Transposition of Three Amino Acids Transforms the HumanMetabotropic Glutamate Receptor (mGluR)-3-Positive AllostericModulation Site to mGluR2, and Additional Characterization ofthe mGluR2-Positive Allosteric Modulation Site□S

Blake A. Rowe, Herve Schaffhauser, Sylvia Morales, Laura S. Lubbers, Celine Bonnefous,Theodore M. Kamenecka, Jeffrey McQuiston, and Lorrie P. DaggettMerck Research Laboratories, West Point, Pennsylvania (B.A.R., H.S., L.S.L.); and Merck Research Laboratories, San Diego,California (S.M., C.B., T.M.K., J.M., L.P.D.)

Received February 20, 2008; accepted April 21, 2008

ABSTRACTGlutamate is a major neurotransmitter in the central nervous sys-tem, and abnormal glutamate neurotransmission has been impli-cated in many neurological disorders, including schizophrenia,Alzheimer’s disease, Parkinson’s disease, addiction, anxiety, de-pression, epilepsy, and pain. Metabotropic glutamate receptors(mGluRs) activate intracellular signaling cascades in a G protein-dependent manner, which offer the opportunity for developingdrugs that regulate glutamate neurotransmission in a functionallyselective manner. In the present study, we further characterize thehuman mGluR2 (hmGluR2) potentiator binding site by showingthat the substitution of the three amino acids found to be required

for hmGluR2 potentiation, specifically Ser688, Gly689, and Asn735,with the homologous hmGluR3 amino acids, inactivates the pos-itive allosteric modulator activity of several structurally uniquemGluR2 potentiators. Based on the characterization of thehmGluR2 potentiator binding site, we developed a novel scintilla-tion proximity assay that was able to discriminate between com-pounds that were hmGluR2-specific potentiators, and those thatwere active on both hmGluR2 and hmGluR3. In addition, wesubstituted Ser688, Gly689, and Asn735 into hmGluR3 and createdan active hmGluR2 allosteric modulation site on the hmGluR3receptor.

Glutamate is the primary excitatory neurotransmitter inthe mammalian central nervous system, and its activity ismediated by two classes of receptors: ionotropic glutamatereceptors, which are multimeric ion channels responsible forfast synaptic transmission, and metabotropic glutamate re-

ceptors (mGluRs), which are seven-transmembrane receptorsthat couple to G proteins to modulate slower synaptic trans-mission through intracellular second messengers. Becauseionotropic glutamate receptors are expressed by nearly alltypes of neurons and mediate fast excitatory neurotransmis-sion throughout the brain, direct pharmacological manipula-tion of this group of receptors has been difficult becauseinhibition could produce widespread disruption of brain func-tion, which could lead to negative side effects. Conversely,mGluRs that activate intracellular signaling cascades offer

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.108.138271.□S The online version of this article (available at http://jpet.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: mGluR, metabotropic glutamate receptor; GPCR, G protein-coupled receptor; SPA, scintillation proximity assay; WT, wildtype; hmGluR, human metabotropic glutamate receptor; GTP�S, guanosine 5�[�-thio]-triphosphate; GPT, glutamate pyruvate transminase;DCG-IV, (2S,2�R,3�)-dicarboxycyclopropyl)glycine; HEK, human embryonic kidney; IP1, inositol monophosphate; HBS, HEPES-buffered saline;MRLSD-650, 2-[(6,7-dichloro-2-cyclopentyl-2-methyl-1-oxo-2,3-dihydro-1H-inden-5-yl) oxy]-N-[4-(1H-tetraazol-5-yl) phenyl] acetamide; MRLSD-230,3�-((2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-yloxy)methyl)biphenyl-4-carboxylic acid; MRLSD-041, 6,7-dichloro-2-propyl-5-(3-((pyri-din-4-ylthio)methyl)benzyloxy)-2,3-dihydro-1H-inden-1-one; MRLSD-973, 2,2,2-trifluoro-N-(pyrimidin-5-ylmethyl)-N-(3-(2-(trifluoromethyl) benzyl)-phenyl) ethanesulfonamide; MRLSD-064, 1-(4-((3�-(1H-tetrazol-5-yl)biphenyl-3-yl)methoxy)-2-hydroxy-3-methylphenyl)-3,3-dimethylbutan-1-one;MRLSD-726, 1-(4-((3�-(1H-tetrazol-5-yl)biphenyl-3-yl)methoxy)-3-bromo-2-hydroxyphenyl)-3-methylbutan-1-one; MRLSD-438, 1-(4-(3-(3-(2H-tetrazol-5-yl)phenoxy) benzyloxy)-3-bromo-2-hydroxyphenyl)-3-methylbutan-1-one; MRLSD-667, 3�-((4-(3,3-dimethylbutanoyl)-3-hydroxy-2-methylphenoxy) methyl)biphenyl-3-carboxylic acid; LY487379, N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethyl-amine; TM, transmembrane domain; PM, point mutation; HTS, high-throughput screening; LY354740, 2-aminobicyclo(3.1.0)hexane-2,6-dicarboxylic acid; LY379268, 2-oxa-4-aminobicyclo(3.1.0)hexane-4,6-dicarboxylic acid; MGS0028, 2-amino-6-fluoro-4-oxobicyclo(3.1.0)hexane-2,6-dicarboxylic acid; CGP7930, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethylpropyl)phenol; PI, phosphoinositide; Ro67-7476, (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)-pyrrolidine.

0022-3565/08/3261-240–251$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 326, No. 1Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 138271/3356012JPET 326:240–251, 2008 Printed in U.S.A.

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an opportunity for developing drugs that regulate glutamateneurotransmission in a functionally selective manner.

mGluRs belong to the G protein-coupled receptor (GPCR)superfamily and have eight known subtypes (mGluR1–8).These receptors divide into three groups based upon se-quence homology, intracellular messengers, and pharmacol-ogy. Group I receptors (mGluR1 and 5) activate phospho-lipase C via Gq proteins and initiate an inositol triphosphate/diacylglycerol second messenger cascade. Group II (mGluR2and 3) and group III (mGluR4, 6, 7, and 8) receptors inhibitadenylate cyclase via Gi/o proteins (Schoepp, 1994; Conn andPin, 1997; Schoepp, 2001).

mGluR2 and mGluR3 are located primarily presynapti-cally in the hippocampus, cortex, striatum, thalamus, andamygdala (Ohishi et al., 1993). Activation of these receptorshas been shown to decrease synaptic transmission and glu-tamate release in the hippocampus (Macek et al., 1996). Thedistribution and prominent functions of group II mGluRs inneuronal excitability and synaptic transmission suggest thatmodulation of these GPCRs is a promising strategy for thetreatment of neurological and neuropsychiatric disorders,such as anxiety, schizophrenia, and pain (Conn and Pin,1997; Chavez-Noriega et al., 2002; Varney and Gereau, 2002;Lorrain et al., 2003). Furthermore, mGluR-mediated gluta-mate activity has been shown to influence calcium and po-tassium ion channels, modulate N-methyl-D-aspartate and�-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid re-ceptor currents, and GABA release (Lea and Faden, 2003).The ability of mGluRs to modulate neuroprotective and neu-rotoxic systems suggests that some mGluRs play a role inneuronal injury following trauma. Indeed, studies haveshown group II and III mGluRs to have a neuroprotectiverole (for review, see Lea and Faden, 2003).

Several lines of evidence suggest that positive allostericmodulators of the mGluRs may offer advantages over classi-cal GPCR agonists by overcoming receptor desensitizationthat often occurs after repeated dosing of agonists (Pin et al.,2001; May and Christopoulos, 2003). Furthermore, in con-trast to full agonists, potentiators can be designed to bedevoid of any agonist activity or act as weak partial agonists.However, when agonist is present, potentiators can signifi-cantly increase potency and/or efficacy of the agonist. Devel-opment of potentiators could also be useful in disease statesin which the level of the endogenous ligand is attenuated.Patients administered a potentiator could achieve normalagonist responses. Due to the high level of conservation of theagonist binding site of mGluRs, developing subtype-specificagonists has proven to be difficult. To date, no agonist hasbeen reported to discriminate between mGluR2 and mGluR3.However, as described in this article and previously, severalmGluR2-specific positive allosteric modulators have beenidentified (Johnson et al., 2003; Schaffhauser et al., 2003; Huet al., 2004; Pinkerton et al., 2004a,b, 2005; Bonnefous et al.,2005).

The present study had three main goals. First, we wanted toprovide additional pharmacological characterizations of a seriesof novel and structurally distinct mGluR2 potentiators. Wetested two indanones (Pinkerton et al., 2005), a biphenyl-indanone (Bonnefous et al., 2005) and a pyrimidine methylanilines (Hu et al., 2004). Second, we wanted to determinewhether this diverse set of mGluR2 allosteric modulators alsobinds to the known mGluR2 potentiator site defined by amino

acids Ser688, Gly689, and Asn735 (Schaffhauser et al., 2003) or todetermine whether another novel potentiator site exists. Third,we wanted to develop a high-throughput transfection systemand dual scintillation proximity assay (SPA) using hmGluR2-wild type (WT) and the hmGluR2_Leu688-, Val689-, and Asp735-expressing cells. With these assays, we could easily identifyhmGluR2-specific potentiators (i.e., positive only in thehmGluR2-WT assay) versus allosteric modulators of bothhmGluR2 and hmGluR3 (i.e., active in both hmGluR2-WT andthe hmGluR2_Leu688, Val689, and Asp735 assays).

Materials and MethodsMaterials. Glutamate, GDP, guanosine 5�[�-thio]-triphosphate

(GTP�S), glutamate pyruvate transaminase (GPT), coenzyme pyridoxalphosphate, and pyruvate were obtained from Sigma-Aldrich (St. Louis,MO). [35S]GTP�S (1250 Ci/mmol), Unifilter GF/B plates, andMicroScint 20 were purchased from PerkinElmer Life and AnalyticalSciences (Boston, MA). (2S,2�R,3�R)-2-(2�,3�)-dicarboxycyclopropyl)gly-cine (DCG-IV) was purchased from Tocris Cookson Inc. (Ellisville, MO).Male Sprague-Dawley rats (250–300 g) were purchased from Harlan(Indianapolis, IN). The pcDNA3.0, pcDNA3.1-based mammalian ex-pression vectors were purchased from Invitrogen (Carlsbad, CA). AQuikChange site-directed mutagenesis kit was obtained from Strat-agene (La Jolla, CA), and an Effectene kit was purchased fromQIAGEN (Valencia, CA). BioCoat Fibronectin 24-well tissue cultureplates were obtained from BD Biosciences (Bedford, MA). Dowex-1-X8(200–300 mesh in the formate form) was obtained from Bio-Rad (Her-cules, CA). Myo-[2-3H]inositol (specific activity 18 Ci/mmol) and RNAbinding YSi SPA beads were purchased from GE Healthcare (Piscat-away, NJ). Protease inhibitor cocktail was obtained from Roche Diag-nostics (Indianapolis, IN). Picoplate 96-well TopCount plates andTopSeal were purchased from PerkinElmer Life and Analytical Sci-ences. The compounds MRLSD-650, MRLSD-230, MRLSD-041, andMRLSD-973 (Fig. 1, A–D) have described previously (Hu et al., 2004;Bonnefous et al., 2005; Pinkerton et al., 2005), and MRLSD-064,MRLSD-726, MRLSD-438, and MRLSD-667 (Fig. 1, E–H) are novel(Cube et al., 2004; Pinkerton et al., 2006a,b; Govek et al., 2006). AllMRLSD-compounds were synthesized at Merck Research Laboratories(San Diego, CA).

Membrane Preparations. Membranes were isolated fromhmGluR2- and hmGluR3-expressing stable cell lines as describedpreviously (Schaffhauser et al., 2003). In brief, confluent cells werewashed into phosphate-buffered saline and harvested by centrifuga-tion (200g). The resulting cell pellet was 1) homogenized in buffer A(20 mM HEPES and 10 mM EDTA, pH 7.4) and centrifuged at40,000g (20 min at 4°C), 2) the resulting pellet was washed one timein buffer A and then one time in buffer B (20 mM HEPES and 0.1mM EDTA, pH 7.4), and 3) resuspended in buffer B. Membraneswere stored at �80°C; protein concentration was �1 mg/ml.

Whole-rat brain membranes were isolated as described previously(Schaffhauser et al., 2003). Briefly, rats were decapitated, and wholebrains were homogenized in 6 volumes (w/v) of 10% sucrose at 4°Cusing a glass-Teflon homogenizer. The homogenate was centrifuged(1000g for 10 min), and the supernatant was removed and centri-fuged again (40,000g for 20 min at 4°C). The pellet was resuspendedin 25 ml of water and centrifuged (8000g for 10 min). The superna-tant and the buffy coat layer were removed and centrifuged (40,000gfor 20 min at 4°C). The pellet was resuspended in buffer C (5 mMHEPES-KOH, pH 7.4), freeze-thawed twice, and then centrifuged(40,000g for 20 min). The resulting pellet was resuspended in bufferC and stored at �80°C at a protein concentration of �3 mg/ml.Protein measurements were determined with the Bio-Rad DC kit.

[35S]GTP�S Binding Assay. This assay was performed as de-scribed in Schaffhauser et al. (2003). Briefly, mGluR2, mGluR3, orrat brain membranes were thawed, further processed, and thenresuspended in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl,

A Three-Amino Acid Change Transforms mGluR3 Potentiator 241

and 3 mM MgCl2) to a final protein concentration of 0.5 mg/ml(hmGluR2- and hmGluR3-expressing membranes) or 0.1 mg/ml (ratbrain). In a 96-well plate (Beckman Coulter, Fullerton, CA), testcompounds were added (300 pM–10 �M) along with 5 �M GDP,membrane (10 �g/well rat brain and 50 �g for recombinant cells),and 0.05 nM [35S]GTP�S (total volume 0.5 ml). Plates were incu-bated at 30°C for 1 h, and then the assay was terminated by rapidfiltration (Unifilter GF/B plate with 96-well cell harvester; BrandelInc., Gaithersburg, MD). Plates were then rinsed, dried, andMicroScint 20 was added to each well, followed by counting in aTopCount scintillation counter (PerkinElmer Life and AnalyticalSciences).

Cloning of Wild-Type hmGluR2 and hmGluR3 cDNAs. Clon-ing of WT hmGluR2 cDNA (Flor et al., 1995; Schaffhauser et al.,2003) and WT hmGluR3 cDNA was performed as described previ-ously (Varney et al., 1998; Schaffhauser et al., 2003) and then clonedinto pcDNA 3.0-based mammalian expression vectors.

Construction of Chimeric and Point Mutations ofhmGluR2/3 and hmGluR3/2 cDNAs. The mGluR2 and mGluR3chimeric receptor cDNAs were generated by site-directed mutagen-esis from WT cDNAs to introduce single or multiple mutations asdescribed previously (Schaffhauser et al., 2003). All of the single and

multiple point mutations (PMs) of hmGluR2/3 receptor cDNAs weresynthesized using the QuikChange site-directed mutagenesis kit andcloned into the pcDNA3.1-based mammalian expression vectors.

Transient Transfection of Chimeric hmGluR2/3 cDNAs intoHEK293 Cells. All generated cDNAs constructs, including WT, werecotransfected with G�16 cDNA (kindly provided by Aurora Pharma-ceuticals, currently Pfizer, San Diego, CA) into HEK293 cells by amodified reverse transfection protocol using a lipid-based transfec-tion reagent (Ziauddin and Sabatini, 2001; Schaffhauser et al.,2003). Briefly, hmGluR2 or hmGluR3, G�16, and enhanced greenfluorescent protein cDNAs were mixed with DNA condensationbuffer plus sucrose (0.3 M). Enhancer solution was added to themixture, mixed, and then incubated at room temperature for 5 min.Effectene reagent was then added to the mixture, mixed, and thenincubated for 10 min at room temperature. Finally, 0.25% glycogenwas added and mixed, and then the resulting DNA-lipid-polymertransfection reagent was evenly added to the bottom of a fibronectin-coated tissue culture plate. The plates were then allowed to incubateovernight at 4°C. The resulting plates were dried under vacuum andseeded with HEK293 cells (0.7 � 106 cells/well) and incubated in a37°C, CO2 incubator for 36 h.

Fig. 1. A to H, chemical structures ofMRLSD-650, MRLSD-230, MRLSD-041, MRLSD-973, MRLSD-064, MRLSD-726, MRLSD-438, and MRLSD-667.

242 Rowe et al.

Measurement of Phosphoinositide Hydrolysis. [3H]Inositolmonophosphate ([3H]IP1) assays were performed as described previ-ously (Schaffhauser et al., 2003). Briefly, transfected HEK293 cellswere labeled overnight with 1 �Ci/well myo-[2-3H]inositol in glu-tamine-free Dulbecco’s modified Eagle’s medium. Subsequently, thecells were washed with HBS buffer containing 125 mM NaCl, 5 mMKCl, 0.62 mM MgSO4, 1.8 mM CaCl2, 6 mM glucose, and 20 mMHEPES, pH 7.4, for 45 min at 37°C. Following the washes, cells wereincubated with HBS buffer containing 10 mM LiCl for 20 min. Afterincubation, 5 �M glutamate alone (EC10), 1 mM glutamate (EC100),or 5 �M glutamate (EC10) in combination with varying concentra-tions of test compounds was added and incubated for 1 h at 37°C. Thereactions were terminated, and accumulated [3H]IP1 was extractedby adding 4°C chloroform/methanol/HCl (4 N). The mixtures weretransferred to new tubes and then extracted with chloroform andH2O. The aqueous phase was separated from the organic phase bysettling for 15 min. The [3H]IP1 fraction was isolated using Dowex-1-X8 and quantified by liquid scintillation counting. Data were nor-malized to the response seen with 1 mM glutamate. For hmGluR3chimera transfections, residual glutamate was removed by preincu-bating and running the assay in HBS and HBS/LiCl buffers contain-ing 3.0 mg of GPT, 3.0 mg of pyridoxal phosphate, and 15.0 mg ofpyruvate per 50 ml. After 20-min incubation in the LiCl-containingbuffer, 10 nM DCG-IV alone (EC10), 3 �M DCG-IV (EC100), or 10 nMDCG-IV (EC10) in combination with varying concentrations of testcompounds was added and incubated for 1 h at 37°C. The reactionswere terminated, and the assay was completed as described above.

SPA for IP1 in Transiently Transfected mGluR2 Cells. Theassay was performed as reported previously (Brandish et al., 2003),with modifications. HEK293 transfected cells were labeled overnightwith 2 �Ci/well myo-[2-3H]inositol in glutamate-free Dulbecco’s mod-ified Eagle’s medium in a 96-well plate. The following day (18 h aftermyo-[2-3H]inositol addition), the medium was removed, and the cellswere washed two times for 40 min in HBS buffer containing 125 mMNaCl, 5 mM KCl, 0.62 mM MgSO4, 1.8 mM CaCl2, 6 mM glucose, and20 mM HEPES, pH 7.4, for 45 min at 37°C. Following the washes,cells were incubated with HBS buffer containing 10 mM LiCl (20min). After incubation, 5 �M glutamate alone (EC10), 1 mM gluta-mate (EC100), or 5 �M glutamate (EC10) in combination with varyingconcentrations of test compounds was added, and cells were incu-bated for an additional hour at 37°C. For hmGluR3 chimeras, weused DCG-IV at 3 �M for the 100% response and 30 nM for �10%response. The wells were aspirated to terminate the incubation, and50 �l of 10 mM formic acid was added to each well and allowed toincubate at room temperature for 20 min. The plate was placed onshaker table for 5 min. In a 96-well Picoplate, 80 �l/well SPA beadswas added to 40 �l of assay lysate, which was mixed with the beadsby pipetting. The plates were sealed with TopSeal and placed ona shaker table for 1 h. Plates were counted in a TopCount-NXT(PerkinElmer Life and Analytical Sciences) after 2-h delay.

Data and Statistical Analysis. All experiments were performedin triplicate and repeated in three separate assays. Data were nor-malized to the response obtained with 1 mM glutamate or 3 �MDCG-IV. The obtained curves were fitted to a four-parameter logisticequation giving EC50 values, Hill coefficient, and maximal effectusing Prism (GraphPad Software Inc., San Diego, CA).

The data were analyzed via one-way analysis of variance as ap-propriate followed by post hoc comparisons. P values 0.05 for alltests and comparisons were deemed significant unless otherwiseindicated. A logarithmic scale was used to satisfy assumptions ofequal variance and normal distribution.

ResultsPharmacological Characterization of MRLSD-650 on

Rat and Recombinant Human Group II mGluRs. Stim-ulation of [35S]GTP�S binding in native and recombinant

hmGluR2 membrane preparations was performed to providea functional measure of G�i-coupled receptors (Lazareno andBirdsall, 1993). Dose-response analysis of MRLSD-650 (Fig.1A) alone determined no stimulation of [35S]GTP�S bindingin membranes prepared from rat brain or from cells express-ing hmGluR2 (Fig. 2, A and B). Conversely, when increasingconcentrations of MRLSD-650 were combined with an EC10

concentration of glutamate (1 �M), there was a significantincrease in the magnitude of the glutamate-induced

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Fig. 2. Concentration-response curves for MRLSD-650 in the absence(open symbol) or presence (closed symbol) of glutamate (EC10) generatedfrom stimulation of [35S]GTP�S binding to membrane from rat brain (A)or cell lines expressing hmGluR2 (B). Concentration-response curves forglutamate in the absence (open symbol) or presence (closed symbol) of 1�M MRLSD-650 generated from stimulation of [35S]GTP�S binding tomembrane from cell lines expressing hmGluR2 (C). Results are expressedas a percentage of the response to 1 mM glutamate, and they are themeans of three individual experiments performed in triplicate.

A Three-Amino Acid Change Transforms mGluR3 Potentiator 243

[35S]GTP�S binding. A similar level of potentiation was seenin both the rat brain and hmGluR2 membrane preparationswhen normalized to the response of 1 mM glutamate, and thepotentiation occurred in a concentration-dependent manner.Table 1 summarizes the EC50 values and Emax values forMRLSD-650 in both rat brain and hmGluR2 membranes.MRLSD-650 increased the potency of glutamate �2-fold inhmGluR2 membranes from glutamate alone: from an EC50

value of 11.61 �M (11.09–12.16 �M) to an EC50 value of 5.15�M (4.90–5.42 �M) for glutamate MRLSD-650 (Fig. 2C).

We also investigated the specificity of MRLSD-650 in othermGluR-expressing cells and determined that MRLSD-650has no detectable glutamate-induced agonist, antagonist, orpotentiator activity in cell lines expressing the hmGluR1,hmGluR3, hmGluR4, hmGluR5a, or hmGluR7a receptor(data not shown). Hence, we determined that MRLSD-650 isa selective mGluR2-positive modulator that increases boththe potency and efficacy of the agonist ligand glutamate.

Positive Allosteric Modulation by MRLSD-650 Is Me-diated by Three Amino Acid Residues in the Trans-membrane Domains of hmGluR2. To determine whetherthe location of the binding site of MRLSD-650 on hmGluR2was the same three residues determined previously to bindthe mGluR2 potentiator LY487379 (Schaffhauser et al.,2003), we investigated the effect of MRLSD-650 on themGluR2/3 chimeric cDNAs reported previously. The variousmutant receptors were prepared by exchanging either seg-ments, single amino acid residues, or multiple amino acidsresidues between hmGluR3 and hmGluR2 (Fig. 3). Transienttransfection of HEK293 cells with WT hmGluR2 in conjunc-tion with G�16 resulted in glutamate-induced production of[3H]IP1 in a concentration-dependent manner, with a geo-metric mean of EC50 � 40 �M (range 30–53 �M). The addi-tion of MRLSD-650 alone (10 �M) increased production of[3H]IP1 4-fold over the response observed with 5 �M gluta-mate (Fig. 3A, black bar). This “basal” increase in [3H]IP1

production seen in the presence of no exogenously addedglutamate most likely results from endogenous glutamatebeing released from the cultured cells due to cell rupture anddeath. We attempted to remove this endogenous glutamatewith multiple media changes before each assay, but it is verydifficult to remove from the media of living cells. However,when 10 �M MRLSD-650 was added in the presence of anEC10 concentration of glutamate (i.e., 5 �M glutamate), wesaw a significant increase in the magnitude of the glutamate-induced phosphoinositide hydrolysis (Fig. 3A, striped bar).

To localize the site of the potentiator interaction, we trans-fected HEK293 cells with three different chimeric constructs

and determined that the exchange of the transmembraneregions TMI-III (Thr550 to Leu656) or a single amino acidresidue in TMVII (Ser817) of hmGluR2 with hmGluR3 se-quences did not significantly effect the potentiation ofMRLSD-650 on glutamate-induced [3H]IP1 accumulation(Fig. 3, B and D, respectively). In contrast, the exchange ofthe mGluR2 amino acid residues present in TMIII-V (Leu656

to Arg750) with homologous hmGluR3 sequences resulted inthe complete loss of the potentiator activity of MRLSD-650(Fig. 3C), and also the loss of the basal activity. Previously,we had identified this same domain as the binding region ofthe mGluR2 potentiator LY487379. The magnitude of gluta-mate-stimulated [3H]IP1 was similar to WT mGluR2, sug-gesting that the loss of effect of MRLSD-650 was mediated byalteration of the amino residues in the allosteric binding site(present between Leu656 and Arg750), and not by alteration ofthe glutamate binding site. Sequence alignments ofhmGluR2 and hmGluR3 in this TMIII-V segment revealedmultiple amino acid differences in residues contained withinthese transmembrane domains and extracellular loop(Fig. 4).

To identify the precise amino acid residues involved in thepositive allosteric modulation of the hmGluR2 by MRLSD-650, we used various multiple or single amino acid residueshmGluR2/3 chimeric constructs described previously inSchaffhauser et al. (2003) (Table 2). We determined by Dun-nett’s post hoc analysis that all of the chimeric constructsresponded to glutamate (P 0.05) and only S688L/G689V(PM2) gave a glutamate stimulation greater than wild type(P 0.05). No other significant differences were seen be-tween the chimeric constructs and WT hmGluR2 in the lim-ited agonist pharmacology profiling performed. TMIV substi-tutions of either the single residue A681F (PM1) or doubleresidues V695S/A696V (PM5) had no significant effect on theactivity of MRLSD-650 (Table 2). A significant reduction ofthe potentiation by MRLSD-650 of glutamate-induced[3H]IP1 accumulation was observed with the hmGluR2S688L/G689V (PM2) receptor mutant (Fig. 5A). Interestinglythe single point mutations S688L (PM3) or G689V (PM4) hadno significant effect individually, suggesting that these resi-dues need to be present together to interact with MRLSD-650and potentiate hmGluR2 in the presence of agonist (Fig. 5A).Further analysis of the amino acid residues that varied be-tween mGluR2 and mGluR3 in the extracellular segments ofTMIV-V TMV confirmed that the single mutation H723V(PM8), G730I (PM9), or A740I (PM10) or double mutationsA710L/P711A (PM6) or V716T/T718I (PM7) had no signifi-cant reduction in the potentiator effect of MRLSD-650 onglutamate-induced [3H]IP1 production. In contrast, we saw asignificant reduction in the positive modulation by MRLSD-650 in the double mutant A733T/N735D (PM11) (Fig. 5B;Table 2). Interestingly, when the A733T and N735D muta-tions were assayed separately, only the single point mutationN735D (PM13) significantly reduced the effect of MRLSD-650, suggesting that Ala733 (PM12) is not involved in thepotentiation of this compound (Fig. 5B; Table 2). The com-bined double mutants S688L/N735D (PM14), G689V/N735D(PM15) or the triple mutant S688L/G689V/N735D (PM16) allcompletely disrupted the activity of MRLSD-650 (Fig. 5C;Table 2). We determined by Western blot analysis that thelack of potentiation by each of these chimeric constructs wasnot due to a different level of protein expression or sensitivity

TABLE 1Activity of MRLSD-650 on glutamate-induced �35S GTP�S bindingConcentration-response curves were determined in the presence of 1 �M glutamatein membranes prepared from rat brain or recombinant cells expressing hmGluR2, or10 nM glutamate for the hmGluR3 preparation. Results are expressed as mean EC50values (lower-upper S.D.) and mean efficacy (Emax) � S.E.M., expressed relative tothe maximal stimulation achieved by 1 mM glutamate alone. Summary data arecalculated from three individual experiments performed in triplicate.

Membrane Preparation EC50 Emax

nM

Rat brain 270 (187–392) 95.6 (86.6–108.6)hmGluR2 435 (354–531) 86.5 (72.6–103.3)hmGluR3 N.E. N.E.

N.E., no effect at concentrations up to 10 �M.

244 Rowe et al.

to glutamate since all constructs expressed protein and re-sponded to glutamate very similarly to WT mGluR2 (Schaff-hauser et al., 2003).

Clearly, the S688L, G689V, and N735D (PM16) substitu-

tions rendered MRLSD-650 inactive (Figs. 5C); interestingly,these are the same residues described previously for theinteraction site of LY487379 with mGluR2 (Schaffhauser etal., 2003). However, this analysis does not definitively prove

Fig. 3. Effect of MRLSD-650 at wild-type hmGluR2 (A), the chimeric recep-tor TMI-III (B), the chimeric receptorTMIII-V (C), and the chimeric recep-tor TMVII (D) on 5 �M glutamate-induced PI hydrolysis. Glutamate (5�M) alone (white bar), 10 �MMRLSD-650 alone (black bar), and 10�M MRLSD-650 5 �M glutamate(striped bar). Results are expressed aspercentage of 1 mM glutamate re-sponse, and they are the means �S.E.M. from three individual experi-ments performed in triplicate. Right,schematic diagram of receptor con-structs indicating the location ofmGluR3 fusion sites (white). ��, P 0.001 for 10 �m MRLSD-650 5 �Mglutamate versus 5 �M glutamate. ##,P 0.001 for 10 �m MRLSD-650 5�M glutamate in TM versus WT.

Fig. 4. Sequence alignment of mGluR2 and mGluR3 between TMIII and TMV. The numbers represent the different PM designations (see Table 2).Highlighted residues represent the amino acids involved in the allosteric binding site.

A Three-Amino Acid Change Transforms mGluR3 Potentiator 245

that these are the only three residues involved in the activityof these compounds. Therefore, to prove that these threeresidues were involved in the potentiation effect, we madeone additional chimeric construct in which these amino acidswere exchanged in hmGluR3 to the hmGluR2 residues (i.e.,L688S/V689G/D735N). HEK293 cells were then transientlytransfected with this chimeric construct or the wild-typehmGluR3 receptor, along with G�16. Glutamate induced theproduction of [3H]IP1 in a very similar concentration-depen-dent manner in the wild-type receptor and the L688S,V689G, and D735N mutant. In WT hmGluR3, the EC50 valuefor glutamate was 360 nM (227–571 nM), whereas the EC50

value for hmGluR3 (L688S/V689G/D735N) was 378 nM (273–525 nM) (Fig. 6A). During these experiments, we found thathmGluR3 transiently transfected cells were very sensitive toglutamate, and any residual glutamate from culturing of thecells was enough to stimulate the cells treated with 10 �MMRLSD-650 to full potentiation. To circumvent this issue, weneeded to add GPT, pyridoxal phosphate, and pyruvate to allwash and assay buffers to remove this residual glutamate.Under these assay conditions, we also used DCG-IV as theagonist (10 nM for EC10 and 3 �M for EC100). As anticipated,when cells transiently transfected with the WT hmGluR3were exposed to MRLSD-650 10 nM DCG-IV, they failed toinduce the production of [3H]IP1 over 10 nM DCG-IV alone(Fig. 6B). In contrast, in cells transiently transfected withhmGluR3 L688S/V689G/D735N mutations, production of[3H]IP1 was �100% of control (Fig. 6C), suggesting that thehmGluR3 L688S, V689G, and D735N mutations created anactive mGluR2-like allosteric modulation binding site on thehmGluR3 receptor.

To further evaluate the hmGluR2 potentiator bindingpocket, we developed a high-throughput screening (HTS) 96-well SPA. In this HTS assay, we can rapidly measure theproduction of IP1 in HEK293 cells transiently transfectedwith either WT mGluR2 or S688L/G689V/N735D chimericconstructs. This assay allowed us to screen four times asmany compounds, at more concentrations and in less timethan with the traditional [3H]IP1 assay using columns. From

these studies, we determined that both of these constructsinduced [3H]IP1 production in response to glutamate in avery similar, concentration-dependent manner (Fig. 7A). WThmGluR2 induced a glutamate EC50 of 57.3 �M (50.5–65.1�M), whereas the hmGluR2 S688L/G689V/N735D chimeraconstruct induced a glutamate EC50 value of 72.2 �M (65.9–79.1 �M). As seen in Fig. 7, B and C, MRLSD-650 and threeadditional selective hmGluR2 potentiators—MRLSD-230(Fig. 1B), MRLSD-041 (Fig. 1C), and MRLSD-973 (Fig. 1D)(Hu et al., 2004; Bonnefous et al., 2005; Pinkerton et al.,2005)—were assayed in this HTS assay using transientlytransfected WT hmGluR2 or hmGluR2 S688L/G689V/N735Dtriple mutant cells (Table 3). We were able to rapidly deter-mine EC50 values that were comparable with those deter-mined in the [3H]IP1 assay and also confirm that these fourcompounds were not active when assayed with cells express-ing the triple mutant chimera. This assay was faster, re-quired much less DNA for transfections, and lent itself to96-well automation systems not available in our other 12-well IP1 assay. Hence, this HTS assay allows for rapidscreening of potentiators in a much more cost-effective andtimely paradigm.

Transmembrane Domain of hmGluR3 Also MediatesPositive Allosteric Modulation of Select Compounds.Interestingly, we also identified other structurally distinctcompounds—MRLSD-064 (Fig. 1E), MRLSD-726 (Fig. 1F),MRLSD-438 (Fig. 1G), and MRLSD-667 (Fig. 1H)—that hadsome hmGluR3 [35S]GTP�S binding activity in cells transientlytransfected with the hmGluR2 S688L/G689V/N735D chimera.These compounds were then assayed using the recombinanthmGluR3 membranes and found to be active with there as well(Fig. 8; Table 4). Hence, we have determined that these com-pounds are both hmGluR2 potentiators (46–197 �M) and weakhmGluR3-positive allosteric modulators (438–950 �M).

DiscussionTo date, various studies have described group II mGluR

receptor-selective agonists, including the heterobicyclic

TABLE 2Effect of glutamate and percentage of potentiation by MRLSD-650 obtained from �3H IP1 accumulation in wild-type and mutated receptorsMutant or wild-type hmGluR2 were transiently co-expressed in HEK293 cells with G�16 and stimulated with or without 5 �M glutamate and/or 10 �M MRLSD-650. Maximalstimulation for wild-type and mutated hmGluR2 was determined using 1 mM glutamate. Summary data are calculated from three individual experiments performed intriplicate and are represent the percentage of potentiation (glutamate 5 �M, and MRLSD-650 glutamate 5 �M, respectively) � S.E.M.

Name (Point Mutation) Mutation EC10 Glutamate (5 �M Glutamate,% of 1 mM Glutamate Response)

EC10 Glutamate MRLSD-650 (5 �MGlutamate 10 mM MRLSD-650,% of 1 mM Glutamate Response)

Wild type None 9 � 1 92 � 4PM1 A681F 5 � 2 80 � 3PM2 S688L/G689V 3 � 1 46 � 6*PM3 S688L 8 � 2 98 � 7PM4 G689V 16 � 2 79 � 10PM5 V695S/A696V 4 � 2 64 � 5PM6 A710L/P711A 9 � 1 81 � 2PM7 V716T/T718I 8 � 2 80 � 5PM8 H723V 7 � 2 79 � 5PM9 G730I 9 � 1 89 � 5PM10 A740I 1 � 1 82 � 10PM11 A733T/N735D 9 � 1 30 � 1**PM12 A733T 14 � 3 88 � 9PM13 N-735D 5 � 1 27 � 2**PM14 S688L/N735D 1 � 1 7 � 1**PM15 G689V/N735D 5 � 1 5 � 1**PM16 S688L/G689V/N735D 8 � 2 9 � 3**

* P 0.05 significantly different from wild type, unpaired Student’s t test.** P 0.001 significantly different from wild type, unpaired Student’s t test.

246 Rowe et al.

amino acids LY354740, LY379268, and MGS0028, which arebelieved to act at the glutamate binding site and activateboth mGluR2 and mGluR3 (Monn et al., 1999; Pin et al.,1999; Kunishima et al., 2000; Nakazato et al., 2000). In thecurrent study, we characterized a novel mGluR2-specific,positive allosteric modulator and identified that the site ofaction of this compound is separate from the agonist bindingsite on the receptor. The presence of another modulatory siteon mGluR2 and mGluR3 proteins allows for the developmentof compounds that selectively potentiate mGluR2 but notmGluR3 or vice versa. MRLSD-650 is a novel, low molecular-weight (mol. wt. � 500.4) compound that acts as a selectivemGluR2-positive allosteric modulator in both native (ratbrain membranes) and recombinant (stably or transientlytransfected) cells expressing human mGluR2.

Group II mGluR receptors have been reported previouslyto stimulate [35S]GTP�S binding in recombinant membranepreparations (Kowal et al., 1998). In the current study,MRLSD-650 alone did not enhance [35S]GTP�S binding inmembranes prepared from rat brain or in membranes pre-

pared from cells stably expressing human mGluR2. However,MRLSD-650 produced very robust, positive modulation ofglutamate-induced responses in both recombinant humanmGluR2 and native rat mGluR2 membranes. These resultsare similar to the results reported for the positive allostericmGluR1 modulator Ro 67-7476 (Knoflach et al., 2001) andthe GABAB(1b/2A) receptor modulator CGP7930 (Urwyler etal., 2001). The potentiation observed was highly selective formGluR2, since no modulatory activity was seen in cells sta-bly expressing human mGluR1, mGluR3, mGluR4, mGluR5,or mGluR7.

The addition of 1 �M MRLSD-650 increased the potency ofglutamate by 2-fold, and MRLSD-650 also increased themaximal response to glutamate. Previous radioligand bind-ing studies (Schaffhauser et al., 2003) support the idea thatthis change in potency might reflect a change in the affinityof the receptor for the agonist. Mechanistically, MRLSD-650might produce this effect by either 1) increasing the affinityof the mGluR2 for the agonist, 2) increasing coupling potencyand or coupling efficiency by enhancing the ability of the

Fig. 5. Effect of MRLSD-650 at hmGluR2 mutatedat S688L, G689V, or both (A); at A733T, N735D, orboth (B); or combined (C) on glutamate-induced PIhydrolysis in HEK293 cells. Glutamate (5 �M) alone(white bar), 10 �M MRLSD-650 alone (black bar),and 10 �M MRLSD-650 5 �M glutamate (stripedbar). Results are expressed as percentage of 1 mMglutamate, and they are the means � S.E.M. fromthree individual experiments performed in tripli-cate. �, P 0.05 significantly different from wildtype, unpaired Student’s t test. ��, P 0.001 signif-icantly different from wild type, unpaired Student’st test.

A Three-Amino Acid Change Transforms mGluR3 Potentiator 247

activated receptor to couple more efficiently to its endoge-nous G protein(s), or 3) allowing more receptors to remain inthe active configuration for longer periods or increasing thetotal number of receptors in the active configuration (Hall,2000; Parmentier et al., 2002; Gilchrist, 2007; Kenakin, 2007;Leach et al., 2007; Schwartz and Holst, 2007). Unfortunately,to date our data have not allowed us to discriminate betweenthese three potential mechanisms, although we did deter-mine that the potentiator increases the affinity of the agonistfor mGluR2 (Schaffhauser et al., 2003).

The site of action of MRLSD-650 was found to be to thesame three amino acid residues that bind the novel mGluR2potentiator LY487379 (Schaffhauser et al., 2003). Our de-

tailed molecular investigation of these mGluR2 and mGluR3chimeric receptors and point mutations also determined thatour transient assay systems (PI and SPA) express high levelsof the mGluR2. Interestingly, MRLSD-650 could evoke a

Fig. 6. Concentration-response curves for glutamate at wild-type hm-GluR3 or triple hmGluR3 L688S, V689G, and D735N mutant receptors(A). WT hmGluR3 (closed symbol); hmGluR3 L688S, V689G, and D735N(open symbol). Effect of MRLSD-650 at wild-type hmGluR3 (B) or triplehmGluR3 L688S, V689G, and D735N receptor mutant (C) on DCG-IV-induced PI hydrolysis in HEK293 cells. DCG-IV (10 nM) only (white bar),10 nM DCG-IV 10 �M MRLSD-650 (black bar). Results are expressedas percentage of 1 mM glutamate or 3 �M DCG-IV, and they are themeans � S.E.M. from three individual experiments performed in tripli-cate. ��, P 0.001 mGluR3_2 chimera versus WT mGluR3.

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Fig. 7. Concentration-response curves for glutamate at wild-type hm-GluR2 or triple hmGluR2 S688L, G689V, and N735D mutant receptors(A) on glutamate-induced SPA [3H]IP1 hydrolysis in HEK293 cells. WThmGluR2 (closed symbol); PM16-hmGluR2 S688L, G689V, and N735D(open symbol). Effect of MRLSD-650, MRLSD-230, MRLSD-041, andMRLSD-973 at wild-type hmGluR2 (B) or triple hmGluR2 S688L, G689V,and N735D mutant receptors (C) on glutamate-induced SPA-PI hydroly-sis in HEK293 cells. Results are expressed as percentage of 1 mM gluta-mate, and they are the means � S.E.M. from three individual experi-ments performed in triplicate.

248 Rowe et al.

small agonist response in these systems in the absence ofexogenous glutamate. Knoflach et al. (2001) reported similareffects with an mGluR1 allosteric modulator. No intrinsicactivity was observed for MRLSD-650 in assays using wellwashed native rat brain membrane preparations or hm-GluR2 membranes from stably transfected cells, in whichcontaminating glutamate levels were very low, suggestingthat the intact transfected cells may have intrinsic glutamateactivity in this transient assay system. Nevertheless, addi-tion of MRLSD-650 in combination with a low level of gluta-mate potentiated the response above that observed with glu-tamate alone, except when the TMIII-IV domain wasexchanged with that of hmGluR3. This work confirms ourprevious findings that the TMIII-V domain is critical forhmGluR2 potentiation.

Previously, we reported that the combination of aminoacids Ser688 or Gly689 in TMIV with Asn735 in TMV, or thecombination of these two residues together with Asn735, werethe critical amino residues in forming the binding pocket forLY487379 (Schaffhauser et al., 2003). This was confirmed bya recent study (Hemstapat et al., 2007). We have now con-firmed that MRLSD-650 also binds to the same residues.Additionally, the importance of these residues was furtherdetermined when we substituted these same three amino

acids, Ser688, Gly689, and Asn735, into the homologous regionof hmGluR3. This hmGluR2-like binding site on mGluR3 waspotentiated by the mGluR2-specific MRLSD-650, suggestingthat we had created a functional hmGluR2-like potentiatorbinding site in the mGluR3. Following the development of theSPA assay, we identified three additional compounds,MRLSD-230, MRLSD-041, and MRLSD-973 (Fig. 1, B–D),that potentiate glutamate activity on hmGluR2, but notwhen the three critical residues were exchanged. In contrastto these mGluR2-specific potentiators, we also identified aseries of compounds that potentiated the agonist response inthe hmGluR2 S688L, G689V, and N735D chimera: MRLSD-064, MRLSD-726, MRLSD-438, and MRLSD-667 (Fig. 1,E–H). These were tested and found to be potentiators inhmGluR3 membranes as well. Thus, the hmGluR2 S688L,G689V, and N735D mutant may be used to discriminatebetween selective and nonselective modulators of hmGluR2in a single assay. This avoids having to use the ultrasensitivehmGluR3 membranes, or hmGluR3 transiently transfectedcells, which can be difficult to work with due to endogenousglutamate issues.

Despite finding MRLSD-064, MRLSD-726, MRLSD-438,and MRLSD-667 to be active in human mGluR2 and humanmGluR3 recombinant membranes as well as in transientlytransfected cells, we did not see biphasic curves with the ratbrain membranes. A recent study (Gu et al., 2008) gives avery detailed report on the distribution of mGluR2 andmGluR3 in the rat forebrain. The two receptors seem to befairly evenly distributed throughout the forebrain, suggest-ing that making large-scale membrane preparations fromspecific brain regions to favor mGluR2 or mGluR3 would bevery difficult. However, it would seem from their study thatsufficient levels of mGluR3 should have been present in themembranes prepared from rat whole brain to see an agonistor allosteric modulator response. Our recombinant mGluR3membranes had an EC50 value of just �100 nM (data notshown), and it is difficult to remove endogenous glutamate inthe rat brain preparations to levels below 1 �M to use formGluR2 testing. It is likely that residual glutamate wasalready near the mGluR3 Emax value before the addition ofglutamate. Therefore, at the 1 �M concentration of gluta-mate we used in the assay, the native rat brain mGluR3would likely have been fully activated by residual glutamate.Any additional mGluR3 activity caused by the compoundswould be above the 100% glutamate response (i.e., from 100to 110%). This activity was probably masked by the muchmore potent mGluR2 potentiator response, where the re-sponse goes from 10% of the mGluR2 glutamate stimulationto 90% or higher. Attempts to further remove the residualglutamate from the rat brain preparations resulted in in-creasing loss of activity.

Novel negative allosteric binding sites have been identifiedfor the noncompetitive mGluR1 antagonist 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile, which involves amino acids inmultiple transmembrane domains (Val757 in TMV; Trp798,Phe801, and Tyr805 in TMVI; and Thr815 in TMVII) (Malherbeet al., 2003), and recently for mGluR2 (Hemstapat et al.,2007). Although the binding pocket for the mGluR2 negativeallosteric modulator has not yet been determined, it does notseem that the negative allosteric binding sites overlap withthe residues identified for positive modulation (Hemstapat et

TABLE 3Activity of MRLSD-650, MRLSD-230, MRLSD-041, and MRLSD-973 onglutamate-induced SPA �3H IP1 hydrolysis on WT mGluR2 in theabsence or presence of 5 �M glutamateResults are expressed as mean EC50 values (lower-upper S.D.) and mean efficacy(Emax) � S.E.M., expressed relative to the maximal stimulation achieved by gluta-mate alone. Summary data are calculated from three individual experiments per-formed in duplicate.

Compound10 �M Compound 10 �M Compound

5 �M Glutamate

Maximum EC50 Maximum EC50

�M

MRLSD-650 0 N.E. 90 1.69 (0.778–3.66)MRLSD-230 0 N.E. 135 1.57 (1.46–1.70)MRLSD-041 0 N.E. 60 2.87 (2.27–3.62)MRLSD-973 0 N.E. 66 1.85 (1.22–2.82)

N.E., no effect.

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A Three-Amino Acid Change Transforms mGluR3 Potentiator 249

al., 2007). Knowledge obtained from the mapping of newallosteric modulators is helping to determine which regionsof the mGluRs are critical for modulation of agonist activity.

The use of highly selective mGluR-positive allosteric mod-ulators in the therapeutic treatment mGluR-related diseasesmight be an attractive approach since these modulatory com-pounds would only be efficacious in the presence of endoge-nous agonist (i.e., use-dependent). They might also elicit lesstachyphylaxis and/or receptor desensitization than competi-tive agonists (Raddatz et al., 2007). Furthermore, it seems tobe easier to find very selective ligands that bind outside of theagonist binding domains, which are highly conserved be-tween all known mGluRs. MRLSD-650 and related com-pounds therefore constitute valuable tools to explore the roleof mGluR2 in neuronal function, and the potential clinicalutility of selective mGluR2 allosteric potentiators.

Acknowledgments

We thank Drs. Ian Reynolds and Fred Hess for critically reviewingof the manuscript and Amy Jackson for technical assistance.

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TABLE 4Activity of MRLSD-064, MRLSD-726, MRLSD-438, and MRLSD-667 on glutamate-induced �35S GTP�S bindingConcentration-response curves were determined in the presence of 1 �M glutamate in rat brain membranes and mGluR2-containing membranes from stably transfected cells.For the hmGluR3-containing membranes from stably transfected cells, 10 nM glutamate was used. Results are expressed as mean EC50 values (lower-upper S.D.) and meanefficacy (Emax) � S.E.M., expressed relative to the maximal stimulation achieved by glutamate alone. Summary data are calculated from three individual experimentsperformed in triplicate.

CompoundhmGluR2 hmGluR3 Rat Brain

Maximum EC50 Maximum EC50 Maximum EC50

�M �M �M

MRLSD-064 115 91 � 27 136 947 � 498 124 452 � 208MRLSD-726 125 55 � 43 130 587 � 572 185 178 � 30MRLSD-438 97 46 � 9 89 845 � 112 243 545 � 184MRLSD-667 114 120 � 71 120 438 � 436 129 415 � 113

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Address correspondence to: Dr. Blake A. Rowe, 770 Sumneytown Pike,P.O. Box 4, West Point, PA 19486-0004. E-mail: blake_rowe@merck.com

A Three-Amino Acid Change Transforms mGluR3 Potentiator 251