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INTRODUCTION |
The norepinephrine transporter
(NET)1 at presynaptic nerve
terminals mediates the uptake of released norepinephrine, resulting in
the rapid termination of synaptic transmission and thereby controlling
the fine tuning of neuronal activities. Psychostimulants, including
amphetamines, and tricyclic antidepressants, such as desipramine, exert
their pharmacological effects by acting on NET (1, 2). Molecular
cloning studies have resulted in the isolation of human NET cDNA
(3), followed by several other monoamine neurotransmitter transporters
from several species (4, 5).
There is a line of evidence suggesting that functional expression of
monoamine neurotransmitter transporters is regulated in different
ways, e.g. direct or indirect protein phosphorylation, long
term modification at the level of mRNA expression, translation, and
processing (4-6). Recently, gene targeting of these transporters by
homologous recombination was utilized to investigate their physiological relevance in vivo. Elimination of the dopamine
transporter (DAT) in knockout mice produces hyperlocomotion and
indifference to cocaine and amphetamines (7).
The regulatory importance of alternative RNA splicing has been
suggested in many genes, including members of the
-aminobutyric acid
(GABA)/norepinephrine transporter family. In the glycine transporter
GLYT1, alternative splicing of 5'-noncoding and -coding sequences
confers differential cell-specific expression of GLYT1 gene products
(8). Two different cDNAs of bovine NET, which display different
COOH termini, have been reported (9, 10). Based on the analysis of
human and mouse NET genes at the exon-intron boundary for this
3'-region (11, 12), it is suggested that the difference may occur from
alternative splicing.
To gain further understanding of the importance of these transporters
in monoaminergic nervous system functions, we isolated and
characterized cDNAs encoding rat NET that displayed RNA splice variants at the 3'-region including both coding and noncoding regions.
One splice form, which codes different predicted COOH-terminal amino
acid sequences, revealed no detectable transport function. However, it
reduced functional expression of another splice variant having a
homologous COOH terminus to human NET.
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EXPERIMENTAL PROCEDURES |
Isolation of Rat NET cDNA--
Double-stranded cDNAs
from rat brain (Sprague-Dawley males, ages 10-12 weeks) ligated to the
adaptor (Marathon ReadyTM cDNA,
CLONTECH) were used to perform the polymerase chain
reaction (PCR)-based rapid amplification of cDNA ends (RACE) (13)
for cloning of 5'- and 3'-cDNAs encoding rat NET. The primers used were based on the following sequences of partial rat NET cDNAs (14,
15): 5'-GAGTAGCAGCATCGATCCATACCGTGGC for 5'-RACE, and 5'-CTGGGCTGTCCTGTTCTTCCTGATGCT for 3'-RACE. PCR conditions were: 5 cycles of 94 °C for 30 s, 74 °C for 4 min, 5 cycles of
94 °C for 30 s, 72 °C for 4 min, and 30 cycles of 94 °C
for 30 s, 68 °C for 4 min. PCR reaction mixtures were purified
and subjected to a second PCR with the nested primers:
5'-GTCAATGTGCAGGTAGGCGTTGATGCC for nested 5'-RACE and
5'-TCTGGGGCTAGATAGCTCAATGGGAGG for nested 3'-RACE. PCR products
were isolated from agarose gels and cloned into pGEM-T or pGEM-T Easy
(Promega), and colony PCR was performed for selection of positive
clones. The resulting cDNAs of positive clones were sequenced.
Full-length cDNA for rat NET was cloned by PCR with reverse
transcribed mRNA from rat brain (Sprague-Dawley male, age 4 weeks) or PC12 cells using pSuperscript II (LifeTech). The primers used were
5'-CTGAATTCGGTGTGTCCCCAGTGCCTCCGAAGTC and
5'-ATGCGGCCGCGAATGATACTTGAACAGTCTGTGACA. PCR
conditions were 94 °C for 2 min, 35 cycles of 94 °C for
30 s, 68 °C for 4 min, and 68 °C for 10 min. The resulting
cDNA was cloned into pcDNA3 (Invitrogen) and completely
sequenced on both strands using the dideoxynucleotide chain termination method.
rNET Gene Analysis--
The rNET gene coding the 3'-region of
rNET that produces alternative spliced isoforms was isolated by PCR
with the primers (P1 and R6, Fig. 1A), cloned into pGEM-T
Easy, and completely sequenced. Exons 1-12 were analyzed by PCR with
specific primers for rat NET located in the regions predicted to be
intron-neighboring exons according to the human and mouse NET genes
(11, 12).
Detection of rNET mRNA Splice Variants by Reverse
Transcriptase-PCR--
Total RNA was isolated from rat whole brain
(except cerebellum) by acid-phenol extraction (16) and used to
synthesize cDNA with avian myeloblastosis virus-reverse
transcriptase and oligo(dT)15 primers (Boehringer
Mannheim). To amplify alternative exons, we used the following PCR
primers for rNETa-L (a and b), rNETa-S (a and c), and rNETb (a and d)
as indicated in Fig. 1, A and C: a,
5'-TACGTCATCTACAAATTCTTCAGC; b, 5'-CGAAAAGAGGTCCTTGTCCTGG; c,
5'-CAATCCTATTTACACAGATGTAG; and d, 5'-CTGTGGGGCAGAGCTGTGGGGT. PCRs were
performed by initial denaturation at 92 °C for 2 min, and then 40 cycles of 92 °C for 30 s, 57 °C for 30 s, and 72 °C for 90 s, with a final extension at 72 °C for 5 min.
Heterologous Expression and Analysis of Transport
Activity--
COS-7 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum at 5%
CO2. Cells at subconfluence were harvested and transfected
with the cDNA by electroporation (17). Parallel transfection with a
pcDNA3 vector alone was performed every time for the negative
control. After electroporation, cells were diluted in the culture
medium and plated in 24- or 48-well culture plates. After the 2-day
culture, cells were washed three times with oxygenated Krebs-Ringer
HEPES-buffered solution (KRH: 125 mM NaCl, 5.2 mM KCl, 1.2 mM CaCl2, 1.4 mM MgSO4, 1.2 mM
KH2PO4, 5 mM glucose, 20 mM HEPES, pH 7.3) and incubated for 10 min at 37 °C with
[3H]norepinephrine (538.72 GBq/mmol, NEN Life Science
Products) in KRH containing an inhibitor of its catalyzed enzymes, 50 µM pargyline and 100 µM ascorbate. After
removal of excess radioligands, the cells were washed three times
rapidly with ice-cold KRH, and any radioactivity remaining in the cells
was extracted with NaOH and measured by liquid scintillation
spectrometry. Nonspecific uptake was determined in the mock-transfected
cells and also in each plate in the presence of 100 µM
cocaine. Uptake of [3H]GABA (1409.7 GBq/mmol),
[3H]glutamate (658.6 GBq/mmol),
[3H]dopamine (888 GBq/mmol), and
[3H]serotonin (1028.6 GBq/mmol) was performed in the same
way (NEN Life Science Products).
Nisoxetine Binding Assay--
Specific binding of nisoxetine, a
specific ligand for NET (18), to the plasma membrane NET was evaluated
using radiolabeled nisoxetine and intact cells on ice (19). Transfected
COS cells were washed with ice-cold KRH and incubated with 2 nM [3H]nisoxetine (3145 GBq/mmol, NEN Life
Science Products) in KRH for 2 h on ice. Nonspecific binding was
examined in the presence of 10 µM nisoxetine. For cold
saturation analysis, cells were incubated in KRH containing 2 nM [3H]nisoxetine and 1-1000 nM
cold nisoxetine. For hot saturation analysis, cells were incubated with
0.1-100 nM [3H]nisoxetine in the presence or
absence of 10 µM nisoxetine. Data were analyzed by
Scatchard plot using kcat (BioMetallics).
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RESULTS AND DISCUSSION |
Cloning and Expression of Splicing Variants of Rat NET--
From
5'- and 3'-RACE and subsequent nested PCR, we obtained from 5'-RACE two
positive groups of clones of different sizes (about 0.7 and 0.9 kb) and
one positive pool of several clones (about 2.0 kb) from 3'-RACE.
Sequence analysis of these amplified fragments indicated that they were
the 5'- and 3'-portions of the rat NET coding and noncoding regions.
Because we failed to amplify the full-length cDNA from
Marathon-ReadyTM cDNA used in RACE, the full-length
cDNA of rat NET was cloned by PCR with reverse transcribed mRNA
from rat brain and PC12 cells. The resulting amplifications showed two
positive bands on agarose gel with lengths of approximately 4.0 and 2.4 kb, respectively. These were cloned into a mammalian expression vector
pcDNA3 (Invitrogen) and analyzed by restriction enzyme
XbaI and by functional expression in COS cells and
[3H]dopamine uptake assay. The results showed that the
shorter band consisted of two different messages, one of which lacked
transport activity. The larger clones displayed
[3H]dopamine transport activities (Fig.
1D). Nucleotide sequence analyses of each clone indicated that there were at least three different forms of rat NET (Fig. 1A). Deduced amino acid
sequences indicate that there are two NET proteins having different
COOH-terminal tails, designated rNETa and rNETb (Fig. 1B).
The COOH-terminal tail of rNETa was homologous to human (3), bovine
(9), and mouse (12) NET but not to another reported bovine NET (10). On
the other hand, the COOH terminus of rNETb showed no similarity to any
known protein, including the latter bovine NET (10). Hydropathy
analysis (20) demonstrated one additional hydrophobic region in rNETb
capable of membrane spanning a structure. The scheme in Fig.
1B displays possible structures of rNETb having a
hydrophobic region in the cytosol or in the plasma membrane. Precise
membrane topology of rNETb needs to be determined using a specific
antibody to the predicted COOH-terminal tail.


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Fig. 1.
Analyses of two different isoforms of rat
NET. A, schematic representation of rat NET mRNAs
and their gene organization. Lines and boxes in
rNET genes represent introns and exons, respectively. Boxes
and lines in rNET mRNAs represent coding and noncoding
regions, respectively. Similar organization of NET genes was observed
in all species, except that the intron between exons 4 and 5 of rat NET
was approximately 10 kb in contrast to 4.5 kb of the mouse or human NET
gene. B, deduced amino acid sequences of rNETa and rNETb at
the COOH-terminal tail, hydropathy analysis of predicted protein of rat
NETs, and the possible structure of their membrane topology.
Boxed amino acid sequences indicate the additional
hydrophobic region of rNETb presented as an intracellular or
transmembrane domain in the scheme. Arrows indicate the RNA
splicing site. C, expression of rNETa and rNETb mRNA in
rat brain assessed by reverse transcriptase (RTase)-PCR.
D, COS cells transfected with five different clones derived
from PCR products were subjected to [3H]dopamine uptake
assay (left). [3H]Nisoxetine binding in intact
COS cells expressing rNETa (rNET2-1) and rNETb (rNET2-4) is shown on
the right. COS cells transfected with rNETa, rNETb, or mock
cDNA (10 µg/107 cells) were incubated with 0.1-20
nM [3H]nisoxetine with or without excess cold
nisoxetine for 2 h on ice, and specific binding of
[3H]nisoxetine was determined by scintillation counting.
*, p < 0.05 versus mock-transfected COS
cells.
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Southern blot analysis using the 3'-flanking region of rNETa (rNET2-1)
and rNETb (rNET2-4) indicated that three variants of rNET are derived
from a single copy of the gene (data not shown). Therefore, it is
suggested that rNETa and rNETb are produced by alternative RNA
splicing. To confirm this, we isolated this region by PCR from rat
genomic DNA using primer sets described in Fig. 1A (P1/R6)
and determined the gene organization. Approximately 4.5-kb PCR products
contained 4 exons (exon 13-16), which were alternatively spliced as
described in Fig. 1A. Notably, rNETb was spliced differently
within exon 13, skipping to the middle of exon 16. On the other hand,
rNETa-S was spliced at exon 13 like rNETa-L but skipped to the middle
of exon 16 at a site different from rNETb. Therefore, these splice
variants used different exo-intronic splicing sites within the exons or introns.
The expression of each rat NET mRNA variant was examined by reverse
transcriptase-PCR using primers complementary to the regions specific
for each variant (Fig. 1A). The majority of the NET mRNA was rNETa-L and, to some extent, rNETb (Fig. 1C). rNETa-S
mRNA was not observed in this condition. Pacholczyk et
al. (3) demonstrated two different sizes of mRNA encoding
human NET having 5.8 and 3.6 kb, the latter being expressed in several
brain regions and therefore a possible glial transporter. The present
findings suggest that the larger mRNA may encode rNETa-L, and the
smaller mRNA may encode rNETb and rNETa-S if any, although the
difference in sequences may be because of alternative polyadenylation
site usage (3). Systematic examination of rNETb mRNA expression in
various regions of the brain or multiple tissues should provide clues regarding its physiological significance.
The functional expression of each clone in the COS cells showed that
[3H]dopamine uptake was observed in rNETa- but not
rNETb-expressing COS cells (Fig. 1D, left). These
results suggest that rNETb lacks transport activity and/or that it is
not expressed in the plasma membrane. To examine the latter
possibility, we performed the binding of [3H]nisoxetine,
a specific ligand for NET (18) in COS cells expressing rNETb under
these conditions using intact cells (19). A small but significant
binding of [3H]nisoxetine was observed in
rNETb-expressing COS cells at 10 nM
[3H]nisoxetine concentration (Fig. 1D, right).
However, further analysis of [3H]nisoxetine binding
kinetics for rNETb could not be determined, because considerable
binding was observed in mock-transfected control COS cells when the
[3H]nisoxetine concentration in the incubation solution
increased. In contrast, rNETa-expressing COS cells showed
[3H]nisoxetine binding with an affinity of 5.99 ± 0.77 nM KD and a capacity of 3.50 ± 0.20 fmol/µg of protein Bmax
(n = 3). It is therefore probable that expression of
rNETb in the plasma membrane is partially restricted and that even
expressed rNETb lacks transport activities.
Dominant Negative NET Isoform--
If rNETa and rNETb are present
in the same cell, they might interact as suggested by DAT that makes
homomultimeric structures (21). First, we examined their interaction in
COS cells by co-expression of rNETa and rNETb. Introduction of both
cDNAs resulted in a reduction of [3H]norepinephrine
uptake and [3H]nisoxetine binding, as compared with those
observed in COS cells expressing rNETa alone (Fig.
2A). At a constant level of
rNETa, introduction of increasing amounts of rNETb cDNA caused
greater suppression of [3H]norepinephrine uptake and
[3H]nisoxetine binding (Fig. 2A). Expression
levels of each mRNA did not change substantially in co-expression,
as assessed by reverse transcriptase-PCR (data not shown). Kinetic
analyses revealed a decrease in both Km and
Vmax, reflecting an increase in apparent
affinity for the substrate and decrease in transport rate for
norepinephrine uptake: 2.46 ± 0.28 µM and 1.45 ± 0.14 µM of Km and 68.8 ± 5.8 fmol/µg of protein/min and 29.6 ± 1.9 fmol/µg of protein/min
of Vmax in COS cells expressing rNETa alone or
rNETa plus rNETb, respectively (Fig. 2B). On the other hand,
an increase in KD and decrease in
Bmax of [3H]nisoxetine binding
were observed in co-expression: 6.52 ± 1.77 nM and
18.28 ± 4.85 nM of KD and
2.00 ± 0.25 fmol/µg of protein and 1.74 ± 0.27 fmol/µg
of protein of Bmax in COS cells expressing rNETa
alone or rNETa plus rNETb, respectively. These results demonstrated the
dominant negative effect of rNETb, suggesting the possible interaction
of rNETa and rNETb, which may alter the properties of substrate
transport and ligand binding.

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Fig. 2.
Functional interaction of rNETa and
rNETb. A, COS cells were transfected with rNETa
cDNA and an increasing amount of rNETb cDNA and subjected to
[3H]norepinephrine (NE, 20 nM)
uptake and [3H]nisoxetine (2 nM) binding.
Nonspecific uptake or binding was determined in the presence of 10 µM nisoxetine. Expression of rNETb alone revealed no
significant specific uptake of [3H]norepinephrine. No
significant increase in specific binding of
[3H]nisoxetine over that in mock-transfected COS cells
was observed in this condition. *, p < 0.05. B,
kinetic analyses of [3H]norepinephrine uptake and
[3H]nisoxetine binding in COS cells expressing rNETa
alone or rNETa plus rNETb. COS cells were transfected with 5 µg of
rNETa cDNA alone or with 10 µg of rNETb cDNA. After a 2-day
culture, cells were subjected for cold saturation analysis; cells were
incubated with 20 nM [3H]norepinephrine and
0.1-10 µM cold norepinephrine or incubated with 2 nM [3H]nisoxetine with 3-100 nM
cold nisoxetine. In this condition for [3H]nisoxetine
binding, specific binding was not observed in COS cells expressing
rNETb alone, possibly because of the presence of endogenous low
affinity binding sites in COS cells (see Fig. 1D).
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To clarify further the interaction mechanism and specificity, rNETb was
co-expressed in COS cells with other rat neurotransmitter transporters
including DAT, serotonin transporter (SERT), neuronal GABA transporter
type 1 (GAT1) and glia glutamate/aspartate transporter (GLAST).
Surprisingly, rNETb reduced the transport function of DAT (Fig.
3A), SERT (Fig.
3B), and GAT (Fig. 3C) but not of GLAST (Fig.
3D). Co-transfection of neurotransmitter transporter
cDNAs, e.g. NET and GAT1 or DAT and GAT1, had no effect
on any function (data not shown). Furthermore, the functionless mutant
of DAT, e.g. D1A in which Asp79 in the putative
1st transmembrane region was replaced by alanine (19), did not change
wild-type DAT activity by co-transfection (data not shown). These
results suggest the possibility that rNETb inhibits functional
expression of the transporter via interaction at some region that is
specific for the subfamily of neurotransmitter transporters.
Alternatively, if each neurotransmitter transporter has its own
alternative spliced form like NET, each transporter may have its own
regulatory mechanism via RNA splicing. It is suggested that membrane
trafficking and sorting of neurotransmitter transporters are dependent
on sequences encoded within the COOH tail as demonstrated in GABA and
betaine transporters heterologously expressed in polarized epithelial
cells (22) and in the wild-type and COOH-terminal mutated DAT
transiently expressed in COS cells (23). Many natural and artificial
mutations have been observed to result in endoplasmic reticulum
retention and degradation, apparently because they affect protein
folding or oligomerization (24). Because oligomeric structure has been
inferred for several neurotransmitter transporters including DAT (21),
these results suggest the involvement of several amino acids at the
COOH terminus in the folding and/or oligomerization process of these
neurotransmitter transporters. To date, there are several reports
indicating that nuclear and plasma membrane hormone receptors, which
were known to form a homo- or heterodimer to act, possessed dominant
negative isoforms produced by alternative RNA splicing (see Refs.
25-27 but Ref. 28 for negative evidence). Our results add a new
example of this phenomenon specific to the nervous system.

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Fig. 3.
Co-expression of rat dopamine transporter
(A), serotonin transporter (B), GABA
transporter (C), or glutamate/aspartate transporter
(D) with rNETb in COS cells. Ligands used for
uptake assays were 20 nM [3H]dopamine with or
without 100 µM cocaine (for nonspecific uptake
determination), 10 nM [3H]serotonin with or
without 100 µM cocaine, 10 nM
[3H]GABA with or without 100 µM nipecotate,
and 20 nM [3H]glutamate with or without 1 mM cold glutamate. 5-HT, serotonin;
DA, dopamine; NS, not significant.
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Physiology and Functional Significance of NET Isoforms--
The
regulation of NET expression in vivo has been investigated
in accordance with the physiological and pathological relevance to
adrenergic neural transmission, but the results are not consistent. Long term treatment with the tricyclic antidepressant desipramine, for
example, has been reported to result in a decrease in norepinephrine uptake sites (29, 30). However, a recent in situ
hybridization study on NET mRNA expression following long term
desipramine treatment showed a significant increase in hybridization
signals in the locus ceruleus of rat brain (31). Taken together, these
suggest that the increase in mRNA expression for the NET may not
lead to an increased transporter protein expressed on the plasma
membrane of norepinephrine neurons. The probe used in the latter
investigation was three endolabeled oligonucleotides complementary to
the nucleotide sequences encoding the amino acids within 600 of the NET
mRNA (3). Therefore, these probes labeled both rNETa and rNETb
mRNAs. Based on the present observations, it is reasonable to
assume that the increase in rNETb mRNA expression causes a
reduction of NET protein on the plasma membrane even though expression
of total RNA for NET increases. We propose that measurement of the mRNA expression ratio of rNETa versus rNETb may offer a
physiological indicator of NET function.
The regulation of alternative splicing of mRNA is brought about by
complex processes in a spatial and/or temporal fashion. Mutation within
a gene can lead to inappropriate pairing of 5'- and 3'-splice sites
resulting in exon skipping or the recognition of cryptic 5'- and
3'-splice sites producing aberrantly spliced RNAs. A recent
investigation of the glutamate transporter GLT-1 in relation to the
neurodegenerative disease amyotrophic lateral sclerosis suggested a
defect in the splicing machinery that leads to the use of inappropriate
5'- and 3'-splice sites (32). These aberrant RNAs function as dominant
negative inhibitors of glutamate transport resulting in glutaminergic
exitoneurotoxicity. However, alternative truncated mRNA of GLT-1
similar to this was observed in normal controls (33). This latter
observation thus suggested that the alternative splicing form of GLT-1
mRNA plays a physiological role in the central nervous system
rather than a pathological one. The finding that alternative splicing
of NET mRNA displays a dominant negative effect provides a novel
mechanism for the regulation of synaptic transmission by
neurotransmitter transporters and highlights the need for further
investigation to learn whether this RNA processing is involved in
neuronal disorders such as depression or vulnerability to drugs of abuse.