(Received for publication, December 15, 1994)
From the
The 6-kilobase (kb) cDNA of pTB11 clone and its 5` fragment of
3.7 kb encoding the canine heart Na/Ca
exchanger (Nicoll, D. A., Longoni, S., and Philipson, K. D.(1990) Science 250, 562-565) were transiently expressed in 293
cells to investigate the role of the 3`-``untranslated''
region. Both fragments yielded high levels of expressed protein that
were well incorporated in the membranes. Cells expressing the 6-kb cDNA
produced rearranged transcripts of smaller than expected size. A
120-kDa polypeptide was produced in cells expressing the modified
exchanger, and Ca
uptake was higher in this type of
transfected cells. A constant stretch of nucleotides located at the 3`
end of the 6 kb cDNA was found to be connected, by alternative RNA
splicing, to four different upstream sequence positions. The deduced
hydrophobic sequence of the spliced-in exon could replace the IX or the
XI trans-membrane domain of the exchanger protein in two spliced
isoforms. The new exon sequence was not completely included in the
pTB11 insert, i.e. these two products were artificially
truncated. The RNA processing of these two alternative 5`-splicing
sites also occurred in tissues, as shown by RNase protection analysis.
In a third type of isoform the splicing took place downstream of the
originally proposed stop codon, whereas in a fourth type a stop codon
was introduced after the V hydrophobic segment in the large
intracellular loop.
The Na/Ca
exchanger (NCE) (
)is an electrogenic transporter that couples the
translocation of Na
and Ca
in
opposite directions across the plasma membrane (see Reeves(1992)).
Partially purified preparations of the heart sarcolemmal exchanger
usually show a major component of about 120 kDa and two other species
of 160 and 70 kDa, respectively (Philipson et al., 1988;
Nicoll and Philipson, 1991). The cDNA sequence of the cloned NCE
encoded a protein of 970 amino acids with a molecular mass of 108 kDa.
The coding sequence was located in the 5`-proximal region (about 3 kb)
of the 6-kb cDNA insert carried in clone pTB11. The full-length NCE
transcript size was estimated at 7 kb (Nicoll et al., 1990).
The deduced protein apparently corresponds to the 120-kDa species, the
difference in size being attributed to glycosylation at Asn-9 (Hryshko et al., 1993). The deduced primary structure of the 108-kDa
polypeptide indicates a cleavable NH
-terminal signal
sequence and 11 trans-membrane domains (TM), interrupted by a large
hydrophilic loop between TM segment V and VI. Cloning of the cardiac
NCE cDNA from several mammalian species has revealed a high degree of
sequence conservation (Nicoll et al., 1990; Aceto et
al., 1992; Low et al., 1993; Kofuji et al.,
1992; Komuro et al., 1992; Lee et al., 1994).
However, tissue-specific isoforms with identical TM domains but with
shortened hydrophilic segments are produced by alternative splicing of
a single gene product (Furman et al. 1993; Kofuji et al. 1993, 1994). Recently a second NCE gene specifically expressed in
brain was also identified (Li et al., 1994).
The exchange
activity of the cardiac NCE expressed in oocytes by injection of the
2.9-kb cRNA coding sequence was found to be similar to that of heart
sarcolemma, whereas an active NCE was not produced by injection of the
6-kb cRNA (Matsuoka et al., 1993), lacking the
poly(A) tail (Huarte et al., 1992). The
exchanger cDNA has been expressed in several heterologous systems, i.e. in the baculovirus Sf9 cell system, in COS-7 and 293
cells, and stably transformed CHO cells by transfection with plasmid
DNA, and in vaccinia virus-infected HeLa cells (Li et al.,
1992; Aceto et al., 1992; Kofuji et al., 1992; Pijuan et al., 1993; Low et al., 1993). In all these studies
a shortened version of the 6-kb cDNA, i.e. a fragment of
3-3.7 kb, that included the proposed coding sequence was used.
The large portion of cDNA downstream of the putative stop codon was
omitted in the construction of all expression vectors. As a result, the
function of this region, traditionally considered to be untranslated,
has remained unknown. The study of its function has been the aim of the
present contribution: specifically, its role in the expression of the
NCE cDNA was explored. The approach used has been the transient
transfection of 293 cells with plasmids carrying different lengths of
the canine cardiac NCE cDNA, the analysis of the expression products,
and the determination of NCE activity in the transfected cells. The
results have led to the conclusion that the 6-kb cDNA is an incomplete
copy of an immature transcript that could produce at least four
differently spliced transcripts when expressed in 293 cells.
Figure 1:
Organization of the NCE expression
vectors and of the amplified DNA fragments. Two constructs of pcDNAI
including the whole 6-kb insert of clone pTB11 (Nicoll et al.,
1990) or the 3.7-kb HindIII-XbaI fragment of canine
NCE cDNA are shown in A, with the EcoRI, HindIII, and XbaI restriction sites as reference. The
position of the eukaryotic cytomegalovirus promoter at the 5` end of
the NCE coding sequence is indicated. The bacterial SP6 promoter at the
opposite site of the polylinker is shown by a triangle,
followed by the pcDNAI splice and poly(A) signal
sequences. In B the position of the primers used for
reverse-transcribed PCR amplification is indicated by arrows.
The SP6 primer at the 3` end of cDNA is indicated by a triangle. In the amplified fragments of 1.3 kb, 1.1 kb, 280
bp, and 260 bp the dashed lines indicate the excised regions.
Consensus sequences in the intron/exons junctions are indicated in lowercase and uppercase letters, respectively. The boxes represent the determined DNA
sequence.
Northern blot analysis of total RNA
extracted from the transfected cells showed that the 3.7-kb fragment
yielded strong hybridization signals when used as a probe in both types
of transfected cells (Fig. 2A, lanes 2 and 3).
The hybridizing mRNA of cells expressing the 3.7-kb cDNA had the
expected approximate size of 4 kb, when also considering the flanking
sequence of pcDNAI included in the 3` end of the transcripts (Fig. 2A, lane 2). By contrast, the mRNA of cells
transfected with the 6-kb cDNA failed to show a prominent band in the
6-kb region, but showed instead a smaller mRNA species (4 kb, lane 3). The similar intensity of the hybridization signals
suggests that transfection with the two NCE constructs produced similar
mRNA levels. This suggestion was supported by the control hybridization
with
-actin shown in lanes 1-3 of Fig. 3. No
hybridizing band was observed with the RNA of 293 cells transfected
with the vector pcDNAI (lane 1), confirming that natural NCE
expression in this cell line is below detection level.
Figure 2:
Northern blotting of NCE transcripts.
Total RNA extracted from 293 cells transiently transfected with pcDNAI
as control (lanes 1) and with the same vector either,
including the 5` region of 3.7 kb (lanes 2) or the total 6 kb
of the NCE cDNA (lanes 3), was loaded in duplicate (10
µg/lane) and analyzed by Northern blotting. The blots were
hybridized separately with the following probes: the 3.7 HindIII-XbaI fragment (A), the 2.3-kb XbaI fragment (B) of the NCE cDNA, and subsequently
with -actin (A and B) as control. The positions
of the hybridizing bands of the expected 6-kb transcript and of the 28
and 18 S ribosomal RNA are indicated.
Figure 3: Immunostaining of NCE polypeptides. Total membranes purified from 293 cells expressing 3.7 (A) or 6 kb (B) of the NCE cDNA and membranes from cells transfected with pcDNAI (C) were analyzed by Western blotting with a specific anti-NCE antibody in parallel with human heart sarcolemmal membranes (D), using 50 µg of protein/lane. The position of the standard molecular mass markers is indicated in kilodaltons.
The RNA samples were also probed separately with the 2.3-kb XbaI fragment to clarify the origin of the 4-kb transcript produced by the transfection with the 6-kb cDNA, (Fig. 2B). This probe produced a clear signal in the region of 4 kb in cells transfected with the 6-kb cDNA template (lane 3). This mRNA species had the size of that revealed by the 3.7-kb probe, although its signal was less intense (Fig. 2A, lane 3). By contrast in cells transfected with the 3.7-kb cDNA, the probe produced a barely detectable signal (Fig. 2B, lane 2) that could have been due to a minimal cross-contamination of the probes. Only traces of the 6-kb NCE mRNA were detected under these conditions, supporting the suggestion that this transcript was unstable in 293 cells (Fig. 2B, lane 3). It could be mentioned at this point that a prominent mRNA species of about 7 kb was previously detected in heart, brain, kidney, and other tissues, although smaller hybridizing species were also revealed by NCE probes (Kofuji et al., 1992; Lee et al., 1994). The Northern blot analysis thus suggested that the 4-kb mRNA produced by transfection with the 6-kb cDNA included portions of the 2.3-kb XbaI fragment. This species therefore appeared to derive from the rearrangement of the 6-kb transcript.
Figure 4:
Sodium-dependent Ca
uptake in NCE-transfected 293 cells. The time course of Ca
uptake (A) was measured in whole cells 24-48 h
after transfection with the 3.7-kb HindIII-XbaI
fragment (A, panel a), with the 6-kb NCE cDNA (A, panel
b), and with the expression vector pcDNAI (A, panel c).
Aliquots containing 100-200 µg of cell protein were taken for
each time point. A typical immunostaining of corresponding samples of
whole cells (150 µg of protein/lane) is also shown (B, lanes
a-c). The values of Ca
uptake are the
average ± S.D. of three to four independent sets of transfection
experiments. Standard molecular mass markers are indicated in
kilodaltons.
The polypeptide pattern revealed by the antibody in transfected cells was similar to that of human heart sarcolemmal membranes (Fig. 3) and consistent with that reported in other expression systems. At least three species were resolved by SDS-polyacrylamide gel electrophoresis in the 120-kDa region (Fig. 3, lanes A, B, and D). The slowest migrating band could correspond to the most prominent species in human heart (lane D) the latter being only slightly larger. This band was very evident in 293 cells expressing the 6-kb cDNA (lane B), but was absent in cells expressing the 3.7-kb cDNA, the faint immunoreactivity sometimes observed being due to a small cross-contamination of the samples (lane A). The two other components of the 120-kDa triplet had approximately the same intensity in the two types of transfected cells (Fig. 3, lanes A and B) and were much more intense than the heaviest component of the triplet. In heart (lane D) the slowest moving component of the triplet was by contrast the most evident. It could thus be that the slowest migrating polypeptide of the triplet was a product of the 6-kb NCE cDNA. Differences in the mobility of the expressed 120-kDa species had been previously observed in COS and CHO cells and tentatively attributed to cell-specific post-transcriptional modification of the NCE proteins (Pijuan et al., 1993).
A second group of polypeptides stained by the
antibody in both transfected cells and in heart membranes had lower
molecular mass (70 kDa; Fig. 3, lanes A, B, and D). These products are generally assumed to be proteolytic
products of the larger bands (Nicoll and Philipson, 1991). A difference
with heart membranes was the presence in 293 cells and in other
NCE-expressing cell lines of a 200-kDa band (Fig. 3, lanes A and B; Fig. 4B, lanes a and b)
which could reflect the dimerization of misfolded overexpressed NCE
proteins. Since the samples were treated with a reducing agent, it
appears likely that the dimerization was due to hydrophobic
interactions. At variance with these cells, heart membranes contained
instead a 160-kDa species also assumed to be a native NCE (Nicoll and
Philipson, 1991). The transfected cells showed additional polypeptides
in the 80-kDa region (Fig. 4B, lanes a and b)
which had not been seen in purified membranes (Fig. 3, lanes
A and B). They could possibly arise from the degradation
of misinserted overexpressed polypeptides.
When the amplification was
performed with primers matching the sequence in the vicinity of the
internal EcoRI sites, in combination with the SP6
oligonucleotide, adjacent to the 3` end of the NCE transcripts (Fig. 1A), an interesting discrepancy in the expected
DNA length was detected. When using a primer from base 3043 in
combination with SP6 (Fig. 1B), a 260-bp fragment was
amplified only in cells expressing the 6-kb cDNA (Fig. 5A,
lane 2). By contrast, this combination of primers amplified a DNA
fragment of the expected size (580 bp) in 293 cells expressing the
3.7-kb cDNA (Fig. 5A, lane 1). Two DNA fragments of 1.3
and 1.1 kb were similarly amplified priming from base 1666 and SP6,
whereas a third fragment of 280 bp was amplified only in low amount
with this primer combination (Fig. 5B, lane 2). These
products were much smaller than expected from the primer positions,
located about 4 kb apart. However, in the system used the upper size
limit of the amplification was 1.8 kb. No DNA fragments were
amplified from the cDNA of 293 expressing the 3.7-kb cDNA with this
primer combination (Fig. 5B, lane 1), the sequence
being too large (2 kb).
Figure 5: Complementary DNA fragments at the 3` end of NCE transcript. Ethidium bromide staining of a 1.2% agarose gel showing the DNA fragments of the canine NCE sequence amplified from base 3043 with oligonucleotide 5`-CCTGCTGATGGAATCCAGCTTCA (A) and from base 1666 with oligonucleotide 5`-AGTGAGAGCATTGGCATCATGGAGG (B) in combination with primer SP6. The cDNAs used for the amplification were from cells expressing the 3.7-kb (lanes 1) or the 6-kb (lanes 2) NCE cDNAs. The size of standard DNA fragments (lanes 3) is indicated in base pairs.
The amplified cDNA fragments, including the
complementary sequences at the 3` end of the shortened transcripts,
were cloned in the TA-PCR vector and further characterized by DNA
sequencing (Fig. 1B). The DNA fragments of smaller than
expected size included at their 3` end a constant stretch of 111
nucleotides that was also found at the 3` end of the 6-kb pTB11 insert.
The 5` end of this sequence contained 47 bp of the NCE cDNA that were
connected with base 3198 (6 bp downstream of the determined canine
exchanger sequence; Nicoll and Philipson, 1990) in the 260-bp fragment
mentioned above (Fig. 1B). This sequence was fused with
a portion of the bluescript polylinker of pTB11 (45 bp, EcoRI-XbaI) followed by 19 bp of the SP6 promoter of
pcDNAI. The approximate size of this modified mRNA was 3.9 kb,
including the flanking vector sequence. It is unlikely that the
predicted amino acid sequence was modified by this rearrangement, since
it occurred downstream of the stop codon after base 2937 of NCE. Thus,
the expression of both NCE inserts produced the canonical 108-kDa
protein (Fig. 6A). Alternatively, the short NCE
sequence adjacent to the splicing site could link up with G-2821 in the
1.3-kb fragment, with G-2620 in the amplified fragment of 1.1 kb or
with G-1844 in the 280-bp fragment (Fig. 1B). The
predicted total size of these three spliced mRNAs is about 3.5, 3.2,
and 2.5 kb. The consensus sequences for RNA splicing in the junctions
of the alternatively spliced intron shown in Fig. 1B include the invariant dinucleotides GU at 5` splice and AG at the
3` splice (Treisman et al., 1983; Krainer et al.,
1984). The deduced amino acid sequence of the adjoining NCE exon found
in frame with the NCE sequence in two spliced isoforms (Fig. 6, B and C) contained a hydrophobic stretch of 16
residues with only one positively charged Lys. The sequence continued
artificially with 16 residues of hydrophilic character, specified by
the polylinker of the vector and terminated with a stop codon in the
SP6 promoter (Fig. 6C). The hydrophobic sequence was
connected to the fifth putative extracellular loop at Gly-931 (Fig. 6B). When taking into account Ile-930 and
1-2 residues of the flanking polylinker sequence, its length
would thus be adequate to replace the XI TM segment of the NCE protein.
The deduced protein sequence could conserve the previously proposed
membrane topology (Nicoll et al., 1990) assuming that the
artificial hydrophilic segment had an intracellular location (Fig. 6B). This spliced isoform had a molecular mass of
103 kDa, including the 32 residues of the NH-terminal
signal peptide. In a third type of spliced transcript, the hydrophobic
stretch was linked up with the fourth extracellular loop at Ile-864 and
similarly could replace the IX TM domain when beginning with Ser-863 (Fig. 6C). This third NCE protein would have a
predicted molecular mass of 96 kDa. Thus the sequence at the 3` end of
the NCE transcripts represents the start of an additional exon only
partially included in clone pTB11.
Figure 6: Schematic representation of the products of alternative splicing at the 3` end of the coding sequence. The predicted topology of the four NCE polypeptides derived by alternative splicing of the 6-kb NCE RNA is shown. Trans-membrane segments are represented by filled bars. The position of the amino (N) and of the carboxyl terminus (C) is indicated. The segment of 47 bp at the 3` end of the canine NCE cDNA is indicated by a filled box. This is connected in four different positions of the NCE sequence: base 3198 downstream of the stop codon of the deduced sequence (Nicoll et al., 1990) in A, base 2820 in B, and base 2621 in C. The deduced amino acid sequence of the adjoining exon is shown in single letter code. Residues encoded by the NCE sequence are indicated in bold (C). The remaining part of the sequence, also indicated by a triangle, is encoded by the polylinker of pTB11 (EcoRI-XbaI) and the SP6 promoter of pcDNAI, where a stop codon in frame with NCE sequence is marked by an asterisk. In D the spliced exon is connected after G-1844 with an in-frame stop codon following a Leu.
A deletion mutant in which the
COOH-terminal amino acids encoded by the vector (Fig. 6C), found in the two NCE isoforms described
above, were completely eliminated was also expressed in 293 cells to
investigate the influence of the artificial sequence on the
Ca uptake activity. In this construct the NCE
sequence continued with a different and shorter sequence encoded by the
vector: PYSIGVT, preceding a stop codon. The replacement of the
artificial COOH-terminal sequence did not modify the uptake activity of
the expressed protein that was in the same range of that measured with
the original construct shown in Fig. 4A, panel b: 18
nmol/mg/min. This indicated that the increased activity of the
expression products was probably due to the modification of the NCE
sequence by the alternative splicing.
An additional type of splicing producing a complete NCE isoform was identified in the DNA fragment of 280 bp (Fig. 1B). This was not abundant in the amplification products shown in Fig. 5B, lane 2, but its small size made it well represented after cloning the total products in the TA-PCR vector. The predicted NCE isoform contained 607 amino acids, including only five TM and terminating in the large intracellular loop. The deduced sequence of this rearranged transcript that was modified after Lys-606 showed a stop codon in frame with the NCE coding sequence following one additional amino acid (Leu) encoded by the spliced exon (Fig. 6D).
Figure 7: RNase protection. A, the full-length RNA probe (855 bases) consisted of 724 bases of NCE sequence and 113 bases of the flanking vector sequence. The NCE fragment of 724 bp was amplified by PCR with the oligonucleotides indicated by the arrows from base 2134 (5`-CTGGGAGAACACACCAAGCTGGAAG) and from base 5863 (5`-AGGAACAGCACAATTAGTGCACTTC) of the canine NCE cDNA. The NCE probe was complementary to the mRNA modified after base 2821, which could protect it completely. It included 687 bases of the alternatively spliced 5` exon, indicated by the shaded rectangles, that could be protected by the unspliced transcript and by that spliced at base 3198. The black box represents 37 bases of the 3` exon and the dashed line the excised intron (not on a linear scale). This probe could be partially protected by the NCE mRNA modified after base 2620 (486 bases). B, total RNA (10 µg) isolated from rat spleen (lanes 1 and 1a) rat brain (lane 2), normal human heart (lane 3), a hypertrophic human heart (lanes 4 and 4a), and 0.5 µg of total RNA of 293 cells expressing NCE (lane 5) were hybridized with the riboprobe. No RNase treatment was performed on rat spleen RNA (lane 1), whereas a RNase dilution 1:100 was used in lanes 2, 3, 4, and 5. A higher RNase dilution (1:200) was used in lane 4a. The protected RNA fragments of 724, 687, and 486 bases are indicated.
A fully protected NCE RNA hybrid of 724 bp was expected when the splicing occurred at base 2821. If this alternative 5`-splicing site would not be processed, a 37-bp shortened hybrid could be expected (Fig. 7A). The heart RNA samples examined (Fig. 7B, lanes 3, 4, and 4a) revealed two equally intense RNA fragments of the correct size (724 and 687 bases). These are most clearly seen in Fig. 7B, lane 4a, in which a lower RNase concentration and a shorter exposure time have been used to improve the resolution. Consistent with previous reports (Holtz, 1993), the human RNA samples from a hypertrophic heart (Fig. 7B, lanes 4 and 4a) showed higher NCE mRNA levels than normal heart tissue (lane 3). The proportion of the protected species was however similar in the two cases, the most abundant RNA fragments being the largest. Also the RNA of 293 cells expressing the 6-kb cDNA (lane 5) protected more efficiently the largest RNA fragments than that of 486 bases. However, these fragments were much more abundant in 293 cells expressing the NCE than in tissues, although much less RNA was used for hybridization in the former. In particular, the 687-base fragment could be the most prominent in 293 cells. This fragment was expected to be protected by the 6-kb mRNA and by the transcript spliced at base 3198. In agreement with the results of Northern blotting that indicated very high levels of a NCE transcripts at 4 kb, the protected 687-base fragment probably represented the mRNA that is spliced at base 3198, with a total size of about 3.9 kb.
The lower content of NCE mRNA in brain (Fig. 7B, lane 2) as compared with heart (lanes 3, 4, and 4a) and possibly also the expression of a second NCE gene in brain (Li et al., 1994) made it impossible to resolve the pattern of heaviest bands. However, traces of the protected species of 486 bases were also visible in brain (Fig. 7B, lane 2). No protection was obtained when rat spleen RNA was used as a negative control (lane 1a), whereas the full-length RNA probe (855 bases) was recovered after incubation with rat spleen RNA in the absence of RNase (lane 1). The results of RNase protection thus indicated that post-transcriptional modifications of the NCE mRNA involving regions coding for the COOH-terminal TM domains also occurred in heart and probably also in brain.
This study has shown that the large 3` end portion of the NCE
cDNAs contains an intron sequence that can be alternatively spliced
when expressed in 293 cells. Thus the 3` region of the cDNA was not
completely untranslated as previously thought. Expression of the 6-kb
NCE cDNA yielded spliced mRNAs encoding polypeptides whose
COOH-terminal domains differed from those of the canonical NCE. The
deduced sequence of the spliced-in domain had predominant hydrophobic
character and was connected to the extracellular loops following TM
segment VIII or X in the accepted NCE topological model (Nicoll et
al., 1990). The newly identified exon, with few adjacent residues,
could replace TM IX or XI. The adjoining NCE sequence was incomplete,
terminating with an hydrophilic stretch of amino acids encoded by the
vector that could represent an artificial cytoplasmic COOH-terminal
domain. Therefore, two of the isoforms produced by transfecting cells
with the 6-kb cDNA represented truncated versions of larger homologues:
they could for example correspond to the 160-kDa NCE protein normally
seen in gels of heart sarcolemmal membranes. On the contrary the
shorter isoform identified in this study was complete, since a
termination signal following one Leu, encoded in an other frame of the
same exon, was introduced by the alternative 5` splicing. This small
NCE protein, including only the five NH-terminal TM domains
and a shortened intracellular loop, could correspond to the 70-kDa
polypeptide revealed by the NCE antibody in sarcolemmal membrane
preparations. The complex immunoreaction pattern in heart and
transfected cells made it difficult to identify each polypeptide.
However, the slowest moving component of the 120-kDa triplet was
produced only in cells expressing the 6-kb cDNA. As discussed under
``Results,'' it is unlikely that the appearance of a 120-kDa
polypeptide was linked to the glycosylation of the expressed protein as
proposed for the heterologous expression in CHO cells (Pijuan et
al., 1993). It could also be added that the splicing of the 6-kb
transcripts would reduce the number of the extracellular loops, thus
eliminating three potential glycosylation sites (Asn-866, Asn-871, and
Asn-796). The individual expression of each isoform will eventually
correlate the deduced polypeptide sequence with the bands detected in
gels.
The mRNA processing producing the two isoforms modified after the VIII or the X TM domains also occurs in heart, as shown by the results of RNase protection. This alternative 5` splicing involving G-2821 or G-2620, is likely to provide an important source of variability for the cardiac NCE protein. The relative amount of the NCE mRNA modified at G-2620 was greater when naturally expressed in heart than in transfected 293 cells. This mRNA encoding the NCE protein modified after the VIII TM domain could also be produced in brain. In addition to the 7-kb transcript, smaller mRNAs are also recognized by NCE probes in heart and other tissues (Kofuji et al., 1992; Lee et al., 1994): particularly abundant is a transcript of about 2 kb that could encode the smaller spliced isoform produced only in low quantity in 293 cells. The full-length 7-kb NCE transcript is evidently more stable in tissues than its shorter homologue (6 kb) was in 293 cells, in which the most abundant species revealed by Northern blotting was of 4 kb. This mRNA was probably spliced at base 3198, as also suggested by RNase protection. A tissue-specific alternative 5`-splicing pattern could be produced by different concentrations of alternative splicing factors (ASF or SF2). The high concentration of SF2 in 293 cells was found to promote the use of the 5` splice site nearest the 3` site in the SV40 tumor antigens expression (Green, 1991). A similar control of alternative 5` splice site selection could also be occurring in the expression of NCE.
The splicing phenomenon described here had an additional point of interest, since it apparently led to the elimination of one, three, or six COOH-terminal TM domains. It is normally assumed that the hydrophobic domains of membrane proteins are essential for their correct insertion in the membrane and naturally, for their proper function. The overexpressed protein described here, and thus presumably its truncated species as well, became nevertheless associated with the membrane. The finding that the COOH-terminal TM composition of the NCE may vary as a result of alternative splicing and that this is accompanied by an increase of the transport function of the protein is interesting and may help to identify TM domains involved in transport regulation. Whether the increased transport activity was due to the elimination of inhibitory domains or whether the spliced-in hydrophobic sequence functioned as an enhancer is not known. However, the possibility that the vector sequence at the COOH terminus of two of the spliced isoforms contributed to the enhanced activity has been ruled out, since deletion of the artificial sequence did not alter the transport function. The variations in the trans-membrane hydrophobic domains brought about by the alternative splicing could influence transport by altering the structural interactions with phospholipid. The NCE activity has been claimed to be regulated by the phospholipid environment of the protein (Hilgemann and Collins, 1992; Collins et al., 1992).