Am. J. Hum. Genet. 51:1078-1083, 1992

Structure and Chromosomal Localization of the Human Antidiuretic Hormone Receptor Gene Anita Seibold, Phillippe Brabet, Walter Rosenthal, and Mariel Birnbaumer Department of Cell Biology, Baylor College of Medicine, Houston

Summary Applying a genomic DNA-expression approach, we cloned the gene and cDNA coding for the human antidiuretic hormone receptor, also called "vasopressin V2 receptor" (V2R). The nucleotide sequence of both cloned DNAs provided the information to elucidate the structure of the isolated transcriptional unit. The structure of this gene is unusual in that it is the first G protein-coupled receptor gene that contains two very small intervening sequences, the second of which separates the region encoding the seventh transmembrane region from the rest of the open reading frame. The sequence information was used to synthesize appropriate oligonucleotides to be used as primers in the PCR. The V2R gene was localized by PCR using DNA from hybrid cells as template. The gene was found to reside in the q28-qter portion of the human X chromosome, a region identified as the locus for congenital nephrogenic diabetes insipidus.

Introduction The vasopressin V2 receptor (V2R) is expressed in the

chief cell of the kidney collecting duct and regulates urine osmolality in response to antidiuretic hormone, arginine vasopressin (AVP). Occupancy of the receptor induces adenylyl cyclase stimulation (Orloff and Handler 1962; Robertson 1974; Harris et al. 1990). The subsequent activation of cAMP-dependant protein kinase promotes, via phosphorylation, the recruitment of preformed water pores into the luminal membrane of the cell (Orloff and Handler 1967), thereby increasing water permeability. Lack of response to AVP in the kidney, such as it is observed in patients with congenital nephrogenic diabetes insipidus (NDI), a sex-linked genetic disease, has been ascribed to mutations in the V2R gene. The disease is characterized by (a) an invariably low concentration of urine, leading to the other two key symp-

Received May 19, 1992; final revision received July 13, 1992. Address for correspondence and reprints: Mariel Birnbaumer, Ph.D., Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. i) 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/S105-0017$02.00

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toms, polyuria and polydipsia, and (b) an increased blood osmolality if the water intake is not sufficient (Culpepper et al. 1983). Genetic linkage studies have localized the NDI locus to the q28-qter region of the X chromosome (Kambouris et al. 1988; Knoers et al. 1988). Cotransfection of human genomic DNA with the thymidine kinase gene into Ltk- cells generated a primary transformant cell line expressing the human V2R gene (Birnbaumer et al. 1990). Secondary and tertiary transformed cell lines were derived from it, and a genomic library was prepared from the tertiary transformant. Analysis of the phages containing human DNA, for their ability to express the receptor in transfected cells, allowed the isolation of the V2R gene and, subsequently, of the corresponding cDNA (Birnbaumer et al. 1992). We report here both the existence of intervening sequences that interrupt the open reading frame (ORF) of the V2R gene and the localization of this gene to the q28-qter region of the X chromosome, by applying PCR to genomic DNA from a panel of characterized somatic cell hybrids. The latter finding strengthens the possibility that a mutation in the V2R gene is the underlying cause of NDI (Bichet et al. 1988; Jans et al. 1990).

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oligonucleotide primers synthesized by the phosphoroamidite method, with a 391 DNA Synthesizer from Applied Biosystems. Primer 33 is a l9mer and has the following composition: 5'GTGGGTGTCCGGATGGGGGC 3'. Primer 28 is a 27mer of the sequence 5'TCATGGCCAGGATCATGTAGGAGGAGG 3'. Prior to PCR analysis the genomic DNA was sheared by passage through 18-gauge and 22-gauge needles. The PCR reaction was performed in a total volume of 100 Rl containing 100 ng of genomic DNA, 10 gl of 10 x PCR buffer (100 mM Tris-HCI pH 8.3,500 mM KCI, 15 mM MgCl2, and 0.01% gelatin), 100 pmol ofeach primer, 0.2 mM of each dNTP, 10 pl dimethylsulfoxide (DMSO) (Pierce), and 2.5 units of Taq polymerase (Cetus). Mineral oil was overlaid, and the reactions were cycled 48 times through the following program: 45 s at 96°C, 2 min at 67°C, and 3 min at 720C. The reaction products were precipitated with an equal volume of 2 M ammonium acetate (pH 5.2) and 2.5 vol of ethanol and were analyzed directly or after digestion with SmaI by electrophoresis in 1.5% Sea Kem agarose (FMC) gels containing 1 gg ethidium bromide/ml in 40mM Tris-acetate and 1 mM EDTA.

Material and Methods DNA Isolation

Genomic DNA was isolated by the method of DiLella and Woo (1987). Cell Lines

Genomic DNA extracted from hamster/human hybrid cells lines was used as template to localize the V2R gene by applying the PCR technique (Saiki et al. 1988). The cells analyzed were line 4.12, which contains a fragile-X chromosome (Nussbaum et al. 1983); cell lines Q 1Z and Q 1N (Warren et al. 1990); and Y 162-Aza (Suthers et al. 1990), which contain different portions of the Xq28 band as referenced in the text and diagrammed in figure 1. DNA from these cells was a gift of Dr. Orly Reiner of the Department of Molecular Genetics, Baylor College of Medicine, Houston. PCR Analysis

Genomic DNA from the hybrid cell lines was analyzed for the presence of the V2R gene by PCR using

Mapping within Xq28 1

Mb j

2

1

3

4

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Y 1 62-Aza FRAXA

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DXS296 DXS295

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IDS DXS304

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-V DXS52 DXS 15

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X qt er F8 DXS I 1 5 DXS64

DXS305 GABRA3 Figure I Diagram of mapping within the Xq28-Xqter region of the human chromosome. The diagram is an adaptation of the one recently published by Poutska et al. (1991) and is almost identical to the data reported by Traupe et al. (1992). The distances (in Mb) and the division of subregions in Xq28 are information transmitted by Dr. S. Warren (personal communication). The hybridization markers DXS1 15, DXYS64, F8 (clotting factor 8), G6PD (glucose 6-phosphate dehydrogenase), RCP and GCP (red and green eye color pigments), DXS15, GABRA3 (gaba A3 receptor subunit), DXS305, DXS304, and DXS296 were located according to the protocol of Poutska et al. (1991). DXS295 and IDS (iduronate-2-sulfatase) were located according to Suthers et al. (1991) and Wilson et al. (1991). DXS52 was located according to the protocol of Oberle et al. (1985). FRAXA (fragile-X syndrome) was located according to the protocol of Suthers et al. (1990).

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Cloning of the V2R Gene

A murine hybrid cell line developed in our laboratory was used to clone the V2R gene by expression (Birnbaumer et al. 1990). A tertiary transformant (HTB-3) cell line expressing the human V2R contained AluI repeats detectable only by a speciesspecific PCR (Lichter et al. 1990). Phages containing human sequences were isolated from a lambda EMBL3 library prepared with genomic DNA from HTB-3 cells and transfected into Ltk- cells. The cells were assayed for AVP-stimulated adenylyl cyclase activity by following our published protocols (Birnbaumer et al. 1990). A positive phage (lambda 14) was identified by this methodology, and, on digestion with BamHI, a 2.2-kb fragment of lambda 14 that expresses the V2R in transfected cells and that exhibits transcriptional activity on its own was identified (Birnbaumer et al. 1992). Screening of a lambda gtlO cDNA library prepared from cells expressing high levels of receptor with the 2.2-kb genomic fragment as a probe identified a 1.7kb cDNA. Both the cDNA and the gene were sequenced in full by the dideoxynucleotide chain termination sequencing method of Sanger et al. (1977). An identical cDNA was isolated from a human kidney lambda gtlO cDNA library (Birnbaumer et al. 1992). Results and Discussion

As illustrated in figure 2, comparison between the sequence of the 2.2-kb genomic transcriptional unit of the human V2R and the sequence of the cDNA iso-

BamHI

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PCR Product 2.2-33 50 nt

H-l

2.2-28

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2.2-28

Diagram of the structure of the gene encoding the Figure 2 human V2R. The structure depicted is based both on the sequence of the complementary DNA isolated from human kidney and on the sequence of the transcriptional unit of the V2R isolated from the tertiary transformant cell HTB-3. The location of the 20 2-mer oligonucleotides 2.2-33 (sense) and 2.2-28 (antisense) is indicated. The lower portion illustrates the position of the diagnostic SmaI restriction site in the PCR product obtained from the V2R gene with the oligonucleotides 28 and 33. Unblackened boxes indicate nonexpressed exons and blackened boxes indicate expressed exons.

lated from the human kidney and cell libraries (Birnbaumer et al. 1992) revealed the existence of three expressed exons framing two intervening sequences. Both introns are unusually short and are flanked by the consensus donor/ acceptor splice sequences described by Breathnach et al. (1977). The first expressed exon is very short (96 bp) and contains the ATG coding for the first methionine. The first intervening sequence, 360 bp long, interrupts the codon that corresponds to the ninth amino acid of the receptor sequence. The second exon contains 840 bp and is followed by the 104-bp-long second intron. The third exon contains 204 bp of ORF followed by 460 bp of 3' untranslated region that terminates at the splicing and polyadenylation signal TAATAAAA. The second expressed exon encodes (approximately 80% of the sequence of the receptor, up to the beginning of the seventh transmembrane region. The sequences of the exon/intron splice junctions are presented in figure 3. Cloning of the genes encoding G protein-coupled receptors has revealed that they are a heterogeneous family in regard to their exon/intron structure. While the first genes cloned were intronless (,B-adrenergic receptors), the cloning of the muscarinic acetylcholine receptors revealed the existence of intervening sequences in the nonexpressed portions of the gene, usually upstream from the beginning oftranslation. Many other G protein-coupled receptor genes contain intervening sequences. Multiple large introns may interrupt the ORF, as is the case in the calcitonin receptor gene; or, as found in genes encoding the receptors of glycoprotein hormones LH, TSH, and FSH, several introns may be present in the segment encoding the large amino-terminal extracellular portion of the protein, while the segment encoding the seven transmembrane regions is intronless. So far, the size and location of the introns of the human V2R gene are unique in that the second exon encodes only six of the seven transmembrane regions, the seventh region being encoded by exon 3. The 2.2-kb genomic fragment encoding the V2R was identified as a fully functional transcriptional unit, by cotransfection with the herpes simplex virus tk gene and by expression in LV2.E4 cells (Birnbaumer et al. 1992). The putative TATAA box 100 bp upstream from the predicted beginning of translation of this mRNA is probably crucial for the expression of this fragment, although we do not know whether the same sequence is responsible for expression of the V2R gene in the human genome.

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Vasopressin Receptor Gene Structure Intron I 360 nt

CACCACTTCCG gtaaggcttgcccc---

---ccgactcctgcccag CTGTGCCTGGGCAT

Intron II 104 nt

ACCTCTGGAAG gtgggtgtagccgt---

---cctccacctccacag GGGCGCCCTTTGTG

Figure 3 Sequence of the exon/intron splice junctions for the intervening sequences that interrupt the human V2R gene. Lowercase letters identify the intron sequences.

To determine the chromosomal localization of the ADH receptor gene in the human genome, we were guided by the accumulated data on the genetic linkage of NDI to the long arm of the X chromosome (Kambouris et al. 1988; Knoers et al. 1988) and by reports that somatic cell hybrids carrying the human locus for NDI express functional vasopressin receptors (Jans et al. 1990). Thus we obtained genomic DNA isolated from the cell lines described in Material and Methods, and we carried out PCR analysis with the methodology and primers described in Material and Methods and located as shown in figure 2. As illustrated in figure 4A, under conditions suitable for amplification the hamster genomic DNA was a poor template for the receptor-specific primers, while human genomic DNA was efficiently amplified. The mixture of human and hamster DNA was employed to rule out an inhibition of the PCR by hamster DNA. The low amount of product obtained with hamster DNA was a prerequisite for the detection, with minimal interference, of the human gene in the somatic cell hybrids. Amplification of the human V2R with primers 33 and 28 yielded the predicted product of 466 bp. The identity of the PCR products was verified by digestion with SmaI, followed by analysis of the fragments by gel electrophoresis. The digestion yielded the predicted fragments of 289 and 159 bp; the additional 18 bp (see fig. 2) SmaI fragment was not detectable in our analysis. As illustrated in figure 4B, the same product was obtained when the template of the reaction was human genomic DNA or DNA extracted from the 4.12 and Q1Z cell lines, which are known to contain a complete fragile X human chromosome (Nussbaum et al. 1983) and the entire human Xq28-qter region (Warren et al. 1990), respectively. As illustrated in figure 4C, two other somatic cell hybrids provided more precise information regarding

the localization of the V2R gene within the q28-qter region of the X chromosome. Analysis of the Q 1N cell (Warren et al. 1990; Traupe et al. 1992) with polymorphic markers revealed a gap in the human Xq28 region present in this cell. Since the St14 (or DX5S2) marker that has been used to identify the NDI locus by genetic linkage analysis (Kambouris et al. 1988; Knoers et al. 1988) is absent in these cells, it was of particular interest to see whether the V2R gene was present. PCR analysis revealed that, although the marker is absent, the V2R receptor gene is present in the human DNA of the Q 1N hybrid (fig. 4C). DNA from the somatic cell hybrid Y1 62-Aza (Suthers et al. 1990; Traupe et al. 1992), which lacks the region hybridizing to St14, as well as the distal portion of Xq28 (see fig. 1), failed to serve as a template in our PCR reaction, indicating the absence of the V2R gene. We also tested, by PCR, the yeast artificial chromosome (YAC) XY845 isolated from the YAC libraries constructed by Wada et al. (1990; also see Abidi et al. 1990). The human DNA insert of XY845, approximately 0.5 Mb long, is known to contain part of the region that hybridizes with the St14/DX552 polymorphic marker. However, this YAC did not contain the V2R gene (fig. 4C). Figure 1 summarizes results shown in figure 4 and compares them with the published data on the presence of different X-chromosome markers in the hybrid cell lines we analyzed. Our results reveal that the gene for the V2R and the St14/DXSS2 polymorphic marker do not reside in the same locus but that they do exist in the vicinity of each other. Although our findings are consistent with the hypothesis that a mutation in the receptor gene is the cause of NDI, they prove that the RFLP detected with the marker is independent from the receptor gene itself. The uncovered information about the gene structure and chromosomal location should facilitate the comparison be-

Siebold et al.

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al. 1990). One is left to speculate whether the RFLP of Stl4 associated with NDI is merely coincidental. More detailed mapping of the Xq28 region and the identification of NDI-causing mutations (coding vs. flanking regions of the V2R gene) will clarify this quandary in the near future.

Acknowledgments

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The authors would like to acknowledge Dr. David Nelson from the Institute of Molecular Genetics at Baylor College of Medicine for helpful discussions and Dr. Stephen Warren from Emory University for sharing unpublished information with our laboratory. This research was supported in part by NIH grants DK-21244 and DK-27685 to M.B., by an INSERM postdoctoral fellowship to P.B., and by a DFGHeisenberg Award to W.R.

References Abidi FE, Wada M, Little RD, Schlessinger D (1990) Yeast artificial chromosomes containing human Xq24-Xq28 DNA: library construction and representation of probe

Localization of the human V2R gene by PCR analyFigure 4 sis. A, Samples (100 ng) of Chinese hamster and human genomic DNAs used as template for the PCR reaction. B, Analysis of PCR products before and after SmaI digest. The templates used were 100 ng of Chinese hamster and human genomic DNA, as well as genomic DNA extracted from the cell clones 4.12 and Q1Z. C, Analysis of PCR products obtained with human genomic DNA as template, as well as with DNA from the cell clone QlN that lacks the estimated 2-3 Mb of DNA in the Xq28-qter region as indicated by the diagram in fig. 1 (see text). DNA from the cell clone Y162Aza, known to have a deletion larger than 4 Mb in the q28 region closest to the telomere (see text), failed to yield any product. Analysis of the YAC XY845 that contains approximately 0.5 Mb of the region positive for the polymorphic marker DXS52 or Stl4 failed to detect the V2R gene in this fragment.

tween the V2R locus in normal subjects and that in individuals affected by NDI. Reports describing the Stl4 marker as a sequence family disperse in the Xq28qter explains the polymorphism of this marker (Feil et

sequences. Genomics 7:363-376 Bichet DG, Razi M, Lonergan M, Arthus M-F, Papukna V, Kortas C, Barjon JN (1988) Hemodynamic and coagulation responses to 1-desamino[8-D-arginine] vasopressin in patients with congenital nephrogenic diabetes insipidus. N EngI J Med 318:881-887 Birnbaumer M, Hinrichs V, Themmen APN (1990) Development and characterization of a mouse cell line expressing the human V2 vasopressin receptor-gene. Mol Endocrinol 4:245-254 Birnbaumer M, Seibold A, Gilbert S, Ishido M, Barberis C, Antaramian A, Brabet P, et al (1992) Molecular cloning of the human antidiuretic hormone receptor. Nature 357: 333-335 Breathnach R, Mandel JL, Chambon P (1977) Ovalbumin gene is split in chicken DNA. Nature 270:314-319 Culpepper RM, Hebert SC, Andreoli TE (1983) Nephrogenic diabetes insipidus. In: Stanbury JB, Wyngaarden JB, Fredrickson DS, Goldstein JL, Brown MS (eds) The metabolic bases of inherited disease, 5th ed. McGrawHill, New York, pp 1867-1888 DiLella AG, Woo SLC (1987) Cloning large segments of genomic DNA using cosmid vectors. Methods Enzymol 152:199-212 Feil R, Palmieri G, d'Urso M, Heilig R. Oberle I, Mandel JL (1990) Physical and genetic mapping of polymorphic loci in Xq28 (DXS15, DXS52, and DXS134): analysis of a cosmid clone and a yeast artificial chromosome. Am J Hum Genet 46:720-728 Harris HW, Handler JS, Blumenthal R (1990) Apical membrane vesicles of ADH-stimulated toad bladder are highly water permeable. Am J Physiol 258:F237-F243

Vasopressin Receptor Gene Structure Jans DA, van Oost BA, Ropers H, Fahrenholz F (1990) Derivatives of somatic cell hybrids which carry the human gene locus for nephrogenic diabetes insipidus (NDI) express functional vasopressin renal V2-type receptors. J Biol Chem 265:15379-15382 Kambouris M, Diouhy SR, Trofatter JA, Conneally PM, Hodes ME (1988) Localization of the gene for X-linked nephrogenic diabetes insipidus. AmJ Med Genet 29:239246 Knoers N, van der Hayden H, van Oost BA, Monnens L, Willems J, Ropers HH (1988) Linkage of X-linked nephrogenic diabetes insipidus with DXS52, a polymorphic DNA marker. Nephron 50:187-190 Lichter P, Ledbetter SA, Ledbetter DH, Ward DC (1990) Fluorescence in situ hybridization with Alu and Li polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines. Proc Natl Acad Sci USA 87:6634-6638 Nussbaum RL, Airhart SD, Ledbetter DH (1983) Expression of the fragile (X) chromosome in an interspecific somatic cell hybrid. Hum Genet 64:148-150 Oberle I, Camerino G, Heilig R, Grunebaum L, Cazenave JP, Crapanzano C, Mannucci PM, et al (1985) Genetic screening for hemophilia A (classic hemophilia) with a polymorphic DNA probe. N Engl J Med 312:682-686 OrloffJ, Handler JS (1962) The similarity of effects of vasopressin, adenosine 3',5'-monophosphate(cyclic AMP) and theophylline on the toad bladder. J Clin Invest 47:11541160 (1967) The role of adenosine 3',5'-phosphate in the action of antidiuretic hormone. Am J Med 42:757-768 Poutska A, Dietrich A, Langenstein G, Toniolo D, Warren S, Lehrach H (1991) Physical map of human Xq27-qter: localizing the region of the fragile X mutation. Proc Natl Acad Sci USA 88:8302-8306

1083 Robertson GL (1974) Vasopressin on osmotic regulation in man. Annu Rev Med 25:315-342 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491 Sanger F, Nicklen S, Coulson AB (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 Suthers GK, Hyland VJ, Callen DF, Oberle I, Rocchi M, Thomas NS, Morris CP, et al (1990) Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines. Am J Hum Genet 47:187195

Suthers GK, Oberle I, NancarrowJ, Mulley JC, Hyland VJ, Wilson PJ, McCure J, et al (1991) Genetic mapping of new RFLPs at Xq27-q28. Genomics 9:37-43 Traupe H, van den Ouweland AMW, van Oost BA, Vogel W, Vetter U, Warren ST, Rocchi M, et al (1992) Fine mapping of the human-byglycan (BGN) gene within the Xq28 region employing a hybrid cell panel. Genomics 13: 481-483 Wada M, Little RD, Abidi F, Porta G, Labella T, Cooper T, Della Valle G, et al (1990) Human Xq24-Xq28: approaches to mapping with yeast artificial chromosomes. Am J Hum Genet 46:95-106 Warren S, Knight SJL, Peters JF, Stayton CL, Conzalez GG, Zhang F (1990) Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage. Proc Natl Acad Sci USA 87:3856-3860 Wilson PJ, Suthers GK, Callen DF, Baker E, Nelson PV, Cooper A, Wraith JE, et al (1991) Frequent deletions at Xq28 indicate genetic heterogeneity in Hunter syndrome. Hum Genet 86:505-508

Structure and chromosomal localization of the human antidiuretic hormone receptor gene.

Applying a genomic DNA-expression approach, we cloned the gene and cDNA coding for the human anti-diuretic hormone receptor, also called "vasopressin ...
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