Genetic Analysis of the Human Type-l Angiotensin II Receptor
Kathleen
M. Curnow*,
Leigh
Pascoe,
and Perrin
C. Whitet
Division of Pediatric Endocrinology Cornell University Medical College New York, New York 10021
receptor (ATl) population of binding sites, whereas CGP 42112A and PD 123177 have a greater affinity for AT2 sites (1, 2). AT1 is the predominant subtype in vascular smooth muscle and human uterus, whereas AT2 is the subtype predominantly expressed in the adrenal medulla and brain. Other tissues such as the adrenal cortex, kidney, and rat uterus have both subtypes of binding sites (3). In addition to being defined by binding studies, the two subtypes of receptor are distinguished by their sensitivity to inactivation by dithiothreitol (DTT), in that the AT1 receptor is inactivated by millimolar concentrations of DTT, whereas the AT2 receptor is resistant to DTT inactivation (4). Although it has proven difficult to purify these receptors, bovine and rat cDNA encoding AT1 were recently isolated by expression screening (5-7). AT1 is predicted to have seven transmembrane segments, thus sharing the common structural frame of the guanine nucleotidebinding regulatory protein (G protein)-coupled receptors. Because angiotensin II is an important effector for maintaining blood pressure, it seems likely that genetic polymorphisms in the receptor(s) for this hormone could be associated with the development of hypertension. As a step in investigating this hypothesis, we characterized cDNA and genomic clones encoding the human AT1 receptor.
Angiotensin II is a potent pressor hormone and a primary regulator of aldosterone secretion. It acts through at least two types of receptors termed AT1 and AT2. We analyzed cDNA and genomic clones encoding the human angiotensin II type-l receptor, ATl. The human AT1 gene was mapped to chromosome 3q by polymerase chain reaction analysis of DNA from a panel of human-hamster somatic cell hybrids. The predicted amino acid sequence is 95% identical to the corresponding rat and bovine receptors and 25% and 22% identical, respectively, to the receptors encoded by the RTA and MAS genes. Characterization of several human cDNA clones demonstrated the existence of two alternate 5’untranslated regions (UTRs) that contain a common initial sequence but differ by the presence or absence of an insertion of 84 base pairs. In the genomit sequence, the coding sequences are contained in a single exon, with an intron occurring in the 5’UTR at the position of insertion of the 84-base pair sequence. The exons encoding the alternate 5’UTRs are located at least 3.8 kilobases away from the exon encoding the protein. Reverse transcription-polymerase chain reaction analysis showed that both forms of 5’-UTR are present in approximately equal abundance in a range of tissues expressing ATl. The reagents developed in this work may be useful in testing the hypothesis that genetic variations in angiotensin II receptor function are associated with a tendency to develop hypertension. (Molecular Endocrinology 6: 1113-l 118, 1992)
RESULTS Human AT1 cDNA A portion of the human AT1 cDNA sequence was obtained by reverse transcription of total adrenal RNA and subsequent polymerase chain reaction (PCR) amplification. The pair of primers used for this purpose (5 and 8 in Fig. 1) was designed on the basis of the published rat and bovine AT1 cDNA sequences (5, 6) such that the sequences chosen encoded stretches of amino acid identity, but more particularly, corresponded to stretches of amino acids that were encoded by the least possible number of codons. Based on the sequence of the PCR-amplified product, two additional primers (6 and 7) were synthesized for the purpose of screening available liver and lung human cDNA libraries by PCR in conjunction with a primer corresponding to
INTRODUCTION Angiotensin II exerts a wide variety of physiological effects on the cardiovascular, endocrine, and central and peripheral nervous systems by binding to specific receptors. Two subtypes of cell surface receptor have been defined by radioligand-receptor binding studies using the peptide antagonist CGP 42112A and the nonpeptide antagonists DuP 753 and PD 123177. DuP 753 has a greater affinity for the type-l angiotensin II 0888-8809/92/l 113-l 118$03.00/0 Molecular Endocmology Copynght 0 1992 by The Endocrine
Society
1113
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
MOL END0.1992 1114
Vol6 No. 7
X-gtl 1 vector sequence. Collated cDNA and predicted amino acid sequences of human AT1 are presented in Fig. 1. The protein sequence of human AT1 is the same length as the bovine and rat proteins (359 amino acids) and shares 95% identity with each. Two different sized PCR products were amplified from a human liver cDNA library using primer 6. They contained the same coding sequence and the same 5’untranslated region (UTR) until position -47, where the sequences diverged; the longer sequence contained an apparent insertion of 84 base pairs (bp) as compared to the shorter. The sequence 5’ of the insertion was notably GC rich (36/41 bp). Chromosomal
Fig. 1. Nucleotide and Deduced Amino Acid Sequences of Human AT1 cDNA and Genomic DNA The insertion sequence in the 5’-UTR is indicated by lower case letters, with an upstream ATG that is in a good context for initiation and a subsequent in-frame stop codon printed in bold and underlined. The genomic intron sequence is printed above the cDNA sequence starting at position -47, and the HindIll site in the 3’-LtTR is bold and underlined. The sequences of the oligonucleotides used in this study are underlined, and arrows beside the numbers identifying each primer indicate whether the primers are sense or antisense. (Sequences have been deposited in Genbank under accession number M93394.)
Localization
DNA
1,066bpt
Mapping of the AT1 Gene to Human Chromosome 3 Specific amplification of the human, and not the hamster, AT1 gene was achieved using oligonucleotides 5-9. A representative set of the PCRs performed on the human-hamster cell hybrid panel is shown. The chromosomal content of each cell line is given in Table 1, which summarizes the complete set of data. Those hybrids containing human chromosome 3 are indicated by an asterisk, and it is noted that cell line 423 contains human chromosome 3 only. Fig. 2.
Table
1. Chromosomal Localization of Human AT1
. .
60 . . 15 .
.
. . D.
.
55.
. . .
. . . E
. . . . .
.
45
.
.
.
.
15
. .
. .
. .
.
.
.
. . . .
.a5
. .
. .
. . .
.
.
. . .
10
. .
. 25 .
A
. .
.
9.6 6.6
. . 45 .
E . . D
.
15
. .
.
.
. .
:
.
. 10 .
45
.
. .
5
.
. .
. .
.
B f 2 E 6.6 2
4.4
. .
2.3
AT1 sequences were amplified (as shown in Fig. 2) from human-hamster hybrid cell lines 423, 860,507, and 1079. This is consistent with a location of human chromosome 3 for the AT1 gene, as indicated by the arrow. Whole chromosomes are contained in cell lines indicated by closed circles, while numbers are given where that percentage of the cell population contains the noted chromosome. D, Deletion at ~15.1-15.2.
2.3 2.0
-
Fig. 3. Genomic Blot Analysis of AT1 Restriction digests of human genomic DNA were probed with 32P-labeled cDNA (the RT-PCR product used was generated using primers 5-8). A, The same DNA was digested with a number of different enzymes; B, DNA from 10 different individuals was digested with Mspl. The DNA in lane 10 of this analysis corresponds to that used in A.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
Genetic
Analysis
of Human
AT1
1115
quently localized to 3q21-3q25 using a somatic cell hybrid regional mapping panel (27) (data not shown). Structure
of the Human Genomic
Exon Usage
A range of tissueswhich express AT1 were examined for the presenceof 5’UTRs with or without the insertion sequence.This analysiswas done by reverse transcription PCR (RT-PCR) (Fig. 4) and does not show preferentialusageof either form of 5’-UTR in any one tissue. Genetic
Polymorphism
In a preliminaryexperiment to identify informative polymorphismsin the vicinity of the AT1 gene, a blot bearingMspl digestsof genomicDNA from 10 unrelated donorswas probedwith a PCR product containingpart of the coding sequence of human AT1 cDNA. This analysis, shown in Fig. 3B, identified a polymorphic Mspl site. The two allelic forms were present at frequenciesof 0.85 (for the 3.4-kb fragment)and 0.15 (for the 4.3-kb fragment).
DISCUSSION Structural
Features
-
50 bp
Gene
A blot of human genomic DNA digested with various restriction enzymes was hybridized with part of the human AT1 cDNA (Fig. 3A). Only one band was detected in each digest (noneof the enzymes chosencut within the probe sequence),indicatingthat there were no relatedgenes of sufficient homologyto cross-hybridize under these conditions. Since the chromosomal locationof the gene had been determined,and a Hindlll site had been identifiedin the 3’-UTR of the cDNA, we screeneda chromosome3 library cloned in the HindIll site of Charon 21A. From the 90,000 recombinants screened, two hybridizing clones were isolated, each containinga 5.1-kilobase(kb) insert. The 5.1-kb HindIll fragment was subclonedinto pBluescriptKS+, and sequence analysisshowed that the genomic and cDNA coding sequenceswere identical down to the HindIll site in the 3’-UTR, 165 bp after the terminationcodon. The entire coding sequence and 3’-UTR were both contained on a singleexon. As would be predicted by the identificationof two alternate5’-UTRs in the cDNAs characterized, an intron spliceacceptor sequencewas located in the 5’-UTR at position -47, the point of divergenceof the translated sequences.No further 5’UTR sequenceswere found on the !%I-kb fragment, indicating that the intron separating them from the coding exon is more than 3.8 kb in length. S-UTR
A
of Human AT1
The cDNA and genomic clones described herein are believedto encode AT1 because the predicted amino
Fig. 4. RT-PCR and Hybridization Analysis Identifying Alternate 5’-UTR Usage in a Range of Tissues A, The positions of the oligonucleotides used for PCR amplification of reverse-transcribed RNA are indicated below a schematic representation of the 5’-end of the AT1 cDNA. 6, The left column shows the ethidium bromide-stained gels of RT-PCR products obtained on various tissues, using the primers and cycle numbers indicated at the left of each gel. The result obtained in a parallel RT-PCR using 02-microglobulin primers (23) is shown at the bottom as an indication of the relative amount and quality of the RNA template used in each reaction. Negative control reactions were performed in parallel on liver RNA, where no reverse transcriptase was added at the cDNA synthesis step, and positive control reactions were performed using the 5’-end PCR products amplified from the liver cDNA library as templates. Note that the positive control template used for PCR with oligonucleotides l-4 does not contain the insertion sequence. In the center column, the blots of the gels at left were hybridized with digoxigenin-labeled oligonucleotide 3, and these blots were then reprobed with an insertion sequence oligonucleotide (primer 2) labeled with [“‘PI, demonstrating that this sequence was present in the 279-bp RT-PCR product in all the tissues studied.
acid sequenceis 95% identicalto bovine and rat AT1 , and no cross-hybridizing genes are detected on genomit blots. The protein sequenceof humanAT1 shares the characteristicfeature of all G protein-coupledreceptors in that seven putative transmembraneregionsare readily identified. As comparedwith the rat and bovine receptors, none of the amino acids believed to play a role in determining the structure and function of this moleculeare altered (5-8). These includefour cysteine residuesat positions18, 101, 180, and 274, and three potential asparagine-linkedglycosylation sites at positions 4, 176, and 188, all of which are located in predicted extracellular domains.Ser and Thr residues at positions 136, 141, 326, 328, 331, 338, 342, 347, and 348 are also conserved, and these amino acids located on the intracellularsurface are suggestedto be sites of regulatory phosphorylation. Also conserved were the Pro residuesin transmembranedomainsIV, V, VI, and VII, and the Asp-Arg-Tyr motif at the intracellularborder of transmembranedomainIll. In general, the best conservation of sequencewas found in the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
MOL 1116
ENDO.
1992
first two intracellular loops and the transmembrane domains. In addition to ATl, three other putative G proteincoupled receptors which may play a role in mediation or modulation of the angiotensin response have been identified by gene cloning (g-12). When injected into Xenopus oocytes, both the MAS and MRG mRNAs markedly increase the electrophysiological response of the endogenous Xenopus oocyte angiotensin receptors to angiotensin peptides. The RTA gene has not yet been found to share any functional similarity with MAS and MRG but is related structurally. The predicted AT1 amino acid sequence was aligned with those of RTA, MAS, and MRG (Fig. 5). Whereas the latter three receptors are about 33% identical to each other in sequence, AT1 is 25% identical to RTA, 22% identical to MAS, and could not be directly aligned with MRG. The 26 positions that were completely conserved and the 54 positions that were functionally conserved among the four receptors were clustered in the transmembrane segments and the second and Cterminal intracellular domains. The remaining intracel-
Vol6
lular and extracellular domains showed limited homology, and the third intracellular domain of AT1 differed considerably, being at least 13 amino acids longer than those of the MAS-related gene family. Overall, the comparison suggests that the latter genes belong to their own separate subfamily. Their physical and functional relationships with the high affinity angiotensin receptors remains unclear but are now amenable to study through cotransfection experiments with ATl. Functional
Significance
of Alternative
5’-UTRs
Transcripts with and without the 84-bp insertion are present at roughly equal levels in all tissues examined that express ATI. An optional 88-bp insertion with a 75% identical sequence has been documented in rat cDNA at an analogous position in the 5’-UTR (-52) (6), and a similar sequence is present in the bovine 5’-UTR, but it is not known whether transcripts without the insertion exist in cattle (5). The relatively high degree of sequence identity suggests a functional role for the insertion. Although there is no open reading frame continuous with the AT1 coding sequence in the insertion in any of the three mammalian species examined, an AUG in a good context for initiation (13), followed by an in-frame stop codon a few codons downstream, is consistently present. Initiation and termination of translation of an mRNA decreases the efficiency of initiation from a downstream AUG (14), thus alternate splicing in the 5’-UTR may be a mechanism for regulating AT1 receptor levels. Polymorphism
Fig. 5. Comparison of the Predicted Amino Acid Sequence of AT1 with Sequences of Three Related Proteins Encoded by the RTA, MAS, and MRG Genes Gaps introduced to optimize the alignment are indicated by dashes. Residues completely conserved in all four proteins are marked with asterisks; residues that are functionally conserved are marked with crosses, and residues that are conserved in three of four proteins are marked with dots. Predicted transmembrane segments are underlined.
No. 7
in the AT1 Gene
Two allelic forms of the AT1 receptor were detected by the restriction enzyme Mspl. Identification of such polymorphisms may be useful markers for genetic analysis of patients with hypertension, as variations in angiotensin II receptor function may play a role in development of this disease. This may be mediated through effects on microvascular growth (15-I 7), vascular smooth muscle cell growth (18), or synthesis of extracellular matrix within vessel walls (19). Altered angiotensin receptor affinities have been suggested to be responsible for the exaggerated pressor and aldosterone responses to angiotensin II in some patients with low renin essential hypertension (20), and could also be responsible for the blunted response to angiotensin II observed in patients classified as having nonmodulating essential hypertension (21). Since this latter form of hypertension tends to occur in families, these families could be examined to determine whether their hypertension shows concordance with an altered AT1 genotype.
MATERIALS
AND METHODS
RNA Preparation Tissues obtained
used for RNA preparation from the Tumor Procurement
were surgical specimens Service at the Memorial
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
Genetic
Analysis
of Human
AT1
Sloan Kettering Cancer Center (New York, NY). RNA extracted using the acid guanidinium thiocyanate-phenol-chloroform method (22).
1117
was
1% SDS, 5x Denhardt’s solution, and 100 pg/ml herring sperm DNA, and washed at a final stringency of 0.3x SSC, 0.5% SDS. at 65 C.
Primers
DNA Preparation
Primers were synthesized on an Applied Biosystems (Foster City, CA) model 391 EP DNA synthesizer. The sequences and uses of all oligonucleotides are given in Fig. 1.
For direct sequencing of PCR products, the fragments of interest were first isolated from agarose gels by electrophoresis onto NA45 paper (Schleicher & Schuell, Keene, NH). Plasmid DNA was prepared by the boiling method and further purified using Strataclean resin (Stratagene, San Diego, CA). Plasmids and PCR products were both sequenced using the dideoxy chain termination method and modified T7 DNA polymerase (Sequenase Version II, U.S. Biochemical Corp.) (24) using templates denatured in the presence of dimethylsulfoxide (25).
RT-PCR RT-PCR was performed as previously described (23) using 0.5 pg total RNA as template. The conditions used for all RTPCRs were a denaturation step at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 72 C for 1 min, with the extension time increasing by 5 set/cycle; the number of cycles performed in the various reactions are given in the figures. Negative control reactions were performed in parallel but differed in that no reverse transcriptase was added at the cDNA synthesis step. PCR Screening
of cDNA
Libraries
Human lung and liver cDNA libraries screened were purchased from Clontech Laboratories, Inc. (Palo Alto. CA), and an additional human liver cDNA librarv was a kind’gift of Frank Gonzalez (NCI, Bethesda, MD). Ail were in the
Kathleen
M. Curnow*,
Leigh
Pascoe,
and Perrin
C. Whitet
Division of Pediatric Endocrinology Cornell University Medical College New York, New York 10021
receptor (ATl) population of binding sites, whereas CGP 42112A and PD 123177 have a greater affinity for AT2 sites (1, 2). AT1 is the predominant subtype in vascular smooth muscle and human uterus, whereas AT2 is the subtype predominantly expressed in the adrenal medulla and brain. Other tissues such as the adrenal cortex, kidney, and rat uterus have both subtypes of binding sites (3). In addition to being defined by binding studies, the two subtypes of receptor are distinguished by their sensitivity to inactivation by dithiothreitol (DTT), in that the AT1 receptor is inactivated by millimolar concentrations of DTT, whereas the AT2 receptor is resistant to DTT inactivation (4). Although it has proven difficult to purify these receptors, bovine and rat cDNA encoding AT1 were recently isolated by expression screening (5-7). AT1 is predicted to have seven transmembrane segments, thus sharing the common structural frame of the guanine nucleotidebinding regulatory protein (G protein)-coupled receptors. Because angiotensin II is an important effector for maintaining blood pressure, it seems likely that genetic polymorphisms in the receptor(s) for this hormone could be associated with the development of hypertension. As a step in investigating this hypothesis, we characterized cDNA and genomic clones encoding the human AT1 receptor.
Angiotensin II is a potent pressor hormone and a primary regulator of aldosterone secretion. It acts through at least two types of receptors termed AT1 and AT2. We analyzed cDNA and genomic clones encoding the human angiotensin II type-l receptor, ATl. The human AT1 gene was mapped to chromosome 3q by polymerase chain reaction analysis of DNA from a panel of human-hamster somatic cell hybrids. The predicted amino acid sequence is 95% identical to the corresponding rat and bovine receptors and 25% and 22% identical, respectively, to the receptors encoded by the RTA and MAS genes. Characterization of several human cDNA clones demonstrated the existence of two alternate 5’untranslated regions (UTRs) that contain a common initial sequence but differ by the presence or absence of an insertion of 84 base pairs. In the genomit sequence, the coding sequences are contained in a single exon, with an intron occurring in the 5’UTR at the position of insertion of the 84-base pair sequence. The exons encoding the alternate 5’UTRs are located at least 3.8 kilobases away from the exon encoding the protein. Reverse transcription-polymerase chain reaction analysis showed that both forms of 5’-UTR are present in approximately equal abundance in a range of tissues expressing ATl. The reagents developed in this work may be useful in testing the hypothesis that genetic variations in angiotensin II receptor function are associated with a tendency to develop hypertension. (Molecular Endocrinology 6: 1113-l 118, 1992)
RESULTS Human AT1 cDNA A portion of the human AT1 cDNA sequence was obtained by reverse transcription of total adrenal RNA and subsequent polymerase chain reaction (PCR) amplification. The pair of primers used for this purpose (5 and 8 in Fig. 1) was designed on the basis of the published rat and bovine AT1 cDNA sequences (5, 6) such that the sequences chosen encoded stretches of amino acid identity, but more particularly, corresponded to stretches of amino acids that were encoded by the least possible number of codons. Based on the sequence of the PCR-amplified product, two additional primers (6 and 7) were synthesized for the purpose of screening available liver and lung human cDNA libraries by PCR in conjunction with a primer corresponding to
INTRODUCTION Angiotensin II exerts a wide variety of physiological effects on the cardiovascular, endocrine, and central and peripheral nervous systems by binding to specific receptors. Two subtypes of cell surface receptor have been defined by radioligand-receptor binding studies using the peptide antagonist CGP 42112A and the nonpeptide antagonists DuP 753 and PD 123177. DuP 753 has a greater affinity for the type-l angiotensin II 0888-8809/92/l 113-l 118$03.00/0 Molecular Endocmology Copynght 0 1992 by The Endocrine
Society
1113
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
MOL END0.1992 1114
Vol6 No. 7
X-gtl 1 vector sequence. Collated cDNA and predicted amino acid sequences of human AT1 are presented in Fig. 1. The protein sequence of human AT1 is the same length as the bovine and rat proteins (359 amino acids) and shares 95% identity with each. Two different sized PCR products were amplified from a human liver cDNA library using primer 6. They contained the same coding sequence and the same 5’untranslated region (UTR) until position -47, where the sequences diverged; the longer sequence contained an apparent insertion of 84 base pairs (bp) as compared to the shorter. The sequence 5’ of the insertion was notably GC rich (36/41 bp). Chromosomal
Fig. 1. Nucleotide and Deduced Amino Acid Sequences of Human AT1 cDNA and Genomic DNA The insertion sequence in the 5’-UTR is indicated by lower case letters, with an upstream ATG that is in a good context for initiation and a subsequent in-frame stop codon printed in bold and underlined. The genomic intron sequence is printed above the cDNA sequence starting at position -47, and the HindIll site in the 3’-LtTR is bold and underlined. The sequences of the oligonucleotides used in this study are underlined, and arrows beside the numbers identifying each primer indicate whether the primers are sense or antisense. (Sequences have been deposited in Genbank under accession number M93394.)
Localization
DNA
1,066bpt
Mapping of the AT1 Gene to Human Chromosome 3 Specific amplification of the human, and not the hamster, AT1 gene was achieved using oligonucleotides 5-9. A representative set of the PCRs performed on the human-hamster cell hybrid panel is shown. The chromosomal content of each cell line is given in Table 1, which summarizes the complete set of data. Those hybrids containing human chromosome 3 are indicated by an asterisk, and it is noted that cell line 423 contains human chromosome 3 only. Fig. 2.
Table
1. Chromosomal Localization of Human AT1
. .
60 . . 15 .
.
. . D.
.
55.
. . .
. . . E
. . . . .
.
45
.
.
.
.
15
. .
. .
. .
.
.
.
. . . .
.a5
. .
. .
. . .
.
.
. . .
10
. .
. 25 .
A
. .
.
9.6 6.6
. . 45 .
E . . D
.
15
. .
.
.
. .
:
.
. 10 .
45
.
. .
5
.
. .
. .
.
B f 2 E 6.6 2
4.4
. .
2.3
AT1 sequences were amplified (as shown in Fig. 2) from human-hamster hybrid cell lines 423, 860,507, and 1079. This is consistent with a location of human chromosome 3 for the AT1 gene, as indicated by the arrow. Whole chromosomes are contained in cell lines indicated by closed circles, while numbers are given where that percentage of the cell population contains the noted chromosome. D, Deletion at ~15.1-15.2.
2.3 2.0
-
Fig. 3. Genomic Blot Analysis of AT1 Restriction digests of human genomic DNA were probed with 32P-labeled cDNA (the RT-PCR product used was generated using primers 5-8). A, The same DNA was digested with a number of different enzymes; B, DNA from 10 different individuals was digested with Mspl. The DNA in lane 10 of this analysis corresponds to that used in A.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
Genetic
Analysis
of Human
AT1
1115
quently localized to 3q21-3q25 using a somatic cell hybrid regional mapping panel (27) (data not shown). Structure
of the Human Genomic
Exon Usage
A range of tissueswhich express AT1 were examined for the presenceof 5’UTRs with or without the insertion sequence.This analysiswas done by reverse transcription PCR (RT-PCR) (Fig. 4) and does not show preferentialusageof either form of 5’-UTR in any one tissue. Genetic
Polymorphism
In a preliminaryexperiment to identify informative polymorphismsin the vicinity of the AT1 gene, a blot bearingMspl digestsof genomicDNA from 10 unrelated donorswas probedwith a PCR product containingpart of the coding sequence of human AT1 cDNA. This analysis, shown in Fig. 3B, identified a polymorphic Mspl site. The two allelic forms were present at frequenciesof 0.85 (for the 3.4-kb fragment)and 0.15 (for the 4.3-kb fragment).
DISCUSSION Structural
Features
-
50 bp
Gene
A blot of human genomic DNA digested with various restriction enzymes was hybridized with part of the human AT1 cDNA (Fig. 3A). Only one band was detected in each digest (noneof the enzymes chosencut within the probe sequence),indicatingthat there were no relatedgenes of sufficient homologyto cross-hybridize under these conditions. Since the chromosomal locationof the gene had been determined,and a Hindlll site had been identifiedin the 3’-UTR of the cDNA, we screeneda chromosome3 library cloned in the HindIll site of Charon 21A. From the 90,000 recombinants screened, two hybridizing clones were isolated, each containinga 5.1-kilobase(kb) insert. The 5.1-kb HindIll fragment was subclonedinto pBluescriptKS+, and sequence analysisshowed that the genomic and cDNA coding sequenceswere identical down to the HindIll site in the 3’-UTR, 165 bp after the terminationcodon. The entire coding sequence and 3’-UTR were both contained on a singleexon. As would be predicted by the identificationof two alternate5’-UTRs in the cDNAs characterized, an intron spliceacceptor sequencewas located in the 5’-UTR at position -47, the point of divergenceof the translated sequences.No further 5’UTR sequenceswere found on the !%I-kb fragment, indicating that the intron separating them from the coding exon is more than 3.8 kb in length. S-UTR
A
of Human AT1
The cDNA and genomic clones described herein are believedto encode AT1 because the predicted amino
Fig. 4. RT-PCR and Hybridization Analysis Identifying Alternate 5’-UTR Usage in a Range of Tissues A, The positions of the oligonucleotides used for PCR amplification of reverse-transcribed RNA are indicated below a schematic representation of the 5’-end of the AT1 cDNA. 6, The left column shows the ethidium bromide-stained gels of RT-PCR products obtained on various tissues, using the primers and cycle numbers indicated at the left of each gel. The result obtained in a parallel RT-PCR using 02-microglobulin primers (23) is shown at the bottom as an indication of the relative amount and quality of the RNA template used in each reaction. Negative control reactions were performed in parallel on liver RNA, where no reverse transcriptase was added at the cDNA synthesis step, and positive control reactions were performed using the 5’-end PCR products amplified from the liver cDNA library as templates. Note that the positive control template used for PCR with oligonucleotides l-4 does not contain the insertion sequence. In the center column, the blots of the gels at left were hybridized with digoxigenin-labeled oligonucleotide 3, and these blots were then reprobed with an insertion sequence oligonucleotide (primer 2) labeled with [“‘PI, demonstrating that this sequence was present in the 279-bp RT-PCR product in all the tissues studied.
acid sequenceis 95% identicalto bovine and rat AT1 , and no cross-hybridizing genes are detected on genomit blots. The protein sequenceof humanAT1 shares the characteristicfeature of all G protein-coupledreceptors in that seven putative transmembraneregionsare readily identified. As comparedwith the rat and bovine receptors, none of the amino acids believed to play a role in determining the structure and function of this moleculeare altered (5-8). These includefour cysteine residuesat positions18, 101, 180, and 274, and three potential asparagine-linkedglycosylation sites at positions 4, 176, and 188, all of which are located in predicted extracellular domains.Ser and Thr residues at positions 136, 141, 326, 328, 331, 338, 342, 347, and 348 are also conserved, and these amino acids located on the intracellularsurface are suggestedto be sites of regulatory phosphorylation. Also conserved were the Pro residuesin transmembranedomainsIV, V, VI, and VII, and the Asp-Arg-Tyr motif at the intracellularborder of transmembranedomainIll. In general, the best conservation of sequencewas found in the
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
MOL 1116
ENDO.
1992
first two intracellular loops and the transmembrane domains. In addition to ATl, three other putative G proteincoupled receptors which may play a role in mediation or modulation of the angiotensin response have been identified by gene cloning (g-12). When injected into Xenopus oocytes, both the MAS and MRG mRNAs markedly increase the electrophysiological response of the endogenous Xenopus oocyte angiotensin receptors to angiotensin peptides. The RTA gene has not yet been found to share any functional similarity with MAS and MRG but is related structurally. The predicted AT1 amino acid sequence was aligned with those of RTA, MAS, and MRG (Fig. 5). Whereas the latter three receptors are about 33% identical to each other in sequence, AT1 is 25% identical to RTA, 22% identical to MAS, and could not be directly aligned with MRG. The 26 positions that were completely conserved and the 54 positions that were functionally conserved among the four receptors were clustered in the transmembrane segments and the second and Cterminal intracellular domains. The remaining intracel-
Vol6
lular and extracellular domains showed limited homology, and the third intracellular domain of AT1 differed considerably, being at least 13 amino acids longer than those of the MAS-related gene family. Overall, the comparison suggests that the latter genes belong to their own separate subfamily. Their physical and functional relationships with the high affinity angiotensin receptors remains unclear but are now amenable to study through cotransfection experiments with ATl. Functional
Significance
of Alternative
5’-UTRs
Transcripts with and without the 84-bp insertion are present at roughly equal levels in all tissues examined that express ATI. An optional 88-bp insertion with a 75% identical sequence has been documented in rat cDNA at an analogous position in the 5’-UTR (-52) (6), and a similar sequence is present in the bovine 5’-UTR, but it is not known whether transcripts without the insertion exist in cattle (5). The relatively high degree of sequence identity suggests a functional role for the insertion. Although there is no open reading frame continuous with the AT1 coding sequence in the insertion in any of the three mammalian species examined, an AUG in a good context for initiation (13), followed by an in-frame stop codon a few codons downstream, is consistently present. Initiation and termination of translation of an mRNA decreases the efficiency of initiation from a downstream AUG (14), thus alternate splicing in the 5’-UTR may be a mechanism for regulating AT1 receptor levels. Polymorphism
Fig. 5. Comparison of the Predicted Amino Acid Sequence of AT1 with Sequences of Three Related Proteins Encoded by the RTA, MAS, and MRG Genes Gaps introduced to optimize the alignment are indicated by dashes. Residues completely conserved in all four proteins are marked with asterisks; residues that are functionally conserved are marked with crosses, and residues that are conserved in three of four proteins are marked with dots. Predicted transmembrane segments are underlined.
No. 7
in the AT1 Gene
Two allelic forms of the AT1 receptor were detected by the restriction enzyme Mspl. Identification of such polymorphisms may be useful markers for genetic analysis of patients with hypertension, as variations in angiotensin II receptor function may play a role in development of this disease. This may be mediated through effects on microvascular growth (15-I 7), vascular smooth muscle cell growth (18), or synthesis of extracellular matrix within vessel walls (19). Altered angiotensin receptor affinities have been suggested to be responsible for the exaggerated pressor and aldosterone responses to angiotensin II in some patients with low renin essential hypertension (20), and could also be responsible for the blunted response to angiotensin II observed in patients classified as having nonmodulating essential hypertension (21). Since this latter form of hypertension tends to occur in families, these families could be examined to determine whether their hypertension shows concordance with an altered AT1 genotype.
MATERIALS
AND METHODS
RNA Preparation Tissues obtained
used for RNA preparation from the Tumor Procurement
were surgical specimens Service at the Memorial
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 30 March 2015. at 13:03 For personal use only. No other uses without permission. . All rights reserved.
Genetic
Analysis
of Human
AT1
Sloan Kettering Cancer Center (New York, NY). RNA extracted using the acid guanidinium thiocyanate-phenol-chloroform method (22).
1117
was
1% SDS, 5x Denhardt’s solution, and 100 pg/ml herring sperm DNA, and washed at a final stringency of 0.3x SSC, 0.5% SDS. at 65 C.
Primers
DNA Preparation
Primers were synthesized on an Applied Biosystems (Foster City, CA) model 391 EP DNA synthesizer. The sequences and uses of all oligonucleotides are given in Fig. 1.
For direct sequencing of PCR products, the fragments of interest were first isolated from agarose gels by electrophoresis onto NA45 paper (Schleicher & Schuell, Keene, NH). Plasmid DNA was prepared by the boiling method and further purified using Strataclean resin (Stratagene, San Diego, CA). Plasmids and PCR products were both sequenced using the dideoxy chain termination method and modified T7 DNA polymerase (Sequenase Version II, U.S. Biochemical Corp.) (24) using templates denatured in the presence of dimethylsulfoxide (25).
RT-PCR RT-PCR was performed as previously described (23) using 0.5 pg total RNA as template. The conditions used for all RTPCRs were a denaturation step at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 72 C for 1 min, with the extension time increasing by 5 set/cycle; the number of cycles performed in the various reactions are given in the figures. Negative control reactions were performed in parallel but differed in that no reverse transcriptase was added at the cDNA synthesis step. PCR Screening
of cDNA
Libraries
Human lung and liver cDNA libraries screened were purchased from Clontech Laboratories, Inc. (Palo Alto. CA), and an additional human liver cDNA librarv was a kind’gift of Frank Gonzalez (NCI, Bethesda, MD). Ail were in the
