Human mineralocorticoid receptor genomic structure and identification of expressed isoforms.

Most of the known effects of aldosterone are mediated by the mineralocorticoid receptor, an intracellular receptor belonging to the steroid/thyroid hormone/retinoic acid receptor superfamily. We determined the genomic structure of the human MR (hMR) and identified 10 exons in the gene, including two exons (1α and 1β) that encode different 5′-untranslated sequence. Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Our results indicate that hMR expression is regulated by alternative promoters perhaps in a tissue- or developmental-specific manner.

Most of the known effects of aldosterone are mediated by the mineralocorticoid receptor, an intracellular receptor belonging to the steroid/thyroid hormone/retinoic acid receptor superfamily. We determined the genomic structure of the human MR (hMR) and identified 10 exons in the gene, including two exons (1␣ and 1␤) that encode different 5-untranslated sequence. Expression of the two different hMR variants is under the control of two different promoters that contain no obvious TATA element, but multiple GC boxes. Our results indicate that hMR expression is regulated by alternative promoters perhaps in a tissue-or developmental-specific manner.
Molecular cloning of a cDNA encoding the human renal mineralocorticoid receptor (hMR) 1 (1) identified it as a member of the nuclear receptor superfamily, which includes the other steroid hormone receptors as well as the thyroid and retinoic acid receptors and a large group of orphan receptors (2,3). Nuclear receptors are highly specialized ligand-dependent transcription factors which interact with specific cis-acting elements to enhance or repress target gene expression (4). They share the same structure-function organization, and their cDNAs are highly conserved particularly in those regions associated with DNA and hormone binding. The mineralocorticoid and glucocorticoid receptor are closely related, and together with the progesterone and androgen receptors they form a subfamily which is closely related both by sequence and functional criteria (3,5).
The adrenal steroids aldosterone and cortisol both bind to the mineralocorticoid receptor (type I corticosteroid receptor); cortisol also binds to the glucocorticoid receptor (type II receptor). Specificity is conferred by the enzyme 11␤-hydroxysteroid dehydrogenase, which converts cortisol to the less active compound cortisone, thus allowing aldosterone to bind to mineralocorticoid receptor (6,7). The mineralocorticoid receptor is expressed in so called "classical" aldosterone target tissues, which are sodium transporting epithelia (kidney, colon, salivary, and sweat glands) and in a variety of non-epithelial target tissues, such as the central nervous system, mononuclear leukocytes, large blood vessels, and the heart (8). In the kidney, the mineralocorticoid receptor regulates the sodium, potassium, and hydrogen ion balance in the distal part of the nephron. In the central nervous system, the mineralocorticoid receptor is expressed in many areas of the forebrain with very high levels of expression reported in the rodent hippocampus. In some, but not all regions in the brain, the mineralocorticoid receptor responds to the diurnal fluctuations in cortisol levels and thus provides, together with the glucocorticoid receptor, a system capable of responding to normal and elevated cortisol concentrations, respectively (9).
Recent studies have reported the presence of multiple variants of the rat mineralocorticoid receptor mRNA, which encode for the same protein but diverge in their 5Ј-untranslated sequences (10). These mineralocorticoid receptor mRNAs are expressed in a tissue-dependent manner and are very likely to be under the control of different promoter sequences, allowing thus an independent regulation of each mRNA isoform.
The hMR gene has been localized to chromosome 4q31.1-4q31.2 (11,12). We have characterized the structure of the hMR gene and determined the location of the hMR splice sites. Two different 5Ј-untranslated exons were identified which splice into a common translated region. Expression of these mRNA species is controlled by two distinct promoters.

EXPERIMENTAL PROCEDURES
Isolation and Characterization of Genomic Clones-A human placental cosmid library (Stratagene, La Jolla, CA), a human placental lambda phage library (13), two human leukocyte cosmid libraries, and a chromosome 4 enriched cosmid library were screened using three different EcoRI restriction fragments of the human mineralocorticoid receptor cDNA following standard colony hybridization techniques (14). Labeling was performed by random priming with nonamers and [␣-32 P]dCTP (Megaprime DNA labeling system, Amersham). Positively hybridizing colonies were picked and passaged until complete purification. DNA of positive clones was prepared following standard techniques (14). Cosmid and phage DNA was digested with different restriction enzymes and characterized by Southern blot analysis using specific [␥-32 P]dATP-labeled oligonucleotides as probes (15,16). Positive restriction fragments were subcloned and sequenced by the dideoxy chain termination method (17) using Sequenase (U. S. Biochemical Corp.). The sequence of the exon 1␤/intron P1 boundary and confirmation of the cDNA sequence was obtained by direct sequencing of the cosmid and by * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM 1 The abbreviations used are: hMR, human mineralocorticoid receptor; hGR, human glucocorticoid receptor; hAR, human androgen receptor; hPR, human progesterone receptor; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s); RACE, rapid amplification of cDNA ends; AP-2, activating protein 2.
Inverse PCR-Inverse PCR was performed according to Ochman et al. (18). Briefly, human genomic DNA was digested with different restriction enzymes (HaeIII, RsaI, MspI, and Sau3A). The purified digests were circularized and submitted to 35 rounds of PCR amplification using exon-specific primer pairs. Oligonucleotide primers (20 -23-mers) were designed, using the predicted exon-intron organization of the hMR (19,20), to be located in the center of each exon to be amplified. PCR primers were as follows (numbering corresponds to the position of the 5Ј-nucleotide according to the published hMR cDNA sequence (1), S identifies primers in sense orientation and A in the antisense orientation): S180 and A83; S2190 and A2187; S2810 and A2803; S2969 and A2942. PCR products were fractionated on a 2% agarose gel, subcloned into Bluescript-KS, and sequenced.
Rapid Amplification of cDNA Ends (RACE)-Mapping of the hMR␣ transcription initiation site by RACE (21) was performed using a commercial kit (5Ј-RACE, Life Technologies, Inc.) following the instructions of the manufacturer. Total human kidney RNA was extracted by standard techniques (22) and submitted to reverse transcription using a reverse primer (23-mer) located at position 480 according to the published mineralocorticoid receptor cDNA sequence (1). The 3Ј-tailed cDNA was submitted to 30 rounds of PCR amplification using an oligo(dC) sense primer and a nested reverse primer (at position 311). PCR included a denaturation step at 96°C for 30 s, an annealing step at 56°C for 30 s, and an elongation step at 72°C for 1 min, after an initial denaturation at 94°C for 5 min and followed by 10 min elongation. 0.05% of the initial PCR product was subsequently reamplified under the same conditions with a sense oligo(dC) primer containing several cloning sites and a second nested reverse primer at position 83. The PCR products were subcloned into Bluescript-KS and screened with an exon 1␣-specific oligonucleotide (position 7); positive clones were sequenced by the dideoxy chain termination method.
Alternative 5Ј variants of the mineralocorticoid receptor mRNA were searched for by RACE using reverse primers located in exon 2 at position 311 and 280 for the first and second round of amplification, respectively. After subcloning PCR fragments, colonies which did not hybridize to an exon 1␣-specific oligonucleotide (position 7) probe were sequenced. Exon 1␤-specific oligonucleotide probes were synthesized and used for direct sequencing of a cosmid containing exon 1␤.
Nucleotide sequences were analyzed using the suite of programs provided by the Australian Genomic Information Service and by CITI 2.

Positions of Cosmid Clones, Phage Clones, and Inverse PCR
Products on the hMR Gene-Four cosmid clones and three phage clones were characterized after the screening for clones containing hMR gene sequences using the hMR cDNA as probe (Fig. 1). Cosmid clones cos25 and cos31 were mapped to the 3Ј-end of the gene using primer A3206 as a probe (primer position according to the published cDNA (1). 9b was characterized using oligonucleotide S1786 and A448 and mapped to the 5Ј-end of the mineralocorticoid receptor gene. Primer S2054, which maps to the DNA-binding domain, was used to characterize clone 14, and phage clone 131 was mapped to the hormone-binding domain of the receptor using oligonucleotides S2381 and A2683. Clones cos72 and cos953 were isolated using EcoRI fragments of the original hMR cDNA (1). Three different inverse PCRs were performed to specifically amplify putative exons not covered by the cosmid/phage clones as indicated (i1-i3). The sequence of the exon for the published 5Јuntranslated and its flanking sequence were determined from both cosmid clone cos72 and also using inverse PCR (ip1). Following the nomenclature used for the rat gene (10), this is designated as the ␣5Ј-untranslated. The cosmid cos72 also hybridized with both exon 2 and rat exon 1␤-specific probes. Exon 1␤ 5Ј-untranslated was identified by RACE which enabled the sequencing of the flanking region and exon-intron junction from the cosmid. Using this strategy, we determined the exonintron boundaries and flanking sequences of the mineralocorticoid receptor gene. As shown in Table I, the intron-exon boundaries have the canonical consensus sequence.
Exon-Intron Organization of the Gene-The hMR gene ( Fig.  1A and Table I) consists of a total of 10 exons and spans over 60 -90 kb, as deduced from Southern hybridization experiments (data not shown). Exons 1␣ and 1␤ are composed only of 5Ј-untranslated sequence. The N-terminal part of the receptor is encoded by exon 2, which contains 2 bp of 5Ј-untranslated region and 1757 bp of coding sequence. Two small exons, exons 3 and 4, encode each of the two zinc fingers of the DNA-binding domain of the receptor. The hormone-binding domain of the mineralocorticoid receptor is encoded by five exons, exons 5-9. Among the glucocorticoid/mineralocorticoid/androgen/protesterone receptor subfamily of nuclear receptor genes (20,23,24) the length of the exons is identical for those encoding both the second zinc finger (exon 4 in the hMR) and the ligand-binding domain (6 -8 in the hMR).
Transcription Initiation Site of hMR␣ and 5Ј-Flanking Region (P1)-Two different hMR 5Ј-mRNA ends were identified, designated as hMR␣ and hMR␤. The 5Ј-untranslated sequence containing exon 1␣ was obtained both from the cosmid clone cos72 and by inverse PCR (Fig. 1). A 2.1-kb HindIII fragment of cos72 shown to hybridize with a rat exon 1␣ probe yielded sequence extending from intron A (ϳ900 bp) through the 5Јuntranslated and into the flanking region (P1:950 bp). The inverse PCR fragment obtained was ϳ1.2 kb in size and similarly extends from intron A to include ϳ750 bp of flanking sequence. Fig. 2 shows the hMR␣ 5Ј-untranslated and flanking A, schematic representation of the hMR mRNA including the two alternative 5Јexons (1␣ and 1␤) and the eight coding exons (2)(3)(4)(5)(6)(7)(8)(9). The location of the translational start and stop codons is indicated. B, genomic organization of the hMR gene. Cosmid (cos72, cos31, cos25, and cos953) and phage clones (9b, 14b, and 131) and inverse PCR products (i1, i2, i3, and ip1) are positioned on the gene and indicated by arrows.
sequences obtained from these clones. No consensus TATA box sequence is present in the putative promoter sequence, and no CCAAT motif is found within the first 100 bp upstream of the transcription initiation site, although two are found at positions Ϫ172 and Ϫ225 (these sequences could be found by chance every 1025 bp). The region is very rich in GC boxes, and computer-assisted analysis revealed seven consensus sequences for the binding of transcription factor Sp-1 (Ϫ33, Ϫ59, Ϫ152, Ϫ254, Ϫ328, Ϫ347, and ;402) (25,26), three very closely clustered activating protein 2 (AP-2)-binding sites (Ϫ32, Ϫ62, Ϫ77) (27,28), as well as a binding motif for transcription factor PEA-3 (Ϫ165) (29). At position Ϫ217 the sequence 3Ј-AGAA-TAnnnTGTTAG-5Ј very closely resembles the GRE consensus sequence 5Ј-AGAACAnnnTGTTCT-3Ј (30,31), although it is inserted in inverse orientation. The 5Ј-untranslated encoded by exon 1␣ shows 90% identity with the rat sequence and the first 400 bp of the flanking region of exon 1␣ show 73% identity with the rat genomic sequence in this region (32).
The transcription initiation site of hMR␣ mRNA was determined by RACE-PCR. The most 5Ј-sequence obtained corresponds to nucleotide positions Ϫ45/Ϫ46, according to the published cDNA sequence (1), which is therefore the putative transcription initiation site (underlined in Fig. 2). By Southern hybridization with an oligonucleotide at position Ϫ45, about 61% of clones were positive for this sequence. Several shorter transcripts could represent minor sites of transcription initiation or truncated artifacts of reverse transcription and amplification.
Cloning of hMR␤ and Its 5Ј-Flanking Region (P2)-We identified a novel 5Ј-sequence that spliced onto the mineralocorticoid receptor mRNA at intron A-exon 2 boundary located 2 bp upstream of the mineralocorticoid receptor translation initiation site (Fig. 3). This sequence shares 85% homology with the first untranslated exon of the rat MR␤ cDNA isolated from rat hippocampus (33). The longest transcripts obtained by subsequent RACE using exon 1␤-specific reverse primers at position 124 and 103 of the cloned cDNA enabled us to determine a putative transcription initiation site of hMR␤ (underlined in Fig. 3). Sequencing of cosmid cos72 with 5Ј-untranslated-specific primers enabled the identification of both the exon1␤intron P1 junction and the region flanking the 5Ј-untranslated (P2). It is possible, given the very GC-rich nature of the region which rendered sequencing difficult, that the RACE products are prematurely terminated in the 5Ј-untranslated. Sequence homology with the rat exon 1␤ cDNA is observed over 216 bp to the putative transcription initiation site of hMR 1␤, after which the sequence similarity decreases. Additional experiments are required to precise whether alternative transcription start sites exist in the human and the rat MR␤ promoter. Computerassisted analysis of P2 revealed two binding sites for transcription factor Sp-1 (Ϫ62, Ϫ150) and one consensus sequence for binding of transcription factor AP-2 (Ϫ26).

DISCUSSION
In the present study we have determined the genomic organization of the hMR gene. The two different mRNA 5Ј-ends isolated by RACE suggested the existence of alternative promoters in the hMR gene, as has been reported for the mouse glucocorticoid receptor (34) and suggested for the rat mineralocorticoid receptor (10,32). The expression of the two mRNAs is not mutually exclusive, since the two transcripts were both found in the kidney. Alternative transcription initiation from exon 1␣ or 1␤ generates two different hMR transcripts, hMR␣ and hMR␤, with the same translation product. A third mRNA isoform was found in rat hippocampus (␥MR) (10), and although we could not detect this mRNA in the human kidney, this and possibly other mineralocorticoid receptor mRNA species might be present in low abundance or restricted to  It is interesting to compare the genomic structure of the hMR, hGR, hAR, and hPR (20,23,24), all belonging to the same steroid receptor subfamily (3,5). Although in these genes the same exon-intron organization is used to assemble functional domains of the receptor protein (eight exons), no structural conservation is found for 5Ј-untranslated exons and regulatory regions. hGR has a unique 5Ј-untranslated exon, whereas only eight exons compose the hAR gene. The expression of both is driven by a unique promoter, whereas the hPR gene (24,35) contains two different hormone-inducible promoters (Fig. 4).
The DNA surrounding the transcription initiation site of hMR␣ (5Ј-CCCTCC ϩ1 TCT-3Ј) resembles the initiator sequence from the terminal deoxynucleotidyltransferase gene 5Ј-CCCTCA ϩ1 TTCT-3Ј, which appears to be important in TATAless promoters, and which has been shown to interact in a position-dependent manner with Sp-1 binding sites to direct high levels of transcription (36,37). This sequence also shares homology with the androgen receptor transcription initiation site II (TISII) surrounding sequence (CCCTC ϩ12 C ϩ13 GAGA), and with the sequence surrounding the putative transcription initiation site of the rat MR␣ (CTCC ϩ1 TGCGC).
As with the promoters directing expression of other steroid receptors (20,24,34,35,38,39), including the rat mineralocorticoid receptor (32), both P1 and P2 do not contain any TATA box or CCAAT motifs within the first 100 bp, though several potential Sp-1-binding sites are present. Promoters with these features are found primarily in housekeeping genes and usually contain several transcription initiation sites as well as several potential binding sites for the transcription factor Sp-1 (36).
The hMR gene is thus regulated by alternative promoters (40,41). This mechanism allows more flexibility in the control of expression and generally, alternative promoters are associated with tissue-and/or developmental-specific gene expression. In the rat, MR␣ is the predominant form expressed in the kidney, whereas in the hippocampus MR␣ and MR␤ are expressed in equal proportions (10). In addition, alternative transcription initiation can affect both the stability of the transcripts and the efficiency of mRNA translation (42). Indeed, mineralocorticoid receptor expression seems to be regulated in a tissue-specific manner by the level of its ligand, although conflicting data are reported in the literature. Whereas there is good evidence for hormonal regulation of mineralocorticoid receptor in the hippocampus (10,43,44), for the kidney both regulation (45) and lack of regulation by corticosteroids have been reported (44). For the rat distal colonic mineralocorticoid receptor, protein and mRNA levels are neither up-regulated after adrenalectomy nor down-regulated in response to a mineralocorticoid receptor agonist (46).
Although mineralocorticoid receptor is known to bind specific consensus sequences, such as the GREs contained in the MMTV promoter (1), no specific mineralocorticoid-responsive element has yet been identified. P1 contains a sequence resembling a GRE in an inverted orientation, which has been shown to confer hormone responsiveness to exogenous promoters in an orientation-independent manner (47). It is worth noting that a significant and selective increase in MR␣ mRNA levels has been reported in rat hippocampus after adrenalectomy, which was reversed by exogenous corticosterone administration, whereas MR␤ mRNA levels did not change. Thus, in the rat, MR␣ may be the hormonally regulated mRNA variant, suggesting that the putative hormone responsive sequence identified in P1 might be of functional significance.
In conclusion, the determination of the genomic structure of the hMR will allow the study of its transcriptional regulation and of the tissue-specific and developmental expression of distinct hMR mRNA species. It will also facilitate the search for mutations in the human mineralocorticoid receptor gene which may be responsible for disorders of salt and water balance, such as mineralocorticoid resistance or hypertension. FIG. 3. DNA sequence of exon 1␤ and its 5-flanking region (P2). An arrow indicates a putative transcription initiation site of hMR␤, which is numbered as ϩ1. The exonic sequence is indicated in bold letters. The 3Ј-intron sequence is indicated by lowercase letters. Sp1-binding sites are boxed, and a consensus sequence for AP-2 is underlined. N indicates ambiguous nucleotide determination due to high GC content of the region.  (20,23,24).