0 1994 by The American Society for Biochemistry and Molecular Biology, Inc THE JOURNAL OF BIOWGICAL CHEMISTRY Vol. 269, No. 16, Issue of April 22, pp. 12290-12297, 1994

Printed in U S A .

Human Norepinephrine Transporter BIOSYNTHETIC STUDIES USING A SITE-DIRECTED POLYCLONAL ANTIBODY*

(Received for publication, October 26, 1993, and in revised form, January 31, 1994)

Haley E. MelikianSs, John K. McDonald+, Howard Gun Gary Rudnickn Kimberly R. Moore+, and Randy D. BlakelySlI From the Wepartment of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322, the $Graduate Program in Neuroscience, Division of Biological and Biomedical Sciences, Emory University, Atlanta, Georgia 30322, and the Wepartment of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

Antibodies have been raised against synthetic pep- tides derived from the predicted primary sequence of the human cocaine- and antidepressant-sensitive nor- epinephrine (NE) transporter (NET). One antibody (N430), raised and purified against a putative intracel- lular human norepinephrine transporter (hNET) epitope, detects hNET expression in a stably transfected cell line (LLC-NET) by indirect immunofluorescence only in the presence of detergent, while no immunore- activity is observed in either the parental cells (LLC- PK,) or in LLC-NET cells incubated with preimmune sera or peptide absorbed antibody. N430 immunoblots of LLC-NET cell extracts reveal two major immunoreac- tive hNET species in these cells, migrating at 80 and 54 kDa, respectively. Pulse-chase N430 immunoprecipita- tion studies confirm that the 54-kDa species is a tran- sient, glycosylated intermediate of a longer lived, more highly glycosylated protein with an apparant M, of 80,000. In contrast, a 54-kDa species is the primary hNET product in vaccinia virus T7-infected HeLa cells, tran- siently transfected with hNET cDNA. PNGase F diges- tion of extracts prepared from LLC-NET- and hNET- transfected HeLa cells convert all immunoreactive species to a 46-kDa form, equivalent to that observed following incubation of whole cells with the glycosyla- tion inhibitor tunicamycin. As transiently transfected HeLa and stable LLC-NET cells exhibit a pharmacologi- cally similar NE transport activity, it appears likely that the additional glycosylation evident in the stable line does not contribute significantly to antagonist sensitiv- ity. On the other hand, NE transport and antagonist ([‘aaI]RTI-55) binding assays on whole LLC-NET cells treated with tunicamycin reveal a pronounced reduc- tion in NE transport activity and hNET membrane den- sity paralleled by an inability of NET proteins to replen- ish the higher M, hNET pool. These findings suggest an obligate role for N-linked glycosylation in hNET biosyn- thetic maturation, stability, and functional expression. In summary, N430 antibody is a useful tool for the visu- alization and characterization of hNET gene products and has permitted the first direct evaluation of biosyn- thetic steps leading to functional catecholamine trans- porter expression.

Grant DA07390 and a Mallinckrodt award (to R. D. B.), National Insti- * This work was supported by National Institute on Drug Abuse

tute on Drug Abuse Grants DA8213 and DA7259 (to G. R.), and Na- tional Institutes of Health Award HD26833 (to J. K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed. Tel.: 404-727-0451; Fax: 404-727-6256.

Many neurotransmitters, including NE,‘ DA, 5-hydroxy- tryptamine, GABA, glycine, and t-glutamate are thought to have their actions constrained by rapid clearance from synaptic spaces by plasma membrane transport proteins (1-3). Recently, it has become clear that at least two distinct gene families, GATl/NET ( G A B M E transporters (3,411 and GLASTl (Gld Asp transporters, (5)) are responsible for extracellular neuro- transmitter clearance. The monoamine transporters within the GATl/NET gene family are high affinity targets for a number of psychoactive agents, including cocaine, amphetamines, and an- tidepressants (6-8). These agents cause an increase in extra- cellular NE, DA, and 5HT concentrations in both the central nervous system and periphery, contributing to their behavioral and autonomic effects. For example, blockade of NETS present in postganglionic sympathetic innervation of the heart may contribute to the acute cardiovascular effects of cocaine and antidepressants, whereas differential blockade of all three transporters in the brain contributes to their behavioral effects (9-11). Although the addictive and/or therapeutic consequences of pharmacologic NE, DA, and 5HT transporter blockade have been clear for decades, molecular details of monoamine trans- porter protein structure and regulation are only beginning to emerge (12, 13).

The expression of antidepressant- and cocaine-sensitive NET from a single cDNA first co-localized psychoactive drug binding sites and transport activity in a single membrane protein (141, findings that were quickly corroborated with the cloning and heterologous expression of homologous DA and 5HT transport- ers (reviewed in Ref. 3). Despite this progress, hNET protein has yet to be directly visualized in situ, and the transporter’s biosynthesis and posttranslational modifications in any prepa- ration remain to be elucidated. Hydropathy analysis of inferred amino acid sequences predicts that NET monomers, like other GATl/NET homologs (3, 12, 14), contain twelve internal TMDs between cytoplasmic NH, and COOH termini, with the latter tails thought not to contribute to substrate or antagonist speci- ficity (15). Conserved and unique sites for phosphorylation are present on putative intracellular domains of hNET, although an inability to isolate hNET protein has limited progress in evaluation of their use. Approximately 20% of amino acid resi- dues in hNET are conserved with GATUNET homologs and distributed nonuniformly throughout the predicted structure,

The abbreviations used are: N E , norepinephrine; NET norepineph- rine transporter; hNET, human norepinephrine transporter; GAT1, y-aminobutyric acid transporter 1; Ab, antibody; KLH, keyhole limpet hemocyanin; N430 peptide, norepinephrine transporter peptide-amino acids 43-44; RTI-55, 3/3-[4-iodophenyll-tropan-2/3-carboxylic acid methyl ester tartrate; PAGE, polyacrylamide gel electrophoresis; TM, tunicamycin; RIPA, radioimmunoprecipitation assay; GABA, y-amino butyric acid; DA, dopamine; TMD, transmembrane domain; PBS, phos- phate-buffered saline.

12290

Antibodies Against the NE Dansporter 12291

concentrated heavily in TMDs 1-2 and 5-8 (3, 12). Like all GATlMET homologs, hNET bears a large hydrophilic region between TMDs 3 and 4 with multiple (three) sites for N-linked glycosylation, which, if utilized, would imply an extracellular localization for this domain. Protein purification and photoaf- finity labeling paradigms (16-20) indicate 5HT and DA trans- porters to be formed from glycosylated monomers of 58-80 kDA. Region- (211, cell-, and species-specific (22) DA trans- porter glycosylation variants have been reported, but, like NET glycosylation, these modifications are of uncertain functional significance. Tunicamycin treatment reduces the NE transport capacity of PC-12 cells (23), suggesting an important role of glycosylation in either NE translocation or NET biosynthesis.

Abs prepared against purified GABA transporter protein and GATl cDNA-derived peptides have proven instrumental in documenting the expression and glycosylation of GATl in vivo and in transfected cell lines (24-261, studies that are largely consistent with the aforementioned topological model. In the present study, we report the preparation and characterization of an anti-peptide Ab directed against a putative cytoplasmic domain of WET. This Ab (N430) detects hNET by indirect immunofluorescence in permeabilized transfected cells, immu- noprecipitates metabolically labeled camers, and recognizes denatured hNET protein on immunoblots of cell and membrane extracts. Our studies with N430 Ab provide support for the hydrophobicity-based topological model of hNET monomers, illuminate biosynthetic and glycosylation kinetics in stably transfected cells, and reveal cell-specific differences in hNET glycosylation. Functional assays conducted in parallel with hNET protein studies support a major contribution of N-linked glycosylation to protein stability and maturation.

EXPERIMENTAL PROCEDURES

Methods Antibody Preparation, Purification, and Characterization-Peptides

were coupled to KLH and bovine serum albumin carrier proteins by maleimide cross-linking to free sulfhydryl groups. KLH or bovine serum albumin (10 mg) were activated by incubating with 2.5 mglml m-male- imidobenzoic acid n-hydroxysuccinimide ester (dissolved in m,m-di- methylformamide) in 0.5 ml of 100 m NaBO, under argon (30 min, 22 "C, stirring). Free maleimide was removed by repetitive spin filtra- tion (Centricon-100, Amicon) in 100 m NaBO, to a final volume of 0.5 ml. Peptides (1.0 ml of 10 mg/ml in 20 IXIM EDTA, pH 9.0) were added to the activated carrier and stirred under argon (4 h, 22 "C) and dialyzed against PBS (138 IXIM NaCl, 2.7 m KCl, 1.5 m KH,PO,, 9.6 m Na,HPO,, pH 7.3). To determine coupling efficiency (which averaged >go%), sulfhydryl groups in uncoupled and coupled peptide were quan- titated using the method of Ellman (27). Primary immunizations of rabbits with peptide-KLH conjugate (1 mg/200 pl) were followed by 14- and 28-day booster injections (0.5 mg/100 4). Thereafter, boosts were 0.5 mg/lOO pl. Animals were routinely bled 1 week after a given boost, beginning after the first boost. Immunoreactivity of sera was deter- mined by enzyme-linked immunoassay as described by Harlow and Lane (28). Affinity chromatography of positive sera was performed by elution over peptide-coupled Sulfolink resin prepared according to the manufacturer's suggestions (2 ml of 1 mg/ml peptide incubated with 2 ml of resin). Sera was heat-inactivated (1 h, 56 "C), diluted 1:l in PBS, and mixed with affinity resin (30 min, 22 "C). Serahesin suspensions were collected and washed with PBS until A,, of wash <0.01. Bound Ab was eluted with 100 m~ glycine, pH 2.8. Fractions (1 ml) were collected into 50 of 1.0 M Tris, pH 9.0; protein-containing fractions were pooled and dialyzed into PBS. Anti-peptide immunoreactivity of fractions was determined by enzyme-linked immunoassay and protein content deter- mined using the Bradford assay (Bio-Rad).

Cell Culture Zkansfections-Cell lines were maintained in monolayer culture in 75-cm2 flasks (Corning) (37 "C, 5% CO,). HeLa cells were grown in Dulbecco's modified Eagle's medium, LLC-PK, and the hNET stable transformant LLC-NET (29) in minimal essential medium, and SK-N-SH cells in RPMI 1640. Media were supplemented with 10% fetal bovine serum, 2 m glutamine, 100 unitdml penicillin, 100 pg/ml strep- tomycin. W E T was expressed in HeLa cells using the vaccinia virus T7 expression system, as previously described (14, 30).

Indirect Immunofluorescence-LLC-PK, and stably transformed LLC-NET cells, grown on glass coverslips to 50% confluency, were fixed in 1.0 ml of ice-cold 4% paraformaldehyde, 100 m~ D,L-lysine, 10 m~ sodium periodate in PO, buffer (50 m~ Na,HPO,, pH 7.4) for 10 min at 22 "C. Following three 10-min washes in PO, buffer, cells were incu- bated with 10% normal goat serum in PTN (0.3% Triton X-100, 0.1% NaN, in PO, buffer) for 45 min at 22 "C to decrease nonspecific binding. Next, this solution was removed, affinity-purified Ab in PTN was added (1-5 pglml final conc), and cells were incubated for 1 h at 37 "C. After washing, as above, fluorescein isothiocyanate-conjugated goat anti-rab- bit Ab (1:50) was added in PTN containing 1% normal goat serum and incubated for 45 min at 22 "C in the dark. Triton X-100 was excluded from all steps of parallel incubations to assess permeabilization depen- dence of immunoreactivity. Following three PO, buffer rinses, coverslips were air-dried and mounted on slides in Entellan rapid mounting me- dium (EM Science), and immunofluorescence was photographed through a 50 x oil immersion lens using a Leitz Laborlux microscope. Control incubations consisted of substitution of N430 antisera with preimmune serum or affinity-purified N430 sera, preincubated with N430 peptide (10 p ~ ) .

Uptake Assays, Membrane Preparation, and Radioligand Binding Studies-Uptake of [2,5,6,3Hlnorepinephrine ([,H]NE) in stably trans- fected LLC-NET and transiently transfected HeLa cells was assessed in triplicate as previously described (14, 30). For analysis of L3H1NE up- take in LLC-NET cells, cells were plated in 24-well plates at a density of 25,00O/well 1 day prior to performing assays. HeLa cells, transfected at lo5 cells/well, were assayed 6-14 h following transfection. Values obtained from assays conducted in the presence of 1 p~ desipramine or with plasmid vector transfection were subtracted from the data to de- fine specific transport. For radioligand binding assays to LLC-NET membranes, confluent 150-mm tissue culture plates were washed three times in 5 ml of PBS, scraped into 5 ml of ice-cold PBS, and homog- enized with a Polytron tissue homogenizer (18,000 rpm, 30 8) . Mem- branes were obtained from the high speed pellet (20,000 x g, 30 min, 4 "C) of samples previously cleared of nonhomogenized debris (700 x g, 5 min, 4 "C). Pelleted membranes were resuspended in PBS and as- sayed for protein (Bio-Rad) with bovine serum albumin as the standard. Binding assays with [12511RTI-55 were performed in duplicate as previ- ously described (31). Briefly, LLC-NET membranes (10 pg) were incu- bated with 100 PM [12511RTI-55, 100 m NaC1,50 m~ Tris-C1, pH 8.0, and unlabeled RTI-55 at a concentration range of 300 PM-300 n~ in a final volume of 1.0 ml for 1 h at 22 "C with agitation. Membranes were collected on glass fiber filters (Schleicher & Schuell, No. 32) coated with 0.1% polyethyleneimine in a Brandel cell harvester and washed three times in ice-cold 100 m~ NaCl, 50 m Tris-C1, pH 8.0, and bound ligand was determined by y radioactivity emission. Values obtained from as- says conducted in the presence of 1 p~ desipramine or with plasmid vector transfection were subtracted from the data to define specific binding. Nonlinear least squares fits of transport and binding data and estimation of kinetic constants were obtained with Kaleidagraph soft- ware.

Immunoblotting and Immunoprecipitation-For immunoblot analy- sis of N E T protein, 400,000 LLC-PK, or LLC-NET celldwell were plated in 6-well plates 1 day prior to harvesting, whereas HeLa cells were transfected as described above. Cells were washed three times with ice-cold PBS, solubilized in 300 pl of ice-cold RIPA buffer (10 m Tris, pH 7.4,150 m NaCl, 1 m~ EDTA, 0.1% SDS, 1% Triton X-100, 1% Na deoxycholate) supplemented with 1 mglml soybean trypsin inhibitor, 1 mM o-phenanthroline, 1 pg/ml leupeptin, 1 m~ iodoacetamide, 1 p~ pepstatin A, and 250 p~ phenylmethylsulfonyl fluoride (30 min, 4 "C, shaking). Solubilized extracts were centrifuged (20,000 x g, 10 min, 4 "C) and protein content of supernatants assessed using the DC pro- tein assay (Bio-Rad) with bovine serum albumin as the standard. SDS- PAGE on 8 or 10% gels was performed according to the method of Laemmli (32). Prior to loading, RIPA extracts were combined with SDS sample buffer containing 10% P-mercaptoethanol(15 min, without boil- ing). Unstained molecular weight markers were electrophoresed in par- allel for immunoblots and prestained markers for immunoprecipita- tions. Prestained markers are reported by their apparent migration and not by their actual molecular weight. Electrophoresed gels were soaked in 200 ml of transfer buffer (50 m Tris, 250 IXIM glycine, 0.01% SDS, 20% methanol, 20 min) and electroblotted (30 V, 16 h, 4 "C) to Hybond ECL nitrocellulose membranes followed by protein staining to verify transfer with 0.1% Ponceau S solution, 5% acetic acid (Sigma). Destained blots, preincubated in 20 ml of blocking solution (5% Carna- tion non-fat dried milk, 0.5% Tween 20, PBS; 45 min, 22 "C) were incubated with primary Ab ( 1 pg/ml in blocking solution, 0.1% NaN,; 45 min, 22 "C) and then rinsed (2 x rapidly, 1 x 15 min, 2 x 5 min, 22 "C in

12292 Antibodies Against the NE Dansporter PBS, 0.5% Tween 20) prior to visualization of bound Ab. Horseradish peroxidase-conjugated goat anti-rabbit Ab was incubated with blots a t 1:3000 dilution in blocking solution (45 min, 22 "C), washes repeated as above, and immunoreactive bands detected by enhanced chemilumines- cence (Amersham Corp.). For immunoprecipitation, cells, plated as de- scribed, were washed with prewarmed MeUCys-free media and labeled with 500 pl of prewarmed Tran3%-label (50 pCi/ml) in MeVCys-free media (3 h, 37 "C). For pulse-chase studies, cells were incubated with 500 pCi/ml Tran36S-label for 10 min that was replaced with unlabeled complete media for the times indicated. Labeled media was removed and whole cell extracts prepared as described above. Sera (5-25 p1) were added to the labeled cell extracts and samples incubated (1 h, 22 "C) with continuous mixing. Protein A-Sepharose beads were prepared as follows: nonspecific binding was blocked by incubation with unlabeled cell extract (1 h, 22 "C), followed by three washes in RIPA buffer and resuspension to 30 mg/ml in RIPA. Blocked beads (100 pl) were added to labeled cell extracts and incubated (either for 1 h at 22 "C or over- night at 4 "C) with continuous mixing, followed by washing as above, and bound protein eluted into 100 pl of Laemmli sample buffer (15 min, 22 "C). Following bead centrifugation, supernatants were electrophore- sed as described above. Gels were soaked in Entensify enhancing solu- tions (DuPont NEN), dried, and exposed to x-ray film at -80 "C for 2-24 h.

Tunicamycin Deatment and Enzymatic Deglycosylation-To assess the impact of glycosylation inhibition on hNET function and protein expression, LLC-NET or transfected HeLa cells were incubated with 10 pg/ml tunicamycin for the times indicated, followed by measurement of C3HINE transport or ['2611RTI-55 membrane binding. Alternatively, cell extracts were prepared as above for Western and immunoprecipitation analyses of hNET protein. To examine glycosylation states of expressed WET in LLC-NET cells or transfected HeLa cells, 50-pg RIPA extracts were treated with PNGase F or neuraminidase, with digestion of fetuin (Sigma) in parallel to validate enzyme activity. PNGase F, cell extracts were reduced by incubation with 0.1 volume of PNGase F denaturation buffer (New England Biolabs, 5 min, 22 "C), followed by addition of 0.1 volume each of 10% Nonidet P-40, 10 x PNGase F buffer, and PNGase F (40 milliunitdml), and samples were incubated for 16 h at 37 "C. Neuraminidase, extracts were directly treated with 0.1 unit/ml neura- minidase for l h, 37 "C, and then subjected to immunoblot analysis or immunoprecipitation as described.

Materials

Cell culture media (Dulbecco's modified Eagle's medium, minimal essential medium, RPMI) were purchased from Fisher, and fetal bovine serum was obtained from Hyclone. Trypsin, glutamine, penicillin, strep- tomycin, OPTI-MEM, and Lipofectin were obtained from Life Technolo- gies, Inc. Vaccinia virus T7 RNA polymerase (VTF,,) was provided by Dr. Bernard Moss, National Institutes on Allergy and Infectious Dis- eases. Peptides corresponding to hNET-inferred amino acids 370-383, 430-444, and 587-601 (13) and containing a COOH-terminal cysteine for carrier conjugation were synthesized and purified by the Emory University Microchemical Facility. KLH and Sulfolink affinity resin were purchased from Pierce Chemical Co. Goat anti-rabbit Abs, SDS- PAGE molecular weight standards, and SDS-PAGE reagents were from Bio-Rad, and fluorescein isothiocyanate-coupled goat anti-rabbit Ab was purchased from Vector Laboratories. Enhanced chemiluminescence reagents and Hybond ECL nitrocellulose membrane were purchased from Amersham. Tran%-label (1131 CYmmol) and MeVCys-deficient media were obtained from ICN; L3H1NE (48.6 CUmmol) was obtained from DuPont NEN. ['2611RTI-55 (2200 Ci/mmol) was a generous gift from Dr. John Boja, National Institute on Drug Abuse. Protein A-Sepha- rose was from Pharmacia LKl3 Biotechnology Inc. PNGase F was pur- chased from New England Biolabs; neuraminidase (Clostridium perfin- gens) was from Boehringer Mannheim. New Zealand White rabbits were purchased and maintained by Spring Valley Laboratories (Sykes- ville, MD). EcoScint H scintillation fluor was obtained from National Diagnostics. Antigenicity analysis of W E T sequence was performed with MacVector software (IBI), and kinetic data from transport and radioligand binding assays was analyzed with Kaleidagraph software (Synergy Software). All other materials were obtained from standard commercial vendors and were of the highest grade possible.

RESULTS

To develop anti-NET Abs, we immunized rabbits with KLH conjugates of three synthetic hNET peptides (N430, W E T 430444; N370, hNET 370-383; hNET CT-1, hNET 586-599).

All three of these antipeptide antibodies were found to immu- noprecipitate hNET protein. In particular, the N430 peptide (GLADDFQVLKRHRKL-C), yielded a high titer Ab that spe- cifically detected hNET by a variety of immunological tech- niques. The sequence of N430 corresponds to a putative intra- cellular region between TMDs 8 and 9 in the proposed model of hNET secondary structure (14). Peptide affinity chromatogra- phy of N430 sera routinely enriched anti-peptide specific activ- ity by 300-500-fold with a 50% recovery of total immunoreac- tivity as assessed by enzyme-linked immunoassay, yielding final purified Ab solutions of 15-50 pg/ml.

As a first test of the utility of N430 Ab, we examined its ability to identify hNET expression in stable transformants (LLC-NET) of the pig kidney epithelial cell line, LLC-PK,. Staining of LLC-NET cells with preimmune serum yielded minimal fluorescence, similar to that observed with N430 se- rum on LLC-PK, cells (data not shown). In contrast, incubation of LLC-NET cells with either N430 serum or aflinity-purified N430 Ab (Fig. IA) resulted in striking fluorescence, including intense border staining, that appeared to be associated with the plasma membrane. Staining was abolished by preincuba- tion of affinity-purified N430 Ab with 10 p 4 N430 peptide (data not shown). N430 immunoreactivity was completely dependent upon the presence of detergent (Fig. lB), as predicted by the intracellular localization of the N430 epitope in the proposed topological model (14). We also observed a considerable level of immunoreactivity associated with intracellular, perinuclear compartments, presumably ER or Golgi membranes, likely to reflect the high expression level of hNET in the stably trans- fected cell line.

Immunoprecipitation of [35SlMet/Cys-labeled LLC-NET cell extracts revealed two broad immunoreactive bands with appar- ent M , of 80,000 and 54,000 Da. These species were absent from parental LLC-PK, cells (Fig. 2 A ) or in LLC-NET extracts im- munoprecipitated with either preimmune sera or peptide-ab- sorbed N430 Ab. To determine whether the two species were distinct subunits that co-precipitated or, alternatively, reflected the same core protein at distinct stages of intracellular proc- essing, we examined LLC-NET extracts directly via immuno- blots. As shown in Fig. 2B, N430 Ab detected both species directly, with signal abolished as before with peptide preincu- bation or with substitution of preimmune sera. Thus, these two species bear the hNET N430 epitope and are unlikely to rep- resent distinct, co-precipitating, transporter subunits. Mem- branes prepared from these cells also exhibit similar immuno- reactivity (data not shown). Notably, preabsorption of N430 Ab with a peptide corresponding to the same region of the rat serotonin transporter did not diminish immunoreactivity in either LLC-NET, further demonstrating the specificity of the N430 Ab for hNET (data not shown). A major band co-migrating with the 80-kDa species was also detected in SK-N-SH cells, the line from which hNET cDNA was originally identified, along with a larger immunoreactive species likely to be a di- meric aggregate.

We also examined the expression of hNET protein in HeLa cells transfected by the vaccinia virus T7 expression system. By 4 h posttransfection, NET activity is measurable in HeLa cells (data not shown) with a temporal profile like that previously described for Na+/glucose transporters (30). Immunoblot anal- ysis of vaccinia-driven hNET expression in HeLa cells revealed the time-dependent accumulation of a single 54-kDa immuno- reactive species (Fig. 3). N430 Ab also immunoprecipitates a single 54-kDa species in hNET-transfected HeLa cells but fails to recognize the rat serotonin transporter, transfected in par- allel in a separate set of cultures, by immunoblot analysis (data not shown).

The differently sized forms of hNET that we observed in

Antibodies Against the NE lFansporter 12293

FIG. 1. Indirect immunofluorescence of N430 Ab i n LLC-NET cells. Cells were cultured, fixed, and stained as described under “Ex- perimental Procedures.” LLC-NET cells were stained with 5 pg/ml af- finity-purified N430 Ab in PTN buffer ( a ) or 5 pg/ml affinity-purified N430Ab excluding Triton X-100 from all incubations ( b ) . The arrows in a indicate positive immunoreactivity associated with cell borders. Scale bars = 10 pm.

LLC-NET cells could represent two distinct populations of hNET glycoprotein due to a lack of clonal homogeneity or could reflect different stages of posttranslational processing acquired by a single hNET gene product. In order to distinguish between these two possibilities, we examined the biosynthesis of hNET in LLC-NET cells utilizing a pulse-chase labeling strategy. Af- ter 30 min of chase with complete media, only the 54-kDa band was evident (Fig. 4). This band remained the major immuno- reactive species, until 2 h after initiation of cold chase, at which time the larger species appeared, and the quantity of material in the 54-kDa band began to decrease. The lower band disap- peared completely by 8 h, whereas the 80-kDa band appeared only slightly diminished by 12 h of chase. Longer chase periods

A

205-

116.5-

8”

49.5-

B

200 -

116”-

97.4 -

66.2 -

a5 -

N430 Ab pre + 1G5M

N430 Ab immune peptide ”

N430 pre immune + 106 M N430 Ab

Ab sera peptide ”

L

munoprecipitation of hNET in LLC-NET cells. Cells were [‘‘SlMetKys- FIG. 2. Visualization of hNET protein expression. a, N430 im-

labeled, immunoprecipitated with N430 serum, and subjected to SDS- PAGE (10%) as described under “Experimental Procedures.” Peptide blocking was performed by preincubating N430 sera with M N430 peptide for 1 h a t 37 “C prior to immunoprecipitation. b, immunoblot of hNET protein expression. Extracts of total protein from LLC-PK, (25 pg), LLC-NET (25 pg), and SK-N-SH (50 pg) were subjected to SDS- PAGE (8%). electroblotted, and incubated with affinity-purified N430 Ab or preimmune serum (1 pg/ml) as described under “Experimental Procedures.” Peptide blocking of N430 immunoreactivity was per- formed as described in a using M N430 peptide. The numbers on the left of the blot indicate the positions of molecular mass markers in kDa.

indicate a half-life of the 80-kDa species of -24 h (data not shown). These results are consistent with the lower M, product being a biosynthetic intermediate of the higher M, species. The differential longevity of the 54- and 80-kDa forms also explains why 3 h of continuous [35S]Met/Cys labeling yields a significant proportion of material in the 54-kDa form, as previously dem- onstrated in Fig. 2A, whereas steady-state assessments of hNET protein in immunoblots reveal largely the 80-kDa form (Fig. 2B).

Substantial posttranslational processing appeared to be in-

12294 Antibodies Against the NE Dansporter

4 hrs - 6 hrs - 8 hrs -

200 - CI*"Crri cr..r* . 4

116-

94.5 -

66.2 -

45 -

FIG. 3. Immunoblot of hNJ3T protein expressed in transiently transfected HeLa cells. Vaccinia T7-infected HeLa cells were trans- fected with hNET cDNA in pBSKII- (14.30) or vector alone as described under "Experimental Procedures." Cell extracts were harvested at the times indicated posttransfection and subjected (30 pg) to SDS-PAGE (8%) prior to immunoblot analysis with affinity-purified N430 Ab (1 pg/ml). LLC-NET extracts (25 pg) were blotted in parallel for compari- son.

Chase time (hours)

0.5 1 2 4 8 24

205-

116-

80-

49.5-

4

4

FIG. 4. Biosynthesis of hNET protein in LLC-NET cells. Cells (400,000) were incubated for 10 min with 500 pCi/ml [D5SlMetfCys label in MetlCys-free media, rapidly washed, and incubated in unlabeled complete media (chase) for the times indicated. Total cell extracts were prepared, immunoprecipitated with N430 serum, and subjected to SDS- PAGE (10%) as described under "Experimental Procedures." Arrows on right indicate the migration of the 80- and 54-kDa products synthesized in LLC-NET cells.

volved in the biosynthesis of hNET in LLC-NET cells. There- fore, we sought to determine the t o t a l contribution of glycosy- lation to protein mobility and assess the functional significance of posttranslational glycosylation by 1) incubation of intact cells with the glycosylation inhibitor TM accompanied by func- tional assay of NE transport and radioligand binding, and 2) treatment of LLC-NET cell extracts with enzymatic glycosi- dases followed by visualization of core hNET protein. Previous studies by Gu et al. (29) have demonstrated transport of [3H]NE in LLC-NET cells with a V,,, of 17 2 0.71 pmol/midmg of protein and K,,, of 0.5 2 0.06 p ~ . Treatment of LLC-NET cells with 10 pg/ml TM for 24 h resulted in a marked reduction in the accumulation of L3H1NE compared with untreated cells (Fig. 5A ). Substrate transport velocity profiles indicated that alter- ations in NE transport rose largely from a reduction in V,,,

B 200

0 10 20 30 40 50 60 70 [RWW nY

FIG. 5. Effect of TM treatment on hNET transport and ligand binding in LLC-NET cells. a , effects of TM treatment on NE trans- port kinetics in LLC-NET cells. Substrate velocity profiles were derived from NE transport studies conducted under initial rate conditions with vehicle (0) or TM-treated (10 pg/ml, 24 h) (0) LLC-NET cells as de- scribed under "Experimental Procedures." Results are the mean 2 S.E. for assays conducted in triplicate, with values adjusted for nonspecific transport, defined as the accumulation of NE in the presence of 1 PM desipramine. b, effects of TM treatment on equilibrium ['2s11RTI-55 binding to LLC-NET membranes. Saturation binding of ['2sIlRTI-55 was performed on membranes prepared from vehicle (0) or TM-treated (10 pg/ml, 24 h) (0) as described under "Experimental Procedures." Results, expressed as pmoVmg of protein, derived from duplicate assays with values adjusted for nonspecific transport, defined as ['2sIIlRTI-55 binding in the presence of 1 p~ desipramine.

(control, 18.94 2 1.26 pmol/midmg of protein; TM-treated, 10.70 2 0.60 pmol/mg of proteidmin) with a decrease in sub- strate K, (control, 0.92 = 0.17 p ~ ; TM-treated, 0.43 = 0.08 p ~ ) . To evaluate whether the reduced capacity for NE transport also corresponded to a decrease in M E T density, we examined equi- librium binding of the cocaine analog ['251]RTI-55 to mem- branes prepared from TM-treated LLC-NET cells. Control LLC-NET membranes exhibited saturable, high affinity RTI-55 binding with kinetic values (K, = 4.0 2 0.71 nM; B,,, = 19.7 2 1.0 pmoVmg of protein) similar to those previously re- ported (29). In contrast, membranes prepared from TM-treated LLC-NET cells exhibited a substantial reduction in membrane transporter density (5.5 e 0.35 pmol/mg of protein) with a small increase in KD (13.6 5 2.3 nM) (Fig. 5B ). Immunoblots of hNET protein in LLC-NET cells (Fig. 6A) revealed that within 8 h of TM treatment, the 54-kDa species had vanished, consistent with previous data, indicating a rapid turnover of this inter- mediate species (Fig. 4) and the absence of further glycosyla- tion to replenish the 54-kDa pool. Thus, it is unlikely that the 54-kDa species from TM-treated LLC-NET cells is the major contributor to transport activity or ['2sI]RTI-55 binding. By 8 h, we detected the presence of a new 46-kDa species, presumably the nonglycosylated core protein. Immunoblots of TM-treated, hNET-transfected HeLa cells also revealed a shift in mobility of hNET protein to the 46-kDA form (data not shown). To confirm that the 80-kDa band in TM-treated cells represented residual hNET synthesized prior to glycosylation inhibition, LLC-NET cells were pulse-labeled in the presence of TM, followed by cold chase and immunoprecipitation. As expected, only the 46-kDa immunoreactive species is formed in LLC-NET cells in the

Antibodies Against the NE Bansporter 12295

A A

0 8 12 16 24 (hrs)

80 kDa &

54 kDa .")

46 kDa 54 kDa "C

46 kDa -+ "I B - +

0.5 4 8 12 24 0.5 4 8 12 24

80 kDa d

54 kDa - 46 kDa- , - I

- TM +TM

FIG. 6. Effects of TM treatment on hNET protein expression in LLC-NET cells. a , immunoblot of extracts prepared from TM-treated LLC-NET cells. Cells were incubated with TM (10 pg/ml, 37 "C) for the times indicated and extracts immunoblotted after SDS-PAGE (8%) with affinity-purified N430 Ab (1 pg/ml) as described under "Experimental Procedures." b, immunoprecipitation of LLC-NET cells pulse-chase la- beled in the absence ( -2") or presence (+?") of TM. Cells, incubated with vehicle or TM in complete media for 1 h (10 pg/ml, 37 "C), were switched to MetICys-free media containing vehicle or TM for 30 min, followed by pulse labeling with 13sS1Met/Cys (300 pCi/ml) for 10 min. Label was removed and cells incubated with complete media containing vehicle or TM. Cells were harvested at the times indicated and under- went immunoprecipitation with N430 sera and SDS-PAGE (8%) as de- scribed under "Experimental Procedures." Sizes of hNET species in a and b indicated with arrows were determined from molecular mass standards electrophoresed in parallel.

absence of core glycosylation (Fig. 6B 1. We also observed that the 46-kDa protein synthesized in the presence of TM has a reduced half-life relative to the 80-kDa species synthesized under control conditions. These data suggest that the reduction in transport and ligand binding in TM-treated LLC-NET cells is due to a depletion of the pre-existing 80-kDa pool of hNET protein.

TM-induced increase in hNET mobility confirms that sub- stantial post- or co-translational glycosylation occurs in the maturation of hNET protein in LLC-NET and transfected HeLa cells. However, sialation does not appear to contribute significantly to hNET mobility as neuraminidase treatment of LLC-NET or transfected HeLa extracts failed to alter W E T mobility (data not shown), despite complete digestion of sialated fetuin controls. In contrast, PNGase F treatment of transfected cell membranes, which should remove all N-linked glycosylation from expressed proteins regardless of sugar con- tent, resulted in the complete loss of both 80- and 54-kDa im- munoreactive bands from LLC-NET cell extracts and the ap- pearance of a lower molecular mass doublet of 46 and 44 kDa

Rnr

46 kDa +

PNGase F treatment of LLC-NET cell extracts ( a ) or hNET-transfected FIG. 7. Deglycosylation of hNET protein in transfected cells.

HeLa extracts ( b ) . Extracts of all cells (50 pg) were incubated with (+) or without (-) PNGase F (16 h, 37 "C), subjected to SDS-PAGE (8%), and immunoblotted with N430 Ab (1 pg/ml) as described under "Experimen- tal Procedures." LLC-PK, cell extracts were blotted in parallel in a as control. Arrows indicate the mobility of hNET species as determined from parallel electrophoresis of size standards.

(Fig. 7A), while PNGase F digestion of the parental cell line LLC-PK, does not induce immunoreactivity. On the basis of the migration of hNET synthesized in the presence of tunicamycin a t 46 kDa (Fig. 6, A and B ), we suspect that the 46-kDa species represents the mobility of the core polypeptide in our gel sys- tem, with the 44-kDa fragment an immunoreactive proteolytic fragment generated in the rather lengthy incubations required for complete PNGase F digestion. Immunoblots of transfected HeLa cell extracts digested with PNGase F also revealed a complete loss of the 54-kDa band and the appearance of a 44-46-kDa species (Fig. 7B ).

DISCUSSION

The isolation and analysis of hNET cDNA provides the first opportunity to directly investigate the structure and biosynthe- sis of hNET protein using polyclonal antibodies, an approach that has proven successful in the examination of both the GABA (24, 33) and DA (34) transporters. Using the predicted hNET amino acid sequence, we generated anti-peptide antibod- ies against three putative hydrophilic hNET domains. Peptides were chosen on the basis of their predicted hydrophilicity, im- munogenicity, and predicted topological localization, as well as divergence from other members of the GATl/NET gene family (3, 12). Of the hNET-directed antibodies, one (N430) proved a useful tool for examination of hNET translation products in both stably and transiently transfected cells using standard immunological techniques. N430 Ab specifically stained stably transfected LLC-NET cells in a detergent-dependent manner but failed to stain if LLC-PK, or peptide-absorbed Abs were substituted. Both immunoprecipitation and immunoblot anal- ysis of LLC-NET cells revealed two hNET species, 80 and 54 kDa, the smaller of which is the biosynthetic intermediate of the larger form. In contrast, only a 54-kDa species was detected

12296 Antibodies Against the NE Dansporter

in transiently transfected HeLa cells. Deglycosylation studies performed with both stably and transiently transfected cells suggest that nonglycosylated hNET protein, synthesized in the presence of TM, is a 46-kDa core protein that is rapidly de- graded. These results are supported by enzymatic digestion of hNET protein with PNGase F, also resulting in a 46-kDa core protein.

Staining of LLC-NET cells with the N430 Ab revealed in- tense border staining in agreement with an expected plasma membrane localization for hNET. When detergent is excluded from antibody incubations, N430 immunoreactivity is abol- ished, consistent with the predicted intracellular localization of the N430 epitope. In a similar manner, differential immunore- activity of peptide-directed Abs in the presence or absence of detergent have yielded data consistent with the established topology of other multi-TMD proteins, including G protein- coupled receptors (35-37). It remains a possibility that the N430 epitope is membrane-embedded or is occluded by another protein, thereby necessitating the presence of detergent to ac- cess the domain. Further analysis of NET topology may be possible using N430 Ab and higher resolution techniques, such as electron microscopy.

In the stably transfected cell line LLC-NET, both an 80-kDa mature protein and a 54-kDa biosynthetic intermediate were detected. In contrast, only a 54-kDa form of hNET was detected in transiently transfected HeLa cells. Even with long expo- sures, we were unable to visualize any HeLa hNET species other than the 54-kDa form. These discrepancies are likely to be the result of cell-specific glycosylation patterns and not due to proteolytic processing of hNET protein, as PNGase treat- ment of LLC-NET and transfected HeLa cell extracts results in a common 46-kDa core protein (Fig. 7, A and B) . Mobility dif- ferences are also not likely to be due to differential sialation, as treatment with neuraminidase caused no shift in the mobility of any of the hNET species detected (data not shown). While functional (38) and structural (19) studies suggest that the brain DA transporter is sialated, neither the GABA transporter (33) or rat DA transporter transiently expressed in COS cells (22) are sensitive to neuraminidase treatment. At present, we do not know whether the difference in glycosylation states we observed between stably transfected LLC-PK, cells and vac- cinia T7-transfected HeLa cells reflect cell- or species-specific (pig versus human) glycosylation of hNET protein or indicate virus alteration in end-stage protein processing. Region- (21), species-, and cell-specific (22) glycosylation differences have been noted for the DA transporter, resulting in M, ranging from 58,000 to r100,OOO. A substantial portion of the rat 5-hy- droxytryptamine transporter expressed using the vaccinia sys- tem also migrates significantly faster on SDS-PAGE than the native brain and platelet carrier (39). Notably, [3Hlxylamine treatment of NET-expressing PC12 cells results in the labeling of a 54-kDa protein in a cocaine-inhibitable manner (40). In contrast, preliminary immunoblot studies have revealed 80- kDa proteins that may represent NETS in sympathetically in- nervated rat tissues.' To rule out viral effects, we attempted to introduce hNET into HeLa cells by calcium phosphate trans- fection and into LLC-PK, cells using VTF,,; however, neither method resulted in detectable levels of immunoreactive protein (data not shown). Additionally, effects of vTF,-, on glycosylation in LLC-NET cells could not be assessed as vaccinia suppresses host cell translation (42). Nonetheless, many proteins ex- pressed using vaccinia virus vectors are processed faithfully when compared with their native proteins (43), and, as yet, we have detected no pharmacologic differences between HeLa and LLC-NET-expressed hNET. We suspect that the differences,

H. E. Melikian and R. D. Blakely, unpublished results.

however achieved, do not contribute substantially to substrate or antagonist discrimination. It remains possible that vaccinia- transfected HeLa cells express a higher M, hNET protein that is responsible for NE transport but that fails to reach levels detectable by our methods. Utilization of surface labeling tech- niques with hNET-transfected cells and N430 Ab should help clarify this issue.

N-Linked glycosylation has been implicated in membrane protein stability, trafficking (44), and function (45). Therefore, the role of N-linked glycosylation on W E T function and bio- synthesis was assessed in LLC-NET cells using the glycosyla- tion inhibitor tunicamycin. Following 24 h of TM treatment, V,,, of L3H1NE transport was reduced by 44%. Similar losses in transport following TM treatment are also noted for the GABA transporter in GAT1-transfected L cells (24) and for the rat NET in PC12 cells (23). Reduced capacity for transport of NE after TM treatment could be due to 1) the instability or poor trafficking to the cell surface of nonglycosylated hNET sub- units, with residual activity reflecting the half-life of transport- ers synthesized prior to TM treatment, 2) altered turnover number of a fixed density of NE carriers, or 3) nonspecific alteration in substrate driving forces (V,,, or ion gradients). Parallel examination of hNET protein during TM treatment demonstrated that in the absence of glycosylation, the 54-kDa biosynthetic intermediate disappears and a short-lived 46-kDa protein appears (Fig. 6A). Little or no increase in the 46-kDa pool is evident over time, and, thus, it is likely that failure to progress in the biosynthetic pathway leads to hNET degrada- tion. This conclusion is supported by a decrease in the stability of the 46-kDa W E T species synthesized in the presence of TM (Fig. 6B). With no further addition to the higher M, pool after TM treatment, the 80-kDa species slowly disappeared as ex- pected for its estimated 24-h half-life. Therefore, residual L3H]NE transport, detected following TM treatment, is likely to reflect the activity of the remaining 80-kDa hNET form of hNET synthesized prior to TM treatment.

['2511RTI-55 binding to membranes prepared from TM- treated LLC-NET cells demonstrated a 72% reduction in B,, compared with control membranes. The slightly larger reduc- tion in B,,, versus V,,, may be representative of either 1) a contribution of immature NET subunits to ligand binding as opposed to transport, or 2) a contribution to binding, but not uptake, of intracellular NET. Gu et al. (29) estimate that the majority of [*251]RTI-55 accessible NET is not on the surface of LLC-NET cells, where it could contribute to transport. Differ- ential sensitivity of the larger intracellular pool of NET to TM could result in a greater loss of binding sites than is apparent from surface measures of transport. Clearly, the 54-kDa spe- cies, which may contribute to antagonist binding but not trans- port, is rapidly lost following TM treatment, (Fig. a).

Glycosidic digestion of hNET using PNGase F confirms that the 46-kDa species observed in the TM studies represents core hNET protein and that canonical sites for N-linked glycosyla- tion are actually utilized. Since all of the predicted sites for N-linked glycosylation are present in the large hydrophilic loop between TMDs 3 and 4, data from TM and PNGase F studies support both the glycosylation of this loop and, by inference, its extracellular localization. Interestingly, the predicted core size for hNET protein inferred from cDNA sequence is 67 kDa, significantly larger than the apparent mass of nonglycosylated hNET. The discrepancy between predicted and observed mobil- ity of hNET core protein is unlikely to arise from proteolysis COOH-terminal to the N430 recognition domain as an addi- tional COOH terminus-directed hNET Ab successfully immu- noprecipitated hNET protein (data not shown). Since we pres- ently have no way to verify the presence of the NH, terminus of hNET in our extracts (at least up to the region of Asn-linked

Antibodies Against the NE Dansporter 12297

glycosylation), some loss of the NH, terminus is possible. More likely, the mobility of hNET protein is simply compromised by the well known exclusion of SDS from highly hydrophobic pro- teins (46). In similar studies, PNGase F-digested native canine and rat brain DA transporters exhibit an apparent mobility of 4548 kDa (19, 21) despite a predicted protein size of 69 kDa (47, 48).

In summary, use of the N430 Ab has permitted the first characterization of hNET protein in transfected mammalian cells. Indirect immunofluorescence data are consistent with the intracellular localization of the N430 epitope, providing sup- port for the hydrophobicity-based model (14). hNET protein was found to contain Asn-linked glycosylation, inferring an extracellular localization of the hydrophilic domain between TM3 and 4, with sequential glycosylation states evident in LLC-NET cells and a single glycosylated 54-kDa form in tran- siently transfected HeLa cells. Inhibition of hNET glycosyla- tion in LLC-NET cells by TM leads to a loss of specific hNET binding sites, decreased desipramine-sensitive NE uptake, ap- pearance of a 46-kDa core protein, and a time-dependent loss of mature hNET. Future cell surface labeling, mutagenesis, and intracellular trafficking studies, making use of the hNET-tar- geted N430 Ab, should further advance our understanding of monoamine transporter structure, function, and regulation.

Acknowledgments-We thank Allan Levey and Larry Rizzolo for ad- vice with immunoblots and Eric Barker for helpful discussions. We gratefully acknowledge the donation of [12511RTI-55 by Dr. John Boja, National Institute on Drug Abuse.

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