INTRODUCTION

Neurotensin (NT) is a tridecapeptide originally isolated andcharacterized by Carraway and Leeman (1973) anddocumented to exert widespread neuromodulatory effects inmammalian central nervous system (see Kitabgi et al., 1985).Three different NT receptors, referred to as NT1, NT2 and NT3have so far been cloned (Tanaka et al., 1990; Chalon et al.,1996; Mazella et al., 1996, 1998). The first two belong to thefamily of G-protein-coupled receptors. The third one (NT3) isa single transmembrane domain receptor with 100% homologywith gp95/sortilin, a protein involved in receptor sorting(Peterson et al., 1997). All three receptors were shown tointernalize upon interaction with NT (Faure et al., 1995a;Chabry et al., 1995; Botto et al., 1998; Beaudet et al., 1998;Navarro et al., 1999). Biochemical and morphological studiesin primary neurons and/or neuronal cell lines in culturedemonstrated that this internalization was both time- andtemperature-dependent and that it was sensitive to theendocytosis blocker phenylarsine oxide (Mazella et al., 1991;Chabry et al., 1993; Faure et al., 1995b; Botto et al., 1998;Navarro et al., 1999). Current biochemical data suggest that,

whereas the NT1 receptor sub-type is not recycled, the NT2largely is (Turner et al., 1990; Hermans and Maloteaux, 1998;Botto et al., 1998). The fate of internalized NT3 receptors isstill unknown.

Confocal microscopic studies carried out using fluorescentNT analogs demonstrated that in neurons endogenouslyexpressing NT1 receptors, the ligand itself was internalizedtogether with its receptor and was subsequently mobilized fromperikarya and dendrites toward the perinuclear region withinsmall vesicular organelles (Faure et al., 1995a,c; Nouel et al.,1997a). Little is still known, however, concerning theintracellular targeting of either ligand or receptor in this modelsystem.

Most of our knowledge concerning the intracellulartrafficking of internalized ligands and/or receptors is derivedfrom studies of single transmembrane domain receptors. Forinstance, the transferrin receptor is constitutively internalizedvia clathrin-coated pits into early endosomes (Pearse, 1982;Larkin et al., 1983; Heuser and Anderson, 1989). In the acidicenvironment of endosomes, iron dissociates from transferrinand both transferrin and its receptor return to the cell surfacein recycling endosomes (Dautry-Varsat et al., 1983; Klausner

2963Journal of Cell Science 113, 2963-2975 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS1346

The neuropeptide neurotensin (NT) is known to beinternalized in a receptor-mediated fashion into its targetcells. To gain insight into the mechanisms underlying thisprocess, we monitored in parallel the migration of the NT1neurotensin receptor subtype and a fluorescent analog ofNT (fluo-NT) in COS-7 cells transfected with a tagged NT1construct. Fluo-NT internalization was prevented byhypertonic sucrose, potassium depletion and cytosolacidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulatedtogether with its receptor in Acridine Orange-positive,acidic organelles. These organelles concentratedtransferrin and immunostained positively for rab 5A,therefore they were early endosomes. After 30-45 minutes,the ligand and its receptor no longer colocalized. Fluo-NTwas first found in rab 7-positive late endosomes and later

in a nonacidic juxtanuclear compartment identified as theTrans-Golgi Network (TGN) by virtue of its staining forsyntaxin 6. This juxtanuclear compartment also stainedpositively for rab 7 and for the TGN/pericentriolarrecycling endosome marker rab 11, suggesting that theligand could have been recruited to the TGN from eitherlate or recycling endosomes. By that time, internalizedreceptors were detected in Lamp-1-immunoreactivelysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that isunique among the G protein-coupled receptors studied todate, and provide the first evidence for the targeting of anonendogenous protein from endosomes to the TGN.

Key words: Neurotensin, NT1, Internalization, Trafficking, Confocalmicroscopy, Trans-Golgi network

SUMMARY

Ligand-induced internalization of neurotensin in transfected COS-7 cells:

differential intracellular trafficking of ligand and receptor

Franck Vandenbulcke 1,*, Dominique Nouel 1, Jean-Pierre Vincent 2, Jean Mazella 2 and Alain Beaudet 1,‡

1Montreal Neurological Institute, McGill University, Montreal, Quebec, H2A 2B4 Canada 2Institut de Pharmacologie Moléculaire, Université de Nice-Sophia Antipolis, CNRS, 06560 Valbonne, France*Present address: Centre de Biologie Cellulaire, Groupe Endocrinologie et Immunité des Invertébrés, UPRES A CNRS 8017, Université des Sciences et Technologiesde Lille, 59655 Villeneuve d’Ascq cedex, France‡Author for correspondence (e-mail: mcin@musica.mcgill.ca)

Accepted 28 June; published on WWW 9 August 2000

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et al., 1983). The low density lipoprotein receptor alsointernalizes constitutively via endosomes. Following liganddissociation, however, it is recycled back to the cell surface,whereas the low density lipoprotein is targeted to lateendosomes and lysosomes in which it is degraded (Goldsteinet al., 1985). The epidermal growth factor receptor internalizesin a ligand-induced fashion, again via clathrin-mediatedmechanisms. In this case, however, both receptor and ligandare degraded into lysosomes (Carpenter and Cohen, 1976;Stoscheck and Carpenter, 1984).

Far less is known about the fate and intracellular targetingof molecules internalized via seven-transmembrane domainreceptors. In fact, it is not even known whether all G-proteincoupled receptor ligands internalize together with theirreceptor. Indeed, it has been proposed that ligands with onlymoderate affinities for their receptors may dissociate in theextracellular space prior to receptor internalization (Koenigand Edwardson, 1997). In the case of neuropeptides, the ligandusually internalizes with its receptor, but little is known of itsfate once sequestered inside the cells except for someneuropeptides, which were shown to be targeted to lysosomesfor degradation (Ghinea et al., 1992; Grady et al., 1995a).

More information is available on the fate of the internalizedreceptors themselves. Most G-protein coupled receptors, suchas the β2-adrenergic receptor, the human thrombin receptor,the thyrotropin releasing hormone receptor, the neurokinin 1receptor, the gastrin-releasing peptide receptor and the A-typecholecystokinin receptor are recycled back to the plasmamembrane following ligand-induced internalization (VonZastrow and Kobilka, 1992; Hoxie et al., 1993; Brass et al.,1994; Ashworth et al., 1995; Grady et al., 1995a,b; Tarasovaet al., 1997). Others, however, such as the luteinizing hormonereceptor, are degraded into lysosomes (Ghinea et al., 1992).

The aim of the present study was to delineate theintracellular migration pathway of NT and its receptorfollowing internalization of the neuropeptide via the NT1receptor subtype, with the aim of providing: (1) further insightinto mechanisms of internalization and routes of trafficking ofinternalized G-protein coupled receptors and their ligandand (2) data on possible pathways through which NTinternalization might induce internalization-dependentsignaling events (Burgevin et al., 1992; Souazé et al., 1997).For this purpose, we monitored in parallel the migration of NTand its receptor in transfected COS-7 cells by confocalmicroscopy and identified their targeting compartments usinga variety of cell markers.

MATERIALS AND METHODS

Antisera, antibodies and reagentsChemicals were obtained from Sigma Chemicals Co. (St Louis, MO,USA) and cell culture reagents from Gibco BRL (Eggenheim, FRG).Iron-saturated human FITC-transferrin and Acridine Orange wereobtained from Molecular Probes Inc. (Eugene, OR, USA). Secondaryantisera were purchased from Jackson ImmunoResearch Laboratories(West Grove, PA, USA) and screened for lack of crossreactivity.Primary antibodies were obtained from the following sources:affinity-purified rabbit antibodies directed against the carboxyterminus of human rab 5A were purchased from Santa CruzBiotechnology Inc. (Santa Cruz, CA, USA). Rabbit rab 7 antiserum(Chavrier et al., 1990) was kindly provided by Marino Zerial (EMBL,

Heidelberg, FRG). Rabbit rab 11 antiserum was purchased fromZymed laboratory (San Francisco, CA, USA) and rabbit NT antiserumfrom Protos Biotech Corporation (New York, USA). Mousemonoclonal and rabbit polyclonal antibodies against human lamp-1(Carlsson et al., 1988) were kindly supplied by Minoru Fukuda (TheBurnham Institute, CA, USA). Mouse monoclonal antibody toSyntaxin 6 was obtained from Transduction Laboratories (Lexington,KY, USA) and rabbit anti-human hemagglutinin (HA) antibody fromRoche Diagnostic, Laval, Quebec. The P5D4 mouse monoclonalantibody, which recognizes the vesicular stomatidis virus (VSV)(Kreis and Lodish, 1986), was generously provided by Thomas Kreis(Université de Genève, Geneva, Switzerland).

Cell cultureCOS-7 cells were grown in Dulbecco’s modified Eagle’s medium(DMEM) containing glutamine and supplemented with 44 mMNaHCO3, 10% fetal calf serum (FCS), 100 U/mlpenicillin/streptomycin. Cells were grown at 37°C in a humidifiedatmosphere of 5% CO2/95% air. 24 hours before transfection, the cellswere plated in 100-mm diameter plastic Petri dishes at a density of106 cells/dish. When semiconfluent (24 hours later), cells weretransfected with cDNA encoding either native NT1 or VSV tagged-NT1 constructs. The NT1 receptor cDNA was kindly provided to usby Dr S. Nakanishi (Kyoto University, Japan) (Tanaka et al., 1990).To generate VSV-tagged cDNA, a 1.45 kilobase (kb) fragment of theNT1 receptor cDNA corresponding to the total reading frame plus the5′ end noncoding sequence was obtained by polymerase chainreaction and standard cloning techniques. A sequence coding apeptide from the vesicular stomatidis virus (VSV) and containing theYTDI peptide signal was incorporated into the cDNA and theconstruct was subcloned into the eukaryotic expression vectorpcDNAI, which contains the cytomegalovirus promoter (Chabry et al.,1995).

All COS-7 cell transfections were carried out by incubating thecells for 30 minutes at room temperature with 1 ml Tris-bufferedsaline containing 2 µg recombinant plasmid and 0.5 mg/mldiethylaminoethyl-dextran. At the end of the incubation, the bufferwas replaced by culture medium containing 100 µM chloroquine andthe cells were further incubated at 37°C for at least 3 hours. They werethen rinsed in tris-buffered saline (TBS), covered with culture mediumand grown at 37°C for approximately 60 hours. For internalizationassays, cells were plated on polylysine-coated glass coverslips (25µg/ml, 15 minutes at room temperature) in 12-mm dishes. After 1-2hours at 37°C, the cells were incubated with fluorescent ornonfluorescent ligand and processed for confocal microscopicanalysis.

Fluo-NT binding and internalization in COS-7 cellsAll studies were performed in Earle’s buffer, pH 7.4, containing 140mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 3.6 mM MgCl2, 0.1% bovineserum albumin (BSA), 0.01% glucose and 0.8 mM 1-10phenanthroline, using Nα-Bodipy-NT (2-13) as fluorescent ligand.This fluorescent ligand, generically referred to here as fluo-NT, wassynthesized and purified as described in Faure et al. (1995a; also seeNouel et al., 1997a). Transfected cells were preincubated for 10minutes at 37°C in Earle’s buffer. They were then incubated forvarious periods of time (5, 10, 15, 25, 30, 45, 60 minutes) with 10-20nM of fluo-NT in the same buffer. For determination of nonspecificlabeling, 1 µM NT was added to the incubation medium. At the endof incubation, cells were rinsed three times with ice-cold Earle’sbuffer. The cells were then either air-dried and mounted on glass slideswith Aquamount or further processed for immunodetection ofintracellular antigens.

Inhibition of receptor-mediated endocytosisTo determine whether fluo-NT internalization proceeded throughclathrin-coated pits, three different treatments were used.

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2965NT and NT1 intracellular trafficking

(1) Hypertonic sucroseAs demonstrated by Heuser and Anderson (1989), hypertonic mediuminhibits receptor-mediated endocytosis. Transfected cells grown onglass coverslips were preincubated in supplemented Earle’s buffer for10 minutes at 4°C. Thereafter, samples were incubated in the samebuffer containing 400 mM sucrose and 10 nM of fluo-NT for 90minutes, first for 60 minutes at 4°C and then for 30 minutes at 37°C.

(2) Potassium depletionThis procedure, which is documented to inhibit the formation ofclathrin-coated pits, was modified from Larkin et al. (1983). Cellswere exposed to hypotonic medium (DMEM/water 1:1) for 5 minutesat 37°C and preincubated for 25 minutes in 50 mM Hepes buffer, pH7.4, containing 100 mM NaCl, 0.2% BSA and 0.8 mM 1-10phenanthroline. They were then incubated at 37°C in the same buffercontaining 10 nM of fluo-NT for 30 minutes. As a control, cellssubjected to hypotonic shock were incubated with fluo-NT in thepresence of 10 mM KCl.

(3) Cytosol acidificationCytosol acidification was shown to prevent pinching-off of coatedpits. The procedure was adapted from Sandvig et al. (1987) and Subtilet al. (1994). Briefly, transfected cells were preincubated for 30minutes at 37°C in DMEM containing 50 mM Hepes, pH 7.2, andsupplemented with 0.2% BSA, 30 mM NH4Cl and 0.8 mM 1-10phenanthroline. Cells were then incubated with 10 nM fluo-NT asdescribed above but in the presence of 1 mM amiloride in order toinhibit the Na+/K+ antiporter of cells loaded with NH4+. As control,cells were subjected to cytosol acidification in the absence ofamiloride in the incubation medium.

In all cases, cells were then rinsed three times in cold Earle’s bufferand washed 2× 2 minutes with a hypertonic acid (pH 4) solutionconsisting of 0.2 M acetic acid and 0.5 M NaCl in Earle’s buffer toremove cell surface-associated fluorescent ligand (Nouel et al.,1997a,b). They were then rinsed again three times in Earle’s buffer,air dried, mounted on glass slides with Aquamount and examined byconfocal microscopy.

Acridine Orange (AO) experimentsTo determine whether fluo-NT was internalized within acidiccompartments, transfected cells were incubated with 10 nM fluo-NTat 37°C as described above. At various time intervals (15, 30, 45 and60 minutes), the binding was terminated by three consecutive rinsesin Earle’s buffer at room temperature. Cells were then stained for 1minute in the same buffer containing 20µM AO, also at roomtemperature. After numerous washes in the same buffer at 20°C, cellswere covered with Earle’s buffer, mounted on glass slides andexamined by confocal microscopy. As control, the AO staining stepwas repeated in the presence of chloroquine (500 µM) or NH4Cl (10mM), two reagents documented to collapse intravesicular pHgradients (Lencer et al., 1995).

Cointernalization of FITC-transferrin and fluo-NTTo determine whether internalized fluo-NT was targeted to earlyendosomes, transfected COS-7 cells were preincubated first in culturemedium deprived of FCS for 1 hour at 37°C, and second insupplemented Earle’s buffer for 10 minutes at 37°C. Cells were thenincubated in Earle’s buffer containing 25 µg/ml of iron-saturatedhuman FITC-transferrin and 20 nM of fluo-NT at 37°C for 10-15minutes. They were then washed three times with ice-cold Earle’sbuffer, fixed with 4% paraformaldehyde, first for 5 minutes on ice andthen for 15 minutes at room temperature. Cells were washed, air dried,mounted on glass slides with Aquamount and examined by confocalmicroscopy.

Dual detection of fluo-NT and of NT1-VSVTo follow fluo-NT and NT1 jointly during and after internalization,

cells transfected with the NT1-VSV construct were labeled as abovewith fluo-NT for various time intervals, rinsed in ice-cold Earle’s bufferand fixed with 4% paraformaldehyde in phosphate buffer (PB) for 20minutes at room temperature. The samples were then rinsed twice withphosphate-buffered saline (PBS), preincubated with PBS containing3% normal goat serum (NGS) for 20 minutes, rinsed again with PBSand incubated for 60 minutes at room temperature with the anti-VSVP5D4 antibody diluted 1:10,000 in PBS containing 1% NGS and0.02% Triton X-100. After rinsing three times (5 minutes each) withPBS containing 1% NGS, cells were incubated with a goat anti-mouseFITC-tagged secondary antibody diluted 1:100 in PBS for 30 minutesat room temperature. The coverslips were then mounted on glass slideswith Aquamount and examined by confocal microscopy.

A similar protocol was used for the identification of fluo-NTinternalization compartments. After various times of incubation withfluo-NT at 37°C, cells were fixed as above and incubated for 60minutes at room temperature with any one of the following antibodies:anti-rab 5A (1 µg/ml); anti-rab 7 (1:500); anti-lamp 1 (1:1000); anti-syntaxin 6 (3 µg/ml); anti-rab 11 (10 µg/ml). After rinsing, the boundantibodies were visualized with the appropriate FITC-labeledsecondary antibodies diluted 1:100.

To determine whether syntaxin 6 selectively identified the TransGolgi Network (TGN), two additional experiments were carried out.(1) Nontransfected COS-7 cells were pretreated with nocodazole (33µM in 0.1% (v/v) DMSO) for 60 minutes at 37°C to disrupt TGNassembly (Ladinsky and Howell, 1992; Mallet and Maxfield, 1999).These cells, together with cells treated with DMSO alone to controlfor the effects of solvent, were then fixed with paraformaldehyde andimmunostained for syntaxin 6 as described above. (2) Cells weretransfected as described above using cDNA encoding an HA-taggedconstruct of the TGN resident protein TGN38 (TGN38-HA,graciously provided by Dr J. Bonifacino, NIH, Bethesda, MD, USA).These cells were dual-labeled 48 hours later for syntaxin 6 and theHA epitope by double fluorescence microscopy (see below). Primaryantibodies were used at concentrations of 2 µg/ml and antigens werevisualized using a 1/150 dilution of Alexa 488-conjugated goat anti-mouse and a 1/100 dilution of Texas Red-conjugated goat anti-ratsecondary antibodies.

Finally, to confirm that the localization of the fluorescent Bodipymoiety reflected that of internalized NT, transfected cells wereincubated with 20 nM fluo-NT at 37°C for 60 minutes andimmunostained for NT (1:3000) using same protocol as above.

Double immunofluorescence microscopyTo identify intracellular compartments of NT1 targeting, transfectedcells were incubated with nonfluorescent NT (20 nM) as describedabove for fluo-NT, washed three times in cold Earle’s buffer and fixedwith 4% paraformaldehyde in PBS (pH 7.4) for 20 minutes. Afterfixation, cells were rinsed twice with PBS and preincubated in thesame buffer containing 3% NGS for 20 minutes. They were thenwashed with PBS and incubated in a mixture of primary antibodiesin PBS containing 1% NGS and 0.02% Triton X-100 overnight at 4°C.The mixture contained the mouse P5D4 VSV monoclonal antibody(1:10,000) and either rab 7 (1:500) or lamp 1 (1:1000) rabbitantibodies. After rinsing 3× 5 minutes, cells were incubated jointlywith goat anti-mouse FITC- and goat anti-rabbit Texas Red-taggedsecondary antibodies diluted 1:100 in PBS for 45 minutes. After threewashes in PBS, the coverslips were mounted on glass slides inAquamount and viewed with a confocal microscope. The absence ofcross-reactivity of the secondary antibodies was verified by omittingone or both primary antibodies during the overnight incubation. Inorder to minimize the intracellular pool of neosynthetized NT1, thesedouble-labeling experiments were also performed after treating thecells with the protein synthesis inhibitor, cycloheximide. For thispurpose, cells were preincubated in culture medium containing 70 µMcycloheximide for 70 minutes at 37°C and incubated with the ligandin the presence of the drug.

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Confocal microscopyLabeled cells were examined under a Zeiss laser scanning microscope(CLSM 410) equipped with an Axiovert 100 inverted microscope andan argon/krypton laser. FITC and Alexa 488 signals were imaged byexciting samples with 488 nm and AO fluorescence was detectedusing a 488 nm band-pass excitation filter and a 575-640 nm passbarrier filter. Bodipy Red and Texas Red signals were detected byexciting samples with 568 nm. Images were acquired sequentially assingle transcellular optical sections and averaged over 32 scans/frame.Images, processed using the Carl Zeiss CLSM software 3.1 version,were stored on Jazz disks, archived and mounted using Photoshop.

RESULTS

Incubation of COS-7 cells transfected with cDNA encoding theNT1 receptor for 0-60 minutes with 10-20 nM fluo-NT resultedin the selective labeling of 20-30% of the cells, in keeping withreported transfection yields in this cell line (Nouel et al.,1997b). This labeling was specific in that it was not observedin nontransfected parent cells or in transfected cells incubatedin the presence of a hundredfold concentration ofnonfluorescent NT. Confocal microscopic examination of fluo-NT-labeled cells revealed that after 0-5 minutes of exposure tothe ligand, the bulk of the fluorescence was membrane-boundand as such was almost entirely removable by hypertonic acid-wash (Fig. 1A,B). At longer time intervals, most of thefluorescent labeling was acid-wash resistant, i.e. wasintracellular. Furthermore, the distribution of this acid-washresistant component varied markedly with time. At short timeintervals (between 5 and 30 minutes), the label formed small‘hot spots’ distributed throughout the cytoplasm of the cells butsparing the nucleus (Fig. 1C). At later time points (>30minutes), these ‘hot spots’ decreased in number and

progressively clustered towards the center of the cell, next tothe nucleus (Fig. 1D).

Mechanisms of fluo-NT internalizationTo determine whether fluo-NT internalization proceededthrough clathrin-coated pits, the above assay was repeatedusing three different protocols previously reported to inhibitthe formation of clathrin matrices: hypertonic sucrose,potassium depletion and cytosol acidification. Hypertonicsucrose and potassium depletion were both documented toinduce abnormal clathrin polymerisation (Heuser andAnderson, 1989). Neither of these two treatments modified theamount or the specificity of fluo-NT binding to transfectedCOS-7 cells (Fig. 2A,C). Both totally prevented internalizationof fluo-NT, however, as specifically bound fluorescenceremained entirely acid-washable at all times (Fig. 2B-D).Furthermore, this cell surface labeling usually predominated atone pole of the cell, in a pattern suggestive of coalescingreceptor-ligand clusters (Faure et al., 1995a; Gaudriault et al.,1997) (Fig. 2A). The effect of potassium depletion wasreversible in that endocytosis could be restored by addingpotassium back to the depleted cells (Fig. 2E). This control alsoconfirmed that the presence of Ca2+ in the medium was notrequired for internalization of fluo-NT.

Cytosol acidification was shown to inhibit internalization bypreventing the pinching-off of clathrin-coated pits (Sandviget al., 1987). In the present study, cells acidified by theammonium chloride/amiloride method no longer accumulatedfluo-NT as indicated by the fact that the bound fluorescenceremained acid-washable at all times (Fig. 2F,G). Thisinhibition, observed after acidification with 30 mM NH4Cl,was rapidly reversed when the internal pH was normalized byomission of amiloride in the incubation buffer (not shown).Taken together, these results demonstrate that the

F. Vandenbulcke and others

Fig. 1. Confocal microscopicimaging of specific fluo-NTbinding to COS-7 cells transfectedwith cDNA encoding the NT1receptor at 37°C. Images wereacquired as single midcellularoptical sections at 32 scans/frame.(A) After 5 minutes of incubation,fluo-NT labeling is confined to thecell surface. (B) This labeling isno longer apparent followinghypertonic acid stripping of cellsurface binding. (C) After 20minutes of incubation, acid wash-resistant label is segregated withinsmall endosome-like particlesdistributed throughout thecytoplasm of the cell. (D) At 45minutes, intracellular fluorescentparticles are less numerous andclustered next to the nucleus. Bar,10 µm.

2967NT and NT1 intracellular trafficking

internalization of fluo-NT proceeds through a clathrin-mediated endocytic pathway.

Fate of internalized receptor-ligand complexesClathrin-mediated endocytosis of ligand-receptor complexes ischaracteristically followed by dissociation of the ligand fromits receptor in the endosomal compartment (see Koenig andEdwardson, 1997). To determine whether the same sequenceof events was triggered by NT internalization, we monitoredin parallel the fate of ligand and receptor in COS-7 cellstransfected with cDNA encoding an epitope-tagged NT1construct.

At all times examined, the distribution of the bound and/orinternalized fluorescent label was similar in COS-7 cellstransfected with the tagged NT1 receptor to that in cellstransfected with the native receptor. Thus, following 5 minutesof incubation with 20 nM fluo-NT, specifically bound ligandwas concentrated at the periphery of the cells (Fig. 3A). Acidwash experiments confirmed that as in cells transfected withnative NT1, the bulk of this eccentric labeling was located at

the cell surface (not shown). Dual localization experiments inwhich the receptor was visualized by immunohistochemistryusing an anti-VSV antibody showed complete ligand/receptoroverlap at this time (Fig. 3A,B).

After 30 minutes of incubation, receptor and ligandmolecules were partly dissociated and were both distributedmore centrally within the cytoplasm of the cell (Fig. 3C,D).After 45-60 minutes of incubation, ligand and receptor nolonger colocalized. Whereas fluo-NT was concentrated inthe core of the cell, next to the nucleus (Fig. 3E), NT1-immunoreactivity was detected peripherally, according to apattern similar to that seen at 5 minutes (Fig. 3F).

Early intracellular trafficking of internalized fluo-NTTo determine whether internalized fluo-NT transited throughthe classical endosomal pathway, confocal microscopicvisualization of the fluorescent label was combined with thedetection of either of two early endosomal markers, transferrinand rab 5A or with that of the pH indicator, Acridine Orange(AO). Transferrin is a well established marker of the

Fig. 2. Effect of endocytosisinhibition on the internalizationof COS-7 cells transfected withcDNA encoding the NT1.Images were acquired as singlemidcellular optical sections at32 scans/frame. (A) Whenincubations are carried out inhypertonic sucrose medium,the labeling is confined to theperiphery of the cells. (B) Thislabeling is no longer apparentafter hypertonic acid wash. (C) After potassium depletion,the labeling usuallypredominates at one pole of thecell. (D) This labeling isentirely acid-washable, i.e.surface bound.(E) Internalization may berestored after potassiumdepletion by adding potassiumto the incubation medium.(F) In cytosol acidifiedconditions, fluo-NT labeling isconfined to the periphery of thecells. (G) This labeling is againentirely acid-washable. Bar,10µm.

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endosomal pathway (Dautry-Varsat et al., 1983; Hopkins andTrowbridge, 1983; Klausner et al., 1983; Goldstein et al.,1985). Transferrin receptors internalize constitutively (i.e.irrespective of ligand binding) and are recycled back to theplasma membrane (Bleil and Bretscher, 1982). After 5-15minutes of incubation of NT1-transfected cells with 20 nMfluo-NT and 25 µg/ml of FITC-transferrin, there was extensivecolocalization of the two markers at the periphery of the cells(Fig. 4A,B). This colocalization could not be attributed tononspecific endocytosis of fluo-NT through constitutivelyinternalizing transferrin receptors since nontransfected COS-7cells present in the same cultures also internalized FITC-transferrin but not fluo-NT (not shown). In contrast, at latertime intervals (>30 minutes), fluo-NT no longer colocalized

with FITC-transferrin. Whereas the former wasconcentrated next to the nucleus, the latterexhibited a much more diffuse distributionthroughout the cytoplasm (not shown).

Following 5-15 minutes of incubationwith fluo-NT, the internalized ligand alsocolocalized with the early endosome markerrab 5A (Fig. 4C,D). Again, dual-labeledendosomes were most prominent at theoutskirts of the cell (Fig. 4C,D). By contrast,at later time intervals, colocalization of fluo-NT labeling and rab 5A immunoreactivity wasconsiderably less extensive than at 5-15minutes (not shown).

AO is a weak base that accumulatesselectively in compartments of low internal pH(Anderson and Orci, 1988). This compoundhas proved a reliable marker of intracellularacidic compartments, including earlyendosomes, late endosomes and lysosomes(Lencer et al., 1995). At short time intervals(

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corresponded to the TGN. First, disruption of the TGN networkby treatment with 33 µM nocodazole resulted in a major

dispersion of syntaxin 6 immunostaining from the juxtanuclearcore to the peripheral cytoplasm (Fig. 6A,B). Second, dual

immunolocalization of syntaxin 6 and ofTGN38-HA gave rise to closely superposablejuxtanuclear labeling patterns (Fig. 6C,D).

As at shorter time points, internalized fluo-NT did not colocalize with lamp 1-positivelysosomal compartments (Fig. 7A,B). Therewas, however, virtually complete overlapbetween fluo-NT and immunoreactive NT (Fig.8A,B), suggesting that the fluorescencetargeted to the juxtanuclear compartmentlargely represented fluo-NT itself and notmerely the dissociated Bodipy fluorophore.

Intracellular trafficking of the NT1receptorThe intracellular mobilization of the taggedNT1 receptor induced by exposure to 20 nMNT was monitored in parallel with that of theexogenous ligand using dual immunolabelingof the receptor and of the intracellularcompartment markers, rab 7 and lamp-1. In anattempt to deplete the pool of intracellularneosynthetized receptors and to focus ourobservations on the fate of receptors associatedwith the plasma membrane at time 0, most ofthese dual immunolocalization studies wereperformed after treating the cells for 70 minuteswith cycloheximide. Cycloheximide treatmentreduced the amount of intracellular receptorspresent in the cells prior to internalization and,by way of consequence, allowed for bettervisualization of internalized receptors. Prior toligand exposure, all cells, including thosetreated with cycloheximide, exhibited bothintense cell surface labeling and a perinuclearcore of intracellular receptors.

After 30 minutes of incubation withexogenous NT, immunoreactive NT1 and rab 7partly colocalized, suggesting that internalizedreceptors were targeted to late endosomes (notshown). This colocalization between NT1 andrab 7 was hardly detectable in cells that had notbeen exposed to the ligand or were exposed tothe ligand for either longer or shorter timeperiods (not shown). After longer incubationtimes (45-60 minutes), the bulk of NT1immunoreactivity was detected in lamp-1-immunopositive lysosomes (Fig. 7C,D). Bycontrast, no colocalization between NT1- andlamp-1 immunoreactivities was observed whencells were incubated with exogenous NT forshorter time intervals (

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the TGN whereas receptors are targeted to lysosomes fordegradation (Fig. 9). Although trafficking of internalizedreceptors to lysosomes has been demonstrated for other G-protein-coupled receptors, the present results provide the firstevidence for the targeting of an internalized ligand to the TGN.

NT has long been known to internalize in a receptor-dependent manner in cells that express NT1 receptors eitherendogenously or ectopically (Mazella et al., 1991; Chabry etal., 1993; Faure et al., 1995a,c; Nouel et al., 1997a).Internalization through this G protein-coupled receptor ishighly efficient and depends on the presence of key threonineand tyrosine residues in the C-terminal tail of the receptor(Chabry et al., 1995). In all cell systems studied to date, NTinternalization was found to proceed through small ‘endosome-

like’ organelles and the internalizedligand to accumulate within the core ofthe cell, next to the nucleus (Faure etal., 1995a,c; Nouel et al., 1997a).Nothing was known, however, of thepathways followed by either ligandor receptor in the wake of theinternalization process. To investigatethis issue, we monitored in parallel thefate of internalized NT and its receptorin epithelial COS-7 cells transfectedwith cDNA encoding the NT1 receptorsub-type.

As previously documented inneurons in culture (Chabry et al., 1993;Nouel et al., 1997a), internalization offluorescent NT within transfectedCOS-7 cells was prevented bytreatment with phenylarsine oxide(data not shown), suggesting that theinternalization process was clathrin-dependent. That this was indeed thecase was demonstrated here by the factthat NT internalization was blockedby treatments known to prevent theformation of clathrin-coated pits,including hypertonic sucrose (Heuserand Anderson, 1989), potassiumdepletion (Larkin et al., 1983), andcytosol acidification (Sandvig et al.,1987; Subtil et al., 1994). Other seven-transmembrane domain receptors suchas β2-adrenergic receptors (VonZastrow and Kobilka, 1992), thrombinreceptors (Hoxie et al., 1993),luteinizing hormone receptor (Ghineaet al., 1992), thyrotropin releasinghormone receptors (Ashworth et al.,1995) and neurokinin 1 receptors(Grady et al., 1995a) were also foundto internalize in a clathrin-dependentmanner.

After short incubations with fluo-NT(0-30 minutes), both ligand andreceptors were detected in acompartment which took up FITC-transferrin, a classical marker of the

early endosomal pathway (Grady et al., 1995a; Ashworth et al.,1995). Additional evidence for selective sequestration of fluo-NT into early endosomes included colocalization with the lowpH indicator AO, as well as with immunoreactive rab 5A, asmall molecular weight GTPase known to be associated withthe membrane of early endosomes (Chavrier et al., 1990;Gorvel et al., 1991; Bucci et al., 1992).

After 30 minutes of incubation, internalized fluo-NT wasshuffled from early endosomes to a compartmentimmunoreactive for rab 7, a small GTP binding proteinimplicated in the membrane transport leading from early to lateendosomes (Feng et al., 1995). The fate of dissociatedreceptors was more difficult to appraise because these couldnot be readily differentiated from reserve receptors which, even

F. Vandenbulcke and others

Fig. 5.Dual localization of internalized fluo-NT and of endosome and TGN markers in COS-7cells incubated with 20 nM fluo-NT at 37°C. After 45-60 minutes of incubation, fluo-NT (A) ismainly concentrated in a rab 7-immunoreactive compartment (B; arrows) next to the nucleus(N). At the same time interval, fluo-NT (C,E) colocalizes extensively (arrows) with the TGNmarker syntaxin 6 (D) as well as with the pericentriolar recycling compartment and TGN markerrab 11 (F). Bar, 10µm.

2971NT and NT1 intracellular trafficking

though depleted by cycloheximide, were similarlyconcentrated within a rab 7-immunoreactive compartment.This rab 7 compartment colocalized syntaxin 6 (not shown),suggesting that it corresponded to the TGN. This finding wassurprising in view of earlier studies in BHK and MDCK cellswhich had shown rab 7 immunoreactivity to be confined to lateendosomes (Feng et al., 1995). Nonetheless, the colocalizationbetween NT1 and rab 7 was more obvious after 30 minutes ofincubation with exogenous NT than in cells that were notexposed to the ligand, suggesting that internalized NT1receptors did transit through late endosomes.

After prolonged incubation with fluo-NT (45-60 minutes),the ligand was heavily concentrated within a small,juxtanuclear compartment. This compartment was rab 7-positive, but stained negatively for AO, indicating that it wasno longer acidic and was therefore unlikely to correspond tolate endosomes. This might explain why earlier experimentsusing a Fluorescein-tagged NT derivative had shown noquenching of internalized ligand (Faure et al., 1994), despite

the fact that the fluorescence emission spectrum of Fluoresceinis responsive to pH (see Anderson and Orci, 1988). It mightalso explain why the ligand reached this compartment intact,as demonstrated by the overlap between fluo-NT and NTimmunoreactivity. This protection from metabolic degradationoccurred whether the experiments were carried out in thepresence of the protease inhibitor 1-10 phenanthroline or not,suggesting that it was physiological.

The size, shape and localization of this late fluo-NT targetingcompartment suggested to us that it may correspond to the TGNor to the pericentriolar recycling endosome, which in a varietyof epithelial cell lines has a distribution and morphology similarto those of the TGN (Gruenberg and Maxfield, 1995).Accordingly, dual-labeling experiments showed internalizedfluo-NT to substantially overlap with both the TGN markersyntaxin 6 (Bock et al., 1997) and the recycling endosomemarker rab 11 (Ullrich et al., 1996). However, because rab 11also labels post-Golgi membranes, including the TGN (Urbé etal., 1993), it remained unclear whether the internalized ligand

Fig. 6. Distribution of syntaxin 6 immunoreactivity in COS-7 cells treated (B) or not (A) with the anti-tubular agent nocodazole or dual-labeledwith syntaxin 6 (C) and HA (D) antibodies 48 hours after transfection with cDNA encoding TGN38-HA. (A,B) Note the dispersion of syntaxin6 immunoreactivity in cells treated with nocodazole (B) as compared to vehicle-treated controls where it forms a tight juxtanuclear cluster (A,arrow). (C,D) Syntaxin 6 immunostaining (C, arrows) colocalizes almost perfectly with TGN38-HA immunoreactivity (D). Bar, 10 µm.

2972

was associated with one or both of these structures. Twoseparate data sets demonstrated that the compartment labeledby syntaxin 6 indeed corresponded to the TGN. First, syntaxin6 immunolabeling was completely dispersed followingnocodazole-induced TGN fragmentation (Ladinsky andHowell, 1992). Second, syntaxin 6 immunostaining closelymatched that of the epitope-tagged TGN resident proteinTGN38-HA (Dell’Angelica et al., 2000). Our results thereforeprovide strong evidence for a targeting of internalized NT to theTGN. Several endogenous proteins have been shown to travelfrom endosomes to the TGN, including the mannose 6-phosphate receptor, TGN38 and furin (Johannes and Goud,1998). Some of these, such as TGN38, appear to bypass lateendosomes and to be delivered from the pericentriolar recyclingendosome to the TGN, whereas others, such as furin, reach theTGN via late endosomes (Mallet and Maxfield, 1999). Furtherexperiments will clearly be needed to determine which of thesetwo paths are taken by internalized NT.

Given that the TGN is an integral part of the regulatedsecretory pathway, it is possible that the ligand targeted to it isactually recycled back to the extracellular space. Indeed,internalized somatostatin was reported to be released back intothe extracellular space following its internalization intransfected epithelial cells (Koenig at al., 1998). It is unknown,however, whether in this case the peptide had first transitedthrough the TGN. Alternatively, internalized NT may berecruited to the TGN for degradation. Gonatas et al. (1977)demonstrated that in neurons, endocytosed horseradishperoxidase-tagged conjugates of lectin accumulate in the TGN

and from there are delivered to lysosomes for catabolism. Inthe present experiments, however, there was no evidence oftargeting of fluo-NT to lysosomes as the ligand nevercolocalized with lamp 1-immunoreactivity. However,endopeptidases E.C.3.4.24.15 and E.C.3.4.24.16, twometalloenzymes implicated in the functional inactivation of NT(reviewed in Checler et al., 1995; Barrett et al., 1995), are bothpresent in the TGN of neuroendocrine cells (Garrido et al.,1999), suggesting that internalized NT might be degradedwithin the TGN itself.

Studies on neurons in culture or rat brain slices have showninternalized fluo-NT to be transported centripetally fromdendrites, axons and perikaria toward a juxtanuclearintracellular compartment, which the present experimentssuggest might have been the TGN (Castel et al., 1991; Faureet al., 1995a,c; Nouel et al., 1997a). Furthermore, a significantproportion of NT internalized within these central neurons wasprotected from metabolic degradation, as seen here in COS-7cells (Castel et al., 1991; Faure et al., 1995a). Because of thisretrograde targeting, internalized ligand and/or receptor havebeen proposed to be involved, through a putative nucleartranslocation process, in long-term genomic effects of NT onits target cells (Laduron, 1992, 1994). The internalization-dependent activation of NT1 transcription observed in HT 29cells following long-term incubation with an NT agonist isconsistent with this hypothesis (Souazé et al., 1997). Althoughthere was no evidence here for nuclear targeting of NT,recruitment of the peptide to the TGN may play a role inintracellular signaling by allowing it to interact with the NT3

F. Vandenbulcke and others

Fig. 7. Intracellular localizationof internalized fluo-NT (A) andof VSV-tagged NT1 receptorimmunoreactivity (C) in relationto that of the lysosomalcompartment marker lamp 1(B,D). After 45 minutes ofincubation with 20 nM fluo-NT(A), there is no colocalizationbetween the fluorescent ligand(arrow) and lamp 1-immunoreactivity (B). Bycontrast, after 45-60 minutes ofincubation with the sameconcentration of nonfluorescentligand, NT1-VSV-immunoreactivity (C, arrow)colocalizes substantially withlamp 1-immunoreactivity (D) ina compartment situated in thecore of the cell, next to thenucleus (arrows). Bar, 10 µm.

2973NT and NT1 intracellular trafficking

receptor, which is concentrated in this structure (Petersen et al.,1997).

Whereas internalized ligand accumulates in the TGN,receptors are targeted to lysosomes in which they arepresumably degraded by acid hydrolases. This targeting ofNT1 receptors to lysosomes is in agreement with biochemicaldata suggesting that the NT1 is not recycled (Turner et al.,1990; Chabry et al., 1993; Hermans et al., 1997; Botto et al.,1998). It does contrast, however, with the fate of the majorityof G-protein-coupled receptors studied to date, including theNT2 sub-type, which are classically recycled back to theplasma membrane (Dautry-Varsat et al., 1983; Klausner et al.,1983; Green and Kelly, 1992; Von Zastrow and Kobilka, 1992;Hoxie et al., 1993; Ashworth et al., 1995; Grady et al., 1995a;Tarasova et al., 1997; Botto et al., 1998). Receptors such as theepidermal growth factor (Stoscheck et al., 1984) and theluteinizing hormone receptor (Ghinea et al., 1992) were foundto be targeted to lysosomes but, unlike the NT1 receptor, theseare sorted together with their respective ligand. It is unclearwhich targeting sequence is actually implicated in thelysosomal recruitment of NT1 receptors. Several copies ofthe lysosomal targeting sequences YXXO (in which ‘O’corresponds to a hydrophobic amino acid; Marks et al., 1996)and YXXI (Rohrer et al., 1996) are present in the NT1sequence, but these are unlikely to be responsible for its

lysosomal targeting since they are also present in the primarysequence of the NT2 sub-type, which recycles efficiently(Botto et al., 1998).

The targeting of internalized NT1 receptors to lysosomessuggests that these receptors are rapidly downregulated from thecell surface unless their degradation is compensated by therecruitment of spare and/or newly synthesized receptors frominternal stores. The rapid reappearance of NT1 immunolabelingobserved here at the cell surface after incubation with fluo-NTindicates that such a mechanism exists. The process likelyinvolves synthesis of new receptors, since biochemical studieshave shown that internalization-induced loss of cell surfacebinding was markedly increased in the presence of proteinsynthesis inhibitors (Hermans et al., 1997).

In summary, the present study demonstrates a fate ofinternalized NT/NT1 ligand/receptor complexes that is so farunique among reported trafficking patterns of G-protein-coupled receptors and their ligands in that (1) the ligand istargeted in largely intact form to the TGN and (2) the receptoris predominantly targeted to lysosomes for degradation ratherthan recycled (Fig. 9). This trafficking pattern is likely to haveimportant implications for the signaling properties ofinternalized NT and for the role of internalization in regulatingcell surface receptor densities.

Fig. 8.Dual localization of fluo-NT and of NT-immunoreactivity inNT1 transfected COS-7 cells incubated for 60 minutes with 20 nMfluo-NT. Note the extensive overlap between fluo-NT (A) andimmunoreactive NT (B), suggesting that the bulk of the Bodipyfluorophore is still attached to the immunoreactive NT moiety. Bar,10 µm.

Fig. 9.Model for the receptor-mediated endocytosis of neurotensin.Fluo-NT and NT1 are internalized together into acidic endosomesvia a clathrin-dependant mechanism. In acidic endosomes, the liganddissociates from its receptor. The receptor is then targeted tolysosomes for intracellular degradation and the ligand is targeted to anonacidic compartment near the nucleus, identified as the TGN.

2974

The technical assistance of Mariette Houle is gratefullyacknowledged. We thank Dr Juan Bonifacino (NIH, Bethesda, MD,USA) for providing us with the TGN38-HA plasmid and AnneMormville for help with the artwork. This work was supported byGrant MT-7366 from the Medical Research Council of Canada to A.B.and by a France-Québec exchange program. from the Fonds de laRecherche en Santé du Québec to A.B. and J.M. F.V. was supportedby a fellowship from the Fondation pour la Recherche Médicale(France).

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