs, Switzerland). l-Glutamate and quisqualate were obtained from RBI (Zu¨rich, Switzerland). l-Cysteinesulfinic acid (l-CSA), l-homocysteinesulfinic acid (lHCSA), ibotenic acid, N-acetyl-l-aspartyl-l-glutamic acid (NAAG), (S)-3,5-dihydroxyphenylglycine (DHPG), l(+)-2-amino-3-phosphonopropionic acid (l-AP3), l(+)2-amino-4-phosphonobutyric acid (l-AP4), l(+)-2amino-5-phosphonopentanoic acid (l-AP5), (1S,3R)-1aminocyclopentane-1,3-dicarboxylic acid (1S,3RACPD), 2(S)-α-ethylglutamic acid (EGLU), 2S,1⬘S,2⬘S2-(2⬘-carboxycyclopropyl)glycine (l-CCGI) and (R,S)α-methyl-4-phosphonophenyl glycine (MPPG) were purchased from Tocris Cookson (Bristol, UK). (2S,2⬘R,3⬘R)-2-(2⬘,3⬘-dicarboxycyclopropyl) glycine (DCG IV), (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY354740) and 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl)propanoic acid (LY341495, mixture of four diastereoisomers) were synthesized at Hoffmann-La Roche Ltd by Dr J. Wichman, G. Adam and T. Woltering respectively. [3H]-LY354740 (s.a. 35 Ci/mmol) was synthesized by Dr P. Huguenin
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at the Roche Chemical and Isotope Laboratories following a synthetic route devised by Dr H. Stadler. 2.2. Subcloning of the rat mGlu2 and mGlu3 receptors cDNAs clones for the rat mGlu2 and mGlu3 receptor genes in the pBluescript II vector were obtained from Professor S. Nakanishi (Kyoto, Japan). Using the PCR techniques, the coding regions of mGlu2 and mGlu3 genes were amplified with a Kosak consensus sequence (CCACCATG) preceded by a BamH I site (in the case of mGlu3) or by a Bgl II site (in the case of mGlu2). Similarly, a Not I site was introduced at the 3⬘ end. The 3.2-kb mGlu2 (Bgl II/Not I) and 3.1-kb mGlu3 (BamH I/Not I) fragments were then subcloned into the BamH I/Not I sites of pSFV2gen vector. 2.3. Virus production and recombinant receptor expression Recombinant Semliki Forest virus particles containing the mGlu2 and 3 receptor genes were generated as described previously (Lundstrom et al., 1994). Briefly, RNAs were transcribed in vitro from linearized pSFV2gen-mGlu2, mGlu3 and pSFV-Helper2 (Berglund et al., 1993) plasmids in 40 mM HEPES/KOH, pH 7.4, 6 mM MgOAc, 10 mM spermidine/HCl in the presence of 1 mM CAP, 10 mM DTT, 1 mM each of ATP, UTP and CTP, 0.5 mM GTP, 50 U RNAse inhibitor and 60 U SP6 RNA polymerase at 37°C for 1 h. The transcripts were co-electroporated into baby hamster kidney (BHK21) cells at 1500 V and 25 µF. The virus stocks were collected 24 h later, and activated by α-chymotrypsin digestion. Confluent BHK cells were infected with the recombinant SFV particles and receptor expression was assayed by [35S]-methionine labeling 16–18 h post-infection. Labeled cell lysates were subjected to 10% SDS– PAGE and the protein expression verified by autoradiography. For large-scale membrane preparations, CHO cells adapted to grow in serum-free suspension cultures (1–4 liters) were infected with recombinant SFV-mGlu particles as described previously (Schlaeger and Lundstrom, 1999). 2.4. Membrane preparation Cells, cultured as above, were harvested and washed three times with cold PBS and frozen at ⫺80°C. The pellet was resuspended in cold 20 mM HEPES-NaOH buffer containing 10 mM ethylene diamine tetraacetic acid (EDTA) (pH 7.4), and homogenized with a polytron (Kinematica, AG, Littau, Switzerland) for 10 s at 10 000 rpm. After centrifugation for 30 min at 4°C, the pellet was washed once with the 20 mM HEPES–NaOH buffer containing 0.1 mM EDTA (pH 7.4), centrifuged and
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resuspended in a smaller volume of a cold Tris–HCl 50 mM, MgCl2 2 mM, pH 7.4 binding buffer. The membrane suspension was frozen at ⫺80°C before use. Protein content was measured by the Pierce method (Socochim, Lausanne, Switzerland) using bovine serum albumin as standard. 2.5. [3H]-LY354740 binding After thawing, the membranes were diluted in binding buffer. Their final concentration in the assay was 25 µg protein/ml. Saturation isotherms were determined by incubating various [3H]-LY354740 concentrations (1– 300 nM) for 1 h at room temperature. For inhibition experiments, membranes were incubated with 10 and 20 nM of [3H]-LY354740 for mGlu2 and mGlu3 receptors, respectively, for 1 h at room temperature in the presence of various concentration of inhibitors. At the end of the incubation, the membranes were filtered onto Whatmann GF/C glass fiber filters and washed five times with cold binding buffer. Non-specific binding was measured in the presence of 10 µM DCG IV. The radioactivity was measured by liquid scintillation after transfer of the filter in plastic vials containing 10 ml of Ultima-gold (Packard, Zu¨rich, Switzerland) in a Tri-Carb 2500 TR counter (Packard). 2.6. Data analysis The inhibition curves were fitted with a four-parameter logistic equation giving IC50 values, and Hill coefficients using the iterative non-linear curve-fitting software Origin (Microcal Software Inc., Northampton, MA, USA). Saturation experiments were analysed with the same program using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, B=(Bmax×[F])/(KD+[F]), where B is the amount of ligand bound at equilibrium, Bmax is the maximum number of binding sites, [F] is the concentration of free ligand and KD is the ligand dissociation constant. Ki values were calculated using the Cheng and Prusoff equation (Cheng and Prusoff, 1973). Each experiment was performed at least three times in duplicate.
3. Results There was no significant [3H]-LY354740 binding on control untransfected CHO cell membranes. Using SFVinfected cell membranes, the saturation analysis provided dissociation constant (KD) values of 20±5 and 53±8 nM and maximal number of binding sites (Bmax values) of 474±161 and 667±89 fmol/mg protein for
mGlu2 and mGlu3 receptors, respectively (Fig. 1). Although the affinity values were statistically significantly different (Student’s t-test, p⬍0.0001), the maximal number of binding sites did not differ between the two receptors. CaCl2 up to 10 mM and NMDA, kainate and DHPG up to 1 mM had no significant effect on [3H]-LY354740 binding to both mGlu2 and mGlu3 receptors. As shown in Fig. 2A and B for selected compounds and Table 1 for Ki values, the most potent compounds for both receptors were the agonist and antagonist from Eli Lilly, LY354740 and LY341495. Glutamate showed Ki values close to 1 and 0.4 µM at mGlu2 and 3 receptors, respectively. DCG IV, LY341495 and ibotenate inhibited the binding with similar potencies on both receptors. l-CCG I, l-AP4, l-AP5, LY354740 and 1S,3R-ACPD were 2to 4-fold more potent inhibitors of [3H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and l-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. All these compounds inhibited the binding to the same maximum level. Finally, the effects of endogenous ligands have been tested and apart from l-CA which inhibited [3H]LY354740 binding to mGlu2 and mGlu3 receptors with Ki values of 21 and 80 µM, respectively, the other compounds, l-aspartate, l-CSA, l-HCSA and l-HCA, were weak inhibitors with poor selectivity (see Table 1). ZnCl2, at 10 mM, inhibited more than 70% of [3H]LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [3H]-LY354740 binding to mGlu3 receptors. The effect of ZnCl2 on [3H]LY354740 binding to both receptors was complex. With mGlu2, the inhibition curve was better fitted with a twosite model (Fig. 3) with a high-affinity site having an IC50 of 60±20 µM which represents almost 60% of the total and a second low-affinity site with an IC50 of 19±8 mM. With mGlu3, this ion gave a first phase of almost 40% of inhibition of [3H]-LY354740 binding at up to 30 µM, followed by a reversal of the inhibition back to the control value with further increase of ZnCl2 concentration (Fig. 3). Finally, GTPγS partially inhibited concentrationdependently [3H]-LY354740 binding to mGlu2 (maximum 50%) with an IC50 of 6.4±0.5 nM without affecting the binding to mGlu3 receptors (data not shown). For LY354740, LY341495, DCG IV, l-CCG I, glutamate, 1S,3R-ACPD, MPPG, l-AP3, l-AP4, l-AP5, EGLU, quisqualate, NAAG and ibotenate we have examined the correlation between the affinities found using mGlu2 and mGlu3 receptors. As shown in Fig. 4 there was an overall good correlation between the Ki values obtained with mGlu2 or mGlu3 receptors. As expected, the outliers were compounds which showed
C. Schweitzer et al. / Neuropharmacology 39 (2000) 1700–1706
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Fig. 1. Saturation analysis of [3H]-LY354740 binding to rat mGlu2 (䊏) and mGlu3 (쐌) receptors from SFV-infected CHO cells membranes isolated and incubated at room temperature for 1 h with various concentrations of [3H]-LY354740. Data are expressed as fmoles of specific bound [3H]-LY354740 per mg of protein and are the mean±standard deviation (bars) of at least three individual experiments performed in duplicate. Inset: Scatchard transform of the data (total [3H]-LY354740/bound [3H]-LY354740 is expressed as a function of the [3H]-LY354740 bound).
some preference for one receptor (preferential 2 under and 3 above the line). The correlation coefficient R2 was equal to 0.83.
4. Discussion We have already reported the binding characteristics of [3H]-LY354740 to rat brain membranes and rat and human brain sections (Richards et al., 1998; Schaffhauser et al., 1998). Using rat brain cortex homogenate this radioactive ligand was found to bind to two sites with Kd values of 5 and 60 nM, respectively (Schaffhauser et al., 1998). In the present study, using transiently transfected cells we found Kd values of 20 and 53 nM for mGlu2 and 3 receptors respectively. The Kd value for the mGlu3 receptor is very close to the value found for the low-affinity site in rat brain cortex membranes, whereas the Kd value of the ligand for the mGlu2 receptor is 4-fold higher than the one found for the high-affinity site in the rat brain membranes. We have observed that the binding of [3H]-LY354740 to mGlu2 receptor is sensitive to GTPγS. This might indicate, as has been described for several agonist ligands of G-protein-coupled receptors (Branchek et al., 1990;
Mazzoni et al., 1993), that the coupling state of the receptor could influence their affinities. We have also measured a higher affinity (Kd value of 10 nM) of this ligand using membranes of CHO cells stably transfected with the mGlu2 receptor and this might indicate again that the level of receptor and the coupling state could influence the affinity for agonist ligands. Interestingly, the [3H]-LY354740 binding to mGlu3 receptors was not sensitive to GTPγS confirming the results of Laurie et al. (1995) who reported the lack of sensitivity to GTPγS of the [3H]-glutamate binding to mGlu3 receptor. Owing to the differential properties of those receptors, our results suggest that the low-affinity site identified in the rat brain cortex membranes would correspond to mGlu3 receptors and that the high-affinity site might correspond to mGlu2 receptors. Moreover, some compounds such as NAAG, EGLU or MPPG, which appeared to be more than 5-fold selective for one or the other receptor, inhibited [3H]-LY354740 binding in rat brain homogenate in a biphasic manner with high and low affinities (Schaffhauser et al., 1998). Furthermore, using functional assays, LY354740 had EC50 values of 5 and 24 nM at mGlu2 and 3 receptors respectively, but showed no effect at mGlu1, 5, 4 and 7 receptors up to 300 µM and displayed only low micromolar affinities for mGlu8a
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Fig. 2. Inhibition of [3H]-LY354740 binding to rat mGlu2 (A) or mGlu3 (B) receptors from SFV-infected cell by: LY341495, DCG IV, glutamate, 1S,3R-ACPD, MPPG, quisqualate and NAAG. Results are expressed as a percentage of [3H]-LY354740 specific bound and are the mean±standard deviation (bars) of three individual experiments performed in duplicate.
and b receptors (Schoepp et al., 1997; Wu et al., 1998; Malherbe et al., 1999). This lends further support to the probability that, in rat brain membranes, [3H]-LY354740 binds only to the group II mGlu receptors. ZnCl2 is a well-known modulator of protein–protein interaction (Berg and Shi, 1996) and the zinc ion has been reported to be stored in neuronal synaptic vesicles and released upon neuron depolarization to reach a concentration of around 300 µM in the synaptic cleft (Assaf and Chung, 1984; Perez-Clausell and Danscher, 1985). In our experiments, this ion had a pronounced differential effect on the two receptors, inhibiting almost 60% of [3H]-LY354740 binding to mGlu2 receptor at 300 µM without any effect on mGlu3 at this concentration. At 10 mM, it inhibited almost 70% of the [3H]-LY354740 binding to mGlu2 without affecting the binding to mGlu3 receptors. ZnCl2 is potentially a very useful tool to discriminate between mGlu2 and 3 receptors using [3H]-LY354740 and its effect will be studied on the binding of this ligand in human and rat brain sections.
MGlu3 has been shown to be stimulated by Ca2+, whereas mGlu2 receptors are insensitive to this ion and the critical site for the determination of the Ca2+ sensitivity was attributed to a serine residue at position 152 of mGlu3 which correspond to an aspartic acid in the mGlu2 receptor (Kubo et al., 1998). Although we have not seen any significant effect of Ca2+ on [3H]LY354740 binding to mGlu2 and 3 receptors, we cannot rule out that part of the differential effect of ZnCl2 is not mediated via this site. The biphasic effect of ZnCl2 on [3H]-LY354740 binding to both receptors strongly suggests that this ion interacts with multiple sites which seem to have different properties on each receptor. Using functional assays in intact cells, potencies of 4– 20 and 4–5 µM have been reported for glutamate at mGlu2 and 3 receptors, respectively (Conn and Pin, 1997). Our results showed that glutamate is slightly more active on mGlu3 than 2 with an affinity close to 400 nM for the former receptor. Interestingly, apart from what was found with glutamate, numerous discrepancies were found when comparing the pharmacology reported using functional assays in intact cells and the results we obtained with binding assays. Quisqualate was reported to have an EC50 value greater than 1 mM on mGlu2, whereas we found a Ki value close to 100 µM using both [3H]-LY354740 and [3H]-DCG IV binding (this study; Cartmell et al., 1998). Using cAMP measurement in intact cells, l-AP4 was reported to be devoid of functional activity at both mGlu2 and 3 receptors (Conn and Pin, 1997). However, this compound was able to inhibit concentration-dependently [3H]-LY354740 binding to mGlu2 receptor with a Ki value of 129 µM. We have previously shown that l-AP3, l-AP5 and l-AP4 were able to antagonize 1S,3R-ACPD-stimulated GTPγ35S binding to mGlu2 receptor-transfected cell membranes with Ki values of 11, 22 and 143 µM, respectively (Cartmell et al., 1998) and the potency of l-AP4 correlates well with its Ki value determined in binding experiment. Interestingly, Laurie et al. (1995) have shown that l-AP4 inhibited [3H]-glutamate binding to mGlu3 receptor with a Ki value of 282 µM which is very close to the Ki value of 216 µM we found using [3H]-LY354740 binding, and our result for l-AP3 (114 µM) is also in agreement with the Ki value of 125 µM reported by this group with the same receptor. It appeared, thus, that the exclusive use of functional assays with intact cells to define receptor pharmacology and compound specificity can be misleading, particularly when uptake phenomena are known to take place. Furthermore, the lack of knowledge of receptor expression and occupancy has to be avoided in order to perform meaningful pharmacological comparisons. In this regard, the use of radiolabeled ligands will become essential and should be more extensively used in the course of mGlu receptors characterization. Finally, the mGlu2 receptor expressed using the SFV vector had characteristics very
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Table 1 Ki and Hill number values±standard deviation for various compounds to inhibit [3H]-LY354740 binding to cloned rat mGlu2 and 3 receptors Compound
mGlu2 Ki (µM)
mGlu2 Hill
mGlu3 Ki (µM)
mGlu3 Hill
LY341495 LY354740 DCG IV l-CCG I Glutamate 1S,3R-ACPD MPPG l-AP3 l-CA EGLU l-AP5 l-HCA l-HCSA Quisqualate l-AP4 Ibotenate NAAG l-CSA l-Aspartate DTT
0.009±0.001 0.013±0.002 0.11±0.02 0.14±0.02 1.2±0.2 4.5±0.5 18±5 19±3 21±5 27±2 51±8 83±27 99±28 113±30 129±25 139±48 236±113 250±48 320±130 453±200
0.96±0.09 0.72±0.05 0.83±0.1 0.78±0.05 1.055±0.07 1.072±0.08 1±0.1 1.15±0.3 0.9±0.1 1.021±0.1 0.8±0.1 1.2±0.4 1±0.2 0.84±0.3 0.99±0.2 0.9±0.2 0.91±0.3 1.2±0.4 1.1±0.5 0.5±0.1
0.01±0.002 0.051±0.003 0.15±0.05 0.3±0.07 0.44±0.05 16±3.6 104±28 114±24 80±19 4±0.6 136±22 51±21 208±37 22±5 216±79 123±50 19±1 320±50 420±40 12850±684
0.82±0.1 0.94±0.04 0.77±0.2 0.86±0.2 1.19±0.1 0.77±0.1 0.73±0.3 0.61±0.1 0.95±0.2 0.6±0.1 1.6±0.4 0.7±0.1 1.2±0.2 0.75±0.1 1.24±0.2 0.91±0.2 0.96±0.05 0.99±0.1 0.9±0.4 0.75±0.3
Fig. 3. Effect of ZnCl2 on [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptor-transfected cell membranes. Results are expressed as a percentage of [3H]-LY354740 specific bound and are the mean±standard deviation (bars) of four individual experiments performed in duplicate.
similar to that of the stably expressed mGlu2 receptor. This SFV-vector system of expression offered several advantages, particularly as their was no need to block the receptor to maintain the expression as was the case for the stable line. In this regard, it seems particularly appropriate to express receptors for which the natural ligand is secreted by the cells.
Fig. 4. Correlation between Ki values for LY354740, LY341495, DCG IV, l-CCG I, glutamate, 1S,3R-ACPD, MPPG, l-AP3, l-AP4, l-AP5, EGLU, quisqualate, NAAG and ibotenate obtained using [3H]LY354740 binding to rat mGlu2 and to rat mGlu3 receptors transiently expressed in CHO cells using SFV vectors (this study). The values have been converted to their negative logarithms.
Acknowledgements We thank Dr E.-J. Schlaeger and M. Fogetta for providing the CHO suspension cultures and A. Nilly and S. Chaboz for their skillful technical assistance.
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