From the
We have raised polyclonal antibodies (N6-28,
L211-226, L371-384, and C590-607) against peptides
corresponding to hydrophilic sequences of the human norepinephrine
transporter (hNET). The antisera immunoprecipitated the
[
cDNA cloning has revealed the primary structure of two families
of Na
Hitherto there has been little direct evidence to
support the topological model for the Na
We have raised
antibodies against N- and C-terminal peptides of the hNET, as well as
against peptides from the putative extracellular loops between TMs 3
and 4 and TMs 7 and 8, to investigate the membrane topology of the
hNET. Our antisera, by specifically labeling these epitopes of the hNET
in immunofluorescence studies, strongly confirm the topological model.
In order to raise antibodies against various sequence motifs
in the proposed model of hNET (Fig. 1), rabbits were immunized with
four synthetic peptides coupled to KLH corresponding to the following
putative domains: an intracellular N-terminal region (N6-28), an
intracellular C-terminal region (C590-607), a region of the large
extracellular loop (L211-226), and an extracellular loop region
between TM 7 and TM 8 (L371-384). In addition antibodies were
raised against a multiple antigenic peptide with eight identical
branches containing an 8-residue N-terminal sequence (MAP49-57;
see ``Materials and Methods''). All antisera were assayed by
ELISA for reactivity against the corresponding uncoupled peptide using
preimmune sera as controls. All KLH conjugates and the multiple
antigenic peptide produced positive antisera in at least one of two
rabbits. The rank order of antigenic activity of the peptides was
N6-28 < MAP49-57 = L371-384 <
L211-226 < C590-607, corresponding to titers between
1:3000 and about 1:30,000. The antisera were tested for their ability
to immunoprecipitate the NET. Antiserum against MAP49-57 was not
able to immunoprecipitate the hNET. The other antisera
immunoprecipitated the [
The utility to identify the NET protein by
immunoprecipitation was first examined in COS-7 cells transfected with
the mammalian expression vector pEUK-C1 containing the hNET cDNA
(6) . As shown in Fig. 2, immunoprecipitation of
[
We could demonstrate that antipeptide antibodies directed
against four hydrophilic domains of the hNET recognize this protein in
the neuroblastoma cell line SKN-SH-SY5Y and in COS-7 cells transiently
transfected with the hNET cDNA. In addition, and as expected from the
high degree of homology (Fig. 1), all four antipeptide antisera
also recognize the recently cloned bovine transporter (bNET; Ref. 16)
in transiently transfected COS-7 cells (Fig. 3, A and
B). In both, SKN-SH-SY5Y neuroblastoma cells and transfected
COS-7 cells, immunoprecipitations identified a 58-kDa hNET or bNET
protein as the predominant species. Interestingly, a polypeptide of
about the same size (53-54 kDa) was identified in rat
pheochromocytoma PC12 cells covalently labeled with tritiated xylamine
(25) and in PC12 and bovine adrenomedullary cells labeled with
tritiated desmethyl xylamine
(26, 27) . On the other
hand, such a protein was not detected in a PC12 subclone deficient in
NE transport
(25, 27) . The earlier studies could not
exclude the possibility that this protein might represent a degradation
product of the NET. The present results, however, clearly rule out this
possibility, since antibodies selective for either N-terminal or
C-terminal regions immunoprecipitated the same 58-kDa species
(Fig. 3 A). This 58-kDa protein represents a glycosylated
form of the hNET. Thus, tunicamycin treatment of transfected COS-7
cells caused a decrease in the intensity of the 58-kDa species with a
concommitant increased appearance of a 50-kDa species, which obviously
represents the core hNET protein. This conclusion is confirmed by
N-deglycosylation of hNET using PNGase F, which converted the
58-kDa species to the 50-kDa species. These two forms of the hNET also
have recently been identified by Blakely and co-workers
(15) as
54- and 46-kDa proteins in stably transfected LLC-NET cells and in HeLa
cells transiently transfected with the hNET cDNA. The glycosylated
58-kDa species, which is equivalent to the 54-kDa species described by
Melikian et al. (15) , must represent a functional form
of the NET, since transfected COS-7 cells expressing only this hNET
form (from 20 to 68 h post-transfection; Fig. 2 B)
exhibited nisoxetine-sensitive transport of [
It
should be noted that our antisera immunoprecipitated a 58-kDa protein
and a 50-kDa form (equivalent to the core protein of the hNET, see
above) in extracts from COS-7 cells transiently transfected with the
cDNAs of bNET or hDAT (Fig. 3 A). In contrast, the amino
acid sequences of these transporters predict a size of about 69 kDa.
These results, together with earlier reports of a relatively low
apparent molecular mass of labeled hNET
(15) or DAT proteins
(7, 28) , indicate that these hydrophobic transporter
proteins migrate anomalously in SDS-PAGE. At least for the hNET
protein, the anomaly is increased when the hNET protein is heated in
electrophoresis sample buffer prior to SDS-PAGE (see
Fig. 2A). Interestingly, a similar discrepancy between
the calculated and the experimentally defined molecular mass has
recently been observed by Tate and Blakely
(10) for the human
serotonin transporter, using the baculovirus expression system. In
Western blots, these authors identified a glycosylated species of 60
kDa and a 54-kDa species representing the unglycosylated form of the
human serotonin transporter, as well as an immunoreactive band of 130
kDa, interpreted to represent a dimer. In COS-7 cells transfected with
the cDNAs of hNET, bNET, or hDAT, in addition to 58- and 50-kDa
proteins, an immunoreactive band of about 105 kDa was also identified
(Fig. 3 A), and this might represent a dimeric aggregate.
Our indirect immunofluorescence studies provide the first evidence
that both termini of the hNET are located intracellularly, as
originally proposed by Pacholczyk et al. (6) . In
addition, we were able to confirm the proposed extracellular
localization of the two hNET epitopes recognized by antisera
L211-226 and L371-384, which labeled SKN-SH-SY5Y cells
without prior permeabilization. Immunofluorescence was very pronounced
in permeabilized COS-7 cells transfected with the hNET cDNA
(Fig. 5 D). Since the rate of [
In summary, our antisera enabled us for the first time to prove that
both hNET termini are located intracellularly and that the loops
between TMs 3 and 4 and between TMs 7 and 8 are located
extracellularly, as originally proposed
(6) .
Immunoprecipitation experiments using antisera against sequences of the
two termini demonstrate that a 50-kDa hNET protein appearing after
inhibition of N-glycosylation by tunicamycin or after PNGase
F-mediated sugar cleavage of hNET represents the hNET core protein.
These antisera, one of which also recognizes the hDAT, should be
helpful in further studies to elucidate the regulation and function of
catecholamine transport proteins.
We are grateful to Drs. S. Amara and M. Caron for
providing cDNA clones, to Dr. D. McCormick for the synthesis of the MAP
peptide, to Drs. H.-G. Sahl and K. Brix for help in immunological
methods, to Bärbel Peschka for carrying out some preliminary
experiments, and to M. Lippoldt for developing the photographs.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
S]Met-labeled hNET. Antiserum L211-226,
directed against a sequence of the putative second (large)
extracellular loop of hNET, also immunoprecipitated the human dopamine
transporter. Antisera N6-28 and C590-607, raised against a
hNET peptide region of the N and the C termini, respectively,
recognized a 58-kDa protein from transfected COS-7 cells expressing the
hNET. This 58-kDa species represents a functional, glycosylated form of
the hNET and not a degradation product. Tunicamycin treatment of
transfected COS-7 cells as well as peptide- N-glycosidase F
digestion of the transporter converted the 58-kDa species to a 50-kDa
form, indicating that the latter represents the hNET core protein. In
indirect immunofluorescence studies, our antisera confirmed the
originally proposed topology of hNET. Antisera N6-28 and
C590-607 detected hNET only in permeabilized cells. In contrast,
antisera L211-226 and L371-384 directed against peptide
sequences of the second and fourth putative extracellular loop
displayed fluorescence signals with the intact cells.
-coupled neurotransmitter transporters, the
sodium- and chloride-coupled transporters and the glutamate family in
which Na
cotransport is coupled to K
countertransport
(1, 2) . The cocaine- and
tricyclic antidepressant-sensitive NE(
)
transporter belongs to the family of sodium- and chloride-coupled
transporters for neurotransmitters such as the monoamines DA and
serotonin (5-hydroxytryptamine) and the amino acids GABA and glycine
(3, 4) . These transporters are plasma membrane
proteins, responsible for the rapid termination of neurotransmission by
reuptake of the corresponding transmitter into presynaptic nerve
terminals or surrounding glial cells. Hydropathy analysis of the
cDNA-deduced primary structure of the first cloned neurotransmitter
transporter for GABA
(5) and of the subsequently cloned human
NET
(6) resulted in the establishment of a topological model
for the proteins belonging to this family. All members of this family
share the following common structural features. Twelve
-helical
transmembrane domains are interrupted by alternating intra- and
extracellular loops. One large, putatively extracellular loop is
positioned between TM3 and TM4 and possesses multiple potential
N-glycosylation sites. Due to the absence of a signal
sequence, N and C termini of the proteins are proposed to be located
intracellularly.
- and
Cl
-coupled neurotransmitter transporters. Consistent
with an extracellular localization of the large loop between TMs 3 and
4 are reports that demonstrated N-linked glycosylation of the
transporters for DA
(7, 8, 9) , serotonin
(10) , and GABA
(11, 12, 13) . First
hints for a functional role of N-linked glycosylation of the
NET were obtained in PC12 cells, which showed reduced NE transport
after treatment with tunicamycin
(14) . Furthermore, very
recently evidence has been presented for N-glycosylation of
the human NET
(15) . Potential protein kinase C phosphorylation
sites predicted for the hNET and bNET at a region between TMs 4 and 5,
and for the bNET additionally at the C terminus
(4, 16) , are also consistent with an intracellular
localization of these domains. Finally, the aforementioned report
(15) showed by means of antipeptide antibodies against an
epitope of the hNET between TMs 8 and 9, that this loop is located
intracellularly, as predicted by the model.
Antisera Preparation and
Characterization
Peptides corresponding to particular regions of
hNET
(6) were synthesized by the solid phase method
(17) , and coupled to KLH (Pierce) by glutaraldehyde
cross-linking
(18, 19) . The conjugate was dialyzed
against PBS (137 m
M NaCl, 2.7 m
M KCl, 8.1 m
M NaHPO
, 1.4 m
M
KH
PO
, pH 7.4) at 4 °C. In addition, an
eight-branched multiple antigenic peptide, whose identical branches
correspond to the hNET N-terminal sequence RDGDAQPRE (MAP49-57),
was synthesized according to a method described by Posnett et al. (20) . Rabbits were immunized according to the following
schedule. On day 1, the KLH conjugate (100 µg) or MAP49-57 (1
mg) suspended in PBS and complete Freund's adjuvant (1:1) was
injected subcutaneously; on day 14, 28, and 56, the injections were
repeated using incomplete Freund's adjuvant. Animals were
routinely bled 10 days after the second and third booster injection,
respectively. Final bleeding was 80 days after the first injection.
Polyclonal antipeptide antisera were diluted 1:10 up to 1:10,000, and
immunoreactivity (titer) was determined by ELISA according to
conventional protocols
(19) on 96-well microtiter plates
(Falcon) coated with 1 µg of uncoupled peptide (and saturated with
bovine serum albumin) using a sheep anti-rabbit immunoglobulin
G-alkaline phosphatase conjugate (Boehringer Mannheim) as secondary
antibody, and the substrate p-nitrophenyl phosphate (Sigma).
Cell Culture, Transient Transfection, and Uptake
Assays
Cell lines were maintained as monolayer cultures in
175-cmNunc flasks (37 °C, 5% CO
). COS-7
cells and SKN-SH-SY5Y cells, a human neuroblastoma cell line from which
hNET cDNA was originally identified
(6) , were grown in minimum
essential medium supplemented with 10% fetal calf serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin (all from Life Technologies,
Inc.). COS-7 cells, grown to about 50% confluence on 60-mm culture
dishes (Falcon), were transfected using about 11 µg of plasmid DNA
and the calcium phosphate method
(21) . The following plasmid
DNAs were used: cDNA encoding the hNET, provided by Dr. S. Amara, and
subcloned into the mammalian expression vector pEUK-C1 (Clontech); bNET
cDNA inserted in the expression vector pSG5
(16) ; the hDAT cDNA
(22) within the vector pRc/CMV (Invitrogen), provided by Dr. M.
Caron; and the vectors without insert. Successful transfection was
examined 2 days post-transfection by a 15-min specific uptake of either
10 n
M [2,5,6-
H]norepinephrine
([
H]NE) or 10 n
M
[
H]DA (both from DuPont NEN) in transiently
transfected COS-7 cells, as described previously
(16, 23) . Specificity of uptake was assessed by
parallel uptake assays in the presence of 10 µ
M nisoxetine
(RBI) or GBR-12909 (RBI), specific inhibitors of NET and DAT,
respectively.
Indirect Immunofluorescence
COS-7 cells 2 days
post-transfection and SKN-SH-SY5Y cells, grown on 12-well tissue
culture plates, were used for indirect immunofluorescent labeling
according to standard protocols
(19) . After washing with cold
PBS, cells were fixed for 20 min in 2 ml of cold 2% (w/v)
paraformaldehyde in PBS or were permeabilized with 2 ml of ice-cold
methanol for 10 min at -20 °C. Following two washes with cold
PBS, cells were incubated for at least 1 h at 4 °C with 0.5 ml of
diluted antisera (1:40 in PBS) or corresponding control sera (preimmune
sera). After three washes with cold PBS, cells were incubated with
diluted FITC-conjugated (Fig. 5 D) or DTAF-conjugated
(Fig. 5, A-C) goat anti-rabbit immunoglobulin G.
After washing with cold PBS, cells were photographed on a Leitz Diavert
fluorescence microscope.
Figure 5:
Indirect immunofluorescence labeling of
SKN-SH-SY5Y cells and transfected COS-7 cells. Immunofluorescence
experiments were carried out in SKN-SH-SY5Y cells ( A-C)
or in COS-7 cells transfected with hNET cDNA ( D2, and
D4) or only the vector pEUK-C1 ( D1 and
D3). Staining was performed using either a
DTAF-conjugated ( A-C) or a FITC-conjugated ( D)
goat anti-rabbit secondary antibody. SKN-SH-SY5Y cells were either
fixed with paraformaldehyde ( C and upper pair of pictures in A and B) or permeabilized with methanol
( lower pair of pictures in A and B) as
described under ``Materials and Methods.'' In the picture
sets A-C, the left-hand panels of each pair of
photographs are phase contrast, and the right-hand panels of
each pair are indirect immunofluorescence. SKN-SH-SY5Y cells were
labeled with one of the following antisera: N6-28 ( A),
C590-607 ( B), L211-226 ( C1 and
C2) or L371-384 ( C3 and C4).
Immunofluorescence photographs of COS-7 cells ( D) were taken
from cells that were 2 days post-transfection and were
methanol-permeabilized prior to incubation with either N6-28
antiserum ( D1 and D2) or C590-607 antiserum
( D3 and D4). Bar, 10
µm.
Immunoprecipitation
Immunoprecipitations were
carried out according to the method described by Keynan et al. (11) using transiently transfected COS-7 cells or
SKN-SH-SY5Y cells grown on 60-mm tissue culture dishes (Falcon). After
removal of the normal culture medium, cells were washed twice with
prewarmed methionine-free culture medium (Life Technologies, Inc.) and
then incubated in this medium for 30 min at 37 °C. Thereafter 25
µCi/ml [S]methionine (Amersham Corp.) was
added to the medium and the incubation was continued for 1 h (COS-7
cells) or 12 h (SKN-SH-SY5Y cells). The cells were then rinsed three
times with ice-cold PBS and solubilized in 800 µl of ice-cold
radioimmune precipitation buffer (150 m
M NaCl, 1 m
M
EDTA, 250 µ
M phenylmethylsulfonyl fluoride, 0.1% (w/v)
SDS, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 10
m
M Tris-HCl, pH 7.4). Following centrifugation of the
solubilized extracts (8.000
g, 15 min, 4 °C),
protein content of the supernatant was determined using DC protein
assay (Bio-Rad) with bovine serum albumin as standard. In general,
preimmune serum (40 µl) was added to the supernatants and the
samples were mixed for 12 h at 4 °C to remove nonspecific proteins.
After addition of 60 µl of Protein A-Sepharose CL-4B beads (Sigma),
shaking was continued for another 4 h. Following bead centrifugation
(8000
g, 15 min, 4 °C), supernatants were mixed
with 10 µl of a given antiserum and incubated overnight at 4
°C. After addition of Protein A-Sepharose CL-4B beads, shaking for
4 h at 4 °C, and centrifugation of the samples, as described above,
pellets were mixed for 15 min at room temperature (or in one experiment
at 95 °C) with 100 µl of SDS sample buffer (containing 5%
-mercaptoethanol) to solubilize bound protein. Following
centrifugation, an aliquot of the supernatant (usually 60 µl/lane)
was used for SDS-PAGE on 8% gels
(24) using Rainbow colored and
[
C]methylated proteins (Amersham) as molecular
size markers. Gels were soaked in Amplify solution (Amersham), dried,
and exposed to x-ray film (Kodak XAR-5) at -70 °C for
12-72 h.
Tunicamycin and PNGase F Treatment
To assess
glycosylation of the hNET protein, the N-glycosylation
inhibitor tunicamycin was applied to COS-7 cells transfected with the
hNET cDNA. Twenty hours after transfection, cells were cultured for
another 24 h in the presence of 10 µg/ml tunicamycin. Thereafter
cells were either used for [H]NE uptake assays or
for immunoprecipitations, as described above. In some experiments,
before immunoprecipitation, hNET protein expressed in lysates of
transfected COS-7 cells was digested for 16 h at 37 °C with PNGase
F (2000 units/ml) according to the protocol of the supplier (New
England Biolabs).
S]Met-labeled NET in
extracts from cells naturally expressing the NET or after successful
transfection measured by uptake of [
H]NE (data
not shown).
S]Met-labeled cell proteins with C590-607
antiserum revealed a single band of immunoreactivity with an apparent
M
of about 58,000, which was absent in extracts of
nontransfected cells (Fig. 2 B), or in control cells
transfected with the vector containing no insert (Fig. 3 A).
This band was shifted to M
of about 50,000 if the
sample was boiled for 15 min prior to SDS-PAGE
(Fig. 2 A). Following transfection, a 58-kDa band became
detectable after about 20 h, exhibited the highest intensity after 44
h, and started to fade 68 h after transfection
(Fig. 2 B). This immunoreactive band was also recognized
by antisera against N6-28, L211-226, and L371-384
(Fig. 3, A and B).
Figure 2:
Immunoprecipitation of
[S]Met-labeled hNET protein by C590-607
antiserum. Nontransfected COS-7 cells and cells transfected with the
hNET cDNA in pEUK-C1 were [
S]Met labeled,
harvested 44 h post-transfection ( panel A) or at the times
indicated ( panel B), and immunoprecipitated with preimmune
serum followed by C590-607 antiserum. Thereafter, cell extracts
were subjected to SDS-PAGE (8%) followed by autoradiography of the gel
as described under ``Materials and Methods.'' Varying from
the usual procedure, in the first lane of panel A, the sample
was boiled for 15 min prior to electrophoresis, resulting in a shift of
the 58-kDa band to 50-kDa species ( arrows). The
numbers indicate the positions of the molecular size markers
in kDa.
Figure 3:
Immunoprecipitation of
[S]Met-labeled hNET, bNET, and hDAT by the four
antipeptide antisera. COS-7 cells, transfected with the cDNA of hNET,
bNET, hDAT, or the vector alone (pEUK-C1 or pSG5), were
[
S]Met-labeled, harvested 44 h
post-transfection, and immunoprecipitated with preimmune serum,
followed by either antiserum C590-607, N6-28 or
L211-226 ( panel A) or antiserum L371-384
( panel B); cell extracts were subjected to SDS-PAGE (8%),
followed by autoradiography of the gel as described under
``Materials and Methods.'' The arrows indicate immunoreactive bands of 105, 58, and 50 kDa,
respectively. The numbers indicate the positions of the
molecular size markers in kDa. The x-ray film was exposed to the gel
for 1 day ( panel A) or for 3 days ( panel B). Note
that only antiserum L211-226 was able to immunoprecipitate
labeled hDAT protein.
As shown in Fig. 1,
the amino acid sequences of the hNET peptides selected as antigens
exhibit 74-100% homology with corresponding sequences of the
bovine NET
(16) . However, corresponding sequences of the human
DAT
(22) show a similarly high homology only within the region
analogous to that of the L211-226 hNET peptide (Fig. 1). It
was therefore of interest to test the specificity of the antipeptide
antisera in extracts of COS-7 cells transfected with either the cDNA of
bNET or the cDNA of hDAT. As shown in Fig. 3, in extracts from
cells expressing the bNET, all antisera identified an immunoreactive
band migrating at about 58 kDa. However, in extracts from cells
transfected with the hDAT cDNA, the antisera directed against N- and
C-terminal domains or the region between TM 7 and TM 8 were inactive
(Fig. 3); only the antiserum against the loop peptide
L211-226 immunoprecipitated a protein of about 58-kDa
(Fig. 3 A). These results indicate that the 58-kDa
species must represent NETs. Interestingly, in addition to the 58-kDa
species, two additional immunoreactive bands of about 50 and 105 kDa
were frequently recognized by the antisera in extracts of COS-7 cells
expressing the hNET, bNET or hDAT (see, e.g.,
Fig. 3A). Identical results were obtained in Western
blots (data not shown). In [S]Met-labeled
SKN-SH-SY5Y cells, C590-607 antiserum, but not preimmune serum,
precipitated a 58-kDa protein and also a 50-kDa species; in addition,
an 81-kDa protein was recognized by this antiserum (Fig. 4 A).
Figure 1:
Model of hNET and epitope-specific
peptides against which antibodies were raised. The heavy black
lines in the hydropathy-based model of Pacholczyk et al. (6) indicate the positions of the four hNET peptides (N6-28,
L211-226, L371-384, and C590-607) against which
antisera were raised. Below the figure, the amino acid sequences of the
hNET peptides as well as homologous sequences of bNET and hDAT are
given. The numbers in parentheses indicate percent
homology to the corresponding hNET peptide, and asterisks indicate that the antiserum caused immunoprecipitation of the
transporter.
To determine whether the 50-kDa species might represent the
nonglycosylated hNET, [S]Met-labeled COS-7 cells
transfected with the hNET cDNA were treated with tunicamycin. In these
cells uptake of [
H]NE was strongly reduced (data
not shown) and only a 48-50-kDa protein was immunoprecipitated by
the C590-607 antiserum (Fig. 4 B), indicating that
this species might represent the hNET core protein. In accordance with
this result, partial PNGase F digestion of membranes from transfected
cells resulted in a marked reduction of the intensity of the 58-kDa
band and appearance of a relatively broad band of about 40-50 kDa
(Fig. 4 B). The breadth of this band might indicate different
degrees of deglycosylation of the hNET. In addition, partial protein
degradation might also have contributed to this broad band.
Figure 4:
Immunoprecipitation of labeled hNET from
SKN-SH-SY5Y cells, and effect of tunicamycin or PNGase F on hNET
expressed in transfected COS-7 cells. SKN-SH-SY5Y cells ( panel
A) were harvested after incubation for 12 h with
[S]Met; hNET-transfected COS-7 cells ( panel
B) were incubated for 1 h with [
S]Met and
harvested 44 h post-transfection. Thereafter cell extracts were treated
with preimmune serum alone ( panel A, lane 2) or additionally
treated with C590-607 antiserum ( panel A, lane 1), and
then subjected to SDS-PAGE (8%) followed by autoradiography of the gel
as described under ``Materials and Methods''. In one
series of experiments ( panel B, lane 2), tunicamycin
(10 µg/ml) was present from 20 h post-transfection until cell
harvesting. For PNGase F digestion ( panel B), cell extracts
were incubated with ( lane 3) or without ( lane 4)
PNGase F (16 h, 37 °C) prior to immunoprecipitation. Arrows indicate immunoreactive bands.
We
examined the proposed topology of the hNET (see Fig. 1) by
indirect immunofluorescence staining of intact and permeabilized cells.
SKN-SH-SY5Y cells and COS-7 cells, transfected with the hNET cDNA, as
well as nontransfected COS-7 control cells were used. Before
immunostaining, the cells were either fixed with paraformaldehyde (for
labeling of extracellular epitopes) or permeabilized with methanol (for
labeling of intra- and extracellular epitopes). Indirect
immunofluorescence staining with antisera N6-28 and
C590-607 was obtained in permeabilized cells expressing the hNET
(Fig. 5, panels A4, B4, D2, and
D4), but not in nonpermeabilized SKN-SH-SY5Y cells (Fig. 5,
panels A2 and B2), or in nonpermeabilized COS-7 cells
expressing the hNET (not shown) or in COS-7 cells transfected with the
vector alone (Fig. 5 D, panels 1 and
3). These results confirm the proposed intracellular
localization of N and C termini of hNET. In contrast,
immunofluorescence labeling was clearly not dependent on
permeabilization of SKN-SH-SY5Y cells when antisera against the
peptides L211-226 (Fig. 5 C, panels 1 and
2) or L371-384 (Fig. 5 C, panels 3 and 4) were used.
H]NE
(data not shown). This conclusion is supported by recently published
results of Melikian et al. (15) , demonstrating
transport of [
H]NE in transiently transfected
HeLa cells expressing only a 54-kDa form of hNET. Whether this also
holds true for the most prominent 58-kDa hNET protein identified in
[
S]Met-labeled SKN-SH-SY5Y cells
(Fig. 4 A) remains to be determined. In these cells an
81-kDa species was also immunoprecipitated. A band of similar size was
occasionally also observed in transfected COS-7 cells (Fig.
4 B). An 80-kDa hNET form was recently identified by Melikian
et al. (15) in SKN-SH-SY5Y cells and in stably
transfected LLC-NET cells, but not in transiently transfected HeLa
cells. These authors proposed the 80-kDa species to represent a mature
and therefore more highly glycosylated form of the hNET. However, in
the present study, the 80-kDa band in COS-7 transfected cells showed no
shift or fading after PNGase F treatment (Fig. 4 B).
Thus, the significance of this 80-kDa species remains unclear.
H]NE
uptake was higher in transfected COS-7 cells than in SKN-SH-SY5Y cells,
the former cells seem to express a higher number of transporter sites
per cell. In SKN-SH-SY5Y cells we recently showed that these cells
express about 150,000 NE transporter sites
([
H]nisoxetine binding sites).(
)
Thus, cells expressing this number of transporter sites are
easily detected with the presented methods. Our immunofluorescence data
strongly support the topological model of the hNET transporter.
Together with recent results from immunofluorescence studies of
Melikian et al. (15) , which confirmed the proposed
intracellular localization of the loop between TMs 8 and 9, the
available data strongly corroborate the proposed model of the hNET.
-aminobutyric
acid; TM, transmembrane domain; PBS, phosphate-buffered saline; FITC,
fluorescein isothiocyanate; ELISA, enzyme-linked immunosorbent assay;
DTAF, 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein; PBS,
phosphate-buffered saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.