Vol.
179,
September
No.
3, 1991
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1991
1485-1490
MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING A HUMAN a,,, ADRENERGIC RECEPTORla'
John
Division State
F. Bruno,
Jonathan and Michael
Whittaker,*Jinfen Berelowitz"
Song
Endocrinology and Metabolism, Medicine and*Physiology/Biophysics University of New York, Stony Brook,
Departments
of
New York
of 11794
Received August 12, 1991 DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previosly cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat IY,* adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 0 1991Academic represents a cDNA for a human Q,* adrenergic receptor. Press, Inc.
The physiological actions of a number of neurotransmitters and neuropeptides are expressed following interactions with specific plasma membrane receptors coupled to guanine nucleotide binding proteins or G-proteins. Molecular cloning of a number of G-protein coupled receptors has revealed that they are members of a diverse superfamily which includes the adrenergic receptors, muscarinic and serotoninergic receptors as well as several peptide receptors (1,2). Sequence comparison of G-protein receptors has revealed that they share a number of structural similarities, the most common being the presence of seven regions of hydrophobic amino acids that are thought to span the plasma membrane (2). lThe nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number M76446. 2 Supported
by NIH grant
#RRO5736.
1485
All
Copyright 0 1991 rights of reproduction
0006-291X/91 $1.50 by Academic Press, IHC. in an>’ form reserved.
Vol.
179, No. 3, 1991
Using to design polymerase receptor similar receptors
BIOCHEMICAL
AND BIOPHYSICAL
the structural similarity degenerate oligonucleotide chain reaction (PCR),
RESEARCH COMMUNICATIONS
found
in transmembrane regions for use in the primers additional members of this
family have recently been cloned approach in an attempt to clone and report the cloning of a human
(3). We have utilized a novel G-protein linked CY,* adrenergic receptor.
Methods Olisonucleotides: Two sets of highly degenerate oligonucleotides were synthesized on an Applied Biosystems 391 DNA Synthesizer (Forster City, CA) with nucleotides encoding Not 1 restriction sites included at the 5' end. one set corresponding to consensus coding sequences for the third (P3) and sixth (P6) transmembrane domains of known receptors was as described (3). The second set was devised from a compilation of sequences corresponding to the second [P2:5'GAATTTGCGGCCGCC(TC)T(TG)G(GC)C(TG)(TG)(TG)GC(ATGC)GA(TC) (AC)T 3'1 and seventh [P7:5tGAATTTGCGGCCGCGTTGA(ATC)ITA(AG)C(AC) (ATGC)AICCA 3'1 transmembrane segments obtained from previously cloned G-protein linked receptors. Polvmerase Chain Reaction: Template DNA from a rat hippocampus cDNA library (Clontech Laboratories, Palo Alto, CA) was subjected to 30 cycles of PCR using primers P2 and P7 under standard conditions using the following protocol: denaturation for 1.5 minutes at 95 "C, annealing for 2 minutes at 55 'C, and extension for 2 minutes at 72 "C. Ten microliters of the first round PCR was subjected to 30 additional cycles under identical conditions using primers P3 and P6. The final reaction products were digested with Not 1 and ligated into Not 1 digested jtZap II vector (Stratagene, San Diego CA). Plaques were screened for recombinant clones by PCR using T3 and T7 primers. Amplified products were sequenced as described below. Screenina of a human hiopocamuus cDNA librarv: Approximately 2 X lo6 pfu of a human hippocampus library in AZap (Stratagene, La Jolla, CA) were screened with a 610 base pair (bp) PCR product labeled with [32P] by nick-translation. This cDNA represented a unique clone amplified from the rat library cDNA that encoded an open reading frame showing sequence homology to G-protein linked receptors. Four cDNA clones were isolated from the human library. One clone (H318/3) containing a 2.5 kilobase pair insert was sequenced. DNA Seouencins: Dideoxynucleotide sequencing was performed by the chain-termination method (4) using Sequenase Version 2.0 (USB, Cleveland, Ohio). Sequencing PCR fragments was accomplished after generating template by single-stranded single-primer reamplification as described (5). The plasmid Bluescript SK(-) was excision-rescued from clone H318/3 by coinfection with R408 helper phage. Restriction fragments of the cDNA were subcloned into plasmid Bluescript II KS(+). Sequence analysis was performed using the GCG software program (6). Results
and Discussion
In order to identify superfamily, we generated of degenerate oligonucleotide
new members of the G-protein receptor a 610 bp cDNA fragment by PCR using sets primers and DNA purified from a rat 1486
Vol.
1
BIOCHEMICAL
179, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IAAALRSYI,,ACYLS ccnaa-mm~-TA~AG
CccGTGcAGGGGDcr~AcAccA-cTA~--ATGGcGGca
100
EWRTPTYRSTEn"*RLRHKA"*Ns~s~AA"GGL rGAG?oGacAaCCcAcGTAcaGraAaGAGA~n2cAG
199
VVSAQGVGVGVFLAAFILnA”AGNLL”ILS”AC GGTGGTGAGCGffiCAGGGC XOGCO-CAGC~-AAC~
AGGCTOAGGAl-CGAGGKCAG
CACAGCA‘ZDXMGGCUiCCGZCGGOUiACP CrrerCAZCCPCPCAG1OCCCTO
99 198 297 396 495
496
ISVDRYVGVRBSLXYPAIXTERKAAAILALLNV CATCTCCfXCGACCGGTACWi’CffiCCACT”iCTCCtZA~~A~AGffi-
BTC-CC-C-T
594 693 792 891 990
1188 1281 1386 1485 1584 1683 1782 MS1 1980
Fig.
1. Nucleotide for
single
and deduced amino acid sequence of the cDNA Amino acids are annotated by the letter amino acid code.
clone
H318/3.
hippocampus cDNA library. This fragment was used to screen over 2 X lo6 plaques of a human hippocampus cDNA library. Four clones were isolated and one clone H318/3 containing a 2.5 kb insert was purified and sequenced. Figure 1 shows the nucleotide and deduced amino acid sequence of clone H318/3. The open reading frame encodes a protein consisting of 501 amino acids. Of the several methionine residues in the NHZterminus, the nucleotides surrounding the codon for the methionine at position 1 contains a sequence matching the consensus sequence proposed by Kozak for translation initiation analysis of the translated amino acid sequence (7) * Hydropathy revealed a seven hydrophobic transmembrane segment structure separated by stretches of hydrophilic residues, consistent with members of the G-protein receptor superfamily. Initial sequence analysis revealed clone H318/3 was most likely related to adrenergic receptors since it contained a number 1487
Vol.
BIOCHEMICAL
179, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
among the adrenergic receptor of conserved residues found family. These include conserved cysteines in the second and aspartic acids in transmembrane regions third extracellular loops, 2 and consensus carboxyl-terminal of 3, the DRY at the transmembrane domain 3, prolines in five of the seven transmembrane and threonine and serine residues spanning regions several representing potential phosphorylation sites in the carboxylterminal region (1,2,8). Clone H318/3 was tentatively identified as a human Q adrenergic receptor since it contained the consensus F-W-
HAR MAAALRSJMMAGYLsEWRTP..........................TYRS
24
RAR
48
..MTF&DILSVTFEGPRSSSSTGGSGAGGGAGTVGPEGGAVGGVPGATGG
HAR T~QR~VQHST............
STAAVGGLWSAQGVGVGVFLA 1~1,11~11,1,111,111, II llllllIlI,IlIIlll,l, RAR GAWGTGSG6DN&'STGEPGAAASGEVNGSAAVGGLWSAQGVGVGvF~
-ml
HAR ~~~MAVAGNLLVILSVACNl?HLQTvTNYFIVNLAVADLLLSATvLPFSA 11,111111,111111111111111111111111,1,, ,111 1,111111,,111111111lIlIlIlllIl,llII,,I RAR AFILTAVAGNLLVILSVACNRHLQTVTNYFIVNLAVADLLLSAAvLPFSA
62 98
TM2 112
,,,,,I ,I,,,,
148
TM3
HAR TMEVLGFF~AFGRAFCD~AAVDVLCCTASILSLCTISVDRYVGVFHSLNY 1111,1,1,111 111111111111111111,11111111111111,,,, 1111,1,1,111 111,111111111111lll11,1111,1,11,,,,,, RAR TMEVLGX;FWAFGRTFCDVWAAVDVLCCTASILSLCTISVDRYVGVRHSLNY
162 198
TM4
HAR PAINTERKAAAILALLWVALWSVGPLLGWKEPvPPDERFCGITE~ ,1,1,,,,1111,lIll lllllllllllllllllllll,l,I,,I, 11,,,1,,,11111l,, 1111~11,111111111111111,1,,1, RAR PAINTERNAAAIIALLWAVALWSVGPLLGWNEPvPPDERFCGITEEVGY
212
II
248
TI45
HAR AVFSSVCSFYLPNAVIWMYCRVYVVARSTTRSLEAGVNRERGKASEWL II Il,I,I,llIllllllllllJJll~,lIl,,,,l~ 1 1 1 llI~llll Il,I,,,IlI,IIl,IIlI1111111~11,,,,11 RAR AIFSSVCSFYLPHAVIWMYCRVYWAP.STTRSLEAGIl%$Pd~&+n;i.
262 298
HAR RIIiCRGAATGADGAHGNRSAHTFRSSI.S7RLLKFSREKKAARTIAIW llIl,l,l, I I ,lllO,,, I I I f It II 1I II lllllI,l,l,l,l,,,,I,lI,,, IllIIIlIIIIIll1l111llI,l, RAR RIHCRGAATSAXGYPGTQSSKGHTLRSSLSvRLLKFSREKKAARTLAIW
312 348
Tl46 TN7 HAR GVFVLCWFPFFFVLPLGSLFPQLNPSEGVFKVIFWLGYFNSCVNPLIYPC ,lIl,,l,,ll~l,lIlllolllIllIl,l,,lJlll,,l,l,llI,,, l~ll,,,,,~lll,lllllIll1lIlIllll,,ll~I~,II,,lllIlI, RAR GVFVLCWFPFFFVLPLGSLFPQLNPSEGVFWIFWLGYFNSCVNPLIYPC
HAR~S~FFRAF~.RLLRCQC ,IJlI,lIl,ll,,lIlllllIl lIIl,,,,I,lll,l,l,I1111 RAR SSREFKRAFLRLLRCQCR
362 398
.RPLNRVYG..HHNRA....STSGLRQ I II LWSLRPPLASLDRRRAFRLRPQPSHRS
HAR DCAPSSGDAPPGAPLALTALPDPDPEPPGTPEMQA...PVASRRSHPAPS II1 II II I II I RAR PRGPSSPHCTPGCGLGRHAGDAGFGLQQSKASLRLREWRLLGPLQRPTTQ HAR ASGGCWGRSGDPRPSCAPKSPACRTRSPPGARSAQ.RQRAPSAQRWRLCP I I II II II I RAR ~WSSLSHKIRSGARRARTACALRSEVEAVSINvPQDGASAVICQAYE Fig.
2.
Alignment of and rat(RAR) acids between broken bars. indicated by
I
405 448
452
I
,
498
501 548
the amino acid sequences of human(HAR) @,A adrenergic receptors. Identical amino the two sequences are connected by Putative membrane-spanning domains are TM1 to TM7 and solid bars.
1488
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
X-G-Y-X-N-S-X-X-N-P-X-X-Y in the seventhtransmembrane domain found in all a adrenerqic receptors cloned to date (9). Sequence comparison with members of the G-protein linked receptor superfamily comfinned that clone H318/3 was a human a! adrenerqic receptor. Figure 2 shows the amino acid alignment of a recently described rat atA adrenergic receptor (RAR) and clone H318/3 (HAR)(lO). Both receptors share a great deal of sequence homology having a 78% amino acid identity overall. The region from the amino terminus of the first transmembrane domain to the carboxyl-terminus of the seventh transmembrane domain are particularly well conserved with an average homology of over 95%. Since amino acid residues within the transmembrane domains of adrenerqic receptors are believed to be involved in liqand binding, one would predict H318/3 and RAR would have a similar pharmacologic profile. The least conserved regions are the aminoand carboxylterminal regions having 50% and 47% identity, respectively. In contrast to the RAR and most G-protein coupled receptor, H318/3 has an amino terminal extracellular region that does not contain potential sites for N-linked glycosylation. This unique structural feature has to date only been observed in an c1z adrenerqic receptor subtype (8,11,12) and two putative G-protein receptors (3). In summary, we have used PCR to clone new members of the Gprotein receptor family. A unique clone was isolated and based on the high degree of sequence homology with a previosly characterized rat a adrenerqic receptor we report the cloning of a human Q,* adrenergic receptor. &knowledgment this
We thank work.
Dr.
Jeffrey
D. White
for
advice
during
the
course
of
References 1) 2)
3) 4) 5) 6)
Dohlman, H.G., Caron, M.G.,and Lefkowitz,R.J. (1987) Biochemistry 26,2657-2664. Strosberg, A.D. (1991) Eur. J. Biochem. 196, l-10. Libert,F., Parmentier, M., Lefort, A., Dinsart, C.,Van Sande, C., Simons, M.J., Dumont, J.E., and Vassart, V. J ., Maenhaut, (1989) Science 244,569-572. Sanqer, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74,5463-5464. Gyllensten, U.B. and Erlich, H.A. (1988) Proc. Natl. Acad. Sci. USA 85, 7652-7656. Devereaux, J., Haeberli, P., and Smithies, 0. (1984) Nucleic Acids Res. 10, 703-795. 1489
Vol. 179, No. 3, 1991
7) 8)
9) 10) 11) 12)
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148. Weinshank, R.L., Zgombick, J.M., Macchi, M., Adham, N., Lichtblau, H., Branchek, T.A., and Hartig, P.R. (1990) Mol.Pharmacol. 38, 681-688. Suryanarayana, S., Daunt,D., von Zastrow, M., and Kobilka, B.K. (1991) Clin. Res. 39, 342A. Lomasney, J.W., Cotecchia, S., Lorenz, W., Leung, W.Y., Schwinn, D.A., Yang-Feng, T.L., Brownstein, M., Lefkowitz, R.J., and Caron, M.G. (1991) J.Biol. Chem. 266, 6365-6369. Lomasney, J.W., Lorenz, W., Allen, L.F., King, K., Regan, R.J. (1990) J-W., Yang-Feng, T.L., Caron, M.G., and Lefkowitz, Proc. Natl. Acad. Sci. USA 87, 5094-5098. Zeng, D., Harrison, J.K., DOAngelo, D.D., Barber, C.M., Tucker, A.L., Lu, Z., and Lynch, K.R. (1990) Proc. Natl. Acad. Sci. USA 87, 3102-3106.
1490
179,
September
No.
3, 1991
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1991
1485-1490
MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING A HUMAN a,,, ADRENERGIC RECEPTORla'
John
Division State
F. Bruno,
Jonathan and Michael
Whittaker,*Jinfen Berelowitz"
Song
Endocrinology and Metabolism, Medicine and*Physiology/Biophysics University of New York, Stony Brook,
Departments
of
New York
of 11794
Received August 12, 1991 DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previosly cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat IY,* adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 0 1991Academic represents a cDNA for a human Q,* adrenergic receptor. Press, Inc.
The physiological actions of a number of neurotransmitters and neuropeptides are expressed following interactions with specific plasma membrane receptors coupled to guanine nucleotide binding proteins or G-proteins. Molecular cloning of a number of G-protein coupled receptors has revealed that they are members of a diverse superfamily which includes the adrenergic receptors, muscarinic and serotoninergic receptors as well as several peptide receptors (1,2). Sequence comparison of G-protein receptors has revealed that they share a number of structural similarities, the most common being the presence of seven regions of hydrophobic amino acids that are thought to span the plasma membrane (2). lThe nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number M76446. 2 Supported
by NIH grant
#RRO5736.
1485
All
Copyright 0 1991 rights of reproduction
0006-291X/91 $1.50 by Academic Press, IHC. in an>’ form reserved.
Vol.
179, No. 3, 1991
Using to design polymerase receptor similar receptors
BIOCHEMICAL
AND BIOPHYSICAL
the structural similarity degenerate oligonucleotide chain reaction (PCR),
RESEARCH COMMUNICATIONS
found
in transmembrane regions for use in the primers additional members of this
family have recently been cloned approach in an attempt to clone and report the cloning of a human
(3). We have utilized a novel G-protein linked CY,* adrenergic receptor.
Methods Olisonucleotides: Two sets of highly degenerate oligonucleotides were synthesized on an Applied Biosystems 391 DNA Synthesizer (Forster City, CA) with nucleotides encoding Not 1 restriction sites included at the 5' end. one set corresponding to consensus coding sequences for the third (P3) and sixth (P6) transmembrane domains of known receptors was as described (3). The second set was devised from a compilation of sequences corresponding to the second [P2:5'GAATTTGCGGCCGCC(TC)T(TG)G(GC)C(TG)(TG)(TG)GC(ATGC)GA(TC) (AC)T 3'1 and seventh [P7:5tGAATTTGCGGCCGCGTTGA(ATC)ITA(AG)C(AC) (ATGC)AICCA 3'1 transmembrane segments obtained from previously cloned G-protein linked receptors. Polvmerase Chain Reaction: Template DNA from a rat hippocampus cDNA library (Clontech Laboratories, Palo Alto, CA) was subjected to 30 cycles of PCR using primers P2 and P7 under standard conditions using the following protocol: denaturation for 1.5 minutes at 95 "C, annealing for 2 minutes at 55 'C, and extension for 2 minutes at 72 "C. Ten microliters of the first round PCR was subjected to 30 additional cycles under identical conditions using primers P3 and P6. The final reaction products were digested with Not 1 and ligated into Not 1 digested jtZap II vector (Stratagene, San Diego CA). Plaques were screened for recombinant clones by PCR using T3 and T7 primers. Amplified products were sequenced as described below. Screenina of a human hiopocamuus cDNA librarv: Approximately 2 X lo6 pfu of a human hippocampus library in AZap (Stratagene, La Jolla, CA) were screened with a 610 base pair (bp) PCR product labeled with [32P] by nick-translation. This cDNA represented a unique clone amplified from the rat library cDNA that encoded an open reading frame showing sequence homology to G-protein linked receptors. Four cDNA clones were isolated from the human library. One clone (H318/3) containing a 2.5 kilobase pair insert was sequenced. DNA Seouencins: Dideoxynucleotide sequencing was performed by the chain-termination method (4) using Sequenase Version 2.0 (USB, Cleveland, Ohio). Sequencing PCR fragments was accomplished after generating template by single-stranded single-primer reamplification as described (5). The plasmid Bluescript SK(-) was excision-rescued from clone H318/3 by coinfection with R408 helper phage. Restriction fragments of the cDNA were subcloned into plasmid Bluescript II KS(+). Sequence analysis was performed using the GCG software program (6). Results
and Discussion
In order to identify superfamily, we generated of degenerate oligonucleotide
new members of the G-protein receptor a 610 bp cDNA fragment by PCR using sets primers and DNA purified from a rat 1486
Vol.
1
BIOCHEMICAL
179, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IAAALRSYI,,ACYLS ccnaa-mm~-TA~AG
CccGTGcAGGGGDcr~AcAccA-cTA~--ATGGcGGca
100
EWRTPTYRSTEn"*RLRHKA"*Ns~s~AA"GGL rGAG?oGacAaCCcAcGTAcaGraAaGAGA~n2cAG
199
VVSAQGVGVGVFLAAFILnA”AGNLL”ILS”AC GGTGGTGAGCGffiCAGGGC XOGCO-CAGC~-AAC~
AGGCTOAGGAl-CGAGGKCAG
CACAGCA‘ZDXMGGCUiCCGZCGGOUiACP CrrerCAZCCPCPCAG1OCCCTO
99 198 297 396 495
496
ISVDRYVGVRBSLXYPAIXTERKAAAILALLNV CATCTCCfXCGACCGGTACWi’CffiCCACT”iCTCCtZA~~A~AGffi-
BTC-CC-C-T
594 693 792 891 990
1188 1281 1386 1485 1584 1683 1782 MS1 1980
Fig.
1. Nucleotide for
single
and deduced amino acid sequence of the cDNA Amino acids are annotated by the letter amino acid code.
clone
H318/3.
hippocampus cDNA library. This fragment was used to screen over 2 X lo6 plaques of a human hippocampus cDNA library. Four clones were isolated and one clone H318/3 containing a 2.5 kb insert was purified and sequenced. Figure 1 shows the nucleotide and deduced amino acid sequence of clone H318/3. The open reading frame encodes a protein consisting of 501 amino acids. Of the several methionine residues in the NHZterminus, the nucleotides surrounding the codon for the methionine at position 1 contains a sequence matching the consensus sequence proposed by Kozak for translation initiation analysis of the translated amino acid sequence (7) * Hydropathy revealed a seven hydrophobic transmembrane segment structure separated by stretches of hydrophilic residues, consistent with members of the G-protein receptor superfamily. Initial sequence analysis revealed clone H318/3 was most likely related to adrenergic receptors since it contained a number 1487
Vol.
BIOCHEMICAL
179, No. 3, 1991
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
among the adrenergic receptor of conserved residues found family. These include conserved cysteines in the second and aspartic acids in transmembrane regions third extracellular loops, 2 and consensus carboxyl-terminal of 3, the DRY at the transmembrane domain 3, prolines in five of the seven transmembrane and threonine and serine residues spanning regions several representing potential phosphorylation sites in the carboxylterminal region (1,2,8). Clone H318/3 was tentatively identified as a human Q adrenergic receptor since it contained the consensus F-W-
HAR MAAALRSJMMAGYLsEWRTP..........................TYRS
24
RAR
48
..MTF&DILSVTFEGPRSSSSTGGSGAGGGAGTVGPEGGAVGGVPGATGG
HAR T~QR~VQHST............
STAAVGGLWSAQGVGVGVFLA 1~1,11~11,1,111,111, II llllllIlI,IlIIlll,l, RAR GAWGTGSG6DN&'STGEPGAAASGEVNGSAAVGGLWSAQGVGVGvF~
-ml
HAR ~~~MAVAGNLLVILSVACNl?HLQTvTNYFIVNLAVADLLLSATvLPFSA 11,111111,111111111111111111111111,1,, ,111 1,111111,,111111111lIlIlIlllIl,llII,,I RAR AFILTAVAGNLLVILSVACNRHLQTVTNYFIVNLAVADLLLSAAvLPFSA
62 98
TM2 112
,,,,,I ,I,,,,
148
TM3
HAR TMEVLGFF~AFGRAFCD~AAVDVLCCTASILSLCTISVDRYVGVFHSLNY 1111,1,1,111 111111111111111111,11111111111111,,,, 1111,1,1,111 111,111111111111lll11,1111,1,11,,,,,, RAR TMEVLGX;FWAFGRTFCDVWAAVDVLCCTASILSLCTISVDRYVGVRHSLNY
162 198
TM4
HAR PAINTERKAAAILALLWVALWSVGPLLGWKEPvPPDERFCGITE~ ,1,1,,,,1111,lIll lllllllllllllllllllll,l,I,,I, 11,,,1,,,11111l,, 1111~11,111111111111111,1,,1, RAR PAINTERNAAAIIALLWAVALWSVGPLLGWNEPvPPDERFCGITEEVGY
212
II
248
TI45
HAR AVFSSVCSFYLPNAVIWMYCRVYVVARSTTRSLEAGVNRERGKASEWL II Il,I,I,llIllllllllllJJll~,lIl,,,,l~ 1 1 1 llI~llll Il,I,,,IlI,IIl,IIlI1111111~11,,,,11 RAR AIFSSVCSFYLPHAVIWMYCRVYWAP.STTRSLEAGIl%$Pd~&+n;i.
262 298
HAR RIIiCRGAATGADGAHGNRSAHTFRSSI.S7RLLKFSREKKAARTIAIW llIl,l,l, I I ,lllO,,, I I I f It II 1I II lllllI,l,l,l,l,,,,I,lI,,, IllIIIlIIIIIll1l111llI,l, RAR RIHCRGAATSAXGYPGTQSSKGHTLRSSLSvRLLKFSREKKAARTLAIW
312 348
Tl46 TN7 HAR GVFVLCWFPFFFVLPLGSLFPQLNPSEGVFKVIFWLGYFNSCVNPLIYPC ,lIl,,l,,ll~l,lIlllolllIllIl,l,,lJlll,,l,l,llI,,, l~ll,,,,,~lll,lllllIll1lIlIllll,,ll~I~,II,,lllIlI, RAR GVFVLCWFPFFFVLPLGSLFPQLNPSEGVFWIFWLGYFNSCVNPLIYPC
HAR~S~FFRAF~.RLLRCQC ,IJlI,lIl,ll,,lIlllllIl lIIl,,,,I,lll,l,l,I1111 RAR SSREFKRAFLRLLRCQCR
362 398
.RPLNRVYG..HHNRA....STSGLRQ I II LWSLRPPLASLDRRRAFRLRPQPSHRS
HAR DCAPSSGDAPPGAPLALTALPDPDPEPPGTPEMQA...PVASRRSHPAPS II1 II II I II I RAR PRGPSSPHCTPGCGLGRHAGDAGFGLQQSKASLRLREWRLLGPLQRPTTQ HAR ASGGCWGRSGDPRPSCAPKSPACRTRSPPGARSAQ.RQRAPSAQRWRLCP I I II II II I RAR ~WSSLSHKIRSGARRARTACALRSEVEAVSINvPQDGASAVICQAYE Fig.
2.
Alignment of and rat(RAR) acids between broken bars. indicated by
I
405 448
452
I
,
498
501 548
the amino acid sequences of human(HAR) @,A adrenergic receptors. Identical amino the two sequences are connected by Putative membrane-spanning domains are TM1 to TM7 and solid bars.
1488
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
X-G-Y-X-N-S-X-X-N-P-X-X-Y in the seventhtransmembrane domain found in all a adrenerqic receptors cloned to date (9). Sequence comparison with members of the G-protein linked receptor superfamily comfinned that clone H318/3 was a human a! adrenerqic receptor. Figure 2 shows the amino acid alignment of a recently described rat atA adrenergic receptor (RAR) and clone H318/3 (HAR)(lO). Both receptors share a great deal of sequence homology having a 78% amino acid identity overall. The region from the amino terminus of the first transmembrane domain to the carboxyl-terminus of the seventh transmembrane domain are particularly well conserved with an average homology of over 95%. Since amino acid residues within the transmembrane domains of adrenerqic receptors are believed to be involved in liqand binding, one would predict H318/3 and RAR would have a similar pharmacologic profile. The least conserved regions are the aminoand carboxylterminal regions having 50% and 47% identity, respectively. In contrast to the RAR and most G-protein coupled receptor, H318/3 has an amino terminal extracellular region that does not contain potential sites for N-linked glycosylation. This unique structural feature has to date only been observed in an c1z adrenerqic receptor subtype (8,11,12) and two putative G-protein receptors (3). In summary, we have used PCR to clone new members of the Gprotein receptor family. A unique clone was isolated and based on the high degree of sequence homology with a previosly characterized rat a adrenerqic receptor we report the cloning of a human Q,* adrenergic receptor. &knowledgment this
We thank work.
Dr.
Jeffrey
D. White
for
advice
during
the
course
of
References 1) 2)
3) 4) 5) 6)
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