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Linkage Mapping of the Angiotensin AT2 Receptor Gene (Agtr2)to the Mouse X Chromosome

LUTZ HEIN,* VICTOR J. DZAU,* AND GREGORY S. BARSH†,1

*Division of Cardiovascular Medicine and †Departments of Pediatrics and Genetics and Howard Hughes Medical Institute,Stanford University School of Medicine, Stanford, California 94305

Received June 19, 1995; accepted September 19, 1995

mouse is an important tool in mammalian genomics,Angiotensin II is a potent regulator of cardiovascu- because it can often provide more precise information

lar homeostasis and binds to two different G-protein- than cytogenetic mapping and because it provides acoupled receptors. While the type 1 receptor (AT1) me- reference landmark with which to evaluate previouslydiates the cardiovascular actions of angiotensin II, the existing mutations in mouse or human pedigrees. Herefunction of the recently cloned type 2 receptor (AT2) we report that Agtr2 maps to the proximal mouse Xremains unknown. We have cloned the mouse AT2 re- chromosome, discuss the implications for X chromo-ceptor gene (Agtr2) and determined its map position some evolution, and consider human diseases for whichby linkage analysis using an interspecific backcross

Agtr2 might be a candidate.(C57BL/6J1Mus spretus). Agtr2 is located on the prox-The genomic DNA for the mouse AT2 receptor wasimal mouse X chromosome between DXMit85 and

cloned from a 129/Sv mouse genomic library (Stra-DXMit49, in a region of conserved synteny with a parttagene, La Jolla, CA) by conventional plaque hybridiza-of the human X chromosome implicated in inheritedtion using the mouse cDNA (17) as a probe. Six differ-forms of premature ovarian failure. The mapping ofent phage clones were isolated, and the structures ofAgtr2 may expand a region of conserved synteny with

human Xq26 that includes Hprt. q 1995 Academic Press, Inc. three of these clones were determined by restrictionenzyme mapping (Fig. 1A). The presence of the Agtr2gene was confirmed by dideoxynucleotide sequencing

The renin–angiotensin system serves as an im- of exons 1, 2, and 3 (up to the ApaI site correspondingportant regulator of cardiovascular homeostasis. The to position 362 of the cDNA) (17), including the intron–effector hormone of this system, angiotensin II, inter- exon borders. The sequence was found to be identicalacts with two different receptor subtypes that are mem- to the one reported recently (8). The open reading framebers of the G-protein-coupled seven-transmembrane is contained entirely on the third exon (Fig. 1A), whichdomain receptor family (9, 15, 16, 21). Angiotensin type is preceded by two small exons. The longest phage1 receptors (AT1) mediate most of the cardiovascular clones isolated contained 7.5 kb of the 5*-flanking re-actions of angiotensin II, i.e., vasoconstriction, aldoste- gion and 9.5 kb of sequence 3 * to the receptor codingrone secretion, and growth stimulation of vascular region.smooth muscle (23). The AT1 receptor is a candidate The genetic map position of the Agtr2 gene was deter-gene for human essential hypertension (2), and it has mined in a mouse interspecific backcross ((C57Bl/6J 1been mapped to human chromosome 3 (22). No physio- Mus spretus)F1 1M. spretus; BSS panel) (20). To iden-logical function has been clearly established for the tify a restriction fragment length variant in the paren-angiotensin type 2 receptor (AT2). The AT2 receptor is tal strains of this backcross, a 450-bp segment in thewidely expressed during fetal development, whereas in 3*-untranslated region of the Agtr2 gene was chosenadult tissues its pattern of expression is restricted to for PCR amplification (Fig. 1A). The primer sequencesa few organs, e.g., brain, adrenal cortex, uterus, and were primer A (5*-AGT GCA AAC TGG CAT GGG-3 * )ovary (24). The gene encoding the AT2 receptor (Agtr2) corresponding to mouse Agtr2 cDNA residues 2227–has been assigned to the human X chromosome by so- 2244 and primer B (5*-AAA ACG CCT GGA ATC TGA-matic cell hybrid analysis (10) and by fluorescence in 3 * ) complementary to residues 2659–2676 (17). Aftersitu hybridization (4). Genetic linkage mapping in the amplification as described (13), a XmnI restriction site

was found to be present in the amplified region of the1 To whom correspondence should be addressed at B 271 Beckman C57BL/6J strain, but not in M. spretus (Fig. 1A). ThisCenter, Howard Hughes Medical Institute, Stanford University

restriction fragment variant was used to type 92 prog-School of Medicine, Stanford, CA 94305. Telephone: (415) 723-5035.Fax: (415) 723-5061. eny from the Jackson Laboratory BSS panel (20) (Fig.

369 GENOMICS 30, 369–371 (1995)0888-7543/95 $12.00

Copyright q 1995 by Academic Press, Inc.All rights of reproduction in any form reserved.

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1B). Agtr2 cosegregated perfectly with the intracis-ternal A-particle proviral locus Iapls1-18 (14), whichlies 6.6 { 3.3 cM away from the most proximal markeryet identified in the BSS cross, DXBir1 (Figs. 1B and2). To anchor the backcross map to that of the MouseChromosome Committee (7), the BSS panel was thentyped for three SSLP markers (DXMit26, DXMit49,and DXMit85). Primers for these markers (ResearchGenetics, Huntsville, AL) were informative for the BSSpanel (Fig. 1B) and revealed a map order and mapdistances (cM) of DXBir1, DXMit26–3.3 { 1.9–DXMit85–3.3 { 1.9–IaplsI-18, Agtr2–3.3 { 1.9–DXMit49 (Figs. 1B and 2). The map location of Agtr2lies in a region on the proximal X chromosome devoidof previously existing mouse mutations. Human homo-logs of genes in this region have been mapped to humanXp11 (which contains MAOA) or to human Xq26 (whichcontains HPRT) (1, 12) (Fig. 2). In the mouse, the mostdistal marker within the Maoa region is Araf (map

FIG. 2. Maps of the proximal region of the mouse X chromosomeposition 6.5), and the most proximal marker within theusing the data obtained from the interspecific BSS backcross (left)Hprt region is Lamp2 (map position 12) (Fig. 2). Thecompared with the cumulative locus map from the Chromosomehuman AGTR2 gene has been previously mapped to Committee (middle) and a cytogenetic map of the human X chromo-some (right). The genetic distances between markers on the BSSmap are presented in Haldane cM { SE. Markers that were typedin this manuscript are highlighted in boldface.

Xq22–q23 (4), which indicates that Agtr2 is part of theHprt region and not of the Maoa region. Comparisonof the human and mouse maps suggests that additionalhuman genes in Xq23–q25 will have mouse homologsthat lie on the proximal X chromosome close to Agtr2(Fig. 2).

In humans, several forms of familial premature ovar-ian failure (POF) have been assigned to different re-gions on the long arm of the human X chromosome.Genetic causes of POF may be secondary to X chromo-some deletions and translocations (11, 18). Based onthese studies, a POF gene was tentatively localized tothe Xq21.3–q27 region (6). It is interesting to speculatethat the Agtr2 gene might be involved in the control ofnormal ovarian function. AT2 receptors are expressed

FIG. 1. Structure and linkage analysis of the angiotensin AT2 in the ovary (3) and are predominantly localized inreceptor gene (Agtr2). (A) Intron–exon structure of the mouse Agtr2 follicular granulosa cells (19). Upregulation of AT2 re-gene isolated from a 129/Sv genomic DNA library. The entire open ceptors during follicle atresia indicates that angioten-reading frame (black box) of the Agtr2 gene is contained on exon 3.

sin II may play a role in the regulation of follicle matu-A restriction map constructed from three overlapping phage clonesration and ovulation (5). These results suggest a poten-is shown by arrows; abbreviations are B, BamHI; E, EcoRI; H, Hin-

dIII; K, KpnI. A XmnI restriction fragment length variant of Agtr2 tial involvement of the angiotensin type II receptorwas detected by PCR amplification of a 450-bp section of the 3 *- gene (Agtr2) in genetic forms of ovarian failure anduntranslated region of the Agtr2 gene. PCR products from M. spretus may provide important insights into the physiologicalor C57BL/6J DNA were incubated with XmnI and separated on a

function of the AT2 receptor.2% agarose gel. The PCR product from C57BL/6J DNA was digestedinto two smaller fragments (218 / 232 bp) by XmnI. The correspond-ing region of the M. spretus gene was not digested by XmnI. (B) REFERENCESGenotype distribution for Agtr2 and three SSLP markers (DXMit26,DXMit85, and DXMit49) in the interspecific backcross panel ((C57Bl/

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