A cDNA clone for a new metabotropic glutamate receptor, mGluR5, was isolated through polymerase chain reaction-mediated DNA amplification by using.
Val. 267, No. 19, Issue of July 5, pp. 13361-13368.1992 Printed in U.S. A.
THEJ O U R N A L OF BIOLOGICAL CHEMISTRY
(0 1992 by The American Society for Biochemistry and Molecular Biology, Inc
Molecular Characterizationof a Novel Metabotropic Glutamate Receptor mGluR5 Coupled to Inositol Phosphate/Ca2+Signal Transduction* (Received for publication, January 31, 1992)
Takaaki AbeS, Hidemitsu SugiharaS, Hiroyuki NawaSQ, Ryuichi Shigemotoy, Noboru Mizunoll, and Shigetada NakanishiS From the tlnstitute for Zmmunolom and YDepartment of Morphological Brain Science, Kyoto University Faculty of Medicine, Kyoto 606,’ Japan . ”
1990). The functional diversity of glutamate results from the A cDNA clone for a new metabotropic glutamate receptor, mGluR5, was isolated through polymerase presence of multiple glutamate receptors which can be catechain reaction-mediated DNA amplification by using gorized into two distinct groups termed ionotropic and metaprimer sequences conserved among the metabotropic botropic receptors (Monaghan et al., 1989). The ionotropic glutamate receptor (mGluR) family and by the subse- receptors for N-methyl-D-aspartate(NMDA)’andfor aquentscreening of a rat brain cDNA library. The amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/ cloned receptor consists of 1171 amino acid residues kainate have recently beencharacterized by molecular cloning and exhibits a structural architecture common to the and have been shown to consist of integral receptor cationmGluR family, possessing a large extracellulardomain specific channel complexes (Hollmann et al., 1989; Keiniinen preceding the seven putative membrane-spanning seg- et at., 1990; Nakanishi et a t , 1990a; Boulter et al., 1990; ments. mGluR5 shows the highest sequence similarity Moriyoshi et al., 1991). The metabotropic glutamate receptors to mGluR1 among the mGluR members and is coupled (mGluRs) are coupled to intracellular signaling transduction t o the stimulation of phosphatidylinositol hydrolysis/ G proteins (Schoepp et al., 1990; Rbcasens et al., 1991). Caz+ signal transduction in Chinese hamster ovaryviacells mGluRs transfected with the cloned cDNA. This receptor also Recent accumulatingevidence has indicated that the are also involved in many central actions of glutamate in the resembles mGluRl in its agonist selectivity and antagonist responses; the potency rank orderof agonists for central nervous system(Rbcasens et al., 1991; Miller, 1991). Recently, we isolated cDNA clones for four different submGluR5 was determined to be quisqualate > L-glutamate 2 ibotenate > trans- 1-aminocyclopentane- 1,3- types of the rat mGluR family (mGluR1-mGluR4) by molecdicarboxylate. Blot and in situ hybridization analyses ular screeningof a rat brain cDNA library (Masu et al., 1991; indicated that mGluR5 mRNA is widely distributed in Tanabe et al., 1992). The mGluR family shares no sequence of G protein-coupled receptors neuronal cells of the central nervous system and is similarity with other members expressed differently from mGluRl mRNA in many and possesses a large extracellular domainpreceding the seven brain regions. This investigation thus demonstrates putative transmembrane segments (Masu et al., 1991; Tanabe thatthere is an additional mGluR subtypewhich et al., 1992). The four mGluRsshow distinct signal transducclosely resembles mGluRl in its signal transduction tion via different G proteins and areexpressed in specialized and pharmacological properties and is expressed in neuronal and glial cells in the central nervous system (Masu specialized neuronal cells in the central nervous sys- et al., 1991; Tanabe et al., 1992). mGluRl is coupled to the tem. stimulation of phosphatidylinositol (PI) hydrolysis/Ca*+ signal transduction (Masu et al., 1991; Houamed et al., 1991), whereas mGluR2 is linked to an inhibitory CAMP cascade (Tanabe et al., 1992). Although the precise signaling pathway Glutamate, a major excitatory neurotransmitter, plays an of mGluR3 and mGluR4 remains be to elucidated, no obvious important role in neuronal plasticity and neurotoxicity and is linkage of these receptors to the PI hydrolysis/Ca2’ cascade thought to be involved in many neuronal functionsincluding has been indicated in theXenopus oocyte expression system memory acquisition, learning, and someneurodegenerative (Tanabe et al., 1992). Thus, among the four cloned mGluRs, disorders such as epilepsy and stroke (Monaghanet al., 1989; mGluRl is the only one that is capable of stimulating PI Collingridge andSinger, 1990; MeldrumandGarthwaite, hydrolysis/Ca2+ signal transduction. However, the presence of different mGluR subtypes linked to the PI hydrolysis/Ca2+ *This work was supported in part by research grants from the cascade hasbeen suggested by pharmacological studies on PI Ministry of Education, Science and Culture of Japan, the Ministry of Health and Welfare, the Yamanouchi Foundation for Research on hydrolysis of various agonists and antagonist effects on gluMetabolic Disorders, theUehara Memorial Foundation, the Cell tamate-induced PI hydrolysis in tissue and cell preparations (Nicoletti et al., 1986a, 198613; Schoepp et al., 1990; Rkcasens Science Research Foundation, and theInamori Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaperhas been submitted to the GenBankTM/EMBLDataBankwith accession number(s) Dl 0891, §Present address:Beckman Neuroscience Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.
The abbreviations used are: NMDA, N-methyl-D-aspartate; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; CHO cells, Chinese hamster ovary cells; D L - A P ~DL-2-amino-3-phosphon, opropionate; bp, base pair; kb, kilobase pair; L-AP~, ~-2-amino-4phosphonobutyrate; mGluR, metabotropic glutamate receptor; PCR, polymerase chain reaction; PI, phosphatidylinositol; PTX, pertussis toxin; t-ACPD, trans-l-aminocyclopentane-1,3-dicarboxylate; EGTA, [etbylenebis(oxyethylenenitrilo)]tetraaceticacid.
13361
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IP/Ca2+-coupled MetabotropicGlutamate Receptor Subtype
et al., 1991). W e thus investigated the multiplicity of mGluR subtypes linked t o PI hydrolysis/Ca2+ signal transduction by extending our molecular studies of the mGluR family. We here report the structure, properties, and expression pattern of a second type of t h e PI hydrolysis-coupled mGluRsubtype,
expressing mGluR5 together with dihydrofolate reductase were isolated as described (Tanabe et al., 1992). Cell lines expressing high levels of mGluR5 were identified by measuring the L-glutamateinduced stimulation of PI hydrolysis. PI hydrolysis and CAMPlevels in mGluR5-expressing cells were measured as described previously (Nakajima et al., 1992). Intracellular concentrations of Caz+ were termed mGluR5. determined with Ca2+-sensitivedye fura-2 as described previously (Ito et al., 1991). Data were analyzed by Student's t test. RNA Blot and in Situ Hybridization Analysis-RNA blot analysis EXPERIMENTALPROCEDURES was carried out by using 10 pg of total RNAs isolated from various Materials-Materials were obtained from the following sources: regions of the brain of adult rats as described (Masu et al., 1991).The quisqualate, ibotenate, trans-l-aminocyclopentane-1,3-dicarboxylate cDNA probe used was the 1824-bp BglII fragment of pmGR5. In situ (t-ACPD), DL-2-amino-3-phosphonopropionate (DL-APS), L-2- hybridization was performed by using 35S-labeledantisense riboprobe amino-4-phosphonobutyrate(L-API), andAMPA from Tocris Neu- corresponding to the 1824-bp BglII fragment or the 547-bp HindIIIramin; L-glutamate, NMDA, and kainate from Sigma; fura-2 from BglII fragment of pmGR5 as described (Masu et al., 1991). Control Dojindo Laboratories; pertussis toxin (PTX) from Kaken Pharma- hybridization experiments were carried out in adjacent sections by ceutical Inc.; Superscript'" RNase H- reverse transcriptase and RNA using the same riboprobe in the presence of an excess of unlabeled ladder from Bethesda Research Laboratories; GeneAmp" DNA am- probe. plification reagent kit from Perkin-Elmer Cetus; pBluescriptI1 and XZAPII from Stratagene. All other chemicals were of reagent grade. RESULTS Polymerase Chain Reaction (PCR)-RNA was prepared from the hippocampus of 6-day-old rats as described (Sambrook et al., 1989). cDNA Cloning and Structure of mGluR5"Thecloned A mixture of oligo(dT)-primed cDNAswas synthesized from the cDNA encoding mGluR5 wasisolated through PCR-mediated hippocampal RNA by using Superscript'" RNase H- reverse tran- DNA amplification by using the mGluR-conserved sequences scriptase. The primers used for PCR were two degenerate oligonuclefor primer extension and by the subsequent screening of a rat otides made according to the amino acid sequences of transmembrane segments I1 and V of the mGluR family, both of which are highly brain cDNA library. The PCR primers used were two degenerate oligonucleotides corresponding to the amino acid seconserved among the four mGluRs reported previously (Tanabe et al., 1992);the regions used corresponded to amino acid residues 612quences of transmembrane segments I1 and V of the four 619 (5' primer) and 756-762 (3' primer) of mGluR1. The sequences mGluRs(Tanabeet al., 1992). The homologous DNA seof the 5' primer andthe 3' primer were TCIAGICGIGA(A/ quences were amplified from a mixture of rat brain cDNAs G)CTITG(C/T)TA(C/T)AT and TTICGIGT(C/T)TT(A/G)AA(A/ by PCR and cloned into pBluescriptI1 KS(+). Thirty-eightof C/G/T)GC(A/G)TA, respectively;the 5' ends of the 5' and 3' primers t h e 55 clones isolated showed restrictionpatterns correspondcontained the EcoRI and HindIII restriction sequences, respectively, for the subsequent cleavage of cloned cDNA fragments. PCR ampli- i n g t o one of the four mGluR cDNAs reported previously fication was performed by using the GeneAmp'" DNA amplification (Tanabe etal., 1992). The remaining 17 clones were sequenced reagent kit according to thefollowing schedule: 94 "C for 1min, 42 "C by the chain-termination method (Sanger et al., 1977), and for 2 min, and 72 "C for 3 min for 50 cycles,followed by further one clone(psMGR.5) showed a nucleotide sequence which has incubation for 10 min at 72 "C. An aliquot of the PCR reaction was an overall similarity to, but is obviously different from, those electrophoresed on a polyacrylamide gel, and the amplified DNA (-450 base pairs (bp)) was excised from the gel and subcloned into of the corresponding regions of a n y of the mGluR1-mGluR4 pBluescriptII KS(+) after digestion with EcoRI and HindIII. Thirty- cDNAs. A rat brain cDNA library consisting of 8.5 x 10' X eight of the 55 clones were identified as one of the previously isolated phage clones was screened by hybridization with the cDNA mGluR cDNA clones, and the remaining 17 clones were sequenced. probe derived from the psMGR5 cDNA under high stringency One clone (psMGR5) was found to show a novel type of the mGluR conditions. Five clones containing the large cDNA inserts cDNA clone. were isolated from the 31 hybridization-positive clones and cDNA Cloning-Poly(A)+ RNA was isolated from 6-day-old rat brains andused for cDNAsynthesis. A cDNA library was constructed were rescued into pBluescript SK(-). The cDNA inserts of from fractions containing more than 4-kilobase pair (kb) cDNAs by these clones were found to share an identical restriction map in t h e 5' end of the cDNA using a XZAPII vector as described (Moriyoshi et al., 1991). Clones except for some size difference (8.5 X lo5)derived from the cDNA library were screened by hybridi- inserts. The nucleotide sequence of a representative clone zation with the 452-bp cDNA fragment of psMGR5. Hybridization containing the largest cDNA insert (pmGR5) was determined was carried out in the formamide (50%)solution at 42 "C as described by the chain-termination method (Sangeret al., 1977). (Sambrook et al., 1989),and filter washing was performed in a solution pmGR5 was determined to be composed of approximately containing 15 mM NaC1,1.5 mM sodium citrate, and 0.1%sodium 8.5 kb. Fig. 1 shows the nucleotide sequence determined for dodecyl sulfate at 65 "C. Thirty-one hybridization-positive clones were isolated, and five clones containing the large cDNA inserts were the protein-coding region and its surrounding 5'- and 3'selected and rescued into pBluescript SK(-). The cDNA inserts of noncoding regions of pmGR5 and the predicted amino acid these clones showed an identical restriction enzyme digestion pattern sequence of mGluR5. The deduced amino acid sequence of except for some length difference in their extreme 5' portions, and mGluR5 consisted of 1171 amino acid residues with a calcuone clone containing the largest cDNA insert (pmGR5) was chosen for further sequence analysis. Because the 5' portion of pmGR5 lated molecular weightof 128,289. T h e hydrophobicity analysis (Kyte and Doolittle, 1982) of mGluR5 showed a profile hybridized with the mGluRl cDNA, the -3.9-kb sequence of the 5' portion of pmGR5 was determined in both strands by the chain- similar t o those of other mGluRs with eight hydrophobic termination method (Sanger et al., 1977). The amino acid sequence segments (Masuetal., 1991; Tanabe et al., 1992): one is located was deduced from the longest open reading frame in the nucleotide at the amino terminusand probably serves as a signal peptide sequence determined for the pmGR5 clone. Electrophysiological (von Heijne, 1983), whereas the others are present at t h e measurements in Xenopus oocytes injected with the in vitro synthesized mRNA were carried out as described (Masu et al., 1991); the carboxylterminus and wouldrepresentsevenmembraneet 1991). mGluR5thus possesses NotI-digested DNA of pmGR5 was usedfor in vitro mRNAsynthesis. spanning domains (Masu al., mCluR5 Expression in CHO Cells and Determination of mGluR5- two large hydrophilic sequences composed of approximately mediated Signal Transduction-The 8.5-kb SalI-Not1 fragment of 560 and 350 amino acid residuesat the amino- and carboxylpmGR5 was inserted into aeukaryotic expression vector pdKCR-dhfr terminal sides, respectively, and this amino-terminal sequence containing the mouse dihydrofolate reductase gene asa selective is very likely to be locatedat the extracellular side preceding marker (Oikawa et al., 1989). This plasmid was transfectedinto Chinese hamster ovary (CHO) cells deficient in dihydrofolate reduc- the seven membrane-spanning domains (Masu et al., 1991). tase activity (CHO-dhfr-) (Urlaub and Chasin, 1980) by the calcium The amino acid sequenceof mGluR5 is highly homologous to phosphate method (Graham and van der Eb, 1973). Clonal cell lines those of the other membersof the mGluR family and is most
IP/Ca2+-coupledMetabotropic Glutamate Receptor Subtype
13363 -241 -121 -1
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1680 580 600 L~uThrClyCyeA~pL~uIloProV.1ClnTyrL.ulrgT~pClyAepP~oCl~~oIl~Al~Al~V~lV~lPh~Al~Cy~L~uClyL~uL~uAl~Th~L~uPh~V~lTh~V~lI CTCACACCCTCTCACTTCATCCCACTCCACTATCTTCCCTCCffiTCACCCTCACCCCATTCCACCTCTCCTCTTTCCCTCCCTCCGTCTCCTACCCACCTTATTCCTTACTCTMTCTTC 1800
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FIG. 1.
Nucleotide and deduced amino acid sequences for mGluR5. The seven putative transmembrane segments (I-VII) were
assigned on the basis of hydrophobicity analysis and the sequence comparison of the four other mGluRs and areindicated by enclosures with solid lines; the termini of these segments were tentatively defined. Other lines and marks are asfollows: a solid line labeled SP, the predicted signal peptide; triangles, potential N-glycosylation sites; stars, possible phosphorylation sites.
.
IP/Ca2+-coupled Metabotropic Glutamate Receptor Subtype
13364 SP mGluR5
MVLLLILSVLLLKEDVRGSA----------QSSERRWAHMGDIIIGALFSVHHQPTVDKVHERKCGAV~Q~GIQRVEAMLHTLERIN I1 1 1 : : :: ::: :: :I::I I I : I l l : l l l l l l l l l l l l : : : l l : l l l l l : : l l l l l l l l l l I I : I II : : I I
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MVRLLLIFFPMIFLEMSILPRMPDRKVLLAGASSQRSVRRGDVIIGALFSVHHQPPAEKVERKCGEIREQ~GIQRVE~HTLDKIN 90
80
SP 0
SDPTLLPNITLGCEIRDSCWHSAVALEQSIEFIRDSLIS-SEEEEGLVRCVDGSSSF---RSKKPIVGVIGPGSSSVAIQVQNLLQLFNI~QIA~~ATSM 176 :II:IIIIlIII:IlIlIIIII:IIIlllIIlIIIIlII ::I::II I I : :: :: 1 : I I I I : I I I /I l l I I I I I I I I / ( ( 1 1 I : [ ( 1 I l l 1 1 1 1 : A D P V L L P N I T L G S E I R D S C W H S S V A L E Q S I E F I R D S L I S I R D E K D G L ~ C L P D G Q T L P P G R T K K P I A ~ V I ~ P ~ ~ ~ ~ V ~190 ~QVQN~~Q~~~~
.
D L S D K T L F K Y F M R V V P S D A o O A R A M V D I V K R Y N W T W S A V H T E G N Y G E S G ~ ~ K D M S ~ E G I C I ~ S Y K I Y S N A G E Q S F D K L L K K L R S H L P ~276 ~AcF IIIIIII:IIl:IIIIII: I I I I I : I I I I I I I I I I l I I I l I l I l I I I I I I : l / I : : : I : I I : I I I I I I I1 I I l l l : l l l : l l : l l I : : [ I ( ( I I [ : I I DLSDKTLYKYFLRVVPSDTLQARAMLDIVKRYNWTYVSAVHTEGNYGESG~AFKE~QEGLCIAHSDKIYSNAGEKSFDRLLRKLRERLPKARVWCF 290 CEGMTVRGLLMRMRRLGLAGEFLLLGSDGWADRYDVTDGYOREAVGGIT~KLQSPD~~FDDYYLKLRPETNLRNP~FQ~F~QH~Q~RLEGFAQEN~K~ 376 IIIIIIIIII IIIIII::IIl I:IIIIIIIl :I::lI: I 1 IllllllIll:I: IIIl:IIl/ :II lIIII:IIIIIllIIII I /I::: CEGMTVRGLLSAMRRLGWGEFSLIGSDGWADRDEVIEGYE~ANGGITIKLQSPEVRSFDDYFLKLRLDTNTRNPWFPEFWQH~QCRLPGHLLENPNF 390
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PEPIAAVVFACLGLLATLFVTVIFIIYRDTPVVKSSSSRELCYIILAGICLGYLCTFCLIAPKQIYCYLORIGIGLSPAMSYSALVTKTNRIARILAGSK 676 I:I I : : I : I l I : I : I I I I I : I l : : I I I I I I / I l l l I l I l 1 l 1 l I I I I I I : l : I I I I I I : : HIHI: : l l l : l l : l l l l l l l l l l l l l l l l l l l IESIIAIAFSCLGILVTLFVTLIFVLYRDTPVVKSSSSRELCYIILAGIFLGYVCPFTLIAKPTTTSCYLQRLLVGLSSAMCYSALVTKTNRIARILAGSX 690
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MYTTCIIWLAFVPIYFGSNYKIITMCFSVSLSATVALGCMFVKVYIILAKPERNVRSAFTTSTVHVGDGKSSSAASRSSSSLVNLWK~GSSGETLS 876 IlIIllII/IIlIIII1111IlII I l : l / I l : I I I I I I I I : / I : 1 1 l : I I l / l l l l l l l l l l : l l l l l l l 1 1 1 ::lI::::I:::I: ::::: : MYTTCIIWLAFVPIYFGSNYKIITTCFAVSLSVTVALGCMFTPKMYIIIAKPERNVRSAFTTSDVHVGDGK---LPCRSNTFLNIFRRKKPGAGNAN 887 SNGKSVTWAQ--NEKSTRGQHLWQRLSVHINKKENP-NQTAVIKPFPKSTENRGPGAAAGGGGSGPGVAGAGNAGCTATGGPEPPDAGPKALYDVAEAEES IIIIII:I:: : ::::III:IIIIIIl::::I:: IIIIIII1::II :::I : : : : : I : :: :::::: 1 : : : : I I : : ::::: : : ::
973
SNGKSVSWSEPGGRQ~KGQH~~RLSVH~TNETACNQTAVIKPLTKSYQGSGKSLTFSDASTKTLYNVEEEEDNTPSAHFSPPSSPSMWHRRGPPVA 987 T
FPAARRPRSPSPIST-LSHLAGSAGRTDDDAPSLHSETAARSSSSSQG-SL~QISSWT~TANISELNS~STAATPGPPGTPICSSYLIPKEIQ-LP 1070 I : ::: ::: : : I : : : : I : :: : ::::: :: I I Il:l: :I I (:I:::)::::: :I::::[ 1 I : ::::: [:::I 1 I TPPLPPHLTAEETPLFLADSVIPKGLPPPLPQoPoPPPQQPPoPKSL~QLQGWTNFGSGIPDFHA-VLAGPGTPGNSLRSLYPPPPPPQHLQMLP1086 TTMTTF-~IQPLPAIEVTGGA---------QGATGVSPAQETPTGAESAPGKPDL--EELVALTPPSPFRDSVDSGSTTPNSPVSESALCIPSSPKYDT ::I I : I : I : :::::: : : : : : : : : : I: :: I : ::I 1: IIIIlIII1III:III::I:IIIIII:lI:I:: :I:: LHLSTFQEESISPPGEDIDDDSE~KLLQEFVYEREGNTEEDELEEEEDLPTASKLTPEDSPALTPPSPFRDSVASGSSVPSSPVSESVLCTPPNVTYAS
1058
LIIRDYTQSSSSL
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1186
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FIG. 2. Amino acid sequence comparison of mGluR5 and mGluR1. The two sequences are aligned with single-letter notation by inserting gaps (-) to achieve maximum homology. Exact matches andconservative substitutions are connected by burs and dots, respectively. Cysteine residues conserved among themGluR family are indicated by black circles. Other symbols are the same asdescribed in the legend of Fig. 1.
closely related to thatof mGluR1, as illustrated inFig. 2. The degree of sequence identity is 60.1% for the mGluRl and mGluR5 sequences, and thesequence similarity between these two sequences increasesto 87.1% if conservative substitutions are included. In addition, mGluR5 shows the structural characteristics of the mGluR family (Tanabe etal., 1992);there is no sequence similarity nor are there any conserved amino acids between mGluR5 and the othermembers of the supergene family of the G protein-coupled receptors (O'Dowd et al., 1989; Nakanishi et al., 1990b); many cysteine residues are conserved, and there are potential N-glycosylation sites (Hubbard and Ivatt,1981) in the putative extracellular regions and many serine and threonine residues for possible regulatory
phosphorylation in thecytoplasmic regions (Kishimoto etal., 1985; Kemp and Pearson, 1990). Thus, mGluR5 has a structural architecture common to the members of the mGluR family with a large extracellular domain preceding the seven transmembrane segments. Characterization of the Properties and Signal Transduction of mGluR5"The signal transduction coupled to mGluR5 was first investigated by examining the electrophysiological responses to application of agonists inXenopus oocytes injected with the mRNA derived from the cloned mGluR5 cDNA. LGlutamate,ibotenete,andt-ACPD evokeda potent longlasting current(Fig. 3), which is characteristic of the response of receptors involved in the stimulationof P I hydrolysis/Ca2+
IP/Ca'+-coupled Metabotropic Glutamate Receptor Subtype lbotenate
+
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FIG.3. Current trace recorded from a Xenopus oocyte injected with the in vitrosynthesized mGluR5mRNA. Responses t o agonists (1 mM each) were recorded under voltage clamp a t -60 mV after 2-day incubation of an oocyte injected with -4 ng of the mGluR5 mRNA. Downward deflection indicates inward current.
0 0.01 0.1
1 10 100 PTX (ng/ml)
FIG.4. Pharmacological and biochemical characterization of mGluR5 in clonal cells expressing mGluR5. A , mGluR5signaltransduction (Dascal et al., 1987). Neithernormal oocytes nor water-injected oocytes showed any response to expressing cells were incubated with the indicated concentrations of quisqualate (O), L-glutamate (A), ibotenate (W), and t-ACPD (A)for the application of L-glutamate. 20 min and then determined for accumulation of total inositol phosSince variability in responses of individual oocytes is inev- phate formation. The inositol phosphate formation is expressed as itable in the Xenopus oocyte expression system, we investi- multiples of inositol phosphate levels in agonist-untreated cells. The gated thedetailed properties andsignal transduction mecha- values are means k S.E. of triplicate determinations. B, intracellular cells in the nism of mGluR5 by DNA transfection and stable expression Ca" ([Ca"],) was measuredformGluR5-expressing of this receptor in CHO cells. CHO(dhfr-) cells were perma- presence (solid line) and absence (dotted line)of CaC12in the external nently transfected with a vector-directing expression of the medium and for cells transfected with the vector DNA alone in the presence of CaCI2 (dashed line);a Ca'+ chelator EGTA (0.5 mM) was cDNA encoding the mGluR5. A mouse dihydrofolate reduc- added to theCaz+-free mediumto eliminate thepossible influence of tase gene was used as a selective marker gene (Urlaub and traceamounts of calcium ion. M L-glutamate was administered Chasin, 1980). We obtained several clonal cell lines which at 0 s to a coverslipof the cells loaded withfura-2. The dataindicated considerably increased PI hydrolysis on incubation with L- were taken from a representative of three experiments. C, mGluR5glutamate. No suchincrease in PI hydrolysis was observedin expressing cells were incubated with the indicated test compounds untransfected CHO(dhfr-) cells or cells transfected with the for 20 min and determined for the formation of total inositol phosIn the experimentin which the antagonist effects of DL-AP3 vector DNA alone (data notshown). ['HIGlutamate, which is phates. and L - A P ~were examined, cells were preincubated with these comthe only available radiolabeled compound for the binding of pounds for 30 minbefore the addition of M L-glutamate. The mGluR5, was not applicable for filtration or centrifugation values are indicated as -fold increases of the formation of total inositol binding assays inmembranepreparations of mGluR5-ex- phosphates in cells which were incubated for 20 min in the absence pressing cells, due to the rapid dissociation of this binding as of any test compounds. The values are means zk S.E. of triplicate determinations. No significant differences ( p < 0.05) from the corexpected from the effective dose value for half-maximal reresponding control values could be obtained for the stimulatory or sponse of this compound (see below) (Young and Fagg, inhibitory effects of DL-AP~ andL-AP4 by statistical analysis. D, 1990). The receptor density of mGluR5 expressed in CHO mGluR5-expressing cells were pretreated with the indicated concencells thus remained to be determined.Specific and appreciable trations of P T X for 7 h. The cells were incubated with (0)or without M L-glutamatefor 20 min andthendetermined for total expression of the mGluR5 mRNA in the receptor-expressing (0) cells, on the other hand, was verified by RNA blot hybridi- inositol phosphate formation. The inositol phosphate formation is zation analysis, whereasno expression of the mGluR5 mRNA expressed as multiples of inositol phosphate levels in cells which were not treatedwith PTX orL-glutamate. The values indicatedare means was detected in untransfected cells or incells transfected with k S.E. of triplicate determinations. *, p < 0.05 compared with Lthe vector DNA (data not shown). glutamate incubation in PTX-untreated cells. We determined dose-response curves of various agonists for the stimulation of PI hydrolysis in receptor-expressing cells from endoplasmic reticulum and increase intracellular Ca2+ (Fig. 4A). P I hydrolysis was measuredby monitoring the even in theabsence of extracellular Ca2+.We labeled mGluR5formation of a mixture of inositol mono-, bis-, and trisexpressing cells with a Ca2+-sensitive dye fura-2 andexamined phosphates, while blocking further dephosphorylation by glutamate-triggered mobilization of intracellular Ca2+in the LiCl. Quisqualate, L-glutamate, ibotenate, and t-ACPD were effective in inducing the stimulation of P I hydrolysis. The presence and absence of Ca2+ in the external medium (Fig. 4B).Application of 1mM L-glutamate to mGluR5-expressing ECso values of the above compounds were 3 X M, 1 X cells caused a rapid and markedincrease in intracellular Ca2+ M, 1 X M, and 5 X M, respectively. Thus, both the rank order and potencies of these compounds in inducing concentration in the Ca2+-containing medium, whereas no P I hydrolysis were very similar between mGluR5 and mGluRl such response was observed in control cells transfected with (Masu et al., 1991; Aramori and Nakanishi, 1992). NMDA, the vector DNA alone. Furthermore, a transient and compaAMPA, and kainate had no effects on PIhydrolysis up to the rable increase in intracellular Ca2+was observed in a Ca2+free medium containing a Ca2+ chelator EGTA, indicating concentration of M (datanot shown). The stimulation of P I hydrolysis is known to release Ca2+ that mGluR5 is linked to the phospholipase C pathway and
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IP/Ca*+-coupled Metabotropic Glutamate Receptor Subtype
elevates intracellularCa2+levels in response to agonist interOrigin - 1 2 3 4 5 6 7 8 9 action. The above functional assay of mGluR5 is very useful for the detailed characterization of the propertiesof this receptor, because there is no ambiguity resulting from the cross-reac9.5 . tivity with adifferent type of mGluR. We investigated several 7.5 properties of mGluR5 in receptor-expressing cells to compare 4.4 with those of the cloned mGluRl and otherpossible mGluRs characterized in tissue and cell preparations. DL-APQ and L2.4 AP4 have been reported to act as antagonistsfor glutamateinduced PI hydrolysis in rat hippocampus slices (Nicoletti et al., 1986a, 1986b; Schoepp and Johnson, 1989a, 1989b). The FIG.5. RNA blot analysis of mGluR5 mRNA. Total RNAs effects of DL-AP3 and L - A P ~on the mGluR5-mediated ino(IO pg each) were as follows: lane 1, thalamus; lane 2, sitol phosphate formationwere determined following the ad- analyzed hypothalamus; lane 3, hippocampus; lane 4, striatum; lane 5,olfactory dition of these compounds in thepresence or absence of bulb; lane 6, cerebral cortex; lane 7, midbrain; lane 8, cerebellum; lane M L-glutamate. Theaddition of M L-glutamate enhanced 9, medulla and pons. The size marker (kilonucleotides) used was the RNA ladder. the inositol phosphate formation about one-third maximal the effectelicited by M L-glutamate. These twocompounds showed no stimulatory or inhibitory effects up to at least is the highest in the striatum and relatively low in the cereM (Fig. 4C). Because DL-AP3 and L - A P ~ similarly showed no bellum and pons/medulla. inhibition on mGluR1-mediated stimulation of P I hydrolysis In situ hybridizationwitha specific riboprobeprepared (Aramori and Nakanishi,1992), neither mGluR5 nor mGluRl from the mGluR5cDNA revealed a wide distribution of seems tocorrespond to the mGluR reported in hippocampus mGluR5 mRNA in neuronal cells throughout the adult and slice preparations. 6-day-old rat brains (Figs. 6 and 7). No significant hybridiThe previous study of the cloned mGluR1 in cDNA-trans- zation was observed in parallel experiments using the same fected CHO cells indicated that the mGluR1-mediated stim- probe in the presence of an excess of unlabeled probe (data ulation of PI hydrolysis was partially but appreciably inhib- not shown). In the adult rat brain, prominent expression of ited(about 40% inhibition) by pretreatment of mGluR1- mGluR5mRNAwas observed mainly in the telencephalic expressing cells with P T X (Aramori and Nakanishi, 1992). regions, including the cerebral cortex, hippocampus, subicuThe effect of PTX on mGluR5-mediated P I hydrolysis was lum, internal granular layer of the olfactory bulb, anterior investigated by treatment of mGluR5-expressing cells with olfactory nucleus, pyramidal cell layer of the olfactory tubervarious concentrations of P T X prior to agonist application cle, striatum, accumbens nucleus, and lateral septal nucleus (Fig. 40). PTX treatment of the mGluR5-expressing cells (Fig. 6a). High expression of this mRNA was also seen inthe showed only a slightinhibition in theL-glutamate-induced P I anterior thalamic nuclei, shell regions of the inferior collicuhydrolysis (about 15% inhibition), and this inhibition was not lus, and caudal spinal trigeminal nucleus. In these regions, further potentiated by the additionof 10 ng ormore of PTX/ most neuronal cells were intensely labeled (Figs. 7, a and b). ml of the medium. Thus, there is an apparent difference in In the hippocampus, hybridization signals were seen in pythe effectiveness of PTX in mGluR1- and mGluR5-mediated ramidal cells throughout CA1-CA4 regions as well as in granule cells of the dentate gyrus. In the cerebellar cortex, only a responses of PI hydrolysis. mGluRl expressed in CHO cells has been shown to mark- small population of Golgi cells was labeled with the mGluR5 edly stimulate cAMP formation inresponse to agonist appli- probe; no hybridization signals were detectable in Purkinje cation, and this stimulation occurred in a rank orderof agonist and granule cells (Fig. 7c). In the6-day-old rat brain, prominent expression of mGluR5 potencies which was in complete agreement with that observed for the activationof P I hydrolysis (Aramori and Nak- mRNA was similarly observed in the telencephalic, diencephalic, and brainstem regions (Fig. 6b). Furthermore, this anishi, 1992). The linkage of mGluR5 tothestimulatory cAMP cascade was examinedinmGluR5-expressing cells. mRNA expression was more widely detected in the6-day-old inferior colliculus, thalamus, and None of the agonists for mGluR5 showed the stimulation of rat brain, particularly in the cAMP formation at the concentrations that induced the stim- basal forebrain regions; prominent expression was also seen ulation of PI hydrolysis (data not shown). The coupling of in theventromedial hypothalamic nucleus. Hybridization sigmGluR5 to the inhibitory cAMPcascade was also examined nals in thefilm images of the telencephalic regionswere more intense in the 6-day-old rat brain than in the adult brain in mGluR5-expressing cells. Incubation withforskolin(10 p ~ evoked ) cAMP accumulation of approximately 30 times (Figs. 6, a and b). This difference is ascribed to high densities above the basal levels. However, this accumulation was not of labeled neurons in the telencephalic regions of the 6-dayreduced by the addition of quisqualate or L-glutamate within old rat brain compared with those of the adult rat; grain the concentration ranges examined (10-s-10-3 M ) (data not densities on individual neurons were similar in bothsections the cortex shown). Thus,mGluR5 has no ability to stimulate or inhibitof the 6-day-old rat and the adult rat. In cerebellar of the 6-day-old rat brain, weak labeling was observed in the the cAMP signal transduction in receptor-expressing CHO Purkinje cell layer and internal granular layer (Fig. 7d). cells. Expression of mGluR5 mRNA-RNA blot hybridization and DISCUSSION in situ hybridization analyses were performed to examine the expression of mGluR5 mRNA. RNA blot analysis gave rise to Our previous investigation demonstrated the existence of a hybridization band with an estimated mRNA size of ap- four related but distinctmembers of the mGluR family (Tanproximately 8.5 kilonucleotides under high stringency condi- abe et al., 1992). mGluRl is primarily coupled to PI hydrolytions (Fig. 5). This size agreed well with that determinedfor sis/Ca2+ signal transduction (Masu et al., 1991; Aramori and the cloned cDNA. The blot analysis showed that mGluR5 Nakanishi, 1992), whereas the other three are linked to difmRNA in adult rats is distributed in many brain regions and ferent signaling cascades (Tanabe et al., 1992). This paper '
'
IP/Ca2+-coupled Metabotropic
Glutamate Receptor Subtype
13367
FIG. 7. Cellular localization of mGluR5 mRNA b y in situ hybridization. Bright-field photomicrographs of emulsion-dipped sections of the adult rat brain (a-c) and the 6-day-old rat brain ( d ) , showing the striatum (a), the pyramidal cell layer of CA1 (b), and the cerebellar cortex (c-d). The arrow in c indicates a labeled Golgi F I ~6.. Localization of mGluR5 mRNA in the adult and the cell. M , molecular layer; P,Purkinje cell layer; G, granular layer; EG, 6-day-old rat b r a i n s b y in situ hybridization. Negative film external granular layer; IG,internal granular layer. Scale bar, 50 pm. images of sagittal sections of the adult rat brain ( a ) and the 6-dayold rat brain (b) are shown. OR, main olfactory bulb; AO, anterior olfactory nucleus; Ac, accumbens nucleus; Tu,olfactory tubercle; St, between mGluRl andmGluR5 may indeed reflect the distinct striatum; Hi, hippocampus; S, subiculum; Cx, cerebralcortex; LS, properties of the two receptor subtypes. This investigation lateral septalnucleus; IC, inferior colliculus; Cb, cerebellar cortex; Sp, thus demonstrates the existence of a novel subtype of mGluR spinal trigeminal nucleus; T,thalamus; V M H , ventromedial hywhich closely resembles mGluRl in its structure, signal transpothalamic nucleus. Scale bar, 4 mm.
duction, andagonist selectivity, and the apparentdifferences of some properties between mGluRl and mGluR5 may be reportsthe presence of anadditionalsubtype of mGluR interesting to investigate for a further understanding of the termed mGluR5 by molecular cloning and characterizationof function of each receptor subtype. a cDNA clone encoding a new receptor. The cloned receptor The mGluR5 mRNA is considerably large (-8.5 kilonucleoshares a high degree of sequence homology with other mem- tides), and this size may have hampered the isolation of the bers of the mGluR family and possesses a large extracellular corresponding cDNA clonein ourprevious study (Masuet al., domain preceding the seven putativemembrane-spanning 1991), despite the fact that mGluR5 mRNA induces apotent domains. This receptor shows the highest sequence similarity electrophysiological response in the Xenopus oocyte expresto mGluRl among the four mGluRs, and consistent with this sionsystem. In situ hybridization analysis indicated that conservation, it iscoupled to the stimulation of P I hydrolysis/ mGluR5 mRNA is widely distributed in neuronal cells of Ca2+signal transduction. Furthermore, mGluR5 indicates a manybrain regions, and this expressionoverlaps that of marked resemblance to mGluRl in agonist selectivity and mGluRl mRNA in some brain regions (Masu et al., 1991; antagonist responses. However, mGluR5, unlike mGluR1, is Shigemoto et al., 1992). Both mRNAs are, for example, comnot capable of inducing the stimulationof the cAMPcascade monly observed in the dentate ~ Y N S and CA2-CA4 regions nor does it undergo an appreciable P T X inhibition in the PI of the hippocampus, striatum,andlateralseptal nucleus. hydrolysis/Ca2+ signal transduction. The promiscuous cou- However, the expressions of the two mRNAs are clearly pling of a certain receptor to the stimulatory cascades of both different in many other regions. mGluRl mRNA is highly P I hydrolysis and cAMP formation often depends on the expressed inthesubstantia nigra and superior colliculus receptor density in transfected cell lines (Ashkenazi et al., (Masu et al.,1991; Shigemoto et al., 1992), where little expres1989; Nakajima et al., 1992). Because the density of mGluR5 sion of mGluR5mRNA is detected. Ontheother hand, in our transfected cells remained to be determined, the pos- prominent expression of mGluR5 mRNA is seen in the ceresibility cannot be excluded that the differing responses of bral cortex, anterior olfactory nucleus, accumbens nucleus, mGluRl and mGluR5 in the stimulatory cAMP cascade re- inferior colliculus, and subiculum, andthis is in marked sults from different densities of the two receptors in our clonal contrast to weak or moderate expression of mGluRl mRNA cell lines examined.With respect to thedifference in the PTX in these regions. Particularly interesting are distinct expressensitivity between mGluRl and mGluR5, there are several sions of the two mRNAs in neuronal cells of the main olfacreports indicating that the PTX sensitivity of glutamate- tory bulb, cerebellum,and hippocampus. In themain olfactory induced PI hydrolysis, and the resulting electrophysiological bulb, mGluR5 mRNA is predominantlyexpressed in internal response are different, depending on the neuronal cells and granule cells, whereas mGluRl mRNA is preferentially lobrain regions examined (Stratton et al., 1990; Sugiyama et al., cated in tufted cells and mitral cells. In Purkinje cells of the 1987). Thus, ourobservation of the different PTXsensitivity cerebellum, no appreciable expression of mGluR5 is seen, in
13368
IP/Ca2+-coupled Metabotropic Glutamate Receptor Subtype
REFERENCES contrast to predominant expression of mGluRl mRNA in I., and Nakanishi, S. (1992) Neuron 8 , 757-765 Aramori, these cells. Furthermore, mGluR5 mRNA, unlike mGluRl Ashkenazi, A,, Peralta, E. G., Winslow, J. W., Ramachandran, J., and Capon, D. J. (1989) Cell 56,487-493 mRNA, shows prominent expression in the hippocampal pyJ., Hollmann, M., O'Shea-Greenfield, A,, Hartley, M., Deneris, E., ramidal cells of CA1. Thus, mGluRl and mGluR5, although Boulter, Maron, C., and Heinemann, S. (1990) Science 2 4 9 , 1033-1037 very similar in their signal transduction and other properties, Collingridge, G. L., and Singer, W. (1990) Trends Pharmocol. Sci. 11,290-296 Dascal. N. (1987) CRC Crit. Reu. Biochern. 22.317-387 are differentially expressed in specialized neuronal cells and Graham, F.'L., and van der Eb, A. J. (1973) Virology 52,456-467 P. P., Wilkins, C. J., Tyler, W., and Watson, S. P. (1988) Br. J. may thusproduce varied and distinct functions in the centralGodfrey, Pharrnacol. 95,131-138 nervous system. Hollmann, M., OShea-Greenfield, A,, Rogers, S. W., and Heinemann,S. (1989) Nature 342,643-648 The existence of two distinct subtypes of mGluRs coupled Houamed, K. M., Kuijper, J. L., Gilbert, T. L., Haldeman, B. A., O'Hara, P. J., Mulvihill, E. R., Almers, W., and Hagen, F. S. (1991) Science 2 5 2 , 1318to the stimulationof P I hydrolysis has been suggested on the 1321 basis of pharmacological studies on agonist selectivity and Hubbard, S. C., and Ivatt, R. J. (1981) Annu. Reu. Biochern. 50,555-583 antagonist effectiveness in the response of P I hydrolysis in Ito, S., Mochizuki-Oda, N., Hori, K., Ozaki, K., Miyakawa, A,, and Negishi, M. (1991) J. Neurochern. 56,531-540 cell and tissue preparations. One is a quisqualate-preferring Keiniinen, K., Wisden, W., Sommer, B., Werner, P., Herb, A,, Verdoorn, T. A,, Sakmann, B., and Seeburg, P. H. (1990) Science 249,556-560 subtype which is more effectively activated by quisqualate Kemp, B. E., and Pearson, R. (1990) Trends Biol. Sci. 15,342-346 than by ibotenate (Schoepp et al., 1990; R6casens et al., 1991). Kishimoto, A,, Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Takeyama, Y., and Nishizuka, Y. (1985) J. Biol. Chem. 260,12492-12499 This receptor subtype is not antagonized by DL-APQ or L- Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 6 7 , 105-132 AP4 and appears to be present in many brain regions. For Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., and Nakanishi, S. (1991) Nature 3 4 9 , 760-765 example, this receptor has been found in cultured striatal Meldrum, B., and Garthwaite, J. (1990) Trends Pharrnacol. Sci. 11,379-387 Miller, R. J. (1991) Trends Pharrnacol. Sei. 12,365-367 neuronal cells (Sladeczek et al., 1985; Weiss, 1989), cerebral Monaghan, D. T., Bridges, R. J.,andCotman, C.W. (1989) Annu. Reu. cortex slices (Godfrey et al., 1988), and forebrain synaptoneu- Pharmocol. Toxicol. 29,365-402 Moriyoshi, K., Masu, M., Ishii, T., Shigemoto, R., Mizuno, N., and Nakanishi, rosomes (Rbcasens et al., 1987) and is expressed in Xenopus S. (1991) Nature 354,31-37 oocytes injected with brain poly(A)+ RNA (Sugiyama et al., Nakajima, Y., Tsuchida, K., Negishi, M., Ito, S., and Nakanishi, S. (1992) J. Bwl. Chern. 267,2437-2442 1987). Both mGluRl and mGluR5 correspond well to the Nakanishi, N., Shneider, N. A., and Axel, R. (1990a) Neuron 5 , 569-581 quisqualate-preferring receptorsubtype. The otherof the two Nakanishi, S., Ohkubo, H., Kakizuka, A,, Yokota, Y., Shigemoto, R., Sasai, Y., and Takumi, T. (1990h) Recent Prog. Horrn. Res. 46,59-84 subtypes is an ibotenate-preferring subtype which is more Nicoletti, F., Iadarola, M. J., Wroblewski, J. T., and Costa, E. (1986a) Proc. Natl. Acad. Sci. U. S. A . 8 3 , 1931-1935 potently activated by ibotenate than by quisqualate and is Nicoletti, F., Meek, J. L., Iadarola, M. J., Chuang, D.M., Roth, B. L., and et al., 1986a, 1986b; Costa, E. (1986b) J. Neurochern. 46,40-46 blocked by DL-APQa n d L - A P(Nicoletti ~ F., Magri, G., Ingrao, F., Bruno, V., Catania, M. V., Dell'Albani, P., Schoepp and Johnson, 1989a, 198913). This receptor appears Nicoletti, Condorelli, D. F., and Avola, R. (1990) J. Neurochern. 5 4 , 771-777 O'Dowd, B. F., Lefiowitz, R. J., and Caron, M. G. (1989)Annu. Reu. Neurosci. t o be located mainly in the neonatal and adult hippocampus, 12,67-83 and ibotenate-stimulated PI hydrolysisseems to bemaxiOikawa, S., Inuzuka, C., Kuroki, M., Matsuoka, Y., Kosaki, G., and Nakazato, H. (1989) Biochern. Biophys. Res. Cornrnun. 164,39-45 mized at the postnatal stage (Nicolettiet al., 1986a). In fact, Rkasens, M., Sassetti, I., Nourigat, A,, Sladeczek, F., and Bockaert, J. (1987) our initial purpose of this investigation was to examine the Eur. J. Pharrnacol. 141,87-93 M., Mayat, E., and Guiramand, J. (1991) in Current Aspects of the existence of an ibotenate-preferringreceptor in the postnatal RBcasens, Neurosciences (Osborne, N. N., ed) Vol. 3, pp. 103-175, Macmillan, New York hippocampus, but the cloned mGluR5 reported in thisinvesSambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A tigation is clearly different from the putative ibotenate-preLaboratory Manuol, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY ferring receptor subtype. It is still possible that there is an Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sei.U. S. A. additional mGluR whichis linked to PIhydrolysis/Ca*+ signal 74,5463-5467 D. D., and Johnson, B. G. (1989a) J. Neurochern. 53,273-278 transduction and possesses a n agonist selectivity different Schoepp, Schoepp, D. D., and Johnson, B. G. (198913) J. Neurochern. 53,1865-1870 Schoepp, D., Bockaert, J., and Sladeczek, F. (1990) Trends Pharrnacol. Sci. 1 1 , from thoseof mGluRl andmGluR5. Furthermore, it hasbeen 508-515 reported that quisqualateevoked a marked stimulation of P I Shi emoto, R., Nakanishi, S., and Mizuno, N. (1992) J. Corn Neurol in press F., Pin,J.-P., RBcasens,M., Bockaert, J., a n t w e i s s , 8. (1985) et al., 1990). Thus, Slajeczek, hydrolysis in cultured astrocytes (Nicoletti Nature 3 1 7 , 717-719 further investigation on the multiplicity of mGluRs coupled Stratton, K. R., Worley, P. F., and Baraban, J. M. (1990) Eur. J. Pharrnacol. 186,357-361 t o P Ihydrolysis will be interesting and importantfor under- Sugiyama, H., Ito, I., and Hirono, C. (1987) Nature 3 2 5 , 531-533 standing the role and functions of this class of the mGluR Tanabe, Y., Masu, M., Ishii,T., Shigemoto, R., andNakanishi, S. (1992) Neuron 8 , 169-179 family. Urlaub, G., and Chasin, L. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,4216-
Acknowledgments-We are grateful to Seiji Ito for help of Ca2' measurements and Akira Uesugi for photographicassistance.
4220 von Hei'ne, G. (1983) Eur. J. Biochern. 133,17-21 Weiss, (1989) Brain Res. 491,189-193 Young, A. B., and Fagg, G. E. (1990) Trends Pharrnacol. Sci. 11, 126-133
d.
