©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Antipeptide Antibodies Confirm the Topology of the Human Norepinephrine Transporter (*)

Michael Brüss (1), Rainer Hammermann (1), Stephen Brimijoin (2), Heinz Bönisch (1)(§)

From the (1) Institut für Pharmakologie und Toxikologie, Universität Bonn, Reuterstrasse 2b, D-53113 Bonn, Federal Republic of Germany and the (2) Department of Pharmacology, Mayo Clinic, Rochester, Minnesota 55905

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 [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.


INTRODUCTION

cDNA cloning has revealed the primary structure of two families of Na-coupled neurotransmitter transporters, the sodium- and chloride-coupled transporters and the glutamate family in which Nacotransport is coupled to Kcountertransport (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.

Hitherto there has been little direct evidence to support the topological model for the Na- 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.

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.


MATERIALS AND METHODS

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 KHPO, 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).


RESULTS

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 [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).

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 [S]Met-labeled cell proteins with C590-607 antiserum revealed a single band of immunoreactivity with an apparent Mof 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 Mof 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.


DISCUSSION

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 [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.

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 [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.

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.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grants Bo 521/8-2 and SFB 400 and by the A. v. Humboldt Foundation (fellowship award to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-228-735431; Fax: 49-228-735404.

The abbreviations used are: NE, norepinephrine; NET, norepinephrine transporter; hNET, human norepinephrine transporter; bNET, bovine norepinephrine transporter; DA, dopamine; DAT, dopamine transporter; hDAT, human dopamine transporter; KLH, keyhole limpet hemocyanine; MAP49-57, multiple antigenic peptide whose identical eight branches correspond to the amino acid of the hNET indicated by the numbers; N6-28, L211-226, L371-384, C590-607, human norepinephrine transporter peptides with a sequence corresponding to the indicated numbers; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide- N-glycosidase F; GABA, -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.

M. Brüss, R. Hammermann, S. Brimijoin, and H. Bönisch, unpublished results.


ACKNOWLEDGEMENTS

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.


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