Neuron,

Vol. 8, 169-179, January, 1992, Copyright

0 1992 by Cell Press

A Family of Metabotropic G lutamate Receptors

Yasuto Tanabe,* Masayuki Masu,* Takahiro Ishii,* Ryuichi Shigemoto,+ and Shigetada Nakanishi* *Institute for Immunology +Department of Morphological Brain Science Kyoto University Faculty of Medicine Kyoto 606 Japan

Summary Three cDNA clones, mGluR2, mCluR3, and mGluR4, were isolated from a rat brain cDNA library by crosshybridization with the cDNA for a metabotropic glutamate receptor (mCluR1). The cloned receptors show considerable sequence similarity with mCluR1 and possess a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR2 is expressed in some particular neuronal cells different from those expressing mCluR1 and mediates an efficient inhibiiion of forskolin-stimulated CAMP formation in cDNAtransfected cells. The mGluRs thus form a novel family of G protein-coupled receptors that differ in their signal transduction and expression patterns. Introduction Glutamate not only acts as a major excitatory neurotransmitter, but also plays an important role in neuronal plasticity and neurotoxicity in the central nervous system (CNS) (Monaghan et al., 1989; Collingridge and Singer, 1990; Meldrum and Garthwaite, 1990; Schoepp et al., 1990). Hence the neurotransmission of glutamate is thought to play an important role in many central neuronal functions, including memory acquisition and learning, as well as in dysfunctions, such as epilepsy, stroke, and some neurodegenerative disorders (Monaghan et al., 1989; Collingridge and Singer, 1990; Meldrum and Garthwaite, 1990). The functional diversity of glutamate is reflected by the presence of disparate glutamate receptors, which can be categorized into two distinct groups termed ionotropic and metabotropic receptors (Monaghan et al., 1989). The ionotropic receptors are thought to contain integral cation-specific ion channels and are further subdivided into four major groups: receptors for N-methyl-o-aspartate (NMDA), a-amino-3-hydroxy-5methyl4isoxazole propionate (AMPA), kainate, and 2-amino4phosphonobutyrate (AP4). The cDNAs for AMPAlkainate receptors have recently been isolated by molecular cloning, and the receptors have been shown to consist of many subtypes of the same receptor family with a ligand-gated ion channel structure (Hollmann et al., 1989; Keinanen et al., 1990; Nakanishi et al., 1990; Boulter et al., 1990). The metabotropic glutamate receptor (mGluR) is both functionally and pharmacologically different

from the family of the ionotropic rec:eptors. The mGluR is coupled to a G protein(s) and evokes a variety of functions by mediating intracellular signal transduction (Sugiyama et al., 1987; Schoepp et al., 1990). It also differs from known ionotropic receptors in agonist selectivity and shares no antagonists with any other glutamate receptors (Sugiyama et al., 1989; Schoepp et al., 1990). Although the physiological importance of the ionotropic receptors has been emphasized, numerous lines of recent evidence have indicated that the mGluR is also involved in the central actions of glutamate, such as long-term potentiation in the hippocampus (Ito et al., 1988; Goh and Pennefather, 1989) and long-term depression in the cerebellum (Ito and Karachot, 1990). However, because there is no selective antagonist, the characterization of the mGluR has been less advanced. Recently, our group (Masu et al., 1991) and Houamed et al. (1991) reported the isolation and characterization of a cDNA for mGluR (mGluR1) by applying a cloning approach using the Xenopus oocyte expression system combined with electrophysiology. This receptor mediates the stimulation of inositol phosphate (IP)/Ca2“ signal transduction. But it has a unique structural architecture, with large hydrophilic sequences in both amino- and carboxy-terminal sides of the seven putative transmembrane domains, and shares no sequence similarity with other members of C protein-coupled receptors (Masu et al., 1991; Houamed et al., 1991). Furthermore, prominent expression of this mRNAwas observed in the hippocampal and cerebellar neuronal cells known to be involved in evoking long-term potentiation and depression (Masu et al., 1991). Because the mGluR not only has a unique structure, but also participates in central actions of glutamate in the CNS, we extended our cDNA cloning analysis of the mCluR to investigate the diversity and functions of this receptor family. We here report the existence and characterization of a family of mCluRs. Results cDNA Cloning and Amino Acid Sequences of Three mGluRs An adult rat brain cDNA library consisting of 4 x 105 clones was screened by hybridization with a cDNA probederived from the mCluR1 cDNAclone (pmCR1) under low stringency conditions. Seventy-three hybridization-positive clones were identified and isolated by repeated purification. They were classified into four groups by restriction enzyme analysis and dot blot hybridization with the mGluR1 cDNA probe under different stringency conditions. One group consisting of 11 clones contained the original mGluR1 cDNA and the additional cDNA that encoded an alternativeform comprisingasmall carboxyl terminusgenerated by different splicing, as described below. Be-

Neuron 170

sides the mGluR1 cDNA clones, three different types of cDNA clones consisting of 17,33, and 8 clones were identified and designated as mCluR2, mGluR3, and mGluR4, respectively.The largest cDNA inserts of representative clones for mGluR2-mGluR4 were subcloned into pBluescript II KS(+), and their nucleotide sequences were determined by the chain termination method (Sanger et al., 1977). Figure la-lc shows the nucleotide sequences determined for the cDNAs encoding mGluR2, mGluR3, and mGluR4 and their predicted amino acid sequences. The cDNA sequences for mGluR2, mGluR3, and mCluR4 consisted of 3294, 3215, and 3704 bp, respectively, and contained the open reading frames of 2616,2637, and 2736 bp, respectively. The observed large open reading frame was the only one without

multiple termination codons in the respective cDNA sequence. In addition, the nucleotide sequence surrounding the initiation codon of each cDNA clone agreed with the consensus sequence (Kozak, 1987). The deduced amino acid sequences of mGluR2, mGluR3, and mGluR4 are composed of 872,879, and 912 amino acid residues, respectively, with calculated molecular weights of 95,770, 98,960, and 101,810, respectively. Among the 11 mGluR1 cDNA clones isolated in this investigation, 1 clone showed a restriction pattern identical to that previously reported (Masu et al., 1991), whereas the IO remaining cDNA clones displayed a different restriction pattern at a limited portion corresponding to the region following the transmembrane segment. The nucleotide sequence of the divergent portion wasdetermined in thecloned

A$tabotropic

Glutamate

Receptor

Family

b

cDNA, and this sequence determination revealed the 85 bp sequence inserted in the original mGluR1 sequence as illustrated in Figure Id. Houamed et al. (1991) reported that the asparagine residue at position 887 of mCluR1 is interrupted by an intron sequence. This position exactly matched the 5’ end of the inserted sequence, strongly suggesting that alternative RNA splicing is responsible for the generation of the above inserted sequence. This sequence contained a translation termination codon in the frame encoding the large extracellular domain and the seven transmembrane segments of mGluR1. This termination thus generates a polypeptide consisting of 906 amino acid residues with a calculated molecular weight of 101,630 and converts the carboxy-terminal sequence

of the original mGluR1 to the 3’ untranslated region in the resultant mRNA sequence (Figure Id). The alternative form of mGluR1 is thus much smaller than the original one, but is very similar in size t:o mGluR2mGluR4. We designated the large and small forms of mGluR1 as mGluRla and mGluRlp, respectively. Figure 2 shows an amino acid sequence alignment of mGluRlg and three newly identified mGluRs. The hydrophobicity analysis (Kyte and Doolittle, 1982) of mGluR2-mGluR4 showed a profile similalr to that of mGluR1 with eight hydrophobic segments (Masu et al., 1991): one is located at the amino terminus and probably serves as a signal peptide (von Heijne, 1983), while the others are located at the carboxy-terminal side and would represent seven membrane-spanning

172

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856 83E8 844 E6CI RQPEFSPSSQCPSAHAQL TTSSL TTSSL PALATKQTYVTYTNHAI Figure 2. Comparison

of the Amino

Acid Sequences

906 87i! 879 912!

of Four mCluRs

The amino acid sequences of the four mCluRs are aligned with single-letter notation by inserting gaps (-) to achieve maximum homology. The amino acids identical in at least three of the four sequences are enclosed. Positions of the putative transmembrane segments of I-VII are indicated above the amino acid sequences; a solid line labeled SP indicates the signal peptide; triangles, potential N-glycosylation sites; asterisks, cysteine residues conserved among the four mGluRs; open circles, possible phosphorylation sites.

Figure 1. Nucleotide

and Deduced

Amino

Acid Sequences

for Three

Newly

Identified

mGluRs

and the Alternative

Form of mGluR1

The amino acid sequences of mCluR2, mGluR3, and mGluR4were deduced from the longest open reading frames of the corresponding cDNA sequences (pmGR2-pmCR4) and are indicated in (a), (b), and (c), respectively. The seven putative transmembrane segments (IVII) were assigned on the basis of hydrophobicity analysis (Kyte and Doolittle, 1982) and the sequence comparison of the four mCluRs. Their termini are tentatively defined. A solid line labeled SP indicates the predicted signal peptide (von Heijne, 1983). The schematic structures of alternative forms of mGluR1 mRNA and the sequence of the inserted region of mGluRlg are indicated in (d). Open boxes indicate the proteincoding regions; solid lines, theSand 3’untranslated regions; black boxes, seven putative transmembrane segments; shaded regions, different carboxy-terminal domains resulting from alternative splicing. The deduced amino acid sequences of mCluRla and mCluRlj3 in the inserted regions are indicated above and below the nucleotide sequence, respectively; the termination codon is underlined.

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A

EXTRACELLULAR

INTRACELLULAR

Figure3. mCluRs

The

Amino

Acid

Sequence

Conservation

of

Four

This model of mGluR consists of seven membrane-spanning domains with a large extracellular amino terminus and an extending cytoplasmic carboxyl terminus and is based on the structural characteristics discussed in the text. The regions where 5 or more of the 10 consecutive amino acid residues are identical in at least three of the four mCluR sequences are shaded.

domains (Masu et al., 1991). The three receptors thus possess large hydrophilic sequencescomposed of approximately 550 amino acid residues at the amino termini, and these amino-terminal sequences are very likely to be located at the extracellular side preceding the seven membrane-spanning domains (Masu et al., 1991). Figure 3 schematically illustrates the highly conserved regions in the sequences of the four mGluRs in light of a transmembrane model of this receptor family. The amino acid sequences of the four mCluRs are highly conserved in not only the membranespanning domains, but also the putative extracellular amino termini. The percentages of amino acid homology in the conserved regions of the four receptors (compared in the sequences corresponding to residues 43-846 of mCluR1) are as follows: RI versus R2, 46%; RI versus R3,44%; RI versus R4,43%; R2 versus R3, 70%; R2 versus R4,43%; R3 versus R4, 47%. The following carboxy-terminal regions are highly conserved between mGluR2 and mCluR3, but diverge

from the others; the overall homology in the whole sequences of mGluR2 and mGluR3 is 67%. The three mGluRs, like mCluR1, show no sequence similarity to any members of the G protein-coupled receptors. Furthermore, although a family of the C protein-coupled receptors possesses many conserved amino acids at mutually corresponding positions (O’Dowd et al., 1989; Nakanishi et al., 1990), almost none of these amino acids were found in the sequences of mGluR2-mGluR4. Thus, both the amino acid sequences and the structural architecture of the members of mGluRs differ from those of conventional G protein-coupled receptors. Several interesting sequence characteristics are also pointed out for the mGluRs. -Among the four receptors, cysteine residues are remarkablyconserved at many positions in the putative extracellular aminotermini, the first and second extracellular loops, and the fifth and sixth transmembrane segments, suggesting the importance of the cysteine residues in the structural formation of mCluRs. -Similar to mGluR1 (Masu et al., 1991), positively charged amino acids predominate in the putative cytoplasmic regions nearthe transmembrane segments. -There are potential N-glycosylation sites conforming to the Asn-X-Ser/Thr sequence (Hubbard and lvatt, 1981) in the amino-terminal regions and many serine and threonine residues for possible regulatory phosphorylation in thecarboxy-terminal regions (Figure2). -We previously pointed out the presence of a stretch of 24 consecutive uncharged amino acids (residues 155-178) in the amino-terminal region of mGluR1 (Masu et al., 1991). This amino acid sequence is well conserved among the four mGluRs. All these findings indicate that the four receptors form a common structural architecture with a large extracellular domain preceding the seven transmembrane segments. Characterization of the Properties of mCluR2 To investigate the signal transduction coupled to mGluR2-mGluR4, we examined electrophysiological responses to L-glutamate in Xenopus oocytes injected with the mRNA synthesized in vitro from the respective cloned cDNA. None of these mRNAs, however, showed electrophysiological responses, suggesting that these receptors are not primarily linked to IP/Ca2+ signal transduction (Dascal, 1987). We attempted the DNAtransfection and stable expression of the individual receptors in Chinese hamster ovary (CHO) cells to investigate the detailed transduction mechanism of these receptors. In a separate experiment designed to establish CHO cell lines stably expressing mGluR1, we found that cell lines incorporating the mGluR1 cDNA showed no expression or generation of aberrant structures of the mRNA when they had been selected and grown in a standard culture medium. This finding suggested somedeleteriouseffectsof mGluR1 expression on cell growth, probably due to constitutive activation of mGluR1 by L-glutamate present in the standard culture medium. We thus removed L-glu-

h$abotropic

Glutamate

Receptor Family

OT 0

' 0.01

0.1

pertussis

1

toxin

10

1 100

(rig/ml)

Agonist(M) Figure 4. Dose-Response Curves for Inhibition of ForskolinStimulated CAMP Accumulation by Agonists in Cells Stably Expressing mGluR2 Agonists added to forskolin-treated cells are as follows: open squares, L-glutamate; open circles, tACPD; open triangles, ibotenate; closed circles, quisqualate. Intracellular CAMP levels in cells treated and untreated with 10 RM forskolin were 380 f 47 and 9.8 + 1.1 pmol per well, respectively. Each point represents the mean * SD of at least two separate experiments done in triplicate.

tamate and reduced the concentration of t-glutamine in the culture medium and succeeded in obtaining several cell lines stably expressing mGluR1 (Aramori and Nakanishi, unpublished data). We applied the same procedure for mGluR2-mGluR4 and succeeded in obtaining cell lines expressing mGluR2, but not cell lines expressing either mGluR3 or mCluR4 for unknown reasons. The detailed pharmacology and biochemistry of mGluR2 were investigated in receptor-expressing CHO cells. L-Glutamate applied to these cells only slightly stimulated IP formation (about a I.&fold increaseatl mM L-glutamateoverthatobservedwithout addition of L-glutamate). This is in marked contrast with the more than 5-fold stimulation of IP formation by mGluR1 expressed stably in CHO cells (Aramori and Nakanishi, unpublished data). mGluR2 had no stimulatory effect on CAMP formation. On the other hand, L-glutamate added to mGluR2-expressing cells resulted in considerable inhibition of the forskolinstimulatedaccumulationof intracellularcAMP(Figure 4). No such inhibition was observed in untransfected cells or those transfected with the vector DNA alone. Furthermore, CHO cells expressing mGluR1 showed no inhibition of CAMP formation with application of L-glutamate. Thus, there is a clear difference in the signal transduction between mGluR1 and mGluR2. Dose-response analysis of mGluR2-mediated inhibition of CAMP formation indicated that effective doses for half-maximal response (EDw) of L-glutamate, transI-aminocyclopentane-1,3-dicarboxylate (tACPD), and ibotenate were 4, 5, and 35 PM, respectively (Figure 4). Quisqualate, which is the most potent agonist for

Figure 5. Effects of PTX on Glutamate-Mediated Forskolin-Stimulated CAMP Accumulation

Inhibition

of

Forskolin-stimulated cAMP levels in cells treated with various concentrations of PTX weredetermined with or without addition of 1 m M L-glutamate; the levels obtained without addition of L-glutamate are taken as 100% at each concentration of PTX indicated. Each point represents the mean + SD of at least two separate experiments done in triplicate.

mGluR1 in inducing IP formation (Masu et al., 1991), was not as potent as L-glutamate for mGluR2. NMDA, AMPA, kainate, and AP4 (1 mM each) had virtually no effect on mGluR2. When mGluR2-expressing cells were treated with pertussis toxin (PTX) prior to addition of forskolin and L-glutamate, glutamate-mediated inhibition of CAMP accumulation was reduced in adose-dependent manner and was almost completely abolished at 1 ng of PTX per ml of medium (Figure 5). Thus, the glutamate response is mediated by a PTX-sensitive, probably G, protein (Gilman, 1984; Ui, 1984). Consistent with our previous observation of mGluR1 expressed in Xenopus oocytes (Masu et al., 1991), the mGluRl-mediated stimulation of IPformation was inhibited by PTX treatment at mGluRl-expressing cells, but this inhibition was partial (about 49% inhibition) even with the treatment at 10 ng of PTX per ml of medium (Aramori and Nakanishi, unpublished data). The characterization of mGluR2 thus unequivocally demonstrates that mGluR1 and mGluR2 are different in both signal transduction and agonist selectivity. Expression of mGluR2 mRNA RNA blot analysis of mGluR2 mRNA gave rise to two hybridization bands with estimated mRNA sizes of about 3.4 and 5.0 kilonucleotides under high stringency conditions (Figure 6). This analysis showed that mGluR2 mRNA is distributed throughout different brain regions and is high in the olfactory bulb and cerebral cortex. In situ hybridization with a specific riboprobe prepared from the mGluR2 cDNA revealed a wide distribution of mGluR2 mRNA in the CNS (Figure 7a). No significant hybridization was observed in parallel experiments using the same probe in the

Neuron 176

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Analysis

of mGluR2

mRNA

Total RNA analyzed was as follows: lane 1, whole brain; lane 2, cerebral cortex; lane 3, hypothalamus; lane 4, midbrain; lane 5, striatum; lane 6, hippocampus; lane 7, medulla/pans; lane 8, spinal cord; lane 9, olfactory bulb; lane 10, cerebellum. The size marker (kilonucleotides) used was the RNA ladder (BRL).

presence of an excess of unlabeled probe (data not shown). Furthermore, a similar hybridization pattern was obtained with a different nonoverlapping probe derived from the mGluR2 cDNA (data not shown). Prominent expression of mGluR2 mRNA was observed in Golgi cells of the cerebellum (Figure 7b), granule cells of the dentate gyrus (Figure 7c), most neuronal cells of the cerebral cortex, and small neuronal cells (probably intrinsic neurons) of the main and accessory olfactory bulbs (Figure 7d). Thus, mGluR2 mRNA is expressed in specific neuronal cells in the CNS.

This paper reports the existence of a family of related but distinct genes encoding mGluRs. Alternative RNA splicing is also involved in the generation of the molecular diversity of mCluR1. The members of this receptor family have a unique structural architecture with a large amino-terminal sequence preceding the seven membrane-spanning domains and share no sequence similarity with other members of the G protein-coupled receptors. The G protein-coupled receptors for large glycoprotein hormones have been shown to possess a large extracellular domain attached to seven membrane-spanning domains (Parmentier et al., 1989; Sprengel et al., 1990). However, there was no example indicating the presence of a large aminoterminal sequence among the G protein-coupled receptors for small molecule neurotransmitters (O’Dowd et al., 1989; Nakanishi et al., 1990). The large aminoterminal sequences are highly conserved and contain many conserved cysteine residues throughout all four members of the receptors. It is thus conceivable that the large extracellular domains of the four mGluRs form a common protein configuration. No functional role of this conserved domain has been elucidated yet, but it may have some important functions such as interaction with a specific protein involved in the regulation of glutamate neurotransmission. Another interesting feature is the sizes and the sequence con-

servation of the third cytoplasmic loops of the mGluRs. On the basis of analysis of mutant and chimeric receptors, the third cytoplasmic loops of the adrenergic and other receptors have been implicated in G protein coupling (O’Dowd et al., 1989; Wong et al., 1990). However, this domain is small and is highly conserved in the mGluR sequences. Because mGluR1 and mGluR2 are linked to different signal transduction pathways, a question arises as to whether the third cytoplasmic loop of the mGluR governs selective interaction with different G proteins or whether other cytoplasmic portions are responsible for G protein coupling. Northern and in situ hybridization analyses indicated a wide distribution of mGluR2 mRNA in the CNS. Prominent expression of this mRNA was observed in Golgi cells of the cerebellum and some particular neuronal cells in other brain regions. mGluRZ mRNA, on the other hand, is predominantly expressed in cerebellar Purkinje cells, CA2-CA3 pyramidal cells of the hippocampus, and mitral and tufted cells of the olfactory bulb (Masu et al., 1991). Thus, the two mRNAs show distinct expression patterns in the cell types of the CNS. Our preliminary in situ hybridization analysis indicated a wide distribution of both mGluR3 and mGluR4 mRNAs in the CNS (data not shown). mGluR4 mRNA is particularly enriched in granulecellsof thecerebellum. Interestingly, mGluR3 mRNA is prominently expressed not only in neuronal ceils of the cerebral cortex and the dentate gyrus, but also in glial cells throughout brain regions. mGluR3 is, however, perhaps different from a glial cell mGluR reported by Pearce et al. (1986) and Nicoletti et al. (1990), because the latter receptor has been shown to mediate the stimulation of IP/Ca2+ signal transduction in glial cells. It is clear from our investigation that the different mGluRs are differentially expressed in specialized neuronal or glial cells. Thefindingofthecouplingof mGluR2totheinhibL tory CAMP cascade was unexpected, because it has been thought that the mGluR is exclusively coupled to IP/Ca2+ signal transduction (Schoepp et al., 1990). The physiological role of this receptor is also interesting, because the receptors linked to the inhibitory CAMP cascade are in many cases involved in suppression rather than excitation of neurotransmission (Andrade et al., 1986; Nicoll, 1988). Because tACPD has been reported to depress excitatory synaptic transmission perhaps via activation of mCluR (Baskys and Malenka, 1991), mGluR2 may indeed mediate suppression of neurotransmission. It is also possible that mGluR2 is located in inhibitory neurons, such as Colgi cells, and ultimately evokes excitation by suppressing inhibitory transmission in these systems. Alternatively, this receptor may be involved in some functions other than neurotransmission, such as synaptogenesis or synaptic stabilization (Monaghan et al., 1989). In conclusion, this investigation has provided the molecular basis for the diversity of mGluRs. Our re-

Metabotropic I77

Glutamate

Receptor Family

a

b

Figure 7. Localization

of mGluR2

mRNA

in the Adult

Rat Brain by In Situ Hybridization

(a) A negative film image of in situ hybridization of a sagittal section is shown. OB, main olfactory bulb; AOB, accessory olfactory bulb; DC, dentate gyrus; Cx, cerebral cortex; T, thalamus; Cb, cerebellum. (b-d) Bright-field photomicrographs of emulsion-dipped sections of the cerebellum (b), the dentate gyrus (c), and the granule cell layer of the accessory olfactory bulb (d) are shown. Colgi cells (b, arrowheads) and granule cells (c and d) are strongly labeled. M, molecular layer; P, Purkinje cell layer; C, granular layer. Bar, 50 pm (b-d).

suits demonstrate that mGluRs form a novel repertoire of G protein-coupled receptors that are diversified not only by mediating distinct signal transduction but also by specializing expression patterns of the individual receptors in the CNS. Additional studies of functions and regulation of diverse members of mCluRs are interesting and important for understanding complex physiological responses of glutamate in the CNS.

Experimental

Procedures

cDNA Cloning An adult rat brain cDNA library was constructed from sizefractionated rat brain poly(A)’ RNA (-3-4 kilonucleotides) by using a kGEM2 vector as described (Tanaka et al., 1990). Clones (4 x IV) derived from thecDNA librarywere screened by hybridization with a 1236 bp PmaCl fragment of the pmCR1 cDNA. Hybridization and filter washing were carried out at 60°C under the conditions described (Sambrook et al., 1989). Seventy-three hybridization-positive clones were isolated by repeated purifica-

Neuron 178

tion and classified into four groups by restriction enzyme analysis and dot blot hybridization. One group consisted of cDNA clones encoding either the original mGluR1 (mGluRla) or an alternative form of mCluR1 (mGluRlJ3) generated by different splicing, while the other groups were composed of three additional types of cDNA clones encoding mGluR2, mGluR3, and mCluR4. Representative clones containing the largest cDNA inserts for mGluR2-mGluR4as well as the one encoding mCluRlB were selected, and each cDNA insert of these clones was excised and subcloned into the EcoRl site of pBluescriptll KS(+) vector (pmCR2, pmGR3, pmCR4, and pmGRlJ3). Nucleotide sequences were determined in both strands by the chain termination method (Sanger et al., 1977). Electrophysiological measurements in Xenopus oocytes injected with the in vitro synthesized mRNA were carried out as described (Masu et al., 1991). Receptor Expression in CHO Cells and CAMP Measurements The3.3 kbpEcoRl fragmentof pmGR2was inserted intoaeukaryotic expression vector (pdKCRdhfr) containing the mouse dihydrofolate reductase gene as a selective marker (Oikawa et al., 1989). This plasmid was transfected into CHO cells deficient in dihydrofolate reductase activity (CHOdhfr-) (Urlaub and Chasin, 1980) by the calcium phosphate method (Graham and van der Eb, 1973). Cell populations expressing mGluR2 together with dihydrofolate reductase were selected in Dulbecco’s modified Eagle’s medium (lacking ribonucleosides, deoxyribonucleosides, and L-glutamate and containing a reduced concentration [2 mM] of L-glutamine), supplemented with 1% proline and 10% dialyzed fetal bovine serum (Urlaub and Chasin, 1980). From these cell populations, clonal cell lines were isolated by single cell cloning. Expression levels of mCluR2 mRNA in clonal cells were determined by Northern analysis. Clonal cells expressing high levels of mGluR2 mRNA were seeded in 12-well plates at a density of 1.5 x 105 cells per well and grown for 3 days. After a 20 min preincubation in phosphate-buffered saline (PBS) containing 1 m M 3-isobuty-I-methylxanthine (IBMX) at 37OC, the cells were incubated with fresh PBS containing 10 FM forskolin, 1 m M IBMX, and test agents for 10 min (Sugama et al., 1989). The medium was aspirated, and the reaction was stopped with ethanol. CAMP levels were measured by a radioimmunoassay kit (Amersham). For PTX treatment, cells were preincubated with varying concentrations of PTX for 11 hr at 37OC. Each experiment was carried out at least twice in triplicate. Northern and In Situ Hybridization Analyses Northern analysis was carried out by using 10 ug of total RNA isolated from various regions of the brain and spinal cord as described (Masu et al., 1991). The cDNA probe used was the 1549 bp BamHl fragment of pmGR2. In situ hybridization was performed as described (Masu et al., 1991). An ?S-labeled antisense riboprobe corresponding to the 629 bp Kpnl-BamHI fragment or the 616 bp Apal-Sacl fragment of pmGR2 was transcribed and hybridized as described (Masu et al., 1991). Sections were exposed to Bmax film (Amersham) for 2 weeks or dipped in NTB2 emulsion (Kodak) diluted I:1 with distilled water, developed after a 4 week exposure, and counterstained with cresyl violet. Control hybridization experiments were carried out in adjacent sections by using the same riboprobe in the presence of excess unlabeled probe. Acknowledgments

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We are grateful to Noboru Mizuno for helpful discussion and Akira Uesugi for photographic assistance. This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.

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