From the M. E. Müller Institute, University of Bern, CH-3010 Bern, Switzerland
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ABSTRACT |
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We have identified a novel
transformation-sensitive mRNA, which is present in cultured
fibroblasts but is lacking in SV40 transformed cells as well as in many
mesenchymal tumor cell lines. The corresponding gene is located on
human chromosome 8 in band 8q13. The open reading frame of the mRNA
encodes a protein of 1119 amino acids forming two distinct domains. The
N-terminal domain consists of 18 repeats that are related to the
cytoskeletal protein ankyrin. The C-terminal domain contains six
putative transmembrane segments that resemble many ion channels. This
overall structure is reminiscent of TRP-like proteins that function as
store-operated calcium channels. The novel protein with an
Mr of 130 kDa is expressed at a very low level
in human fibroblasts and at a moderate level in liposarcoma cells.
Overexpression in eukaryotic cells appears to interfere with normal
growth, suggesting that it might play a direct or indirect role in
signal transduction and growth control.
Fibroblasts transformed by tumor viruses are often used in the
laboratory as a model system to investigate transformation-associated changes in the phenotype and in the adhesive properties of the cells
(1, 2). Usually these fibroblasts exhibit a roundish shape and show
reduced adhesion to their substratum similar to authentic tumor cells.
In contrast to spontaneous cancer cells, virally transformed
fibroblasts have the advantage that the normal counterpart, the
original cell, is available for comparative studies. A set of
transformation-sensitive proteins that are expressed by normal
fibroblasts but specifically repressed after oncogenic transformation
has emerged from these studies. The best known example is fibronectin,
an important adhesion protein of the extracellular matrix (3). It is
likely that the down-regulation of fibronectin and the other
transformation-sensitive proteins contributes to the establishment of
the transformed phenotype and to the unrestricted growth of tumor cells.
We have recently used this model system in combination with a
subtractive cDNA cloning approach to identify novel
transformation-sensitive proteins (4, 5). Our efforts led to the
isolation of more than 40 cDNA clones that showed a dramatic
reduction in their relative synthesis after oncogenic transformation.
The proteins encoded by these clones could be grouped into four
distinct classes (5): cytoskeletal proteins (e.g. vinculin,
SM22), extracellular matrix proteins (e.g. fibronectin,
collagen VI), proteolytic enzymes (e.g. collagenase,
urokinase), and regulatory proteins (e.g. IGFBP-5, myosin
kinase). Six novel gene products were discovered during our study,
including a GTP-binding protein (4), a zyxin-like protein (6), and a
novel serine protease (7).
Here we set out to characterize one of the new cDNA clones. This
clone codes for an integral membrane protein with 18 ANK repeats. The
polypeptide appears to combine the features of the cytoskeletal protein
ankyrin with those of an ion channel. The function of the novel protein
remains obscure, although there is circumstantial evidence that it
might be involved in a growth regulatory pathway.
Molecular Cloning and Sequencing--
A cDNA clone of 207 bp1 that originated from a
subtracted cDNA library (4, 5) was labeled with
[
The sequences of the isolated cDNA clones were determined on both
strands by the dideoxy chain termination technique (11) using Sequenase
2.0 (U. S. Biochemical Corp.). To resolve ambiguities observed in the
5' region, suitable restriction fragments were subcloned into the
sequencing vectors M13mp18 and M13mp19 and subsequently sequenced in
the presence of dITP instead of dGTP. All sequences were analyzed with
the software computer package of the Genetics Computer Group
(University of Wisconsin, WI) using default settings unless otherwise
stated. The sequences were compared with all entries of the EBI data
bank (release 55.0) and the Swissprot data bank (release 36.0).
Northern Blotting--
Total RNA was extracted from confluent
cell layers by the SDS/proteinase K method (12). For some experiments
this RNA was further purified by chromatography on oligo(dT)-cellulose
utilizing an mRNA isolation kit (Amersham Pharmacia Biotech). The
RNA (15 µg total RNA or 2 µg of poly(A+) RNA/lane) was
resolved on 1% agarose gels in the presence of 1 M
formaldehyde and transferred to nylon membranes by vacuum blotting
(10). The membranes were hybridized with radiolabeled cDNA probes
at 42 °C in a buffer containing 50% formamide. After being washed
at regular stringency (10), the membranes were exposed to BioMax MS
film (Kodak, Rochester, NY) or analyzed with a phosphorimager (Storm
840, Molecular Dynamics, Sunnyvale, CA).
The multiple tissue Northern blots used in this study contained
poly(A+) RNA from 16 adult human tissues (MTN I, MTN II,
CLONTECH) and total RNA from 4 fetal human tissues
(Northern Territory Human Fetal Tissue, Invitrogen, San Diego, CA). All
these blots were processed as described above.
Quantitative PCR--
Competitive PCR was performed essentially
as described by Gilliland et al. (13). Poly(A)+
RNA (0.25 µg) was transcribed into cDNA with 25 units of avian myeloblastosis virus reverse transcriptase using a cDNA synthesis kit (Boehringer Mannheim). The reaction (total volume 20 µl) was primed with an oligonucleotide corresponding to positions 3115-3137 of
the cDNA sequence. Aliquots of the single-stranded cDNA (0.5 µl) were amplified with AmpliTaq polymerase (Perkin Elmer) in the
presence of 2.5 mM MgCl2 through 35 cycles of
1' at 94 °C, 1' at 50 °C, and 45" at 72 °C. The upper primer
for this PCR corresponded to positions 2780-2802 of the cDNA, the
lower primer was identical compared with the one used for first strand
synthesis. For quantitative experiments, the PCR was competed with
serial 3-fold dilutions of a competitor template. This competitor
consisted of the SacI/PstI restriction fragment
derived from the cDNA (positions 2662-3366) which contained, in
its EcoRV site (position 2919), 52 unrelated nucleotides
from pcDNA3.1 (positions 2003-2054). Amplification of this
competitor template yields a 410-bp fragment, whereas amplification of
the authentic cDNA gives a 358-bp fragment. The PCR products were
resolved on a 5% polyacrylamide gel and stained with ethidium bromide.
The initial amount of cDNA was estimated by the amount of
competitor DNA that had to be added to the PCR in order to obtain
equimolar levels of the 358- and 410-bp products.
Cell Culture--
Normal human fibroblasts as well as various
transformed cell lines were purchased from the American Type Culture
Collection (ATCC; Manassas, VA): WI38 (CCL75), IMR90 (CCL 186), WI38
VA13 (CCL 75.1), A204 (HTB 82), A673 (CRL 1598), Hs729T (HTB 153), RD
(CCL-136), HT1080 (CCL 121), Hs913T (HTB 152), MG63 (CRL 1427), SW1353
(HTB 94), SW872 (HTB 92), SK-LMS-1 (HTB 88), SK-UT-1 (HTB 114), and
COS-1 (CRL-1650). All cells were grown at 37 °C under an atmosphere
of 5% CO2 in DMEM supplemented with 9% fetal calf serum,
100 µg/ml streptomycin, and 100 units/ml penicillin.
For immunological analysis the cells were scraped with a rubber
policeman into an isotonic buffer containing various protease inhibitors and lysed by freezing and thawing. Cellular proteins were
further fractionated into water soluble cytoplasmatic proteins, detergent soluble membrane proteins, and nuclear proteins as described (14).
Gene Localization--
The localization of the p120 gene was
determined by FISH as described (15, 16). Metaphase spreads of human
chromosomes were prepared from synchronized lymphocytes and fixed on
glass slides. One of the cDNA clones (3,863 bp) was labeled with
biotinylated dATP and hybridized overnight at 37 °C to the denatured
chromatin in a buffer containing 50% formamide. After being washed,
the slides were stained with fluorescein isothiocyanate-conjugated avidin and counterstained with DAPI. The FISH signal and the DAPI banding pattern were separately recorded (15, 16).
Immunological Procedures--
Antibodies were raised against a
synthetic peptide corresponding to amino acids 1105-1119 of p120
(Neosystem, Strasbourg, France). A tyrosine residue was introduced at
the N terminus of the peptide to enable coupling to the carrier protein
ovalbumin. The peptide-ovalbumin conjugate was injected into two
rabbits at 14-day intervals (17, 18). After the fourth injection, blood
was collected and the antibodies were purified from the serum by
affinity chromatography following standard procedures (18).
For this purpose an affinity column was prepared that carried the C
terminus of p120 expressed as a bacterial fusion protein. A
SacI/EcoRI restriction fragment (nucleotides
3361-3888 corresponding to amino acids 1063-1119) was ligated into
the expression vector pGEX-5X (Amersham Pharmacia Biotech) in the
correct reading frame. This construct was transfected into competent
bacteria (Escherichia coli BL21) and expressed after
induction with isopropyl thiogalactopyranoside (final concentration 0.1 mM). The bacteria were collected by centrifugation and
lysed by sonification in 0.1 M MOPS, pH 7.5. After washing of the cell debris with the same buffer, the fusion protein was extracted with 2% SDS, 0.1 M MOPS, pH 7.5. This extract
was coupled to Affi-Gel 10 (Bio-Rad, Richmond, CA) according to the
instructions of the supplier.
The purified antibodies were tested in an enzyme-linked immunosorbent
assay as described (17, 18). For immunoblotting, cell culture proteins
were separated on 3-10% gradient polyacrylamide gels (19) and
transferred to nitrocellulose (17, 20). After residual sites had been
blocked with bovine serum albumin, the blot was incubated with
antibodies at a dilution of 1:50 (relative to initial volume). Bound
antibodies were detected with a phosphatase-conjugated secondary
antibody (Sigma), followed by development with bromochloroindolyl phosphate/nitro blue tetrazolium substrate.
Transfection Studies--
A full-length construct was created by
ligation of selected restriction fragments derived from our cDNA
clones into the expression vector pcDNA3.1(+) (Invitrogen). The
final construct contained nucleotides 63-3888 inserted into the
KpnI/XbaI site of the plasmid. Identity and
reading frame were verified by DNA sequencing. The construct was
transfected into monkey cells (COS-1) for transient expression or into
human cells (A204 and HT1080) for stable expression. To this end, the
cells were grown in DMEM supplemented with serum to about 50%
confluency (approximately 105 cells/10 cm2
dish), and the plasmid DNA (2 µg/10 cm2 dish) was added
along with 8 µl of Lipofectin in a total volume of 2 ml of Opti-MEM
(both from Life Technologies, Gaithersburg, MD). After an incubation
period of 12 h, the medium was replaced with DMEM supplemented
with serum, and the cells were allowed to recover for 48 h.
Resistant colonies were selected in the presence of 800 µg/ml
Geneticin G418 sulfate (Life Technologies) over a period of 4-8 weeks
(10). Total RNA was extracted from resistant colonies with the RNeasy
kit from Qiagen (Hilden, Germany). Expression of the p120 gene was
analyzed by Northern blotting using the full-length cDNA for p120
as a probe. Expression of the G418 resistance gene APH was detected
with a radiolabeled PstI/NcoI restriction
fragment derived from pcDNA3.1 (nucleotides 2314-2702).
Molecular Cloning of p120--
We have previously reported on the
preparation of a subtracted cDNA library enriched for clones that
are expressed by normal but not by SV40 transformed human fibroblasts
(4, 5). One of the clones derived from this library attracted our
attention because its expression was completely down-regulated upon
oncogenic transformation. This clone was 207-bp long and appeared to
code for a novel ankyrin-like protein.
The clone was now used as a probe to screen a commercial cDNA
library prepared from human fibroblasts with the intention to isolate
the full coding region for the novel ankyrin-like protein. After four
rounds of screening we obtained 21 independent
Altogether, the 32 cDNA clones covered a contiguous sequence of
4230 bp (Fig. 1). This sequence started
with a 5' untranslated region of 174 bp that was particularly rich in
the nucleotides G and C. The first ATG codon at position 175-177 was
preceded by two in frame stop codons. This putative translation start
site was followed by an open reading frame of 3357 nucleotides that terminated in the stop codon TAG (position 3532-3534). After the stop
codon, there was a 3' untranslated sequence of 708 nucleotides which
harbored two consensus polyadenylation signals AATAAA at position
3842-3847 and 4218-4223. The sequence of the probe used for screening
was found at position 1607-1813.
Amino Acid Sequence--
The open reading frame predicts a protein
of 1119 amino acids with a molecular mass of 127.4 kDa and an
isoelectric point of 7.0 (Fig. 1). In the following, this polypeptide
will be termed p120. It contains five potential glycosylation sites of
the form NXT/S, but only two sites are likely to be used for
modification (positions 747 and 753) because only these sites are
situated in the extra-cytoplasmic portion of the novel protein (see
below). Detailed computer analyses revealed that the novel polypeptide can be divided into two parts, an N-terminal part that is related to
the cytoskeletal protein ankyrin and a C-terminal part that is related
to transmembrane proteins.
The N-terminal part encompasses 18 repeats of 33 amino acids each
(Fig. 2A). Fifteen of these
repeats share 21-52% identity (or 27-55% similarity if conserved
amino acid substitutions are included) with the consensus motif
XGXTPLHLAARXGHVEVVKLLLDXGADVNAXTK derived from ankyrin-like proteins (21-23). Three repeats contain only
part of the ANK motif (in particular, the Leu-His sequence) but are
otherwise barely related to the ANK consensus sequence. Given this
repetitive structure, it seems likely that the N-terminal portion of
the novel protein evolved from an ancestral ANK motif that was
duplicated 15-18 times during evolution. ANK motifs occur in a large
variety of proteins with vastly different functions (21-23), including
cytoskeletal proteins (ankyrin), transcriptional regulators (NF
The C-terminal part of the novel polypeptide encompasses seven
hydrophobic segments of about 20 amino acids each, which may act as
transmembrane domains. A hydrophobicity plot according to Kyte and
Doolittle (24) locates these segments at positions 722-742, 767-787,
806-824, 850-870, 873-893, 941-961, and 1010-1030 (Fig.
2B). Another program based on the algorithm of Engelman et al. (25) also predicts seven hydrophobic segments, but
this program places one of them at position 897-917 instead of
position 850-870. It is therefore possible that only six of the
hydrophobic segments function as transmembrane domains, while one of
them (probably the segment at position 850-870 which contains several charged residues) does not span across the entire membrane but enters
the lipid bilayer partially. This structure is reminiscent of some ion
channels, which possess six transmembrane domains and a pore loop
gating the channel (26, 27). Since the polypeptide does not contain a
typical signal peptide at its N terminus, an internal segment must
serve as signal peptide as found for example with the anion exchanger
of the red blood cell membrane (28, 29). The topological orientation of
membrane proteins can be predicted by analysis of positive charges
adjacent to the first transmembrane domain (30). These predictions
suggest that the novel protein will assume a type II orientation, in
which the N-terminal domain with all the ANK repeats lies at the
cytoplasmic side of the cell membrane as proposed in Fig.
2C. In this model, the two glycosylation signals found at
positions 747 and 753 occur in the first extracellular loop and should
therefore be accessible for modification by N-linked
carbohydrates. It is obvious that this proposed structure awaits
verification by experiments.
In addition to the ANK repeats and to the transmembrane domains, the
predicted protein contains an N-terminal segment of 60 residues and a
C-terminal segment of 90 residues. Both of these segments do not reveal
substantial similarity to any known protein motif.
Similarity to Other Proteins and to Expressed Sequence
Tags--
The amino acid sequence of the novel protein was compared
with all entries of the Swissprot data bank. Numerous hits were found
that corresponded to the cytoskeletal protein ankyrin and to other
proteins containing ANK repeats. In addition to these hits, we found
one protein that had emerged from the genome project of the nematode
Caenorhabditis elegans (C29E6.2, accession number Z72504).
Using a GAP creation weight of 9 and an extension weight of 3, an
alignment with 28% identity or 37% similarity resulted and extended
over the entire length of the two proteins, including the ANK repeats
and the transmembrane domains (not shown). Nothing is known about the
function of the C. elegans protein as it was simply
predicted by computer analysis of the genomic sequence. Nevertheless,
the striking conservation of the primary structure between two such
distantly related species may indicate a fundamental function of the
new protein.
The cDNA sequence of p120 was also compared with all
expressed sequence tags stored in the EST data bank. Three partial
cDNA clones were found that matched our cDNA sequence (not
shown), namely AA502409 (441 bp), AA972567 (284 bp), and AA873172 (580 bp). Interestingly, all these clones were derived from human tumor
samples (colon, lung, and kidney), which were analyzed in the course of
the Cancer Genome Anatomy Project (CGAP). The significance of this
finding is not yet clear.
Expression of p120 in Cells and Tissues--
The expression of the
novel gene was analyzed by Northern blotting experiments. On a blot
containing RNA from different batches of human fibroblasts (WI38,
IMR90), our cDNA clones hybridized to a single band of 4.6 kilobases (Fig. 3). This size is in
agreement with the length of our cDNA sequence (4230 bp) assuming a
poly(A) tail of about 300 nucleotides. With RNA from SV40 transformed fibroblasts (VA13) no signal was obtained, indicating that
transcription of the corresponding gene was completely turned off after
oncogenic transformation.
To determine whether expression of the p120 gene was repressed
exclusively in virally transformed fibroblasts or whether this down-regulation was a common feature of transformed cells, we analyzed
11 different cell lines derived from spontaneous mesenchymal tumors
(Fig. 3). Cells from four rhabdomyosarcomas, two fibrosarcomas, an
osteosarcoma, and a chondrosarcoma did not possess any traces of the
mRNA. When two different leiomyosarcoma cell lines were examined,
the p120 mRNA was detected at a low level in one, but not in the
other. Only cells from a liposarcoma contained large amounts of the
p120 mRNA. Rehybridization of the same Northern blot with a probe
for GAPDH demonstrated that all lanes contained similar amounts of
intact RNA. Thus, the repression of the p120 mRNA is a common
feature of most, but not all, mesenchymal tumor cells.
We now asked the question what tissues might express the novel
mRNA. To this end our probes were hybridized to commercial Northern
blots containing mRNA from various adult human tissues, including
skeletal muscle, heart, lung, placenta, kidney, liver, pancreas,
spleen, thymus, brain, prostate, testis, ovary, small intestine, colon,
and cultivated leukocytes. To our surprise, we were not able to detect
any signal that was stronger than background (not shown). Likewise, a
Northern blot containing RNA from four fetal human tissues (brain,
liver, lung, and skeletal muscle) did not yield any distinct signal.
Rehybridization of the same blots with probes for
We therefore used a very sensitive PCR approach to detect expression of
the p120 gene in normal human tissues (Fig.
4). The mRNA was transcribed into
cDNA and used as template for competitive PCR. A pair of
strand-specific primers was selected, which allowed amplification of a
358-bp fragment corresponding to the fourth and fifth putative
transmembrane domain. These domains were chosen because we have
evidence that they are encoded by two separate exons.2 For quantitation, a
competitor template was used as an internal standard. This template
consisted of the same 358-bp sequence but harbored additionally an
unrelated sequence of 52 bp (total length 410 bp) in its center to
permit distinction between competitor DNA and the 358-bp cDNA on a
polyacrylamide gel. The competitor template was added to the reactions
at serial 3-fold dilutions. At a high cDNA to competitor ratio,
amplification of the 358-bp fragment was observed, whereas at a low
ratio, amplification of the 410-bp competitor occurred. The absolute
amount of cDNA in the reaction mixture could thus be estimated from
the transition point where competitor DNA and cDNA fragment were
obtained in equimolar amounts. Applying this technique to mRNA from
cultivated fibroblasts, the expected band of 358 bp was obtained (Fig.
4). Equimolar amounts of the 358-bp fragment and the 410-bp competitor were reached when 3.3 × 106 copies of the competitor
template were added to the reaction mixture. This number would
correspond to approximately 500 copies of p120 mRNA per fibroblast.
When RNA from a 12-week old human embryo was analyzed in a similar way,
the diagnostic band of 358 bp was also obtained, indicating that the
p120 gene was in fact expressed in human tissues. Competition
experiments suggested that these tissues contained at least a 1000-fold
lower level of p120 mRNA than cultivated fibroblasts. RNA from
embryonic muscle possessed an even lower level, which was barely
detectable by this sensitive method (not shown). The same results were
obtained when another set of PCR primers was used. Thus, the p120 gene is faithfully transcribed in human tissues but at an extremely low
level.
Localization of the p120 Gene--
To further characterize the
p120 gene, we set out to determine its localization in the human genome
by the FISH technique. When a biotinylated probe prepared from our
cDNA clones was hybridized to metaphase spreads of human
chromosomes, a clear signal was observed on the long arm of chromosome
8 (Fig. 5). Among 100 mitotic spreads
examined, 89 showed specific signals on at least one pair of
chromatids. The exact position as determined by superimposing the FISH
signal with the DAPI banding pattern was found to be band 8q13. No
additional locus was observed. The gene for the novel protein is
therefore situated at a single locus on human chromosome 8. It might be
of interest to note that the gene for ankyrin 1 is also located on this
chromosome, in region 8p11.2 (23).
Initial Characterization of the Protein--
In an effort to
characterize the protein encoded by the p120 gene, polyclonal
antibodies were raised against a synthetic peptide comprising 15 amino
acids from the C terminus of the predicted polypeptide (residues
1105-1119). In enzyme-linked immunosorbant assays, the antibodies
recognized their antigen even at high dilution (not shown). On the
other hand, they did not work for radioimmunoprecipitations or indirect
immunofluorescence studies probably because they were directed against
a sequential epitope, which may not be accessible in the folded
protein. When tested on Western blots, the antibodies reacted readily
with a bacterially expressed fusion protein spanning the intracellular,
C-terminal domain of p120 (Fig. 6). We
therefore used Western blotting to analyze protein extracts from
liposarcoma cells, which possess a relatively high level of the p120
mRNA as demonstrated above. A total cell extract did not yield any specific immunoreactive band, but an extract enriched for membrane proteins showed a major band with an apparent molecular mass of 130 kDa
and several minor bands with molecular masses smaller than 60 kDa (Fig.
6). It is likely that the 130-kDa band corresponds to the full-length
product of the p120 mRNA, whereas the minor bands may represent
unrelated proteins cross-reacting with our antibodies as they were
obtained also with all other samples examined. When a membrane extract
from fibroblasts was analyzed in a similar way, a very faint band of
130 kDa was detected (not shown). In contrast, no bands in the 130-kDa
region were observed with membrane extracts from SV40 transformed
fibroblasts or HT1080 fibrosarcoma cells. Thus, liposarcoma cells and
fibroblasts produce p120 at a low but clearly detectable level.
Overexpression in Eukaryotic Cells--
For a functional analysis
of the novel protein, we tried to express large amounts of p120 in
human cells. To this end, a full-length cDNA construct was
transfected into A204 and HT1080 cells. Positive transfectants were
selected by their resistance to the antibiotic G418, and resistant
colonies were tested on Northern blots for their expression of the p120
mRNA (Fig. 7). Only a very few
resistant colonies were obtained, and more surprisingly, only one of
them (n = 32) was found to express a mRNA related
to p120. However, a closer inspection revealed that even this colony
did not express the full-length mRNA, but rather a truncated form
of less than 3000 nucleotides that cannot encode a functional protein
(Fig. 7, lane 9). Rehybridization of the same Northern blot
with a cDNA probe for the G418 resistance gene APH demonstrated
that all resistant cells did in fact express the APH mRNA. When a
batch of freshly transfected cells was investigated prior to
subcloning, a weak signal corresponding to the p120 mRNA was
initially observed. However, this signal gradually disappeared after
subcultivation, whereas the APH signal persisted. Similar results were
obtained when transient expression studies were performed with COS-1
cells. We therefore concluded, that expression of the p120 mRNA is
selectively turned off in transfected cells, either by specific removal
of the p120 cDNA or by selective inactivation of the incorporated gene. One possibility to explain these results is that the uncontrolled expression of p120 is not compatible with the normal growth of human
cells.
By subtractive cDNA cloning, we have identified a novel gene
on human chromosome 8 that codes for a transformation-sensitive protein
termed p120. Although we have utilized state of the art immunological
and molecular biological techniques, we have not yet been able to
demonstrate the exact function of this protein. The biochemical
characterization was made difficult by the unusual regulation of the
novel gene product as follows.
The mRNA for p120 was expressed by cultured fibroblasts, but it was
specifically repressed after oncogenic transformation. Based on a
quantitative PCR analysis, we estimate that a fibroblast contains about
500 copies of the mRNA, which corresponds to a gene that is
expressed at moderate level. In contrast a variety of mesenchymal tumor
cell lines did not express p120 at all, with the exception of
liposarcoma cells that contained a relatively high level of the mRNA.
The protein encoded by the p120 mRNA could barely be detected in
fibroblasts, but it was found at a moderate level in liposarcoma cells.
Since these cells possess a fairly high level of the mRNA, the
protein must either turn over very quickly or it is translated with
very poor efficiency. There is evidence in favor of the latter possibility. Analysis of the mRNA reveals an extremely high
proportion of rare amino acid codons along the entire sequence.
In contrast to cultivated fibroblasts, p120 was expressed at a very low
level in human tissues. These levels could not be detected by Northern
blotting, but required a very sensitive PCR approach for detection.
Based on a competitive PCR experiment, we estimate that the tissues of
a human embryo possess a 1000-fold lower level of the mRNA than
cultivated fibroblasts. It is possible that the gene is transcribed
only at a very restricted area by specialized cells. However, our
Northern blotting experiments with 20 adult and embryonic tissues and
our preliminary studies with in situ
hybridization2 do not support this idea. It seems more
likely that most cells express the gene at a very low level and/or for
only a very short time during their life span.
The low expression of p120 is reflected by the fact that the
comprehensive data bank of expressed sequence tags contains only three
partial clones that overlap with our sequence. All these clones are
derived from human tumor samples.
Overexpression of the p120 cDNA in normal and transformed cells has
not been successful. Even utilization of an ecdysone-inducible system2 did not overcome the problem. A careful analysis of
our transfection experiments suggested that the uncontrolled expression
of the novel protein might be toxic to the transfected cells.
The low expression of p120 in human tissues might indicate a crucial
function of the novel protein played in an as yet unknown regulatory
pathway. This hypothesis is supported by the remarkable conservation of
the protein sequence between distantly related species such as humans
and nematodes. Why the mRNA is found at a moderate level in
cultured fibroblasts, but only at very low level in human tissues, is
difficult to explain. Even fetal lung, the tissue from which the
fibroblasts had been derived, contain a very low level of the mRNA
that was not detectable by Northern blotting experiments. It therefore
appears that removal of the cells from their natural surrounding and
subsequent cultivation in vitro leads to the specific
up-regulation of the p120 mRNA, which is then barely translated
into protein. This up-regulation seems to be counteracted by oncogenic
transformation, as SV40 transformed fibroblasts as well as most
mesenchymal tumor cells do not express the p120 mRNA. The major
differences between normal and transformed cells as well as between
fibroblasts grown in tissues and fibroblasts cultivated in
vitro are related to the morphological and adhesive properties of
the cells (1, 2). In a preliminary study we have therefore analyzed the
expression of p120 under different cultivation conditions.2
Yet we could not detect any changes in the mRNA level between fibroblasts grown on plastic, fibroblasts grown on collagen-coated dishes, and fibroblasts grown within a contracted collagen gel. The
peculiar regulation of the p120 gene is therefore not simply controlled
by the shape or the attachment of the cells.
A clue to the function of p120 might be obtained from a detailed
analysis of its primary structure. The N-terminal part of the protein
contains 15-18 ANK repeats, a structural motif that is found in some
cytoskeletal proteins, some transcription factors, and some membrane
receptors (for review, see Refs. 21-23). A comparable number of ANK
repeats, however, occurs only in the cytoskeletal protein ankyrin,
which contains 22-24 repeats, and in an insect toxin (latrotoxin).
Transcription factors and membrane receptors possess 2-6 ANK repeats.
It is generally assumed that the ANK motif plays an important role in
protein-protein interactions. Ankyrin 1 from erythrocytes has been
characterized in great detail. It has been demonstrated to interact
with a variety of other proteins, including spectrin, tubulin, and
several integral membrane proteins. A typical example of the latter
group is the anion exchanger of the erythrocyte membrane which is
anchored via ankyrin to the cytoskeleton (28).
The C-terminal part of p120 contains six putative transmembrane domains
and a pore loop structure, features that are reminiscent of many ion
channels (26, 27). It is therefore conceivable that p120 functions as
an ion channel that is bound to a cytoskeletal element similar to the
anion transporter mentioned above (28). If so, p120 would be unusual in
that it combined the feature of a transporter and that of a
cytoskeletal anchor within a single polypeptide chain. Since the gene
is expressed at an extremely low level under physiological conditions,
it might play a regulatory function. These characteristics are
compatible with the idea that it acts as an ion channel involved in
signal transduction.
All the structural features of p120 are now shared by members of a
loosely defined family of proteins, the TRP-like molecules (for review,
see Refs. 31 and 32). Originally, TRP and its related protein TRPL were
discovered in the fruit fly Drosophila as
G-protein-regulated channels that mediate the light-activated conductance in the visual system. Like p120, TRPL is composed of about
1100 amino acids, it lacks an N-terminal signal peptide, but contains
six trans-membrane domains and a pore loop, and it possesses four ANK
repeats in its N-terminal domain. Recent evidence suggests that TRP and
TRPL form part of a capacitative (or store-operated) channel (31, 32).
Such channels are activated by depletion of Ca2+ from
internal stores. They are widely distributed in invertebrates and
vertebrates and seem to be located in the plasma membrane of most
excitable and nonexcitable cells. Several TRP-related proteins have
also been identified in humans (e.g. TRPC1 and TRPC3). All
these TRP-like molecules have a topology similar to p120, although they
show a rather low similarity at the amino acid level. TRPC3 (htrp3,
Ref. 33) taken as example shares 27% identity or 39% similarity with
p120 over a region of 364 amino acids corresponding to part of the ANK
repeats and part of the transmembrane domains. This similarity may
suggest that p120 represents a novel member in the heterogeneous family
of TRP-like molecules.
To conclusively demonstrate that p120 is indeed an ion channel, it is
inevitable to express our novel protein in a eukaryotic expression
system. So far, all our efforts in this direction have been fruitless
since the uncontrolled expression of p120 did not seem to be compatible
with normal growth of the transfected cells. However, it is conceivable
that a selected domain (rather than the entire protein) could
successfully be expressed in eukaryotic cells. Thus, experiments
performed with suitable fragments derived from our cDNA clones
might eventually shed light on the function and regulation of this
interesting protein.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-32P]dCTP by the random primed oligo-labeling method
(8). This probe was used to screen approximately 0.5 × 106 recombinant phages of a commercial cDNA library,
which had been prepared from human lung fibroblasts (HL1011,
CLONTECH Laboratories) by the plaques hybridization
technique (9). Positive colonies were picked, amplified, and subcloned
into the plasmid pUC19 following standard procedures (10). To obtain
the 5' end of the cDNA, the RACE technique was applied using methyl
mercuric hydroxide-denatured mRNA from human fibroblasts (IMR90)
and the AmpliFINDER RACE kit (CLONTECH). Two
synthetic oligonucleotide primers were prepared for this purpose
corresponding to the cDNA sequence (reverse primer nucleotides
523-547, nested primer nucleotides 439-466).
RESULTS
clones, but none of
them reached all the way to the 5' end of the corresponding mRNA.
The RACE technique was therefore utilized to amplify the 5' end, which
resulted in the isolation of 11 additional clones.
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Fig. 1.
Nucleotide and derived amino acid sequence of
clone p120. Cysteine residues are encircled, and
potential glycosylation sites are marked by triangles.
Open symbols indicate glycosylation sites located at the
cytoplasmic side of the plasma membrane, and closed symbols
those located at the extracellular side.
B,
I
B, bcl-3), and membrane-bound receptors (Notch).
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Fig. 2.
Topological model of p120. A,
alignment of ANK repeats present in the p120 polypeptide. Identical
residues are boxed. The repeats are ordered according to
similarity (dendrogram). B, hydropathy plot of
the p120 polypeptide calculated according to Kyte and Doolittle.
Putative transmembrane domains are shaded. C,
putative model of p120 in the cell membrane. ANK repeats are shown as
shaded circles, and sequences sharing only partial
similarity with the ANK consensus sequence are shown as
open circles. Transmembrane domains are indicated by
white bars. Asn-linked carbohydrates are denoted by
"Y".
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Fig. 3.
Northern blot analysis. Total RNA from
two different batches of normal human fibroblasts as well as from 12 transformed human cell lines was resolved on an agarose gel,
transferred to nylon membranes, and hybridized with a radiolabeled
cDNA probe for p120 (top) or for GAPDH
(bottom). The migration positions of the ribosomal RNA
subunits are indicated in the right margin.
-actin or GAPDH
produced strong signals, indicating that all the blots contained normal
amounts of undegraded mRNA. It therefore appears that all the human
tissues investigated above produce extremely low amounts of the p120
mRNA, which cannot be traced by Northern blotting.
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Fig. 4.
Quantitative PCR analysis. RNA from
human fibroblasts (IMR90) and from a 12-week old human embryo was
transcribed into cDNA and amplified by PCR. A set of
strand-specific primers was used that yielded a diagnostic band of 358 bp. The reactions were competed with a competitor template that
contained the same sequence plus 52 nucleotides of an unrelated
sequence (410 bp). The copies of this competitor added per reaction are
indicated at the top. The PCR products were separated on a
5% polyacrylamide gel and stained with ethidium bromide.
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Fig. 5.
Chromosomal localization of the p120
gene. Metaphase spreads of human lymphocytes were hybridized with
a labeled cDNA probe for p120 (A). The chromosomes were
counterstained with DAPI (B). The gene was assigned to
chromosomal band 8q13 by superimposing the FISH signal and the DAPI
banding pattern (C).
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Fig. 6.
Detection of p120 by antibodies. A crude
preparation of a fusion protein containing amino acid residues
1063-1119 (lanes 1 and 4), as well as two cell
membrane extracts, one obtained from liposarcoma cells (SW872,
lanes 2 and 5), the other from SV40 transformed
fibroblasts (VA13, lanes 3 and 6) were resolved
on a 3-10% gradient SDS-polyacrylamide gel and either stained with
Coomassie Blue (lanes 1-3) or transferred to nitrocellulose
and stained with polyclonal antibodies (lanes 4-6). The
antibodies recognize a sequential epitope of p120 within amino acids
1105-1119. The migration positions of globular protein standards are
shown in the left margin.
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Fig. 7.
Transfection experiments. A full-length
construct for p120 was transfected into A204 cells. The entire
population of freshly transfected cells (lane 3) as well as
individual colonies selected by their resistance to G418 (lanes
4-9) were analyzed by Northern blotting. The blot was hybridized
with a cDNA probe for p120 (top) or a cDNA probe for
the APH resistance gene (bottom). The migration positions of
the ribosomal RNA subunits are indicated in the right
margin. RNA from IMR90 fibroblasts (lane 1) and from
A204 cells (lane 2) was included as control.
DISCUSSION
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ACKNOWLEDGEMENT |
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We thank Dr. K. H. Winterhalter for continuous support.
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FOOTNOTES |
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* This study was supported by grants from the Swiss National Science Foundation (31-50571.97) and the Bernese Cancer Liga.The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) Y10601.
M.E. Müller Institute, University of Bern, P. O. Box 30, CH-3010 Bern, Switzerland. Fax: +41 31 632-4999; E-mail:
trueb{at}mem.unibe.ch.
2 T. Schenker and B. Trueb, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: bp, base pair(s); APH, aminoglycoside phosphotransferase; DAPI, 4,6-diamino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; TRP, transient receptor potential protein; TRPL, TRP-like protein; MOPS, 3-(N-morpholino)propanesulfonic acid.
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REFERENCES |
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