journal of 268, no. 16, of 5, pp. 1993 for biology, inc ...the journal of biological chemistry 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 16, Issue of June 5, pp. 12033-12039.1993 Printed in U.S.A.

Primary Structure and Cell-type Specific Expression of a Gustatory G Protein-coupled Receptor Related to Olfactory Receptors*

(Received for publication, February 1, 1993)

Keiko Abe, Yuko Kusakabe, Kentaro TanemuraS, Yasufumi Emorig, and Soichi Arai From the Department of Agricultural Chemistry and the $Department of Veterinary Anatomy, Faculty of Agriculture and the §Department of Biophysics and Biochemistry, Faculty of Science, the Uniuersity of Tokyo, Bunkyo, Tokyo 113, Japan

We have reported on the partial structures of a mul- tigene family encoding GTP-binding protein (G pro- tein)-coupled, seven-transmembrane receptors ex- pressed in the tongue (Abe, K., Kusakabe, Y., Tane- mura, K., Emori, Y., and Arai, S. (1993) FEBS Lett. 316, 253-266). Here we describe a full-length cDNA clone encoding a tongue cell-type specific receptor. The encoded protein consists of 312 amino acid residues. In overall structure, the protein is similar to known G protein-coupled, seven-transmembrane receptors such as an olfactory receptor (56% identity) but is signifi- cantly different in part, particularly in NHP-terminal extracellular and COOH-terminal cytoplasmic domain structures. Northern analysis showed that the mRNA for this protein is expressed only in the epithelium of the tongue, not in other organs. In situ hybridization experiments clearly indicated that the mRNA is ex- pressed exclusively on the tongue apical surface, not on the reverse side of the tongue nor in its muscle layer. Expression was also detected in the taste buds and surrounding cellular tissues of the fungiform and circumvallate papillae. It is suggested that this gusta- tory receptor structurally related to olfactory recep- tors may be a candidate for a taste receptor.

Various sensory systems are able to recognize numerous stimulants and also to differentiate their intensities. In the visual system, opsin functions as a receptor to catch light signals for final transduction to optical neurons (1-4). For the sense of smell, a recent finding has disclosed the existence of olfactory receptors possibly functioning to interact with odorant molecules on the surface of microvilli (5). These receptors in the visual and olfactory systems are characterized by having seven-transmembrane domain motifs and being coupled to GTP-binding proteins (G proteins). Thus, it is possible to propose that sensory signal transduction is in general initiated by the action of G protein-coupled receptors. However, little is known about the molecular biology of gus- tatory receptors.

Physiologically, several lines of evidence have been devel- oped (6). According to these, signal transduction triggered by tastant molecules takes place first on the surface of the taste cells in fungiform, foliate, and circumvallate papillae of the tongue (7-10). It is also krown that the taste cells involved in synapse-mediated transduction of taste stimuli are those

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "Oduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper ~ Q S been submitted

012820. to the GenBankTM/EMBL Data Bank with accession number(s)

differentiated from nonsensory epithelial cells (11). To elucidate the existence of taste receptors and their

molecular entity, we have used a reverse transcription-polym- erase chain reaction (RT-PCR)' to identify a gene family with more than 60 members expressed in tongue epithelium, which could encode membrane receptors similar to olfactory recep- tors (12). Here we report that a full-length cDNA clone (GUST27) encodes a protein structurally related to olfactory receptors and that the GUST27 mRNA is expressed in taste buds and the surrounding cellular tissues of rat tongue pa- pillae.

EXPERIMENTAL PROCEDURES

Materials-Restriction enzymes, other enzymes, and plasmid vec- tors pUC18 and pBluescript were obtained from Takara Shuzo and Toyobo. The Sequenase sequencing kit was from Toyobo. Hybond- N, [cY-~ 'P]~CTP (9.25 MBq/mmol), [y3'P]ATP (9.25 MBq/mmol), the rapid hybridization kit, and the multiprime labeling kit were obtained from Amersham Corp. The oligo(dT)-cellulose and cDNA synthesis kits were from Pharmacia LKB Biotechnology Inc. The digoxigenin RNA labeling kit was from Boehringer. Other reagents were from Nakarai and Wako Chemical.

Construction of a cDNA Library and Sequencing-Total RNA was prepared from rat tongue epithelia by the standard method (13). Poly(A+) RNA was purified on an oligo(dT)-cellulose column. Dou- ble-stranded cDNA, synthesized according to Gubler and Hoffman (14), was ligated to X g t l O phage vector. After in uitro packaging (Stratagene), the phages were plated with Escherichia coli C600hfl. Recombinant plaques were transferred onto nylon filters (Hybond- N), prehybridized for 16 h at 60 "C as described previously, and hybridized for 24 h at 60 "C with a mixed probe of the previously reported RT-PCR clones, PTE 01, 03, 33, 38,45, and 58 (12) labeled with [cY-~'P]~CTP. The filters were finally washed in 2 X SSC containing 0.1% SDS at 60 "C.

Nucleotide Sequencing-DNA was extracted from the recombinant phages, digested with EcoRI, and subcloned into pUC18 vector (15). Nucleotide sequences were determined by a dideoxy sequencing method (16) using modified T7 DNA polymerase (Sequenase).

Genomic Southern Blot Hybridization-Rat genomic DNA was isolated from liver according to the method of Maniatis et al. (13). About 10 pg of DNA was digested with EcoRI, HindIII, and BamHI, fractionated on agarose gels, denatured, and transferred onto a nylon filter (Hybond-N) according to the method of Southern (17). Filters were prehybridized at 65 "C and hybridized at 65 "C with a 32P-labeled cDNA clone (GUST27) (see "Results") as a probe. The filters were washed at 68 'C in 0.1% SSC buffer containing 0.1% SDS.

RNA Blot Hybridization-Total RNA was extracted from rat tis- sues, and poly(A+) RNA (2 pg) was isolated, denatured, and electro- phoresed in formaldehyde-containing agarose gels. After electropho- resis, the RNA was transferred onto nylon membranes (Hybond-N) and hybridized with 32P-labeled cDNA at 65 "C in rapid hybridization solution. The filters were finally washed at 65 "C in 0.1 X SSC containing 0.1% SDS.

In Situ Hybridization-The EcoRI-HincII fragment (nucleotides -132 to 150 in Fig. 1) of GUST27 (see "Results" and Fig. 1) and the

' The abbreviation used is: RT-PCR, reverse transcription-polym- erase chain reaction.

12033

12034

FIG. 1. Nucleotide and deduced amino acid sequences of the cDNA clone, GUST27. Nucleotides are num- bered in the 5'-3' direction starting with the first methionine of the longest open reading frame. The inferred positions of putative seven-transmembrane domains are underlined (cf. Fig. 2). An in-frame termination codon in the 5"noncoding sequence is marked by asterisks.

Gustatory G Protein-coupled Receptor

AGCGTCATTTTGTGATATGTCCGGTGCTATTATTTGGAMCTAMCTTAMTTTTCTTTA ACAGATACAAMGTCATATGGAM~AGMCCACACMTGAGMCAGMTTTCACATCC

TGCGTCTCTCAGATGATCCTGMCTGCMCCCATTCTCTGGACTGTTCCTGTC~TGTAT t

H I L N C N P F S G L F L S H Y

CTGGTCACAGTGCTTGGGMCTTGCTCATCATCCTGGCTGTCAGCTCTMTTCACATCTC L V T V L G N L L I I L A V S S N S H L

CACMCCTCATGTATTTCTTCCTCTCCMTCTGTCCTTTGTTGACATCTGTTTCATCTCA H N L H Y F F L S N L S F V D I C F I S

ACCACMTACCAAMATGCTAGTOMCATACATTCACAGA~ACATCTCCTA~TA T T I P K H L V N I H S Q T K D I S Y I

GMTGCCTTTCACAGGTATATTTTTTMCTACTTTTGGTGGMTGGATMTTTTTTACTC E C L S Q V Y F L T T F G G H D N F L L

ACTTTMTGGCCTGTCATCGCTATGTAGCCATCTGCCACCCCCTCAACTACACTGTMTC T L H A C D R Y V A I C H P L N Y T V I

ATGMCCTTCAGCTGTGTGCCCTTCTGATTCTGA~TTT~GTTMTCATGTTCTGTGTC H N L Q L C A L L I L H F W L I H F C V

TCCCTGATTCATGTTCTATTGATGMTGMTTGMCTTCTCCAGAGGCACAGMATTCCA S L I H V L L H N E L N P S R G T E I P

CATTTCTTCTGTGMCTGGCTCAAGTTCTTMGGTAGCCMTTCTGACACTCATATCMT H F F C E L A Q V L K V A N S D T B I N

MTGTCTTCATGTATGTGGTGACTTCCCTACTAGGACTGATCCCTATGA~MTACTT N Y F H Y V V T S L L G L I P H T G I L

A T G T C T T A C T C A C A G A T T G C T T C A T C C T T A T T A M G A T G T C J f S Y S Q I A S S L L K H S S S V S K Y

MGGCCTTTTCCACCTGTGGATCTCACCTCTGTGTGGTCTCTTTATTCTATGGGTCAGCA K A F S T C G S B L C V V S L P Y G S A

A C T A T A G T T T A C T T C T G C T C T T C T G T G C T C C A T T C T A C A C T I V Y E C S S V L H S T H K K H I A S

TTGATGTACACTGTMTCAGCCCCATGCTGMCCCCTTTATCTATAGCCTGAGAMCMG L H Y T V I S P H L N P F I Y S L R N K

GATGTAMGGGTGCCCTTGGAAMCTTTTCATCCGAGTTGCCTCTTGCCCATTGTGGAGC D V K G A L G K L F I R V A S C P L W S

AMGACTTTAGACCTAMTTCATACTAAMCCTG~GG~GTTTATAMCAMCCTC K D F R P K F I L K P E R Q S L *

TCCTGGGTCATTTGTATCATTATATGCCTMTTTACACTATTCTAAMGTATATAT AGCTTGTCATTTGTGTACTTTCTACAAAMATATTTTMTTCCCTATGCATATTGTTTM MTTTGCMTTCTTGTTATGTC

I

II

111

IV

V

VI

VI1

-73 -13

48 16

108 36

168 56

228 76

288 96

348 116

408 136

468 156

528 176

588 196

648 216

708 236

768 256

828 276

888 296

948 312

1008 1068 1090

six RT-PCR products (12) were each subcloned into SK' or KS' pBluescript vector. Digoxigenin-labeled RNA probes were synthe- sized by T3 or T7 RNA polymerase. The tongue of a five-week-old rat (Fischer) was perfused through the heart first with Ringer solution containing 0.01% diethyl pyrocarbonate and then with Bouin solution fixative (Sigma). A tongue tip specimen was dehydrated in a graded series of ethanol containing 0.01% diethyl pyrocarbonate and em- bedded in paraffin. Cross-sections (2-pm thickness) of the tongue tip were prehybridized for 3 h and then hybridized overnight with digox- igenin-labeled RNA probe (1 mg/ml) under stringent (50 " C ) and less stringent (45 " C ) conditions (18). After washing, the cross-sections were incubated with peroxidase-conjugated anti-digoxigenin antibody (Boehringer) for 1 h. The immunostained samples were visualized by treatment with a mixture of 0.01% H202 and 0.05% 3,3-diaminoben- zidine tetrahydrochloride in 0.01 M phosphate-buffered saline for 10- 15 min.

RESULTS

Isolation of cDNA Clones-Using six RT-PCR clones (PTE 01, 03, 33, 38, 45, and 58) encoding parts (transmembrane domains 11-VII) of putative seven-transmembrane receptors

(12), we screened a cDNA library prepared from rat tongue epithelium and finally obtained five clones out of 2.5 x lo6 plaques. These five cDNA clones of gustatory origin were found to have mutually different restriction enzyme maps (data not shown). This indicates that they should be generally similar but partially distinct proteins (12). Since four of the five cDNA clones contained incomplete coding sequences, we analyzed only the complete clone, termed GUST27; its total coding sequence was as shown (Fig. 1). The analysis showed that GUST27 contains an open reading frame spanning 936 base pairs encoding 312 amino acid residues preceded by an in-frame termination codon (-33 to -31) but lacking a poly(A) sequence in the 3"noncoding region (Fig. 1).

Overall Structure and Sequence Homology-The protein encoded by GUST27, a putative gustatory receptor, most resembles PTE33 among the previously isolated RT-PCR clones, with 80% identity in amino acid sequence. As shown in Fig. 2, the GUST27 protein has seven major hydrophobic regions and shows structural similarity in whole to various

Gustatory G Protein-coupled Receptor 12035

3.6

0 . 0

- 3 . e 35 1

FIG. 2. Hydropathy profile of GUST27. The left uerticd anis indicates the hydropathic index; the horizontal axis represents amino acid position. Seven major hydrophobic regions are indicated by bars I-VI%

2 0 4 0 . . . . . . . . . . . . . . . . . . . . . . . . . " . . . . . . " " . . . . " ' ' ' . ' ' ' I

GUST27 OLFF3 MDSSKHPRVSEFLLLGFVENKDLQPLI HGMPO73 MLLCFRfGNOSMKRFNPILITUFVFPGFSSFilPOOITL Rhodopsin MNCTEGPNFYVPFSNK7GVVRSPFEAPGYYLAEPWQLSMLAAY TXR MWI'NGSSLGPCFRPTNI~I ,~ : -ERRLlASPWFAA--- - - "I MNTS--VPPAVS-PNIT-VI.AI""""-

6 0 80 1 0 0 1 2 0 1 4 0

, . , . 1 ,,..

. . . . . . . . . . . . . ...'E*". . . . . . . . . . . . . . IV

GUST27 -

Rhodopsin F

. . . . . . . . . . ' ~ ~ ' . ' , ' ' . ' ' , '

160 180 ? O O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

GUST77 NF N SRGT OLFF3

L#bTHI, HGMPO7J F I' AR-

Rhodopsin ---- S R Y I P

"_""_"___""""""""""""~ """__"""_"""""""~""""~ _"______""_"""""""""""" OI'VF1'V __""____""__ """"""""

......................... 2 7 0 1 4 0 2 6 0

. . . . . . . . . . . . . . . . . . . . . . VI I ""_""" """"_" """""_

. . . . . 1 3 . . . . . . . . . . . . . . . . . . . . . . . 2 8 0 3 0 3

GUST2 7 OLP'F'3 HCMPO7J

Lt!FFLVLCIII.I'CFII.'CY

Rhodopsin GFRhiCMVTTLCCGKN*DUEQS'TTVSKlET APA TXR -FRR&LRRi QPRl.@rRPR L S L

: ) ~ I ~ I I , K ~ R & I ,

"I . , R , , , F , , , I ; . L , , K , R l ? b r i ' ~ ~ ~ ~ ~ ~ ~ ~ S R Q C . . . . . . . . . . . . . . . . . . . . 1 4 . . . . . . . . . . . . . . . . . . . . .

FIG. 3. Alignment of the amino acid sequences of the GUST27 protein and several known G protein-coupled receptors: rat olfactory receptor (OLFF3). human germ cell receptor (HGMPO7J), bovine rhodopsin, human thromboxane A2 receptor (TXR), and mouse M1 muscarinic acetylcholine receptor (MMZ). Amino acid numbering is based on the sequence of the GUST27 protein. Gaps were inserted to maximize the number of matches. White letters in black boxes denote amino acids identical to the GUST27 protein. Solid ouerlines indicate seven-transmembrane domains I-VI1 inferred from the hydrophathy profile of the GUST27 protein (Fig. 2) as well as from those of other receptors (Refs. 2, 5, and 19-21). Dotted Eines aboue and below the alignments show the inferred positions of four extracellular side, El-E4, and four inner domains in the cytoplasmic side, 11-14, respectively.

receptors with seven-transmembrane domain motifs (Fig. 3). receptor (19). A certain degree of homology was also observed In particular, its highest homology (56%) is to OLFF3 protein, in comparisons with rhodopsin (2) and other receptors (20, one of the olfactory receptors (5), and it has significant 21) in terms of amino acid sequences of some transmembrane homology (33%) to HGPO7J protein known to be a germ cell domains (Fig. 3).

12036 Gustatory G Protein-coupled Receptor

kb E H B

23.1

6.6 . 9.4 *

4.4.

2.3 - 2.0 -

0.6 -

FIG. 4. Identification of the gene corresponding to GUST27. Total rat liver DNA (10 pg) was digested with EcoRI ( E ) , Hind111 (H), or BarnHI (E). The blotted membrane was hybridized with 3ZP-labeled probe for GUST27 as described under “Experimental Procedures.”

28s - 18s -

FIG. 5. Expression of the GUST27 mRNA in various rat tissues by Northern blot analysis. Poly(A+) RNA (2 pgllane) was electrophoresed on a 1% denatured agarose gel containing 2.2 M formamide. The RNA samples were prepared from rat tongue epithe- lium, whole tongue, testis, retina, brain, liver, kidney, lung, pancreas, small intestine, skeletal muscle, and smooth muscle. The blotted membrane was hybridized with a 32P-labeled cDNA insert of GUST27. The positions of 28 S and 18 S ribosomal RNA are indicated.

The GUST27 protein contains several particular amino acid residues commonly conserved in all other receptors listed. These are Cys” and CyslG0, which possibly form a disulfide bond (22-24), Leu47 and Leuw, probably involved in some hydrophobic interaction, and Pro”’ and Pro2=, which may each play a role in the formation of a bent structure (25, 26). In particular, the GUST27 protein shows similarity to OLFF3 and HGMP07J proteins in terms of such partial amino acid sequences as Met-Tyr-Phe-Phe-Leu-Ser-Asn-Leu-Ser, Phe- Leu-Leu-Thr-Leu-Met, Phe-Ser-Thr-Cys-Gly-Ser-His-Leu- Cys-Val-Val, and Tyr-Thr-Val-Ile-Ser-Pro in transmembrane domains 11, 111, VI, and VII, respectively, and Asp-Arg-Tyr- Val-Ala-Ile-Cys and Tyr-Thr-Val-Ile-Met-Asn in the I2 do- main (the second cytoplasmic domain) and Pro-His-Phe-Phe- Cys in the E3 domain (the third extracellular domain). Also, a major characteristic commonly found in these three recep- tors is the size of the I3 domain containing a G protein

coupling site (27-29). Each of the three has a short I3 domain comprising 19 amino acid residues, whereas the I3 domains of rhodopsin, TXR, and MM1 are composed with 29,28, and 157 amino acid residues, respectively (2, 20, 21). A unique finding in the structure of the GUST27 protein is that it bears a particular cysteine residue (CYS~~’), which might have under- gone palmitoylation as in the case of rhodopsin (30).

Identification of the Gene for GUST27-To identify the gene for GUST27, rat liver chromosomal DNA was digested with restriction enzymes and subjected to a total genomic Southern experiment using GUST27 as a probe. Fig. 4 indi- cates that the gene should be of a single copy. However, multiple bands were observed when the hybridization was conducted under less stringent conditions (data not shown), which means that there are multiple genes as reported in our previous paper on RT-PCR clones (12).

Expression of GUST27 mRNA in Various Tissues-As shown in Fig. 5, a single band of approximately 2 kilobases was detected only in tongue epithelium. No detectable expres- sion was observed for whole tongue mRNA. Also, the mRNA was not expressed in any other organs tested (Fig. 5). Thus, the GUST27 mRNA was concluded to be expressed specifi- cally in tongue epithelium.

In Situ Hybridization-To obtain more precise information about the expression of this gustatory gene, in situ hybridi- zation experiments were carried out using digoxigenin-labeled RNA probes. When an antisense RNA probe was used, posi- tive signals were detected as a brownish color (Fig. 6a), whereas no signal was observed with the sense RNA used as a control (Fig. 6b). The expression was specific to epithelial cells on the apical surface of the tongue (Fig. 6a, upper); no positive expression was detected on the reverse side of the tongue in the muscle layer, horny layer, or connective tissue of the tongue (Fig. 6a, lower). In some experiments where the hybridization conditions were made relatively stringent, the resulting signals predominated in the taste buds of the fun- giform papillae (Fig. 6c), although in other experiments, sig- nals were observed in almost all epithelial cells including taste bud cells (Fig. 6d) . The GUST27 mRNA was also expressed in the circumvallate papillae (Fig. 6, e and f ) . Thus, the expression of the GUST27 mRNA was specific to epithelial cells including taste buds.

We have shown that multiple genes related to GUST27 are expressed in the tongue (12). However, since the cell-specific distribution of these mRNA in the tongue remains unknown, we next carried out a similar, in situ hybridization experiment using the RT-PCR clones. As shown in Fig. 7, a-d, the antisense RNA probes originating from the four RT-PCR clones gave essentially the same results, indicating that most, if not all, of the gustatory receptor genes identified here and previously (12) are specifically expressed in the epithelium of the tongue, particularly in the taste bud cells. Such expression was observed neither in other parts of the tongue nor in other organs.

DISCUSSION

Our preceding paper identified a gene family with more than 60 members and revealed partial structures suggesting possible seven-transmembrane proteins expressed in rat tongue epithelia. In the present study, we actually obtained from a cDNA library prepared from rat tongue epithelium a full-length cDNA clone, GUST27, closely related to each of the RT-PCR clones of gustatory origin (12). The results of in situ hybridization indicate that tongue epithelial cells express multiple genes each encoding a G protein-coupled receptor. This feature of gene expression in the tongue closely resem-

Gustatory G Protein-coupled Receptor 12037

FIG. 6. Identification of the mRNAa for the G protein-coupled receptors in epithelial cells by in situ hybridization with digoxigenin-labeled RNA probe of GUST27 origin. a and b, whole tongue sectione hybridized with GUST27 antisense (a) and sense (b ) probes and stained with haematoxylin (30 times magnification in each picture). c, apical surface layer hybridized with GUST27 antisense probe under stringent conditions (40 times magnification). d, apical surface layer hybridized less stringently with GUST27 antisense probe (160 times magnification). e and f, surface layer (containing circumvallate papillae) hybridized less stringently with GUST27 antisense probe and shown at 30 times (e) and 340 times ( f ) magnification. Arrows indicate taste buds.

bles the case where a multiple gene family exists to express a large number of olfactory receptors in the nose. However, clear structural differences can be found between the GUST27 protein and the olfactory receptors in three respects. First, the NHz-terminal extracellular domain, El, of the GUST27 protein consists of only 10 amino acid residues and is smaller than those of any G protein-coupled receptors known. This domain lacks any potential N-glycosylation site (Fig. 11, whereas in the OLFF3 protein as a representative of the olfactory receptors, the El domain consists of as many as 28 amino acid residues and contains a potential N-glycosylation site (5). Second, there is a great difference in transmembrane structure. Comparing transmembrane domains IV and V of the GUST27 protein with those of the OLFF3 protein, low

identities of 27 and 35%, respectively, are found. Much lower identities are found when the extracellular halves of trans- membrane domain IV in both receptors are compared. The same is true for the extracellular half of transmembrane domain V. These differences may be very important in light of the experimental data of Strader et al. (31) who showed by site-specific mutagenesis of &-adrenergic receptor that the amino acid residues located on the a-helix nearest the extra- cellular surface are involved in ligand binding. There is also a greater difference in the amino acid sequence between the E4 domain of the GUST27 protein and that of the OLFF3 protein (18% identity), which may be somehow involved in determining the specific ligand binding. Third, domain I4 of the GUST27 protein has very low similarity to that of the

12038 Gustatory G Protein-coupled Receptor

FIG. 7. IdemtXkation of the m R N h for the G protein-coupled receptors in epithelial cella by in situ hybridization with digoxigenin-labeled RNA probea of RT-PCR clone origin (12). a, apical surface layer hybridized with PTE 01 antisense probe under stringent conditions and stained with haematoxylin (45 times magnification). b, apical surface layer hybridized with F'TE 03 antisense probe under stringent conditions (40 times magnification). c, surface layer (containing circumvallate papillae) hybridized with PTE 45 antisense Drobe under stringent conditions (60 times mamification). d, auical surface layer hybridized less stringently with PTE 58 antisense probe 1160 times magnification).

- . . -

OLFF3 protein or to those of other seven-transmembrane receptors. It is also important that the GUST27 protein differs largely from the OLFF3 protein in its I3 and I4 domain structures, since seven-transmembrane receptors are known to function in intracellular signaling through interaction with G proteins at some sites in the I3 and I4 domains, as dem- onstrated for rhodopsin-transducin interaction (28). Thus, such characteristic structures in these cytoplasmic loops (I3 and 14) of the GUST27 protein may be related to its specific G protein coupling. It is thus likely that the possible tongue epithelial receptors exemplified by the GUST27 protein, though closely related in whole to olfactory receptors, have some particular gustatory functions. Also, the overall similar- ity of the GUST27 protein to other G protein-coupled, seven- transmembrane receptors indicates that this protein itself has a similar receptor function; but, with respect to specific ligand binding and G protein coupling, its function is thought to be bate-specific in light of the significant differences observed here.

Meanwhile, taste physiology has progressed to present a great deal of data on signal transduction. For salty taste transduction, it is now postulated that sodium and potassium ions are involved in the depolarization of cell membranes of tongue papillae by ion-gated channeling (32-34). It is also reported that both K+ and Ca" channels mediate the proton- dependent transduction of sour taste (34-37). On the other hand, it is indicated that CAMP levels increase in papillae exposed to both sweet and bitter taste stimuli (38-40). Most of these physiological observations suggest the involvement of some G protein-coupled receptors in initiating taste signal

transduction, because in other tissues, seven-transmembrane receptors and G proteins coupled to these receptors are known to mediate intracellular signaling (reviewed in Ref. 41). In fact, gustducin has been reported as a taste cell-specific G protein of the Gi type (42). In the case of sweet and bitter taste transduction, the possibility exists that other G proteins, especially those of the G, type, function in tongue papillae by coupling with gustatory receptors such as those reported here and previously (12). It can also be predicted that a great many such receptors exist, since the senses of sweet and bitter tastes can recognize and differentiate among innumerable numbers of sweet and bitter compounds, each having a delicately different taste nuance. This prediction is supported by elec- trophysiological studies on taste reception (38,43-45).

In the present study, we demonstrated by in situ hybridi- zation that the GUST27 mRNA is expressed in taste buds of fungiform and circumvillate papillae (Figs. 6 and 7). This finding, together with the consideration that the GUST27 protein is structurally related to proteins identified as olfac- tory receptors (5), strongly indicates the existence of func- tional taste receptors in sensory cells of taste buds. However, we detected the expression of GUST27 mRNA not only in the taste buds but also in other epithelial areas of the papillae, suggesting the following two notions. One is that the mRNA is expressed in both sensory and nonsensory cells, since both are cognate in the tongue epithelium (11). In this case, the proteins expressed in sensory cells should be functional for taste signaling. The other notion concerns the involvement of the GUST27 protein and related receptors in a wide range of responses triggered by foods including the recognition of

Gustatory G Protein-coupled Receptor 12039

some physical stimuli usually understood to induce nonchem- ical senses of taste.

The molecular biology of taste transduction is just begin- ning to provide clues to understanding the cellular responses triggered by tastants and their transduction to taste neurons. The information provided here concerning the GUST27 pro- tein as a probable candidate for a taste receptor will contribute to developing this new approach from the aspect of molecular biology. Further evidence will lead to the elucidation of the entire molecular mechanism of taste transduction in mam- mals.

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