(Received for publication, May 18, 1995; and in revised form, June 27, 1995)
From the
Two distinct cDNAs encoding a human mercurial insensitive water
channel (hMIWC) were cloned from a fetal brain cDNA library. The
longest open reading frame of cDNA clone hMIWC1 encoded 301 amino acids
with 94% identity to rat MIWC (Hasegawa, H., Ma, T., Skach, W.,
Matthay, M. M., and Verkman, A. S. (1994) J. Biol. Chem. 269,
5497-5500). A second cDNA (hMIWC2) had a distinct 5`-sequence
upstream from base pair (bp) -34 in clone hMIWC1 and contained
two additional in-frame translation start codons. Expression of hMIWC
cRNAs in Xenopus oocytes increased osmotic water permeability
by 10-20-fold in a mercurial insensitive manner. Cell-free
translation in a reticulocyte lysate/microsome system generated single
protein bands at 30 kDa (hMIWC1) and 32-34 kDa (hMIWC2) without
glycosylation. Northern blot and polymerase chain reaction/Southern
blot analysis showed expression of mRNA encoding hMIWC in human brain
muscle heart, kidney, lung, and trachea. Analysis of hMIWC
genomic clones indicated two distinct but overlapping transcription
units from which multiple hMIWC mRNAs are transcribed. The promoter
region of hMIWC1 was identified and contained TATA, CAAT, AP-1, and
other regulatory elements. Primer extension revealed hMIWC1
transcription initiation at 46 bp downstream from the TATA box. There
were three introns (lengths 0.9, 0.2, and 6 kilobases) in the hMIWC1
coding sequence at bp 381, 546, and 627. A distinct 5`-sequence in
clone hMIWC2 suggested an alternative upstream transcription initiation
site. Two alternatively spliced, nonfunctional hMIWC transcripts with
exon 3 deletion and partial exon 4 deletion were identified. A
poly(A)
signal sequence was identified at 138 bp
downstream of the translation stop codon. Genomic Southern blot
analysis indicated the presence of a single copy hMIWC gene;
chromosome-specific polymerase chain reaction and in situ hybridization localized hMIWC to human chromosome 18q22. The
structural organization of the hMIWC gene represents a first step in
definition of hMIWC differential expression, regulation, and possible
role in human disease.
Several water-selective channels (aquaporins) have been
identified and cloned in mammals, including channel-forming integral
protein (CHIP28)(1, 2) , water channel-collecting duct
(WCH-CD or AQP-2)(3) , mercurial insensitive water channel
(MIWC) ()(4) , and possibly glycerol intrinsic
protein (GLIP or AQP-3)(5, 6) . CHIP28 is widely
expressed in epithelial and endothelial cells in fluid-transporting
tissues, including kidney proximal tubule and thin descending limb of
Henle, choroid plexus, ciliary body, colonic crypt, and
others(7, 8, 9) . WCH-CD is expressed
exclusively in the apical membrane and subapical vesicles in collecting
duct principal cells, where it functions as a vasopressin-regulated
water channel(10, 11) . GLIP is expressed in the
basolateral membrane of collecting duct principal cells and in several
extrarenal tissues(5, 6, 12, 13) .
MIWC is unique in that it encodes a water-selective channel that is
not inhibited by high concentrations of mercurial compounds such as
HgCl(4) . Rat MIWC is a 301-amino acid hydrophobic
protein that spans the membrane six times with its NH
and
COOH termini in the cytosol(14) . An unusual feature of rat
MIWC is the tissue-specific expression of both a full-length transcript
encoding the functional protein and a short transcript, which had a
165-bp deletion and did not appear to be translated(4) .
Immunohistochemistry with MIWC peptide-derived antibodies localized rat
MIWC protein to the basolateral membrane of kidney collecting duct,
ependymal cells lining brain ventricles, astrocytes in brain and spinal
cord, and epithelial cells in stomach, trachea, bronchi, ciliary body,
colonic villi, salivary gland, and lacrimal
gland(12, 13) . Immunoblot analysis detected a 30-kDa
band in kidney, stomach, and lung and two bands (at 30 and 32 kDa) in
brain. Interestingly, MIWC was also expressed strongly in the
plasmalemma of skeletal muscle, where immunoblot showed a single band
at 26 kDa(13) . The functional analysis and tissue distribution
of MIWC suggests an important physiological role in the urinary
concentrating mechanism, cerebrospinal fluid reabsorption, airway
hydration, and glandular secretions.
We report here the cDNA and genomic cloning of a human mercurial insensitive water channel (hMIWC) with greatest mRNA expression in human brain and skeletal muscle. Two distinct cDNAs with different 5`-sequences (hMIWC1 and hMIWC2) were identified that encoded functional water channels. Analysis of cDNA and genomic sequences defined the promoter/transcriptional unit corresponding to hMIWC1, including upstream genomic regulatory elements, transcription initiation site, intron/exon gene structure, and a downstream polyadenylation signal sequence. A single copy hMIWC gene was localized to chromosome 18q22. Several interesting features of hMIWC expression included the expression of two protein isoforms corresponding to distinct transcriptional units and the presence of short, nonfunctional mRNAs with exon 3 or partial exon 4 deletions. The genomic analysis reported here should permit the examination of hMIWC regulatory mechanisms and the role of hMIWC in a subpopulation of cases of congenital nephrogenic diabetes insipidus, which are not associated with mutations in the V2 receptor or AQP-2 proteins.
Two hMIWC cDNA clones with different 5`-nucleotide sequences were isolated from a human fetal brain cDNA library probed by the coding sequence of rat MIWC. One clone (hMIWC1, 1.9 kb) had the longest open reading frame encoding a 301-amino acid protein with 94% identity to rat MIWC (Fig. 1A). The amino acid sequence contained NPA and other amino acid motifs conserved in MIP family members(18, 19) . There were three consensus sites for N-linked glycosylation and four sites for phosphorylation by protein kinases A and C. As in rat MIWC, residue 188 of hMIWC, corresponding to the mercurial sensitive residue C189 of CHIP28, was alanine rather than cysteine. Two short forms of the hMIWC transcript were identified (see below), corresponding to indicated deletions of bp 547-627 and bp 628-697. A polyadenylation signal sequence AATAAA was found 138 bp downstream of the translation stop codon.
Figure 1:
cDNA and deduced amino acid sequence of
hMIWC. A, sequence of clone hMIWC1 showing consensus sites for N-linked glycosylation (*) and phosphorylation by protein
kinases C () and A (
). Amino acids in parentheses are for rat MIWC (4) where they differ from hMIWC. Intron
positions are indicated. Boxes correspond to deletions in exon
3 (solid) and a portion of exon 4 (dashed). An
in-frame stop codon in the 5`-untranslated region and a downstream
poly(A)
signal sequence are underlined. B, different 5`-sequence of clone hMIWC2. The sequence distal
to the leftbracket is identical with that of hMIWC1
in A. An upstream in-frame stop codon is underlined,
and two additional consensus sites for phosphorylation by protein
kinase C are shown.
A
second cDNA clone (hMIWC2, 1.5 kb) had identical DNA sequence to hMIWC1
from position -34 onward but a different upstream sequence. The
additional 5`-sequence encoded two in-frame methionines, designated M1
and M2 (Fig. 1B), which extended the amino terminus by
40 and 22 amino acids, respectively. The extended hydrophilic amino
terminus contained two consensus sequences for phosphorylation by
protein kinase C and three cysteine residues. The ATGs in hMIWC1 and
hMIWC2 had favorable Kozak's sequences for
translation initiation, whereas hMIWC2
had a poor
Kozak's sequence (T at position -3).
cRNAs encoding
three full-length forms of hMIWC (hMIWC1, hMIWC2, and
hMIWC2
) and two truncated forms (hMIWC1[
3]
and hMIWC1[
4]) were in vitro transcribed and
expressed in Xenopus oocytes. hMIWC2
and
hMIWC2
refer to cDNA constructs in which M1 or M2
comprised the amino terminus (see ``Materials and Methods'').
hMIWC1[
3] and hMIWC1[
4] indicate cDNAs
with exon 3 or partial exon 4 deletion. Fig. 2A shows
that osmotic water permeability (P
) was strongly
increased in oocytes expressing hMIWC1, hMIWC2
,
hMIWC2
, and rat MIWC compared to control water-injected
oocytes, although expression of hMIWC2
conferred a lesser
increase in P
. Both short forms of MIWC did not
increase oocyte water permeability. Oocyte P
was
not inhibited by HgCl
in the three groups of
hMIWC-expressing oocytes. Measurements of
C-urea and
glycerol uptake of control and hMIWC-expressing oocytes indicated that
hMIWC functioned as a water-selective channel (data not shown).
Cell-free translation of hMIWC1 (Fig. 2B) in rabbit
reticulocyte lysate generated a single protein band of 30 kDa that did
not become glycosylated when pancreatic endoplasmic reticulum-derived
microsomes were present during translation. Interestingly, translation
of hMIWC2
and hMIWC2
produced single bands of
34 and 32 kDa, respectively, without a 30-kDa band.
Figure 2:
Functional analysis and tissue-specific
expression of hMIWC. A, expression of hMIWC cRNAs in Xenopus oocytes. Left, time course of swelling in
oocytes microinjected with water or 5 ng of cRNA encoding rat MIWC and
full-length hMIWC1 and hMIWC2. Where indicated, 0.3 mM HgCl
was added. Right, mean and S.E. for P
of various full-length and truncated
forms of hMIWC. Numbers of oocytes are shown in parentheses. B, cell-free translation of hMIWC cRNAs in rabbit reticulocyte
lysate in the absence(-) and presence (+) of pancreatic
microsomes (labeled memb). C, Northern blot of human
tissues (2 µg of mRNA/lane) probed with a 903-bp DNA
corresponding to the hMIWC1 coding sequence. D, PCR-Southern
analysis of hMIWC tissue distribution. Reverse transcriptase-PCR was
carried out using cDNAs from human brain, kidney, trachea, and skeletal
muscle as template and PCR primers derived from exons 1 and 4 in hMIWC1
(see ``Materials and Methods''). The Southern blot was probed
with hMIWC1 cDNA. Arrow indicates a 0.43-kb full-length PCR
fragment.
The tissue
distribution of hMIWC was determined by Northern and PCR-Southern blot
hybridization. Northern blot analysis showed strong expression of a
5.5-kb mRNA in brain and muscle, with two less intense bands at 3.2 and
1.4 kb (Fig. 2C). Prolonged film exposure revealed a
similar pattern in heart, kidney, and lung (not shown). To identify
spliced hMIWC transcripts, PCR-Southern analysis was carried out using
as template cDNAs from human brain, kidney, trachea, and skeletal
muscle and exon-derived primers corresponding to exon 1 (bp +325
to +345) and exon 4 (bp +753 to +733) (Fig. 2D). PCR products were blotted and hybridized
with a DNA corresponding to the hMIWC1 coding sequence. A major band of
430 bp and a band of smaller size were revealed. Sequence analysis
of the subcloned bands revealed a full-length 428-bp hMIWC fragment, as
well as two short forms of hMIWC with an (81 bp) exon 3 deletion or a
partial (70 bp) exon 4 deletion (see Fig. 1A). The
deleted segment in exon 3 corresponds to a hydrophilic segment between
the 5th and 6th transmembrane domain; the partial deletion in exon 4
corresponds to the 6th transmembrane domain of MIWC and results in a
shift in reading frame(14) . Immunolocalization studies were
performed in human tissues with a rabbit polyclonal antibody raised
against a synthetic COOH terminus peptide as previously
described(12, 13) ; hMIWC protein localized to
basolateral membrane of principal cells in kidney-collecting duct and
to skeletal muscle sarcolemma, similar to results reported in rat (data
not shown).
Screening of a human genomic library using a DNA probe corresponding to the hMIWC coding sequence yielded two overlapping fragments of 20 and 18 kb, designated G4 and G7, respectively (Fig. 3). Initial maps of the coding regions were constructed by Southern hybridization after digestion with multiple restriction enzymes. Exon-intron boundaries were determined by sequence analysis of subcloned genomic fragments with exon-specific primers. All exons and introns 1 and 2 were fully sequenced from a subcloned 7-kb XbaI fragment of clone G4 and a 3.6-kb XbaI fragment from clone G7. The intron locations and sizes are summarized in Table 1and intron-exon boundary sequences are given in Fig. 4A. The three introns in the hMIWC coding sequence were of class 0 and followed the gt-ag rule. Sequence analysis of 860 bp into the 5`-flanking region revealed a TATAAAA element (TATA box) at 385 bp upstream from the ATG translation initiation codon of hMIWC1. In addition, one CAAT box, two E-boxes, and SP1, AP-1, two AP-2, and APRRE elements were identified. A polyadenylation signal sequence AATAAA was found at 138 bp downstream of the translation stop codon, identical to that found in cDNA clone hMIWC1. Because cDNA clone hMIWC1 contained 604 bp downstream from the polyadenylation signal sequence, additional downstream polyadenylation sites must exist.
Figure 3: Genomic structure of hMIWC. Overlapping human genomic clones G4 and G7 were analyzed. The restriction map of the hMIWC gene is shown. The rectangles indicate exon segments that constitute coding (filled) and untranslated (open) sequences. The stripedrectangle denotes an alternative upstream exon segment corresponding to the alternate 5`-flanking sequence of cDNA clone hMIWC2. Rightarrows indicate transcription initiation sites. H, HindIII; X, XbaI; E, EcoRI; A, ApaI.
Figure 4:
Genomic sequence of hMIWC and
identification of the transcription initiation site. A, DNA
sequence of hMIWC showing the 5`- and 3`-flanking regions and
intron-exon boundaries. Upstream regulatory elements and a downstream
poly(A) signal sequence are indicated in boxes, and the transcription initiation site (+1) is
shown in bold. The upwardarrow indicates a
splice site involved in the generation of cDNA clone hMIWC2, and the doubleunderline is a consensus splicing acceptor
site. B, mapping of the transcription state site by primer
extension. 8% polyacrylamide gel showing sequencing ladder was
generated using a subcloned 7-kb XbaI fragment from genomic
clone G4 as template, and primer 5`-TCAAAGATCATCCAGTTTCAC-3`
corresponding to bp +101 to +81 (bp -233 to -253
in hMIWC1 cDNA). Lanepe, the same primer was
P-end labeled, hybridized to human brain mRNA, and
extended with reverse transcriptase. Lanec, control
with yeast tRNA as template. See ``Materials and Methods''
for details.
The 5`-untranslated sequence in the hMIWC gene upstream from the translation initiation codon was identical to that of cDNA clone hMIWC1. The transcription initiation site was determined by primer extension using human brain mRNA as template. A single transcription start site was identified at a T residue 332 bp upstream from the translation start of hMIWC1 (Fig. 4B). The different 5`-flanking sequence in cDNA clone hMIWC2 indicates the existence of a splice site at 34 bp upstream from the translation initiation codon (position 300 of the hMIWC gene in Fig. 4A, arrow), where an A-G splicing acceptor consensus sequence was found.
To determine the copy number of the hMIWC gene in the human genome, genomic Southern analysis was performed using human genomic DNA digested with EcoRI, XbaI, SacI, and ApaI. The blot was hybridized with a probe corresponding to the hMIWC1 cDNA coding sequence. Fig. 5A shows two positive bands in the XbaI lane (7 and 3.6 kb), EcoRI lane (15 and 0.4 kb), and ApaI lane (15 and 12 kb), consistent with the restriction map of the overlapping G4 and G7 genomic clones (Fig. 3). A single positive band of 10 kb was found in SacI lane, consistent with the absence of a SacI site in both the hMIWC cDNA and genomic introns analyzed. The genomic DNA hybridization pattern indicated that hMIWC gene is present as a single copy per haploid human genome.
Figure 5: Chromosomal mapping of hMIWC. Panel A, genomic Southern blot. Human DNA was digested with indicated restriction enzymes and probed with an hMIWC1 cDNA corresponding to the coding sequence. Each lane contains 10 µg of DNA. Panel B, chromosomal assignment by PCR analysis using the NIGMS chromosomal panel. PCR was carried out using primers flanking the exon 1 coding sequence (see ``Materials and Methods''). Lanes1-24 contain DNA from the chromosomal panel. G, G4 genomic clone (positive control); C, Chinese hamster genomic DNA (negative control); H, human genomic DNA; M, mouse genomic DNA. PanelC, chromosomal localization of hMIWC by in situ hybridization summarizing data from 12 metaphase chromosome spreads.
Chromosomal assignment using the NIGMS human/rodent somatic cell hybrid mapping panel 2 indicated localization to chromosome 18 (Fig. 5B). The hMIWC sequence was confirmed in the positive band obtained with chromosome 18 DNA as template. The same positive band was observed with genomic clone G4 as template (laneG) and with total human genomic DNA (laneH). Chromosomal localization by fluorescence in situ hybridization using a 7.0-kb hMIWC genomic fragment as probe indicated localization to chromosome 18q22 (Fig. 5C).
This study reports the cDNA and genomic cloning of a human mercurial insensitive water channel (hMIWC) with 94% amino acid identity to a rat homolog (rMIWC) cloned previously from our laboratory (4) . hMIWC and rMIWC transcript expression differed in several respects. Northern blot analysis revealed hMIWC transcripts with three different sizes (5.5, 3.2, and 1.4 kb) in brain and skeletal muscle, whereas a single mRNA band was found for rMIWC. Two nonfunctional short forms of hMIWC were identified by reverse-transcriptase PCR analysis with exon 3 and partial exon 4 deletion, whereas only a single short form of rMIWC was found with an exon 2 deletion. Furthermore, two additional upstream translation start codons were identified in cDNA clone hMIWC2 from human brain. The different 5`-sequence extended the amino terminus of hMIWC1 protein by 22 and 40 amino acids and indicated a different but overlapping transcription unit. The hMIWC1 and hMIWC2 cDNAs probably explain the two distinct MIWC bands reported on immunoblots of brain tissue (13) and results of the cell-free translation of rat brain cRNA(20) . Interestingly, cRNAs corresponding to each of the three ATG codons were in vitro translated as single protein bands and functioned as water channels when expressed in Xenopus oocytes. The presence of multiple transcription units and translation initiation sites has not been found for other proteins in the water channel family.
Table 1compares the genomic organization of human MIP and three human water channels examined to date. The hMIWC gene spans >9 kb and contains three introns at positions identical to those of hCHIP28(21) , hWCH-CD(22) , and hMIP26(23) , suggesting origin from a common ancestor (19) . Genomic Southern blot and fluorescence in situ hybridization indicated that hMIWC is present as a single copy gene at chromosome location 18q22.
Two distinct hMIWC cDNA clones, hMIWC1 and hMIWC2, corresponded to distinct transcriptional units. Primer extension using an antisense oligonucleotide corresponding to 5`-cDNA flanking region of hMIWC1 revealed a single transcription initiation site at a T residue 332 bp upstream from the translation start codon. The 5`-sequence contained a TATA box 46 bp upstream from the transcription initiation site, as well as CAAT and E-boxes, and other regulatory elements. These findings, taken together with the identity in the upstream sequences of the hMIWC1 cDNA and hMIWC gene, suggested that this region constitutes the promoter for transcription of hMIWC1 mRNA. Primer extension using oligonucleotides corresponding to 5`-cDNA flanking region of hMIWC2 was not successful, possibly due to the long 5`-untranslated region of hMIWC2. However, a splice site with an A-G consensus acceptor sequence was suggested by the identity of the hMIWC1 cDNA and hMIWC genomic sequences downstream from position -34 in hMIWC1. The transcription initiation site and promoter corresponding to the hMIWC2 transcription unit is thus located upstream of the hMIWC1 promoter.
Transcription initiation from alternative promoters has been previously reported. Two different size mRNAs of the myosin light chain were transcribed by two promoters located 10 kb apart. mRNAs made from distinct transcription initiation sites encoded proteins with different amino terminus sequences in different muscle cell types(24) . Similarly, for the angiotensin-converting enzyme, different amino terminus sequences were transcribed from a single gene by a tissue-specific choice of alternate transcription initiation sites (25) . Transcription of hMIWC gene from alternative initiation sites appears to produce proteins of different size with presumably different amino termini. The reason for synthesis of different hMIWC isoforms is unclear. The data here indicate that both isoforms are functional water channels. Because hMIWC2 contains additional consensus sites for protein kinase C phosphorylation, the existence of two hMIWC isoforms may be related to regulation of hMIWC protein targeting and/or expression.
Alternative choice of transcription termination and
polyadenylation sites (26) appears to be another mechanism to
produce multiple hMIWC mRNAs. A poly(A) signal
sequence AATAAA was found at an identical positions (138 bp downstream
of the translation stop codon) in hMIWC cDNA and genomic clones. The
shortest hMIWC transcript on Northern blot of 1.4 kb probably
corresponds to an mRNA initiated at the hMIWC1 transcription initiation
site and terminated at this poly(A)
signal sequence.
The presence of an additional 604 bp downstream of this
poly(A)
signal in the hMIWC1 cDNA clone indicates
additional downstream polyadenylation sites corresponding to the longer
mRNAs.
The mRNA diversity of hMIWC related to alternative choices of transcription initiation and polyadenylation is increased further by alternative splicing. In contrast to the alternative splicing pattern of rat MIWC mRNA in which a 165-bp coding sequence corresponding to exon 2 is deleted, shorter forms of hMIWC were identified with an (81 bp) exon 3 and a (70 bp) partial exon 4 deletion. In the latter case, a splicing donor site was identified in exon 4. However, protein was not translated in vitro, and cRNA expression did not confer increased water permeability in Xenopus oocytes. The mRNAs containing specific deletions, which appear to be produced in a tissue-specific manner(4) , may provide a mechanism at the post-transcriptional level to regulate the expression of functional hMIWC water channels.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33013[GenBank], U34844[GenBank].