(Received for publication, March 20, 1995; and in revised form, July 12, 1995)
From the
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) 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 5`-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 5`-untranslated exons were identified which splice into a common translated region. Expression of these mRNA species is controlled by two distinct promoters.
Alternative 5` 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.
Figure 1:
Structure of the hMR gene. A,
schematic representation of the hMR mRNA including the two alternative
5`-exons (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.
Figure 2:
DNA sequence of the region upstream of
exon 1 (P1). The transcription initiation site is indicated by an arrow. Consensus sequences for Sp1-binding sites are boxed, and those for transcription factors Ap-1 and PEA3 are underlined; consensus sequences for AP-2 are indicated by a line above the sequence. The exonic sequence is indicated in bold letters. Numbering is relative to the transcription
initiation site of hMR
.
The transcription
initiation site of hMR mRNA was determined by RACE-PCR. The most
5`-sequence obtained corresponds to nucleotide positions
-45/-46, 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 -45, 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.
Figure 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
+1. The exonic sequence is indicated in bold letters. The
3`-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.
In the present study we have determined the genomic
organization of the hMR gene. The two different mRNA 5`-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 particular tissues.
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 5`-untranslated exons and regulatory regions. hGR has a unique 5`-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).
Figure 4: Comparison of the genomic organization of hMR, hGR, hAR, and hPR. The locations of the translational start and stop codons are indicated(20, 23, 24) .
The DNA
surrounding the transcription initiation site of hMR
(5`-CCCTCC
TCT-3`) resembles the initiator sequence
from the terminal deoxynucleotidyltransferase gene
5`-CCCTCA
TTCT-3`, which appears to be important in
TATA-less 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
C
GAGA), and with the
sequence surrounding the putative transcription initiation site of the
rat MR
(CTCC
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]and U30816[GenBank].