ARTICLE |
Correspondence to: C. Llorens-Cortes, INSERM U36, College de France, 3 rue d'Ulm, 75005, Paris, France. E-mail: c.llorens-cortes@college-de-france.fr
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Summary |
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Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1EGFP receptor in a dose-dependent fashion (EC50 = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs. (J Histochem Cytochem 48:15531563, 2000)
Key Words: neurotensin, green fluorescent protein, internalization, cell-based assay, orphan receptor
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Introduction |
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Recent efforts in genome research have led to the identification of a growing collection of cDNA sequences encoding G-protein-coupled receptors (GPCRs). In the human alone, over 200 GPCRs have already been reported, and up to 1000 more are purported to exist (
Until now, research strategies for the identification of GPCR endogenous ligands have mainly resorted to G-protein-mediated signaling as an indicator of receptor activation. Thus, the endogenous ligand for the orphan opioid-like receptor ORL1 was isolated from rat (
Although clearly successful in these specific instances, signal transduction-based approaches have the disadvantage of being highly dependent on the successful prediction of the transduction pathways used by the orphan GPCR. In addition, the orphan receptor may have to be expressed in a variety of cell lines before one is found to exhibit viable coupling (
A major characteristic of GPCRs is their property of internalizing into their parent cells on ligand exposure. This phenomenon, which is also referred to as receptormediated endocytosis, is time- and temperature-dependent and involves clustering and sequestration of receptor-ligand complexes into clathrin-coated pits. Internalization is clearly dissociated from G-protein signaling (
Our proposed assay consists of confocal microscopic monitoring of the fate of green fluorescent protein-tagged GPCRs in transfected cells after exposure to HPLC fractions of pre-purified tissue extracts. This approach is based on findings from several recent studies that reported successful C-terminal tagging of GPCRs with autofluorescent proteins without substantially altering pharmacological and internalization properties of the tagged GPCRs (
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Materials and Methods |
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Plasmid Construction of the NT1EGFP Fusion Protein
To construct NT1-EGFP, the rat NT1 cDNA (a gift from Dr S. Nakanishi; Institute for Immunology, Kyoto University Faculty of Medicine, Japan) was first amplified from plasmid pcDNA I by polymerase chain reaction using as 5'-oligonucleotide primer 5'-CTT AAG CTT ATG CAC CTC AAC AGC TCC GTG-3' [containing the nucleotides CTT, the Hind III restriction site sequence and nucleotides 121 of the rat NT1 cDNA (
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Preparation of Frog Brain Extracts
Adult male frogs (Rana ridibunda) were obtained from a commercial source (Couétard; St-Hilaire de Riez, France). The brains of 2500 animals (215 g wet weight) were collected and kept frozen. The tissue was boiled for 15 min in 0.5 M acetic acid and homogenized in a Waring blendor. After centrifugation (4000 x g for 30 min at 4C), the supernatant was pumped at a flow rate of 2 ml/min through 10 Sep-Pak C18 cartridges (Waters Associates; Milford, MA) connected in series. Bound material was eluted with 70% (v/v) acetonitrile in water. The eluted material was then partially evaporated in a rotavapor (Büchi; Flavil, Switzerland) and centrifuged at 13,000 x g for 5 min at 4C. The supernatant was pumped at a flow rate of 2 ml/min onto a 1 x 25-cm Vydac 218TP1010 C18 reversed-phase HPLC column (Separations Group; Hesperia, CA) equilibrated with 0.12% trifluoroacetic acid. The concentration of acetonitrile in the eluting solvent was raised from 14 to 42% over 40 min at a flow rate of 2 ml/min and then raised again to 56% over 60 min at a flow rate of 1 ml/min. One-minute fractions were collected and the absorbance was measured at 215 and 280 nm.
Stable Transfection of CHO Cells
CHO-K1 (American Type Culture Collection; Rockville, MD) were maintained in Ham's F12 medium supplemented with 7.5% fetal calf serum, 1 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Boehringer Mannheim; Mannheim, Germany). To establish pure cell lines expressing the NT1EGFP receptor, 2.6 x 106 CHO-K1 cells were trasnfected with 8 µg of the plasmid, using a liposomal transfection reagent (DOSPER Liposomal Transfection Reagent, Cat. No. 1781995; Boehringer Mannheim). Transfected cells were grown in a humidified atmosphere of 5% CO2/95% air and selected during 1 week for their resistance to 750 µg/ml geneticin G418 (Gibco; Cergy-Pontoise, France).
Resistant cells were dissociated by trypsin (Gibco) and analyzed by fluorescence-activated cell sorting (FACS) on an Epics EST flow cytometer equipped with an Autoclone cell sorter (Coulter France; Marjency, France), made available through the Service de Cytometrie en Flux at the Jacques Monod Institute (Paris, France). Whenever possible, 10,000 gated events were collected for analysis. Dead cells and debris were excluded from analysis based on forward angle and side-scatter light gating. Excitation was achieved using an argonion laser emitting 488 nm light and emission was detected using a 520/30 bandpass filter. Untransfected cells were used as negative controls to exclude non-transfected CHO cells from the selection window (Fig 1B, left). Sorting was performed to distribute cells on a 96-well microplate, one cell per well. After 7 days in culture, final selection of clones was achieved by assessing the intensity of membrane fluorescence in each clone using an Olympus BX 60 fluorescent microscope equipped with a standard FITC filter set. A subsequent FACS analysis (Fig 1B, right) was performed to verify the purity and fluorescence intensity of the selected stable cell line (no. 18, NT1EGFP CHO), which was then used for all experiments.
Radioligand Binding Assay
Stable transfected CHO cells expressing the NT1EGFP receptor were seeded onto 48-well plates at a density of 5 x 104 cells per well. After 1 night in culture, medium was discarded and cells were washed once with 250 µl of PBS and incubated with increasing concentrations (10 to 2000 pM) of 125I-labeled Tyr3-neurotensin (2000 Ci/mmole; Amersham, Les Ulis, France) at 4C overnight in 100 µl of 50 mM Tris-HCl buffer, pH 7.5, containing 2 mM MgCl2, 20 mM HEPES, 0.1% BSA, and 0.8 mM 110 phenanthroline. Nonspecific binding was determined by adding 1 µM non-radioactive neurotensin (NT). At the end of the incubation, cells were washed twice with 500 µl of the same buffer, lysed in 250 µl of 1 N NaOH, and bound radioactivity was measured with a gamma counter. Scatchard analysis of the binding data was performed using the GraphPad Prism program (GraphPad Software; San Diego, CA).
Inositol Phosphate Production
The production of total IPs by stably transfected CHO cells expressing the NT1EGFP receptor was determined as previously described (
Internalization Assay
Trypsinized NT1EGFP-transfected CHO cells were diluted to obtain 105 cells/ml in Ham's F12 medium, seeded (300 µl/well) on polyallylamine-hydrochloride-coated (Aldrich, Madison, WI; 0.1 mg/ml for 30 min) 16-well glass slides (Lab-Tek Chamber Slides, Nalge Nunc; Rochester, NY), and grown overnight in a humidified atmosphere of 5% CO2/95% air. The volume of incubations (except for the pulse period) and washings was 250 µl per well. Ninety minutes before the beginning of the experiment, the medium was supplemented with cycloheximide to a final concentration of 70 µM (SigmaAldrich; St Quentin, France). Cells were then preincubated in ice-cold Earles' buffer, pH 7.4 (140 mM NaCl, 5mM KCl, 1.8 mM CaCl2, 3.6 mM MgCl2), complemented with 0.1% bovine serum albumin, 0.01% glucose, and 0.8 mM 1-10 phenanthroline for 10 min. Cells were then incubated for 30 min at 4C (pulse period) with either (a) known concentrations of NT, (b) 20 nM fluorescent NT (N-Bodipy-NT(213), Fluo-NT (a generous gift of J.-P. Vincent; Nice, France), or (c) individual HPLC fractions of frog brain extracts diluted in complemented Earle's buffer to a final volume of 50 µl. At this point, some cells were subjected to a hypertonic acid buffer wash (0.2 M acetic acid solution containing 0.5 M NaCl in Earle's buffer, pH 4, for 2 min) to dissociate surface-bound ligand. Internalization was promoted by replacing the incubation medium with complemented Earle's buffer at 37C and placing the cells at 37C for 2030 min (chase period). At the end of the chase period, cells were rinsed in cold Earle's buffer, fixed for 10 min with 4% paraformaldehyde dissolved in 0.1 M PBS, pH 7.4, rinsed again in cold Earle's buffer, and coverslipped using Vectashield (Vector Laboratories; Burlingame, CA) for confocal microscopic examination. In some experiments, cell nuclei were labeled by adding 1.5
propidium iodide (SigmaAldrich) to the mounting medium.
Confocal Microscopy
Cells were examined with a Leica TCS SP (Leica Microsystems; Heidelberg, Germany) confocal laser scanning microscope mounted on a Leica DM IRBE inverted microscope equipped with an argonkrypton laser. EGFP fluorescence was detected with 100% excitation at 488 nm, using an RSP 500 (dichroic) mirror and the spectrophotometer set to acquire emission between 530 and 560 nm. Fluo-NT was detected with 100% excitation at 568 nm, using a DD 488/568 dichroic mirror and the spectrophotometer set to acquire emission between 590 and 660 nm. PI was detected with excitation at 568 nm, using a DD 488/568 dichroic mirror and the spectrophotometer set to acquire emission between 580 and 700 nm. Optical sections (1024 x 1024) of individual cells were taken at the equatorial level (level of the nucleus; Fig 3), using a x63 1.32 NA oil-immersion objective. Acquisition settings were kept constant for all cells.
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Quantification of EGFP Fluorescence Internalization
Digital image analysis was performed on a Macintosh computer using the NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/) Microsoft Excel 5.0 (Microsoft; Redmond, Washington) and in-house-developed macro programs (submitted to the NIH Image Internet server and available at ftp://codon.nih.gov/pub/nih-image/user-macros/internalization_macros.txt). For each cell, after gray-scale conversion and median filtering, gray-scale density of pixels was measured along 12 radial axes (Fig 4A) and plot data were transferred into Excel. To better assess the amount of cell surface vs intracellular fluorescence, the gray-scale value of each pixel was multiplied by its radial distance from the measurement center, because the probability for a pixel of being sampled by this radial axis method is inversely proportional to its distance from the measurement center, and this probability is a simple function of the radius. Subsequently, Excel calculated the mean pixel density of membrane (M, corresponding to the mean gray-scale density of the most distal 30 pixels = 1 µm) and intracellular (I, mean gray-scale density for the remaining intracellular pixels) EGFP fluorescence for each cell (Fig 4B). M/I and I/M ratios were then derived for each cell from the mean of the 12 M and 12 I values.
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Radioimmunoassay for Neurotensin on Pre-purified Frog Brain Fractions
Neurotensin-like immunoreactivity was measured using an antiserum (kindly supplied by Dr. G. Tramu; University of Bordeaux 1, Talence, France) raised against the C-terminal fragment of pig NT and 3-[125I]-iodotyrosyl-NT as a radioligand (Amersham; Poole, UK). The IC50 of the assay was 187 pg/tube and the minimal detectable amount of peptide was 30 pg/tube.
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Results |
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Pharmacological Characterization of the NT1EGFP Receptor
Stable transfection of CHO cells with an expression vector containing the rat NT1EGFP cDNA conferred to these cells the ability to bind 125I-labeled Tyr3-NT in a saturable and displaceable manner (Fig 1C). Scatchard analysis of the binding data (inset in Fig 1C) indicated that the radioligand interacted with a single class of binding sites with an apparent affinity (Kd) of 299 ± 50 pM and a high binding capacity (Bmax) of 342,700 ± 3500 sites/cell.
Effect of Neurotensin on Inositol Phosphate Production in NT1-EGFP Cells
Incubation of NT1EGFP-transfected CHO cells with increasing concentrations of rat NT (10-1110-6 M) resulted in a dose-dependent increase in IP formation in CHO NT1EGFP cells (Fig 1D). The concentration of rat NT eliciting a half-maximal increase in total IP production (EC50) was 1.3 ± 0.5 nM. The maximal stimulation (626% over basal level) was observed at 30 nM. Frog NT also induced an increase in IP production with an EC50 similar to that of rat NT, whereas no stimulation was observed in non-transfected CHO cells (data not shown).
Internalization of the NT1-EGFP Receptor in the Presence of Rat and Frog Neurotensin
In the confocal microscope, cells incubated either with buffer alone or with buffer containing low concentrations (<0.1 nM) of rat or frog NT showed an intense concentration of NT1EGFP fluorescence at the level of the plasma membrane (Fig 2A and Fig 3a3h). Incubation of NT1EGFP-transfected cells with 1 nM1 mM rat or frog NT resulted in a marked decrease of surface-associated fluorescent label and the appearance of many fluorescent intracytoplasmic vesicles, 0.6 µm in mean diameter (Fig 2B and Fig 3i3p). This change in labeling pattern was no longer apparent when the cells were subjected to acid wash at the end of the pulse period (Fig 2C), demonstrating that it was induced by the binding of NT to NT1EGFP receptors during the pulse period. Dual labeling experiments using Fluo-NT as a ligand showed that the newly formed intracytoplasmic NT1EGFP-containing vesicles (Fig 2D) also contained Fluo-NT (Fig 2E; merged image in Fig 2F), indicating that the observed vesiculization of NT1EGFP was due to ligand-induced internalization of receptorligand complexes. Quantification of membrane/intracellular fluorescent ratios (M/I) indicated that this internalization was dose-dependent, with an EC50 = 0.98 nM (Fig 4C).
Internalization of NT1EGFP Receptors Induced in the Presence of HPLC Fractions from Frog Brain Extracts
In a first set of experiments, 0.5-µl aliquots from each of 10 consecutive fractions were pooled (total number of fractions 120, yielding 12 pools of 10 fractions) and each pool was completed to 50 µl with Earle's buffer. Of these 12 pools, only the second one, containing fractions 1120, promoted internalization of NT1EGFP (data not shown). In a second set of experiments, fractions of Pool 2 and Pool 3 (used as negative controls) were tested individually (0.5 µl of each fraction completed to 50 µl with Earle's buffer). Of these, only fractions 15, 16, 17 and 18 promoted internalization of NT1EGFP (Fig 5A). This internalization was not detected when the cells were subjected to an acid wash at the end of the pulse period (data not shown), indicating that the internalization was ligand-induced.
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Stimulation of IP Production by HPLC Fractions From Frog Brain Extracts on NT1EGFP Cells
In a first set of experiments, 5-µl aliquots from each fraction (total number of fractions 120) were pooled in pairs and completed to 50 µl with distilled water. Five microliters from each of these 60 pools was diluted in a final volume of 200 µl and checked for the capacity to stimulate IP production. Only Pools 8 and 9, containing fractions 15, 16 and 17, 18, respectively, induced an increase in IP production comparable to that observed with a maximal dose of rat or frog NT (10-7 M) (data not shown). In a second set of experiments, 5 µl each of HPLC fractions 1419 and 2428 (used as negative controls) was checked individually. Only fractions 15, 16, 17, and 18 stimulated IP production (Fig 5B).
RIA Measurement of Neurotensin in HPLC Fractions From Frog Brain Extracts
Characterization of NT-like immunoreactivity contained in Sep-Pak-pre-purified frog brain extracts was carried out by combining HPLC analysis and RIA detection (Fig 5C). The immunoreactive material eluted from fractions 1518, with a major peak detected in fraction 16. The amounts of NT-like immunoreactivity measured in the whole fractions 15, 16, 17, and 18 were 141 ng, 350 ng, 285 ng, and 118 ng, respectively.
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Discussion |
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We describe here a novel single cell-based assay that should be applicable to the identification of endogenous ligands of orphan receptors. In contrast to currently used signal transduction-based screening methods, which monitor changes in the generation of second messengers (
For this purpose, we established a stable CHO cell line expressing the NT1 receptor fused with EGFP. The pharmacological properties of this NT1EGFP receptor were similar to those of the wild-type NT1 receptor. Accordingly, incubation of NT1EGFP-expressing CHO cells or transiently transfected COS-7 cells (unpublished results) with exogenous NT resulted in a dose-dependent internalization of the tagged NT1 receptor. By confocal microscopy, this internalization was characterized by a progressive depletion of cell surface receptors and by the formation of small intracellular fluorescent clusters. This internalization pattern was similar to that previously observed in these as well as in other cell types that express the NT1 receptor (
Four of the 120 frog brain fractions tested on NT1EGFP-expressing CHO cells induced receptor internalization as monitored by confocal microscopy. The same four fractions, and only these four, were found to stimulate the production of IP in our heterologous transfection system. This finding confirmed that the internalization-promoting fractions were the only ones to contain an NT1-activating factor. Because NT was the endogenous ligand most likely to be present in these fractions, we tested individual fractions using a radioimmunoassay for frog NT and did indeed find the peptide to be present within each of the active ones. Although we cannot exclude the possibility that other peptides documented to act at the NT1 site, such as neuromedin N or xenopsin (
The method devised here to quantitate the amount of ligand-induced internalized EGFPNT1 receptor proved highly sensitive because it enabled us to detect nanomolar concentrations of ligand in the incubation medium. Given that the Kd for NT binding to the NT1 receptor is in the same concentration range, it appears that the affinity of the ligand for its receptor is the major determinant for the sensitivity of the proposed technique. Thus, in the present assay we detected femtomolar quantities of neurotensin from 0.5 µl of the initial HPLC fractions. Furthermore, because the observation of a single cell is theoretically sufficient for testing a single HPLC fraction from brain extracts, further miniaturization of the procedure should be possible to help economize precious starting material. To our knowledge, this method represents the first quantitative approach for assessing GPCR activation using such a minimal assay system as a single transfected cell.
The present method offers several advantages over the activation-based methods previously devised for the same purpose. First, it is completely independent of signaling mechanisms downstream of the receptor and therefore does not rely on a search for the appropriate transduction pathway, specific coupling of individual GPCRs being notoriously difficult to predict from their primary sequence. In addition, in the absence of knowledge of the effector molecules downstream of the orphan GPCR, transduction-based methods often require expression of the receptor in different cell lines before viable coupling can be established.
Second, internalization of G-protein-coupled receptors is triggered only by their specific ligands. This latter property is particularly important for the screening of biological samples such as brain extracts, which may contain several hundreds or thousands of different peptides. Therefore, simultaneously activated (and perhaps also internalized) but non-tagged endogenous receptors will remain invisible and will not interfere with the detection of tagged receptor internalization. By contrast, generation of second messengers in transduction-based assay systems, acidification of the extracellular milieu in the cytosensor microphysiometer system (
Third, ligand-induced internalization appears to be an almost universal property of GPCRs. Hence, the advantage of the present approach over systems based on downstream events is demonstrated such as the translocation of fluorescently tagged ß-arrestin-2 after GPCR activation (
Autofluorescent proteins are not the only molecular tags suited to visualize receptor internalization. For example, it is possible to engineer a receptor presenting an epitope that is detectable by subsequent immunohistochemistry. However, the direct fluorescent label of the orphan GPCR confers some additional advantages to the method. First, the success of cell transfection and the correct plasma membrane expression of the labeled orphan GPCR can easily be verified, because mutant versions of the green fluorescent protein (GFP) are easily detectable using standard fluorescence microscopic equipment. Second, establishment of stable cell lines is largely facilitated by the expression of a marker that is easily detectable in living cells (
In conclusion, the present internalization-based assay system offers a simple, selective, and highly sensitive quantifiable method for the detection of ligands acting on GPCRs. Therefore, this technique should allow reliable detection of an endogenous ligand for an orphan GPCR in a biological sample and consequently, by using successive purification and evaluation steps, should enable pure peptide to be obtained, which could be subsequently identified by sequencing. Other possible applications include screening of potential agonists and antagonists of GPCRs or detection of femtomolar quantities of known ligands dissolved in microvolumes of biological or non-biological solutions. This approach should not be limited to GPCRs, because a large group (Class II) of receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor, undergo similar ligand-induced internalization (
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Acknowledgments |
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Supported by grants from the Institut National de La Santé et de la Recherche Médicale (APEX no. 4X011E to C.LC) and from the Conseil Regional de Haute Normandie to C.N., B.B., H.V., from the Medical research Council of Canada to A.B., and from the Ministère de l'Education du Quebec to Z.L.
We thank Mariette Houle for expert technical assistance and Charles Parnot for the help on Excel macros.
Received for publication December 29, 1999; accepted June 7, 2000.
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