Grant DA07390 and a Mallinckrodt award (to R. D. B.), National Insti- ...... Goldstein, D. S., Brush, J. E., Eisenhofer, G., Stull, R., and Esler, M. (1988). 10.
THEJOURNAL OF BIOWGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc
Vol. 269, No. 16, Issue of April 22, pp. 12290-12297, 1994 Printed in U S A .
Human Norepinephrine Transporter BIOSYNTHETIC STUDIES USINGA SITE-DIRECTED POLYCLONAL ANTIBODY* (Received for publication, October 26, 1993, and in revised form, January 31, 1994)
Haley E. MelikianSs, John Randy D. BlakelySlI
K. McDonald+,Howard Gun Gary Rudnickn Kimberly R. Moore+,and
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 peptides derived from the predicted primary sequence of the human cocaine- and antidepressant-sensitivenorOne antibody epinephrine (NE) transporter (NET). (N430), raised and purified against a putative intracellular 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 immunoreactivity is observed in either the parental cells (LLCPK,)or in LLC-NET cells incubated with preimmune sera or peptide absorbed antibody.N430 immunoblots of LLC-NET cell extracts reveal two major immunoreactive hNET species inthese cells, migrating at 80 and 54 kDa, respectively. Pulse-chase N430 immunoprecipitation studies confirm that the 54-kDa species is a transient, glycosylated intermediate of a longer lived, more highlyglycosylated protein with anapparant M, of 80,000. In contrast, a 54-kDa species is the primary hNET product in vaccinia virus T7-infected HeLa cells, transiently transfected with hNET cDNA. PNGase F digestion of extracts prepared from LLC-NET- and hNETtransfected HeLa cellsconvert all immunoreactive species to a 46-kDa form, equivalent to that observed following incubation of whole cells with the glycosylation inhibitor tunicamycin. As transiently transfected HeLa and stable LLC-NET cells exhibit a pharmacologically similar NE transport activity, it appears likely that the additional glycosylation evident in the stable line does notcontribute significantly to antagonist sensitivity.On the other hand, NE transport and antagonist ([‘aaI]RTI-55)binding assays on whole LLC-NET cells treated with tunicamycin reveal a pronounced reduction in NE transport activity and hNET membrane density paralleled by an inability of NET proteins to replenish the higher M, hNET pool. These findings suggest an obligate rolefor N-linked glycosylationin hNET biosynthetic maturation, stability, and functional expression. In summary, N430 antibody is a useful toolfor the visualization and characterization of hNET gene products and has permitted the first direct evaluation of biosynthetic steps leading to functional catecholamine transporter expression.
Manyneurotransmitters,including NE,‘ DA, 5-hydroxytryptamine, GABA, glycine, and t-glutamate are thought to have their actions constrained by rapid clearancefrom 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 ( G l d Asp transporters, (5))are responsible for extracellular neurotransmitter clearance. The monoamine transporters within the GATl/NET gene family are highaffinity targets for a number of psychoactive agents, including cocaine, amphetamines, and antidepressants (6-8).These agents cause an increase in extracellular NE, DA, and 5HT concentrations in both the central nervous system andperiphery, 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,whereasdifferential blockade of allthree transporters in the brain contributes to their behavioral effects (9-11). Although the addictive and/ortherapeutic consequences of pharmacologic NE, DA, and 5HT transporterblockade have been clear for decades, molecular details of monoamine transporter protein structure and regulation areonly beginning to emerge (12, 13). The expression of antidepressant- andcocaine-sensitive NET from a single cDNA first co-localized psychoactive drug binding sites and transportactivity in a single membrane protein (141, findings that were quickly corroborated with the cloning and heterologous expression of homologous DA and 5HT transporters (reviewed in Ref. 3). Despite this progress, hNET protein has yet to be directly visualized in situ, and the transporter’s biosynthesis and posttranslationalmodifications in any preparation remain to be elucidated. Hydropathy analysisof inferred amino acid sequences predicts that NETmonomers, like other twelve internal TMDs GATl/NET homologs (3, 12, 14), contain between cytoplasmic NH, and COOH termini, with the latter tails thoughtnot to contribute to substrate or antagonist specificity (15).Conserved and unique sitesfor phosphorylation are present on putative intracellular domains of hNET, although a n inability to isolate hNET protein has limited progress in evaluation of their use.Approximately 20% of amino acid residues in hNET are conserved with GATUNET homologs and distributed nonuniformly throughout the predicted structure,
* This work was supported by National Institute on Drug Abuse Grant DA07390 and a Mallinckrodt award (toR. D. B.), National Institute on Drug Abuse Grants DA8213 and DA7259 (to G. R.), and National Institutes of Health Award HD26833 (to J. K. M.). The costs of publication of this article were defrayedin part by the payment of page charges. This article must thereforebe 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.
The abbreviations used are:N E , norepinephrine; NET norepinephrine transporter; hNET, humannorepinephrine 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, phosphate-buffered saline.
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Antibodies Against the NE Dansporter concentrated heavily in TMDs1-2 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 photoaffinity labeling paradigms (16-20) indicate 5HT and DA transporters to beformedfrom glycosylated monomersof 58-80 kDA.Region-(211, cell-, and species-specific (22) DA transporter 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 preparedagainst purified GABA transporter proteinand GATlcDNA-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, immunoprecipitates metabolically labeled camers, and recognizes denatured hNET protein on immunoblots of cell and membrane extracts. Our studies with N430 Ab providesupportfor the hydrophobicity-basedtopologicalmodelof 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. EXPERIMENTALPROCEDURES Methods Antibody Preparation, Purification, and Characterization-Peptides were coupled to KLH and bovine serum albumin carrier proteins by maleimide cross-linkingto free sulfhydryl groups. KLH or bovine serum albumin (10 mg) were activated by incubating with 2.5 mglml m-maleimidobenzoicacidn-hydroxysuccinimide ester (dissolved in m,m-dimethylformamide) in 0.5 ml of 100 m NaBO, under argon (30 min, 22 "C, stirring). Free maleimide was removed by repetitive spin filtration (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 quantitated using the method of Ellman (27). Primary immunizations of rabbits with peptide-KLH conjugate (1mg/200 pl)were followed by 14and 28-day booster injections (0.5 mg/100 4).Thereafter, boosts were 0.5 mg/lOO pl. Animals were routinely bled 1week after a given boost, beginning after the first boost. Immunoreactivity of sera was determined 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 1mg/ml peptide incubated with 2 ml of resin). Sera was heat-inactivated (1h, 56 "C),diluted 1:l in PBS, and mixed with affinity resin (30 min, 22 "C). Serahesin suspensions were collectedand washed with PBS until A,, of wash
