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THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1987 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 262, No. 29, Issue of October 15, pp. 14241-14249,1987 Printed in U.S.A.

Molecular Cloning of Five GTP-binding Protein cDNA Species from Rat Olfactory Neuroepithelium*

(Received for publication, May 1, 1987)

David T. Jones$ and Randall R. Reed From the Howard Hughes Medical Institute Laboratory of Genetics, Department of Molecular Biology and Genetics, Johns Hopkins Uniuersity School of Medicine, Baltimore, Mavland 21205

Biochemical studies in vertebrate olfactory tissue indicate that certain odorants stimulate adenylyl cyclase in a GTP-dependent manner. Additionally, immunochemical and toxin-labeling studies demonstrate the presence of several GTP-binding protein (G-protein) species in vertebrate olfactory epithelium. To identify the G-protein(s) responsible for olfactory signal transduction, we screened a rat olfactory cDNA library with an oligonucleotide probe and isolated 32 recombinant clones encoding five distinct types of G- protein a subunits. The majority of the clones encoded Gas, while the remaining clones encoded Ga,, Gail, GaiP, and a novel species, Gaia. Messenger RNA corresponding to each Ga was detectable in all tissues examined; however, the levels for a given Ga varied in a tissue- specific manner. In olfactory tissue, Gas was the most abundant of these messages and in combination with the biochemical studies suggests that Gas is the G- protein component of the olfactory signal transduction cascade.

The guanine nucleotide binding proteins (G-proteins) are a family of proteins that couple extracellularly activated membrane receptors to intracellular second messenger enzymes (1, 2). The G-proteins mediate signal transduction in visual, hormonal, and neurotransmitter systems and are cytoplasmic, membrane-associated heterotrimers composed of an a subunit (36-52 kDa), a @ subunit (35-36 kDa), and a y subunit (8-10 kDa). The Ga subunits bind guanine nucleotides and exist in two alternative activation states. They are inactive when bound to GDP and associated with the GPy subunits. Stim- ulated receptor activates a Ga subunit by catalyzing the exchange of GTP for GDP. The activated Ga subunit (Ga- GTP) dissociates from the G@r subunits and directly regu- lates second messenger proteins. Additionally, Ga subunits have an intrinsic GTPase activity and spontaneously return to the inactive state (GaGDP-Pr) in the absence of activated receptor. While some evidence suggests that G@r subunits may directly regulate effector proteins (3), experimental evidence from most systems suggests that these subunits play a passive role and that specificity for interaction with receptor and effector proteins resides with the Ga subunits (4-6).

In the best characterized transduction system, the visual system, photorhodopsin catalyzes the exchange of guanine nucleotides bound to the visual G-proteins, transducin I (Ta,,

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

$ Supported by a National Science Foundation Predoctoral Fellow- ship.

39 kDa) in rod cells and Tar, (39 kDa) (7) in cone cells, leading to activation of cGMP-phosphodiesterase (8). Similarly, in the @-adrenergic system, agonist-bound receptor catalyzes guanine nucleotide exchange on the stimulatory G-protein, G. ( a = 45 and 52 kDa), which then activates adenylyl cyclase (9). Another G-protein, Gi (a = 41 kDa), acts antagonistically to G. and inhibits adenylyl cyclase when activated (Ga,-GTP) (10-14). Other Ga subunits (39-42 kDa) have been implicated in the regulation of several other effector proteins, including phospholipase C (15-17) and cation channels (18-21).

Recently, molecular cloning techniques have allowed the isolation and characterization of cDNA clones encoding several Ga subunits as well as the GP and Gy subunits. cDNA clones encoding two different forms of Ga, (45 or 52 kDa) have been identified (22-26). In addition, Itoh et al. (25) have identified an amino-terminal truncated murine Ga, cDNA clone (a G-protein abundant in brain but with no known function). Furthermore, although previous biochemical evidence suggested a single Gai species, two different Gai-encoding cDNA clones have been identified in bovine (27, 28) and murine (24, 25) tissues, respectively. The identification of multiple Gai cDNA clones that encode proteins of similar predicted biochemical properties has made their assignment to particular proteins or functions tenuous. The resolution of this situation would be facilitated by the isolation and characterization of each of these Ga clones from a single species and tissue source.

In the vertebrate olfactory system, morphologic and elec- trophysiologic data suggest that the initial events of olfactory signal transduction occur in the cilia of the primary sensory neurons. Recently, Pace et al. (29) and Sklar et al. (30) have measured odorant-stimulated, GTP-dependent adenylyl cyclase activity in a crude membrane preparation of frog olfactory cilia. Their observations suggest that odorant signal transduction occurs via a second messenger system initiated by a coupled cascade of receptors, G-proteins, and adenylyl cyclase. Immunocytochemical analyses (31) and toxin labeling studies (32) have shown that Gas and other Ga species are expressed in the olfactory epithelium. These biochemical studies and the apparent analogy of olfaction to vision transduction led us to hypothesize that the olfactory system may have evolved a distinct G-protein (e.g. “olfactory transducin”). To identify and characterize G-proteins that may be involved in olfactory signal transduction, we have isolated cDNA clones encoding five Ga subunits from rat olfactory tissue. These cDNAs encode Ga., Ga,, and three distinct forms of Gai. We report the characterization and tissue distribution of these five cDNA clones.

MATERIALS AND METHODS

cDNA Library: Construction and Screening-The cDNA library was constructed by E. Barbosa (Johns Hopkins University) using the

14241

14242 Five G-Protein-encoding cDNA Clones method of Gubler and Hoffman (33). The first cDNA strand was synthesized by oligo(dt)-primed reverse transcription of poly(A)+- enriched rat olfactory RNA. The second strand was synthesized by treatment with RNase H and DNA polymerase I and the ends of the cDNA were made blunt by brief treatment with nuclease S1. Internal EcoRI sites were methylated, EcoRI linkers were ligated to the ends, and the cDNA was digested with EcoRI and cloned into the unique EcoRI site of X-gtl0. The library contained 2 X lo7 independent recombinant plaques. Oligonucleotide probes synthesized with an Applied Biosystems 380B automated synthesizer were 5'-end-labeled with [-y-32P]ATP (7000 Ci/mmol) and hybridized to replica filters. The filters were washed in tetramethylammonium chloride (Aldrich) at 50 "C as described by Wood and co-workers (34) and subjected to autoradiography at -70 "C with x-ray film and an intensifying screen.

Isolation of RNA and Northern Analys&"otal RNA for cDNA library construction and Northern analysis was isolated by guanidin- ium CsCl centrifugation (35) from the dissected tissue of 3-&month- old Sprague-Dawley rats. Glyoxal-treated total RNA was resolved on 1% agarose gels and transferred to Nytran filters (36). Filters were prehybridized at 42 "C for 6-12 h in hybridization solution (50% deionized formamide, 5 X SSC (1 X = 150 mM NaC1, 15 mM sodium citrate), 5 X Denhardt's solution (5 X = Ficoll, bovine serum albumin, and polyvinylpyrollidone, each at 1 mg/ml), 0.1% sodium dodecyl sulfate, and 100 pg/ml sonicated, denatured salmon sperm DNA). DNA fragments, recovered from agarose gels, were radiolabeled with (CI-~'P]~CTP (3000 Ci/mmol) by nick translation or mixed oligo labeling (37). Filters were hybridized at 42 "C in hybridization solution containing denatured probe at 1-3 X lo6 dpm/ml for 24-36 h. The filters were then rinsed 10 min at 65 "C in 2 X SSC, 0.1% sodium dodecyl sulfate and washed twice for 20 min at 65 "C in 0.2 X SSC, 0.1% sodium dodecyl sulfate or 0.5 X SSC, 0.1% sodium dodecyl sulfate. DNA size standards (1-kilobase ladder, Bethesda Research Laboratories) were similarly treated with glyoxal and detected on the filters with nick translated probe. Autoradiography was performed with x-ray film and an intensifying screen at -70 "C.

cDNA Sequencing-Sequencing was performed with [cI-~'S]~ATP (500 Ci/mmol) by the method of Sanger et at. (38). Specific oligonu- cleotides complementary to the ends of the bacterial transposon, 7 6 , were used to prime synthesis from random transposon insertions into the cDNA clones (39, 40). Alternatively, restriction fragments were subcloned into M13mp18 or M13mp19 and sequenced. Sequencing of GC-rich regions was facilitated by substitution of 7-deaza-dGTP (Boehringer Manneheim) for dGTP in the sequencing reactions (41).

Alignments-The predicted protein sequences were aligned using the DFASTP algorithm developed by Pearson and Lipman (43) and optimized by inspection. Nucleotide alignments are not shown but were performed with the NNCALN algorithm developed by Wilbur and Lipman (42).

RESULTS

Isolation of cDNA Clones-Five distinct Ga-encoding cDNA clones were isolated from a rat olfactory cDNA library with an oligonucleotide probe directed against a highly conserved Ga region. Previous amino acid sequence analysis of the known Ga subunits revealed that they all shared an identical 18-amino acid stretch (44, 45). After consideration of available nucleotide and amino acid sequence data (44, 46) and codon usage frequencies (47), we synthesized a 32-fold degenerate, 34-base oligonucleotide directed toward the latter half of the 18-amino acid stretch (5'-TCATC/TTGCTTCA- CAATGGTA/GCTC/TTTC/TCCA/GGATTC-3'). This oligonucleotide was used to screen a A-gtl0 rat olfactory cDNA library. Screening of 160,000 recombinant plaques yielded 32 hybridizing clones. Radiolabeled probes prepared from puri- fied EcoRI inserts of several of the clones were individually used to screen the entire collection at high stringency. This analysis revealed that the 32 clones comprised five independent classes: 24 encoded Ga,, 2 encoded Ga,, and two clones were identified for each of three distinct forms of Gai. The largest cDNA insert of each class (G2, GIG, GI89 G27, and G d was subcloned into the unique EcoRI site of the plasmid pGEM-2 for further analysis.

Identification and Analysis of cDNA Clones-The strategy

used to determine the primary structures of the cDNA inserts is diagrammed in Fig. 1.

Gz Encodes Ga.-Gz is a full-length cDNA clone that encodes the larger form of Ga, (52 kDa) (Fig. 2). Th' IS was determined by comparison of its nucleotide sequence with bovine (22, 23), mouse (24), human (26), and rat (25) cDNA sequence. The two rat Gas-encoding clones (Gz and Ref. 25) are identical within the coding region, nearly identical in the 3"nontranslated region (98%), and differ markedly in the 5'- nontranslated region (84%). These two rat clones were isolated from different tissues and the origin of these discrep- ancies may be biologically significant or merely the result of cloning or sequencing artifacts. Between species, the Ga, nucleotide sequences are highly conserved within the coding region (bovine versus rat, 94%; mouse versus rat, 98%; and human versus rat, 95%) with most codon differences occurring at the third position. The nontranslated regions have greater interspecies diversity (5' bovine versus rat, 67%; 3' bovine or mouse versus rat, 77-80%; and 3' human versus rat, 91%).

At the protein level, comparison of corresponding residues between the human and rat Gas proteins indicates that they are identical. Similar comparison between the mouse and rat proteins reveals that they differ at 2 residues. Likewise, the bovine and rat Gas sequences differ at only 2 (23) or 4 (22) residues. Not surprisingly, the Ga. protein coding regions have been under more stringent selective pressure than the nontranslated regions.

In order to further characterize the 24 Ga, clones we isolated, the recombinant phage were probed with a 30-mer oligonucleotide (5 '-GCTGTTGCTCCTTGCAGCCTGC- GGGTCCTC-3') based on the rat sequence corresponding to the 14-amino acid exon that distinguishes the 45- and 52-kDa forms of Ga. (48). This oligonucleotide hybridized to 14 out of the 24 Gas-encoding clones (data not shown) confirming the presence of both forms of Ga, in the library. However, this approach was not sufficiently sensitive to determine whether all four types of Gas, as described by Bray et al. (26), were present in this library. None of the putative Ga,-45 kDa encoding clones were sequenced.

G31 Encodes Gao"G31 encodes Ga, as determined by comparison to the truncated rat Ga, cDNA sequence reported by Itoh et al. (25), (Ga,-Kaziro). However, G31 encodes the ad- ditional 44 amino-terminal amino acid residues absent from the previously reported sequence. The nucleotide sequence and predicted protein sequence for G31 are shown in Fig. 3. The initiator methionine was identified by homology to the amino termini of Gz, GI, and GZ7. The three ATG sites upstream of the initiator ATG are each shortly followed by a translation termination codon. The comparable protein sequences for GSi and Ga,-Kaziro are identical while the nucleotide sequences differ at a single position.

Gla and G27 Encode Gaiz and Gail"G18 (Fig. 5 ) and G27 (Fig. 4) encode two distinct forms of Gai. GI8 was identified as a Gai species by comparison of its cDNA nucleotide sequence to mouse Gai (24) and rat Gai (25) sequences. Similarly, Gz7 was identified by resemblance of its cDNA sequence to homologous bovine cDNA sequences (27,281.

Comparison of the two rat nucleotide sequences (G18 and Ref. 25) shows that they share identical coding and 3'-nontranslated regions but diverge in the 5' nontranslated region (92%). As with Gas, the source of the 5' diversity between the Gai rat clones is not clear. Interspecies comparison of GI8 and the corresponding mouse cDNA sequence (24) indicates that the coding regions are highly conserved (96%) while the nontranslated regions are divergent (5', 76%; 3', 36%). At the

Five G-Protein-encoding cDNA Clones 14243

5 ' 3

FIG. 1. DNA sequencing strategy. The arrows indicate the regions sequenced. All coding regions and noncoding regions were sequenced on both strands, except for parts of the 3'-nontranslated region of GI+ The restriction sites are as follows: B, BarnHI; H , HindIII; N , NcoI; R, EcoRI; S, SphI; V , EcoRV; and X, XbaI. The dotted lines with double arrows indicate the regions used to generate clone-specific probes for Northern analysis. GI, and G3, were derived from similar messages but diverge in the 3' nontranslated regions, the thick black line on G,, represents DNA from a random fusion event. We believe GI6 represents the normal cDNA for this message. GI, was not sequenced in its entirety but all sequenced overlapping regions of GI, and G,, were identical; the sequence reported for GI, is a composite of G16 and G33 sequences.

protein level, the rat and mouse sequences differ at only 3 amino acid residues.

Interspecies nucleotide comparisons between G2, and the analogous bovine Gai sequences indicates that the coding regions are relatively conserved (88%) while the 5' and 3' regions are more divergent ( 5 ' , 58%; 3', approximately 67%). The bovine and rat (G2,) predicted protein sequences differ at only 1 (27) or 2 (28) residues.

Thus, as with Ga,, the Ga, protein coding regions for both GIs and GZ7 have evolved under more stringent selective pressure than the nontranslated regions. In accordance with the nomenclature introduced by Gilman (l), Gls and G2, have been designated Gai2 and Gail, respectively.

G I , A Novel Go, Species-The nucleotide sequence and predicted amino acid sequence for GI, suggest it represents a third distinct form of Gai (Fig. 6). We have assigned the initiation methionine of GI, by homology with the other G- protein amino termini. The predicted protein sequences for G16 and G2? (Gail) are highly homologous (94% identity), while the nucleotide sequences share only 56% homology overall and 71% homology within the coding region. Although the message sizes for GIG and GZ7 are similar, the observation that coding sequence differences reside primarily in codon third positions combined with the diversity in the nontranslated regions strongly suggests that the two cDNA clones are derived from separate genes. Therefore, we have classified GI, as a novel Gai species and shall refer to it as Gai3.

Protein Homologies-We have aligned the predicted protein sequences of each of the rat cDNA clones reported here and of bovine Tal (45,49,50) and Tar, (44) and shaded the regions of identity as indicated (Fig. 7). Gas is the most divergent,

retaining approximately 40% identity with any of the other six. The Gais and Ga, share greater homology. Ga, shares 73% identity with Gail (G2,), 68% with Gai2 (GIB), and 70% with Gai3 (GI,). Gai2 (GIs) shares 88% identity with Gail (GZ7) and 85% identity with Gai3 (GI,). Gail (G27) and GW,~ (GI6) are the most homologous with only 22 differences out of 354 residues (94% identity). It is notable that the 18-amino acid stretch implicated in guanine nucleotide binding (Ga, 43-60) is perfectly conserved in all of the G-proteins even though the first 7 of these amino acid residues were not constrained by the oligonucleotide probe used to screen the cDNA library. The most divergent region between all of the G-proteins occurs between amino acid residues 65 and 150.

Northern Analysis-The message sizes and tissue distributions of the five rat clones were determined by Northern analysis. Total rat RNA was treated with glyoxal, resolved on agarose gels, transferred to filters, and probed with radiolabeled full-length cDNA inserts or clone-specific restriction fragments under conditions of high stringency. The message size for each clone is constant in each of the seven tissues examined. The Ga, (G2) probe hybridizes to a 1.85-kilobase message likely to encode the two forms of Ga, protein as described by Robishaw et al. (48). Hybridization with a Gaiz (GIs) probe detects a message of 2.35 kilobases while Gai, (G2,) and Gq3 (GIG) probes hybridize to similarly sized messages of 3.5 kilobases. Probes were prepared from unique sequence regions of Gail (GZ7) and Gai3 (G16) (see Fig. 1) to establish that the similar message size was not an artifact resulting from the high homology shared in some regions of these two clones. A Ga, (G,,)-specific probe (see Fig. 1) hybridized to two messages, 4.1 and 4.5 kilobases; the origin

14244 Five G-Protein-encoding cDNA Clones

64 CCGCGCGCTTGCCTTAGTCCGAGCCGCCACCCTCCCCGCGTTCCCCGCTTTCCCGGCCCGCCCGAGGCCGCCCGCCCCGTCCCCGCCGCCCCGCAGCCCGGCCGCGCCCCGCC~ 182 1 GCAGCAGCAGCTCCCGCGGCTCCTGCTCTGCTCCGCCTCGGCCCCGGAGCGAGGGGCGGAGAG 63

183 -C TGC CTC GGC AAC AGT AAG ACC GAG GAC CAG CGC AAC GAG GAG AAG GCG CAG CGC GAG GCC AAC AAA AAG ATc GAG AAG CAG CTG 272 i MET Gly Cys Leu Gly Asn Ser Lys Thr Glu Asp Gln Arq Asn Glu Glu Lys Ala Gln Arq Glu Ala Asn Lys Lys Ile Glu Lys Gln Leu

273 CAG RAG GAC AAG CAG GTC TAC CGG GCC ACG CAC CGC CTG CTG CTG CTG GGT GCT GGA GAG TCT GGC AAA AGC ACC ATT GTG AAG CAG ATG 362 31 Gln Lys Asp Lys Gln Val TYK Arq Ala Thr H i s Arq Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys c!n MET

363 AGG ATC CTA CAT GTT AAT GGG TTT AAC GGA GAG GGC GGC GAA GAG GAC CCG CAG GCT GCA AGG AGC AAC AGC GAT GGT GAG A A G GCC ACC 452 61 Arg 110 Leu H i s Val Asn Gly Phe Asn Gly GlU Gly GlY GlU Glu ASP Pro Gln Ala Ala Arq Ser Asn Ser Asp Gly Glu Lys Ala ;hr

453 AAA GTG CAG GAC ATC AAA AAC AAC CTG AAG GAG GCC AT1 GAA ACC ATT GTG GCC GCC ATG AGC AAC CTG GTG CCC CCC GTG GAG CTG GCC 542 91 Lys Val Gln Asp Ile Lys Asn A m Leu Lys G1U Ala lle Glu Thr Ile Val Ala Ala MET Ser Asn Leu Val Pro Pro Val Gld Leu la

543 AAC CCT GAG AAC CAG TTC AGA GTG GAC TAC ATT CTG AGC GTG ATG AAC GTG CCA AAC TTT GAC TTC CCA CCT GAA TTC TAT GAG CAT Ccc 632 121 Asn Pro Glu Asn Gln Phe Arg Val Asp Tyr Ile Leu Ser Val MET Asn Val Pro Asn Phe Asp Phe Pro Pro Glu Phe Tyr Glu 91s Ala

151 Lys Ala Leu Trp Glu Asp Glu Gly Val Arq Ala cys Tyr Glu Arq Ser Asn Glu Tyr Gln Leu Ile Asp Cys Ala Gln Tyr Phe Leu ~ s p 633 AAG GCT CTG TGG GAG GAT GAG GGA GTT CGT GCC TGC TAC GAG CGC TCC AAC GAG TAC CAG CTG ATC GAC TGT GCC CAG TAC TTC CTG SAC 122

723 AAG ATT GAT GTG ATC AAG CAG GCC GAC TAC GTG CCA AGT GAC CAG GAC CTG CTT CGC TGC CGC GTC CTG ACC TCT GGA ATC TTT GAG ACC 812 181 Lys lle Asp Val lle Lys Gln Ala Asp Tyr Val Pro Ser Asp Gln Asp Leu Leu Arq Cys Arq Val Leu Thr Ser Gly Ile Phe Glu Thr

813 AAG TTC CAG GTG GAC AAA GTC AAC TTC CAC ATG TTC GAT GTG GGC GGC CAG CGC GAT GAA CGC CGC AAG TGG ATC CAG TGC TTC AAT GAT 902 211 Lys Phe Gln Val Asp LyS Val Asn Phe H i s MET Phe ASP Val Gly Gly Gln Arg Asp Glu Arg Arg LyS Trp Ile Gln C y 3 Phe Asn Asp

903 GTG ACT GCC ATC ATC TTC GTG GTG GCC AGC AGC AGC TAC AAC ATG GTC ATC CGG GAG GAC AAC CAG ACC AAC CGT CTG CAG GAG GCT CTG 392 241 Val Thr Ala Ile Ile Phe Val Val Ala Ser Ser Ser Tyr Asn MET Val lle Arg Glu Asp Asn Gln Thr ASn Arq Leu Gln Glu Ala Leu

993 PAC CTC TTC AAG AGC ATC TGG AAC AAC AGA TGG CTG CGT ACC ATC TCT GTG ATC CTC TTC CTC AAC AAG CAA GAT CTG CTT GCT GAG AAG 1082 271 Asn Leu Phe Lya Ser Ile Trp Asn Asn Arg Trp Leu Arq Thr 110 Ser Val Ile Leu Phe Leu ASn Lyi Gln Asp Leu Leu Ala Glu LYS

1083 GTC CTC GCT GGG AAA TCG AAG ATT GAG GAC TAC TTT CCA GAG TTC GCT CGC TAC ACC ACT CCT GAG GAT GCG ACT CCC GAG CCC GGA GAG 1172 301 Val Leu Ala Gly Lys Ser Lys Ile Glu Asp Tyr Phe PC0 Glu Phe Ala Arq Tyr Thr Thr Pro GlU ASP Ala Thr Pro Glu Pro Gly Glu

1173 GAC CCA CGC GTG ACC CGG GCC RAG TAC TTC ATC CGG GAT GAG TTT CTG AGA ATC AGC ACT GCT AGT GGA GAT GGA CGT CAC TAC TGC TAC 1262 331 Asp Pro Arq Val Thr Arq Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile ser Thr Ala Ser Gly Asp Gly Arg H i s Tyr Cy3 Tyr

1263 CCT CAC TTT ACC TGC GCC GTG GAC ACT GAG PAC ATC CGC CGT GTC TTC AAC GAC TGC CGT GAC ATC ATC CAG CGC ATG CAT CTT CGC CAA 1352 361 Pro H i s Phe Thr Cys Ala Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cyr Arg Asp Ile Ile Gln Arq MET His Leu Arq Gln

1353 TAC GAG CTG CTC TAA GMGGGAACGCCCAAATTTMTTCAGCCTTMGCACMTTAATTAAGAGTG~CGCAATCGTACAAGCAGTTGATCACCCACCATAGGGCATGATCAA 1466 391 Tyr Glu Leu Leu END

1467 CACCGCAACCTTTCCCTTTTCTCCCCAGTGATTCTGARAACCCCCTCTTCCCTTCAGCTTGCTTAGATGTTCCAAATTTAGTAAGCTTAAGGCGGCCTACAGAAGAAAAAGAAAAAGAA 1585 1586 AAAGGCCRCIIRAAGTTCCCTCTCACTTTCAGTAAAT 1705 C A T T A A A A A A T C A T T M T G A G Po y A

t - - ~ A G C A A C A A A C A G A g g T g g g G ~ T G A ~ T G h A A C T C A A A A T G ~ T A T T G T G T T G T G C A G 1704

FIG. 2. Rat Ga. cDNA sequence. The nucleotide and predicted amino acid sequence of rat Ga. (G2) is shown. The eukaryotic translation initiation consensus sequence (57) is boxed, and possible polyadenylation signals are underlined. The predicted protein contains 394 amino acid residues.

231 112

350 469 588

707 1

797 31

887 61

977 91

1067 121

1157 151

1247 181

1337 211

1427 241

1517 271

1607 301

1697 331

1791

2029 1910

CTGCCCAGCCCTGCCCTGCGCGCGGGGGTCGGAGAAGGCGCCGGGACGCACCGACGGCCGAGGAGCGGCGATGCACATGCACTAGCGGCACCCCCTAACTCACTCCCTCCACACCCCCG 1 CTCTCGCGCTCTCCCTGTCTCCTGTCCGCTCCGCCGAGCGATGCGAGTTCTTGGCCCCGGCGACGCCGCCTCCAGCTAGAGATCTGCACCCCTCACCCCCGGCCCGGCCCT

CCGCCGCCGCCGCCACCGCCTCCGCCTCCGCCTCCTCCTCCGCCTCCGGCAGCCGCGGCAGAAGGACCCACCCTGCCCCCCACCCCACCCTCCGCCGGCTCCGGCTGCGGATCCAGCCT CGACTCCTATTTTATTTATTTTGGGTCGTGCACTAGTCTCGGTGCCTGCAACCCGCGCCTCCCGGGCCCGCGGGCGCCTCCTCTCTCGGCTCCGGAGCCCCAGACCCCGGCCACCCTCA CCTCGACACCCCCAGACCCCAGCCAGCCGCCGCTAATCTTCGCCGCTGGAATCTTGATAGAGGCTGTCCTTTTGGGGGGATTCTGGTCTTTCGACAATTTTGTTCCCAACCAAGGAAAG GATATCGTGATTTTCTCCCCTTTGAGCCCAGGCTCTGCTCTGTGGGGGGGTGGGGGGCGCGCCGACCCGAGGAGTCGTGCCAGCCGAGTCGTGCGGGCTGTGGCAGGGAAGGG~

-A TGT ACT CTG AGC GCA GAG GAG AGA GCC GCC CTC GAG CGG AGC AAG GCG ATT GAG AAA AAT CTC A M GAA GAT GGC ATC AGC GCC MET Gly Cys Thr Leu Ser Ala Glu Glu Arg Ala Ala Leu Glu Arq Ser Lys Ala Ile Glu Lys Asn Leu Lys Glu Asp Gly Ile Ser Ala

GCC AAA GAC GTG AAA TTA CTC CTG CTG GGG GCT GGA GAA TCA GGA AAA AGC ACC ATT GTG M G CAG ATG AAG ATC ATC CAT GAA GAT GGC Ala Lys Asp Val Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln MET Lys Ile Ile H i s Glu Asp Gly

TTC TCT GGA GAA GAC GTA AAG CAG TAC AAG CCT GTC GTC TAC AGC AAC ACC ATC CAG TCT CTG GCA GCC ATT GTG CGG GCC ATG GAT ACT Phe Ser Gly Glu Asp Val Lys Gln Tyr Lys Pro Val Val Tyr Ser Asn Thr Ile Gln Ser Leu Ala Ala Ile Val Arg Ala MET Asp Thr

CTG GGC GTG GAG TAT GGT GAC AAG GAG AGG AAG GCA GAC TCC AAG ATG GTG TGT GAC GTG GTG AGT CGC ATG GAG GAC ACT GAA CCA TTC Leu Gly Val Glu Tyr Gly Asp Lys Glu Arq Lys Ala Asp Ser Lys MET Val Cys Asp Val Val Ser Arg MET Glu Asp Thr Glu Pro Phe

TCT GCA GAA CTG CTT TCT GCC ATG ATG CGA CTC TGG GGC GAC TCG GGG ATC CAG GAG TGC TTC AAC CGA TCT CGG GAG TAT CAG CTC AAC set Ala Glu Leu Leu ser Ala MET MET Arq Leu Trp Gly ASP ser Gly Ile Gln Glu cys Phe Asn Arg ser Arg Glu Tyr Gln Leu Asn

GAC TCT GCC AAA TAC TAC CTG GAC AGC TTG GAT CGG ATT GGA GCC GCT GAC TAC CAG CCC ACC GAG CAG GAC ATC CTC CGA ACC AGG GTC Asp Ser Ala Lys Tyf Tyr Leu Asp Ser Leu Asp Arq Ile Gly Ala Ala Asp Tyr Gln Pro Thr Glu Gln Asp Ile Leu Arg Thr Arg Val

AAA ACA ACT GGC ATC GTA GAA ACC CAC TTC ACC TTC AAG AAC CTC CAC TTC AGG CTG TTT GAC GTT GGG GGC CAG CGA TCT GAA CGT AAG Lys Thr Thr Gly Ile Val Glu Thr H i s Phe Thr Phe Lys Asn Leu H i s Phe Arq Leu Phe Asp Val Gly Gly Gln Arg Ser Glu Arg Lys

AAG TGG ATC CAC TGC TTC GAG GAT GTC ACG GCC ATC ATC TTC TGT GTC GCA CTC AGC GGC TAT GAC CAG GTG CTC CAC GAG GAC GAA ACC Lys Trp Ile H i s Cys Phe Glu Asp Val Thr Ala Ile Ile Phe Cys Val Ala Leu Ser Gly Tyr Asp Gln Val Leu H i s Glu ASP G1U Thr

ACG AAC CGC ATG CAC GAG TCT CTC ATG CTC TTC GAC TCC ATC TGT AAC AAC AAG TTT TTC ATC GAT ACC TCC ATC ATT CTC TTC CTC AAC Thr Asn Arg MET H i s Glu Ser Leu MET Leu Phe Asp Ser Ile Cys Asn Asn Lys Phe Phe Ile Asp Thr Ser Ile Ile Leu Phe Leu Asn

AAG AAA GAC CTC TTT GGC GAG AAG ATT AAG AAG TCA CCC TTG ACC ATC TGC TTT CCT GAA TAC CCA GGC TCC AAC ACC TAT GAA GAC GCA LYS Lys Asp Leu Phe Gly Glu Lys Ile Lys Lys Ser Pro Leu Thr Ile CYS Phe Pro G1U Tyr Pro Gly Ser Asn Thr Tyr GlU ASP Ala

GCT GCC TAC ATC CAR ACA CAG TTT GAA AGC AAA AAC CGC TCA CCC AAC AAA GAA ATT TAC TGT CAC ATG ACT TGT GCC ACA GAC ACG AAT Ala Ala Tyr Ile Gln Thr Gln Phe G1u Ser Lys Asn Arg Ser Pro Asn Lys Glu Ile Tyr Cys H i s MET Thr Cys Ala Thr Asp Thr Asn

ART ATC CAG GTG GTA TTC GAC GCC GTC ACC GAC ATC ATC ATT GCC AAC AAT CTC CGG GGC TGT GGC TTG TAC TGA CCTCTTGTCCTGTATAGCA Asn Ile Gln Val Val Phe Asp Ala Val Thr Asp lle Ile Ile Ala Asn Asn Leu Arq Gly Cy3 Gly Leu Tyr END

ACCTATTTGACTGCTTCATGGACTCTTTGCTGTTGATGTTGATCTCCTGGTAGCATGACCTTTGGCCTTTGTAAGACACACAGCCTTTCTGTACCAAGCCCCTGTCTAACCTACGACCC CAGAGTGACTGACGGCTGTGTATTTCTGTAGAATGCTGTAGAATACGGTTTTAGTTGAGTCTTTACATTTAGAACTTG~GGATTTAAA~CATTTCTCATGTGCTTTGT AGCTTTAAAAAGGAAAACTCACCATTTCATCCATATTTCC 2068

230 111

349 468 587 706

796

886

97 6

1066

1156

1246

1336

1426

1516

1606

1696

1130

1903 2028

FIG. 3. Rat Ga, cDNA sequence. The nucleotide and predicted amino acid sequence of rat Ga, ( G d is shown. The eukaryotic translation initiation consensus sequence is boxed. The 3’ end is not polyadenylated and is presumably truncated. The predicted protein contains 354 residues.


loo TAGCAGACCTCGCCTCCAGCTTGCCGGAGCTGAGGACCTAGCGGGGCCGCCTCCGGGCCAGCCTCTCCCCGCTCTGCCAGCCGGCCCCGGCGCCGAGGGCGCGGCGACGCTCG~ 218 1 GCGGCGGCGGTACCGAGCCTAGCGGTCGCCGGGACTCGCGGGCGCGGATCGTAGAGCGTCGCGGGCGGGGACGGCCGCCCGAGGCGGCGGCGTATCGGT 99

219 -C TGC ACA CTG AGC GCT GAG GAC AAG GCG GCC GTG GAG CGC AGC AAG ATG ATC GAC CGC AAC CTC CGG GAG GAC GGA GAG AAG GCA 308 1 MET Gly Cys Thr Leu Ser Ala Glu Asp Lys Ala Ala Val Glu Arg Ser Lys MET Ile Asp Arg Asn Leu Arg Glu Asp Gly Glu Lys Ala

309 GCG CGC GAG GTC AAG CTG CTG CTG CTG GGT GCT GGT GAA TCC GGG AAG AGC ACA ATT GTG AAG CAG ATG A M ATT ATC CAC GAG GCT GGC 398 31 Ala Arg Glu Val Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln MET Lys Ile Ile H i s Glu Ala Gly

399 TAC TCA GAG GAA GAG TGT AAG CAG TAC AAA GCA GTG GTC TAC AGC AAC ACC ATC CAG TCC ATC ATT GCC ATC ATT AGA GCC ATG GGG AGA 488 61 Tyr ser Glu Glu Glu Cys Lys Gln Tyr Lys Ala Val Val Tyr ser Asn Thr Ile Gln Ser Ile Ile Ala Ile Ile Arg Ala MET Gly Arg

489 TTG AAA ATC GAC TTT GGA GAC GCT GCT CGT GCG GAT GAT GCT CGC CAA CTC TTC GTG CTT GCT GGG GCT GCA GAG GAA GGC TTT ATG ACC 578 91 Leu Lys Ile Asp Phe Gly Asp Ala Ala Arg Ala Asp Asp Ala Arg Gln Leu Phe Val Leu Ala Gly Ala Ala Glu Glu Gly Phe MET Thr

579 GCG GAG CTC GCC GGC GTC ATA AAG AGA CTG TGG AAG GAC AGC GGT GTG CAA GCC TGC TTC AAC AGA TCC CGG GAG TAC CAG CTG AAC GAT 668 121 Ala Glu Leu Ala Gly Val Ile Lys Arg Leu Trp Lys Asp Ser Gly Val Gln Ala Cy3 Phe Asn Arg Ser Arg Glu Tyr Gln Leu Asn Asp

669 TCG GCG GCG TAC TAC CTG AAT GAC TTG GAC AGA ATA GCA CAA CCA AAT TAC ATC CCA ACC CAG CAG GAT GTT CTC AGA ACT AGA GTG AAA 758 151 Ser Ala Ala Tyr Tyr Leu Asn Asp Leu Asp Arg Ile Ala Gln Pro Asn Tyr Ile Pro Thr Gln Gln Asp Val Leu Arg Thr Arg Val Lys

759 ACG ACG GGA ATT GTG GAA ACC CAC TTT ACT TTC AAA GAT CTT CAT TTT AAA ATG TTT GAC GTG GGA GGC CAG AGA TCA GAG CGG AAG AAG 848 181 Thr Thr Gly Ile Val Glu Thr H i s Phe Thr Phe Lys Asp Leu H i s Phe Lys MET Phe Asp Val Gly Gly Gln Arg Ser Glu Arg Lys Lys

849 TGG ATT CAC TGC TTT GAA GGC GTG ACT GCC ATC ATC TTC TGT GTG GCC CTG AGT GAC TAT GAC CTG GTT CTT GCT GAG GAT GAA GAA ATG 938 211 Trp Ile H i s Cys Phe Glu Gly Val Thr Ala Ile Ile Phe Cys Val Ala Leu Ser Asp Tyr Asp Leu Val Leu Ala Glu Asp Glu Glu MET

939 AAC CGG ATG CAT GAA AGC ATG AAG CTG TTC GAT AGC ATA TGT AAC AAC AAG TGG TTT ACG GAC ACA TCC ATC ATC CTT TTC CTG AAC AAG 1028 241 Asn Arg MET H i s Glu Ser MET Lys Leu Phe Asp Ser Ile Cy3 Asn Asn Lys Trp Phe Thr Asp Thr Ser Ile Ile Leu Phe Leu Asn Lys

1029 AAG GAC CTC TTC GAA GAG AAG ATC AAA AAG AGT CCC CTC ACG ATA TGC TAT CCA GAA TAT GCA GGC TCA AAC ACA TAT GAA GAG GCG GCT 1118 271 Lys Asp Leu Phe Glu Glu Lys Ile Lys Lys ser Pro Leu Thr Ile Cys Tyr Pro Glu Tyr Ala Gly Ser Asn Thr Tyr Glu Glu Ala Ala

1119 GCG TAT ATC CAG TGT CAG TTT GAA GAC CTC AAT AAA AGG AAG GAC ACA AAG GAA ATT TAC ACC CAC TTC ACT TGC GCC ACG GAT ACG AAG 1208 301 Ala Tyr Ile Gln Cys Gln Phe Glu Asp Leu Asn Lys Arg Lys Asp Thr Lys Glu Ile Tyr Thr H i s Phe Thr Cys Ala Thr Asp Thr Lys

1209 AAT GTG CAG TTT GTG TTC GAT GCT GTA ACG GAC GTC ATC ATA AAG AAT AAC CTA AAA GAC TGT GGT CTC TTC TAA GCTCTGCAGTGGGGTAGTA 1302 331 Asn Val Gln Phe Val Phe Asp Ala Val Thr Asp Val Ile Ile Lys Asn Asn Leu Lys Asp cys Gly Leu phe END

1303 AAAATGCATTTTCAAACCAGACGAGTACTTATATATGGATCTCTCTAGATTAGAGTCTCCCAGCAACAGAGAATGTAGTGTACAGCAAATGCATCCTGGACCTGACCAGAGTTCTGACC 1421 1422 AAAGTTGTTCTGTATCGTGTCTTTAATAGAAGGACCTTTCGAGAAGGCGAAAGATGGTCCTGGAGTGTGAGAGGGAGGGTTGATGTTGAAGCTGGGGCTCTAGTGTACTGATGATTTCT 1540 1541 GTGTAAGTGTAAATATGCAAATGTAAGATGTATTTATMCTTCTGATTTCCCACGTTATGTTTATGTTGTMGAGTGGCAACTTATGGTCCTAGTGCGATGGCTTTGGG~CATAAC 1659 1660 TGTTAAGTACCTTGTACGGAATGACAGATTGGTTCTGTGTTTGGCAGTTTTGATCAGCTTTATTTATGTTCGTACCCCTAAATTTTAGGCACCATAATTAACATTAGGAAATGTCAACC 1778 1779 CCCTTACCCTGATTCTATGTTTACTTATACTCAT~TGTTATTTGTATAAACGTTGCACAGACTATTTTAGTACCATGATTTGTATACAGGCTTTTGATTCATAGGG~CTAGTTTG 1897 1898 AGGTTCATTACTTTATGTCATGACTTCTTTTGCATTAGGTATTC 1945

FIG. 4. Rat Gail cDNA sequence. The nucleotide and predicted amino sequence of Gail (GZ7) is shown. The eukaryotic translation initiation consensus sequence is boxed. The 3’ end is not polyadenylated and is probably truncated. The predicted protein contains 354 amino acid residues.

32 CGGAACTGCGGACCTGAGAGCTTCCCGCAGAGGGCCGGCGGTGGGAGCGGAGTGGGTCGGGCGGGGCCGAGCCGGGCCGTGGGCCGTGTGGGGGCCAGGCCGGGCCGGCGGACGGCBI;G 150 1 GGCGGGCGGGAAGGCGCCTCCCGCAGTCGCT 31

151 m C TGC ACC GTG AGC GCC GAG GAC AAG GCG GCA GCC GAG CGC TCT AAG ATG ATC GAC AAG AAC CTG CGG GAG GAC GGC GAG AAG GCG 240 1 MET Gly Cys Thr Val Ser Ala Glu Asp Lys Ala Ala Ala Glu Arg Ser LyS MET Ile Asp LyS Asn Leu Arg Glu Asp Gly Glu LyS Ala

241 GCA CGG GAG GTG AAG TTG CTT CTG TTA GGT GCT GGA GAA TCA GGG AAG AGC ACC ATC GTC AAG CAG ATG AAG ATC ATC CAC GAG GAT GGC 330 31 Ala Arg Glu Val Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln MET Lys Ile Ile His Glu Asp Gly

331 TAC TCA GAG GAG GAG TGC CGG CAG TAC CGT GCG GTT GTC TAC AGC AAC ACC ATC CAG TCT ATC ATG GCC ATC GTC AAA GCC ATG GGC AAC 420 61 Tyr Ser Glu Glu Glu Cys Arg Gln Tyr Arg Ala Val Val Tyr Ser Asn Thr Ile Gln Ser Ile MET Ala Ile Val Lys Ala MET Gly Asn

421 CTG CAG ATC GAC TTT GCT GAC CCC CAG CGT GCG GAT GAT GCC AGG CAG CTG TTC GCA CTG TCC TGT GCT GCC GAG GAG CAA GGC ATG CTT 510 91 Leu Gln Ile Asp Phe Ala Asp Pro Gln Arq Ala Asp Asp Ala Arg Gln Leu Phe Ala Leu Ser Cys Ala Ala Glu Glu Gln Gly MET Leu

511 CCG GAA GAC CTG TCG GGC GTC ATC CGG AGG CTC TGG GCT GAC CAT GGT GTG CAA GCC TGC TTT GGC CGC TCA CGG GAA TAT CAA CTC AAT 600 121 Pro Glu Asp Leu ser Gly Val Ile Arg Arg Leu Trp Ala Asp His Gly Val Gln Ala Cys Phe Gly Arg Ser Arg Glu Tyr Gln Leu Asn

601 GAC TCA GCC GCT TAC TAC CTG AAT GAC CTG GAG CGC ATA GCA CAG AGT GAC TAT ATC CCT ACA CAG CAG GAT GTG CTG CGG ACC CGT GTG 690 151 Asp Ser Ala Ala Tyr Tyr Leu Asn Asp Leu Glu Arg Ile Ala Gln Ser Asp Tyr Ile Pro Thr Gln Gln Asp Val Leu Arg Thr Arg Val

691 AAG ACC ACA GGC ATC GTC GAA ACA CAC TTC ACC TTC AAG GAC TTA CAC TTC AAG ATG TTT GAT GTG GGT GGT CAG CGA TCT GAG CGG AAG 180 181 Lys Thr Thr Gly Ile Val Glu Thr His Phe Thr Phe Lys Asp Leu His Phe Lys MET Phe Asp Val Gly Gly Gln Arg Ser Glu Arg Lys

781 AAG TGG ATC CAC TGC TTT GAG GGT GTC ACG GCC ATC ATC TTC TGT GTC GCC TTG AGC GCG TAC GAC TTG GTG CTG GCT GAG GAT GAG GAG 870 211 ~ y s ~ r p Ile His cys Phe Glu Gly Val Thr Ala Ile Ile Phe cys Val Ala Leu Ser Ala Tyr Asp Leu Val Leu Ala Glu Asp Glu Glu

871 ATG AAT CGC ATG CAT GAG AGC ATG AAG CTG TTT GAT AGC ATC TGC AAT AAT AAG TGG TTC ACA GAC ACC TCC ATC ATC CTC TTC CTC AAC 960 241 MET Asn Arg MET H i s Glu Ser MET Lys Leu Phe Asp ser Ile Cys Asn Asn Lys Trp Phe Thr Asp Thr Ser Ile Ile Leu Phe Leu Asn

271 Lys Lys Asp Leu Phe Glu Glu Lys Ile Thr Gln Ser Pro Leu Thr Ile Cys Phe Pro Glu Tyr Thr Gly Ala Asn Lys Tyr Asp Glu Ala 961 AAG AAG GAC CTG TTT GAA GAG AAG ATC ACA CAG AGC CCC CTG ACC ATC TGT TTC CCT GAG TAC ACA GGG GCC AAC AAG TAT GAC GAG GCA 1050

1051 GCC AGC TAC ATC CAG AGC RAG TTT GAG GAC CTG AAT AAA CGC AAA GAC ACC AAG GAG ATC TAC ACG CAC TTC ACA TGC GCC ACC GAC ACC 1140 301 Ala Ser Tyr Ile Gln ser Lys Phe Glu Asp Leu Asn Lys Arg Lys Asp Thr Lys Glu Ile Tyr Thr His Phe Thr Cys Ala Thr Asp Thr

1141 AAG AAC GTG CAG TTT GTG TTT GAT GCC GTC ACT GAC GTC ATC ATC AAG AAC AAC CTG AAG GAC TGT GGC CTC TTC TGA GGGGCAGTGGGCCTG 1233 331 Lys Asn Val Gln Phe Val Phe Asp Ala Val Thr Asp Val Ile Ile Lys Asn Asn Leu Lys Asp Cys Gly Leu Phe END

1234 GCAGGATGGGCCACCGCTGACTCTGCTCCCCACTACCTGTGAGGAAGATGGGGGCAAGAAGACCATGCTCCCTGCCTGTTCCCCCAGCTGCTTCTCCCCTGTCTCTTCTCTCTGTTCTC 1352 1353 AGCTCCCCTGTCCCCTCCCTCGGCTCTAGACTTGGGGGAGGGGTTGCCACAGGCCTCCCTGTCTAAAACCCACCTTTGTCTGAGGTGCCGGGAGTGGCCATGGTACCTCCTTACTGGGC 1471 1472 ATCCATTCGGGTTTTCTAACCATTGTCTTGTTCTGGGGGTGAGCGGGGAGCGCATGCAGAGTTTCCCAAGGCCTATGTCTGGAGGAGTACCAATTCCTCCAGCCTAGACCCCTGGCTTT 1590 1591 GTCCAACACCAGCCCTGACCCAAGTCCAAATGTTTACAGGGAGCCTCCTGCCTACCCCACTCTCTGCCGCTTGGAGGCC~CAAAGG-AGCACAAGAAGCGTGAGAGACACCACCAT 1709 1710 TCCTGGAGACAAAGCCCACCTGCTCATTCTCGTAGCTTT--poly A

FIG. 5. Rat Gqz cDNA sequence. The nucleotide and predicted amino acid sequence of Ga, (GIs) is shown. Residues identical with the consensus eukaryotic translation initiation site are boxed. The 3’ end is polyadenylated although there are no apparent upstream polyadenylation sites. The predicted protein contains 355 residues.

and significance of the two Ga, messages is not known. Gas is expressed at highest levels in heart and kidney tissue The same probes were used to assess the relative abundance while Ga, is abundantly expressed in brain, kidney, and

of each of the five G-protein messages in several different intestinal tissue. The level of expression of the three types of tissues (Fig. 8). Messenger RNA corresponding to each clone Gai varies dramatically. Gail (G27) is highly expressed in brain is expressed at a detectable level in all of the tissues examined. and kidney tissue but barely detectable in olfactory, liver, and

14246 Five G-Protein-encoding cDNA Clones

1 CAGMGACGCGTCTCAGCCGGTGTGTCGCGGTTCCCCGCGGTGTGTGAGTGAGCCAGGGCCCGGTCCCCTCTCCGGCC~ 84

85 D C TGC ACG TTG AGC GCC GAG GAC M G GCG GCG GTG GAG CGG AGT M G ATG ATC GAC CGC M C TTG CGG GAG GAC GGA GAG AAA GCG 174 1 MET Gly Cys Thr Leu Ser Ala Glu ASP LYS Ala Ala Val Glu Arq Ser LYS MET Ile Asp Arq Asn Leu Arg G1U Asp Gly Glu Lys Ala

175 GCC A M G M GTG M G CTG CTG CTG CTC GGC GCT GGA G M TCT GOT AAA AGT ACT ATT GTG AAA CAG ATG AAA ATC ATT CAT GAG GAT GGC 264 31 Ala Lys Glu Val Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln MET Lys Ile Ile H i s Glu Asp Gly

265 TAT TCC GAG GAC GAG TGT M G CAG TAT AAA GTT GTC GTC TAC AGC M T ACC ATT CAG TCC ATC ATT GCA ATC ATA AGA GCC ATG GGA CGG 354 61 Tyr ser Glu Asp Glu cys LYS Gln Tyr Lys Val Val Val Tyr ser Asn Thr Ile Gln ser Ile Ile Ala Ile Ile Arg Ala MET Gly Arq

355 TTG AAG ATT GAT TTT GGG G M GCT GCC AGA GCG GAT GAT GCC CGA CAG TTA TTT GTT TTA GCT GGC AGT GCT GAG G M GGA GTC ATG ACT 444 91 Leu LYS Ile Asp Phe Gly GlU Ala Ala Arg Ala Asp ASP Ala Arq Gln Leu Phe Val Leu Ala Gly SeK Ala Glu Glu Gly Val MET Thr

445 TCA G M CTA GCA GGC GTG ATT AAA CGT TTA TGG CGA GAT GGC GGG GTG CAG GCA TGC TTC AGC AGG TCC AGG G M TAT CAG CTC M T GAT 534 121 ser Glu Leu Ala Gly Val Ile Lys Arg Leu Trp Arq Asp Gly Gly Val Gln Ala Cys Phe Ser Arq Ser Arg Glu Tyr Gln Leu Asn ASP

151 ser Ala Ser Tyr Tyr Leu Asn Asp Leu Asp Arq Ile ser Gln Thr Asn Tyr Ile Pro Thr Gln Gln Asp Val Leu Arq Thr Arg Val Lys

181 Thr Thr Gly Ile Val Glu Thr His Phe Thr Phe Lys Glu Leu Tyr Phe Lys MET Phe Asp Val Gly Gly Gln Arq ser Glu Arq Lys Lys

211 Trp Ile His cys Phe Glu Gly Val Thr Ala Ile Ile Phe cys Val Ala Leu ser Asp Tyr Asp Leu Val Leu Ala Glu Asp Glu Glu MET

535 TCT GCT TCA TAT TAC CTA M T GAT TTG GAT AGA ATA TCC CAG ACC AAC TAC ATT CCA ACT CAG C M GAT GTT CTT CGG ACG AGA GTG M G 624

625 ACT ACA GGC ATT GTG GAG ACC CAC TTC ACC TTC M G GAG CTC TAC TTC AAA ATG TTT GAT GTA GGT GGC C M AGA TCC G M CGG AAA M G 714

715 TGG ATC CAC TGT TTT GAG GGA GTG ACA GCA ATT ATC TTT TGT GTG GCT CTC AGT GAT TAC GAC CTT GTT CTG GCT GAG GAC GAG G M ATG 804

805 M C CGA ATG CAT G M AGC ATG AAA TTG TTT GAC AGC ATT TGT M C M C AAA TGG TTT ACA GAC ACT TCA ATC ATT CTC TTC CTT M T M G 894 241 ASn Arq PlET His Glu Ser MET Lys Leu Phe Asp Ser Ile Cys A m Asn Lys Trp Phe Thr Asp Thr Ser I18 Ile Leu Phe Leu Asn Lys

895 AAA GAC CTT TTT GAG G M AAA ATA M G AGG AGT CCA TTA ACA ATC TGT TAT CCA G M TAC ACA GGT TCC M T ACG TAC G M GAG GCA GCT 984 271 Lys Asp Leu Phe Glu Glu Lys Ile Lys Arg Ser Pro Leu Thr Ile CyS Tyr Pro Glu Tyr Thr Gly Ser Asn Thr Tyr Glu Glu Ala Ala

985 GCT TAC ATC CAG TGC CAG TTT G M GAT CTG M C CGA AGA AAG GAC ACC M G GAG GTC TAC ACT CAC TTT ACC TGT GCC ACT GAC ACC AAA 1074 301 Ala Tyr Ile Gln Cys Gln Phe Glu Asp Leu Asn Arq Arq Lys Asp Thr Lys Glu Val Tyr Thr H i s Phe Thr Cys Ala Thr Asp Thr Lys

1075 AAC GTG CAG TTT GTT TTT GAT GCT GTT ACA GAT GTC ATC ATT AM M C M C TTA M G G M TGT GGG CTT TAC TGA GAGGATGGCATAGTAAAAG 1168 331 ASn Val Gln Phe Val Phe Asp Ala Val Thr ASP Val Ile Ile Lys Asn Asn Leu Lys Glu cys Gly Leu Tyr E m

1169 CTACTACAGGGAGGMTGTTGAGACCAGACATCATCTACTGTCTCTTGGGTCAGCGACMGCAT~CAGGACCMGGMTGGCMCA~ATGCAGMTCTTTAGCACMTCTTCTGTAT 1287 1288 TAGGAMTGTTTMTTGGCATGAGMGAGATGATCGAGTCAGACATGAAATTGGAC~TGTAAAGTATGACCGGATCATCAGG~ACCGCTTGGACTTT~TCTCMTGTTTAGGGC 1406 1407 ATATTGMGTCGAGGTGCTGCATTCCAGMCTTAAACTTGTAGCTTACTGTTCCCCGTCTTTTTMCAAATGACCAGTAGTTMTTTCTMGGTTTTTCTCAT~GAGMCMTACCT 1525 1526 AAAAACTCTTACTTGTTTGCAAAAGMTCTTCTTCTGffiG~TCAGTCTTMCTAT~ACACMTGTA~CGG~ATCTTGAAATATTCCTCTTAGTAffiMCTGTTTGTTTTTMCTCTTG 1644 1645 GMTGGTCAGGAGTMTATTCCACAGCT~TCTTCCTGGTTATTGGGACTATACTMTAGAGCTTTTT~MTGMGTTTATGTTACTGTCCTGTTT~GTATCACMTGGCTTC 1763 1764 TAAACAGCATTCACTTCGGAGAGCTGTACAGTMGATTCTA~TCTCGTTTCTTMGCTTACGGTTGGAG~MCCTTGTTTGT~TGATTACCAAATTACTAGATGMTACTGATGTA 1882

2002 TTTGCCTGCTCGTCTCAGGCGTGTffiGTTGTAT~GGT~CCCCTTGTTTTCCATCTTTGT~CTTTTT~TAGGAGACGTCTMGAGTATMCMCTGTGCATGCCTTTCCTTTGT 2120 1883 ATATGATAAACTGCTCTTGGGTACAGACACACGCAGGATAGCTGTCAGTCAGAGACGCA~CTGCTMffiTGAGMGCAGTGACTT~TGCTACAGATCTACCGA~TGGCCACACTGA 2001

2121 AAATGTGCTGCCAAATCCCTGCTTGCTGTCATTGTCACTGTATCGATGGTMCTGCAT~CffiCCCTTGCTGTGTTCTTTTTCTMGACTTGCATTCTffiGACTCTGTTCCACCTTGTC 2239 2240 ACGTGTACAGACTTTMTCTGTTACAGCTGGCAGCAGTATTMC~GGTGAGTAAA~GCCCAG~TTGCTCCTGTTTGTMCCGATCCTTTCTGGM~CCTGCCTTCTGTTTCTTTT 2358 2359 TGCCCCTGCTGAGTGTTCTTTAGCCTTTGAGAGTGGGTTTAffiTAG~GTGTTTTT~T~~TTCCTTGATGTMTAGTGATATCAAATCTAGffiCT~GCTTTATGTCCA 2477 2478 GTCTGTGCMGTCATACMGTGTCCGATGTGTACAGTTTGCTT~GCT~~GCTTC~GTCCTC~GTACAGACTGTATCAAATGTCATTAAACACTGAGTGGACCACTGA~GACGGC 2596 2597 GAAACAGAGGATGCATTTTGAGCATTTCAGATCAGTAGTTTGATCAGTGCCTTTTGAGGTCCAGTCTTTGTffiATTGGATCCGTTCATCTTTTGATGTTACCTCCTACTCATTTGAAAG 2715 2716 MGGTTGGTGGTCAGMCTGCTTTGGGM~MGCCACGGT~TCAGTGCCTTCATT~~TTTTACCATTTTTMTTCTTACGTGTCTTTGCGAAATCATTATTAT~AGACAGTA 2034 2835 TMCTGTTATCTTACAAATATTTCCATAG~TMGAAAGTATCACTTGAAAG-GTATT~~TCMTCAGCGATTGACTCMTTCCCACAGTGTAGGGGTTMTMGTCTTACTC 2953 2954 TCAATTTATTGTTCCTTTGTTGTATTCTGTMTATMGMCATCTffiGMT~GMCTAGTffiMTATGTTTTCATMTTTTTCTATACTTTGGGTTTMT-TGGTGTTGATMTC 3072

FIG. 6. A novel Gai species-Ga,s cDNA sequence. The nucleotide and predicted amino acid sequence of Gaia (GI6) is shown. The conserved residues of the eukaryotic translation initiation consensus sequence are boxed. The 3' end is long but does not contain a poly(A) tail or suitable polyadenylation sites and is probably truncated. The predicted prGtein contains 354 residues.

heart tissue while Ga,O (GIB) is expressed abundantly in lung tissue and at low levels in olfactory and liver tissue. Gais (G16) message is expressed at approximately the same level in all tissues except brain, where it is poorly expressed. It should be noted that, although the specific activities of the probes were comparable (1-3 X 10' dpm/pg), the sizes of the labeled fragments and length of exposure times varied for each clone; therefore, the relative message abundance between clones cannot accurately be determined from these data. Generally, the relative message levels of each clone in olfactory tissue approximate their abundance in the library. Specifically, Gaa is the most prevalent Ga message detected in olfactory tissue.

DISCUSSION

Recent biochemical evidence suggests that, at least for some odorants, olfactory signal transduction is mediated by specific receptors coupled to adenylyl cyclase via a G-protein (29,30). This type of transductory cascade is analogous to those previously described for visual, hormonal, and neurotransmitter systems (1). In an effort to identify the G-protein(s) mediating olfactory signal transduction, we screened a rat olfactory cDNA library with an oligonucleotide probe based on a highly conserved G-protein amino acid sequence and isolated five independent G-protein-encoding clones. Further analysis demonstrated that these clones encoded Gas, Ga,, and three distinct forms of Gai. Alignment of the predicted protein sequences of these rat clones and the two forms of bovine transducin clearly demonstrates that G-proteins comprise a highly conserved protein family (Fig. 7). Specific functions

(i.e. interaction with guanine nucleotides. G@y, receptor, and effector molecules) have tentatively been assigned to internal G-protein domains (44, 45, 51). The plausibility of these assignments is discussed here in the context of a large family of G-protein-encoding clones derived from a single species.

Nucleotide Sequence Comparisons-The five rat cDNA clones are highly homologous, yet the nucleotide differences are not clustered but are distributed evenly throughout the sequences. These clones each hybridize to a unique set of genomic fragments by Southern analysis' and specific mRNA species by Northern analysis. Together, these results strongly suggest that the five different cDNA clones are derived from five separate genes and do not result from alternative splicing of messages transcribed from a single gene.

Although the five rat Ga subunits share considerable homology, the conservation of a particular Ga subunit between species is far more dramatic. Comparison of the nucleotide sequences for a given clone between species reveals that both coding and noncoding regions are conserved and that most codon differences are redundant third position changes. The significance of this observation will be discussed below.

Protein Sequence Comparisons-We have aligned the predicted protein sequences from the five rat cDNA clones along with the two forms of bovine transducin (Fig. 7). Ga. is the most divergent and contains several insertions the others lack, while the other six members form a more highly conserved set. Indeed, sequence comparisons suggest that Ga, should be grouped with the Gai species and, in the absence of

D. T. Jones and R. R. Reed, unpublished data.


* . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FIG. 7. Predicted protein sequence alignments. The predicted protein sequences of the five rat cDNAs and the predicted bovine transducin amino acid sequences (rod transducin (Tar (44) and cone transducin (Tal,) (45)) have been aligned. Only identical amino acid residues are shaded. Shading was determined by choosing the most common reside in a column or, in cases where 2 residues were equally represented, choosing the most common residue with a majority in the G*s. In addition, the guanine nucleotide binding domains A, C, E, and G (55) are indicated above the sequences and the predicted effector, receptor, and subunit binding domains (51) are underlined.

rigorous biochemical evidence, one may wish to avoid assigning functional roles to Ga, or an individual Gai.

Comparison of each of the predicted protein sequences from rat with those from other species indicates that minimal divergence occurred during speciation. Specifically, corresponding Ga subunits across species share greater than 98% identity, whereas different G a subunits within a species share only 40-94% identity. Thus, protein sequence comparisons also indicate that individual Ga subunits are more highly conserved across species than are any of the different Ga subunits within a species.

The identification of four highly homologous but distinct types of G-proteins (Ga,, Gil, Gai2, and Gai3) invites specu- lation on their functions. For example, these differences may derive from cell-type-specific expression of functionally identical G-proteins. Alternatively, each type of G-protein may be a component of a distinct second messenger cascade.

The primary structures of several other highly conserved protein families are known. For example, the a- and P-tubulin gene families encode proteins that are differentially expressed in various tissues and are typically greater than 90% identical (excluding the marginal band 6-tubulin) (52, 53). Similarly, the actin gene family encodes proteins that are differentially expressed and generally greater than 95% identical (54). The extreme conservation of these protein families may reflect the functional requirement that different isotypes must form pol- ymers. In neither of these systems is there experimental evidence of functional diversity within the protein family. Perhaps the greater diversity of the G-proteins, relative to the actins and tubulins, reflects their conserved functions in disparate signal transduction systems.

Several G-protein regions share considerable homology with the guanine nucleotide binding regions of elongation factor Tu and p21"" (55). Consistent with this role, these regions (Fig. 7 , labeled A, C, E, and G) are all highly conserved

within the five rat G-proteins and the two bovine transducins. Additionally, regions interspersed between the guanine nucleotide binding domains have been implicated in receptor, GB?, and second messenger protein interactions (Fig. 7 ) (51). These regions are not as conserved as the guanine nucleotide binding regions, perhaps reflecting their role in the independent modulation of different G-proteins in distinct signal transduction pathways.

Immunochemical Considerations-The high degree of homology among the seven G a subunits suggests that caution must be used when distinguishing among the various species by immunochemical methods. This is especially important in the case of Gail (GZ7) and Gai3 (GIG) which have only 22 out of 354 amino acid differences. Mumby et al. (56) have shown that monospecific antisera can be prepared using peptide antigens; nevertheless, the specificity of polyclonal or mono- clonal antisera should be well characterized for definitive immunoanalysis.

Northern Analysis-We have examined the message sizes and tissue distributions of the five rat G-protein-encoding clones by Northern analysis (Fig. 8). The message size corresponding to a given clone was constant for each of the tissues examined. Additionally, all five clones were expressed in each of the tissues examined and the level of expression varied in a tissue-dependent manner. The significance of this differ- ential expression between tissues is not clear but may be related to the numbers and types of receptors and/or effectors that are expressed in each of the cell types comprising the tissue.

Although each of the rat Ga clones was expressed in all of the tissues examined here, further screening by Northern analysis may identify a tissue or cell type that expresses only one of these G-proteins. This should aid considerably in assigning a given clone to a given G-protein and in determining the functional role of that G-protein. Alternatively, similar


O B K L i L u H I

FIG. 8. Northern analysis. Determination of message levels for each of the five rat G-proteins. Each lane contains 10 pg of total rat RNA as follows: 0, olfactory; B, brain; K, kidney; Li, liver; Lu, lung; H , heart; and I, intestine. Filters were probed with radiolabeled full- length cDNAs for Ga, and Gaiz (G,) or radiolabeled restriction fragments for Gao, Gail (GZT), and Gaia (G16) (indicated in Fig. 1). Filters were subsequently probed with radiolabeled frog rRNA in order to verify that equal amounts of RNA were loaded in each lane (data not shown).

information may be obtained by introducing suitable G-protein expression vectors into cells and examining the properties of the corresponding protein products.

We have described the isolation and characterization of five distinct G-protein-encoding clones from rat olfactory tissue. It is not known if any of the predicted proteins actually play a role in olfaction. All five of these clones are expressed in several different tissues in addition to olfactory tissue, a property not anticipated for olfactory transducin. Unlike the visual system, the olfactory system may not have evolved a distinct signal-transducing G-protein. The preponderance of Gas message and protein (31) as well as biochemical evidence for GTP-dependent stimulation of adenylyl cyclase by odorants strongly implies that Ga, is olfactory transducin. We are determining the distribution of each of the five G-proteins in the various cell types of rat olfactory mucosa by in situ hybridization. This technique should allow us to determine if Gas message is appropriately expressed at high levels in olfactory sensory neurons.

Acknowledgments-We would like to thank C. Moser for excellent technical assistance, B. Sollner-Webb for the frog rRNA clone, and

our colleagues for helpful discussions and critical reading of the manuscript.

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