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Endocrinology Vol. 145, No. 6 2876-2885
Copyright © 2004 by The Endocrine Society

Ligand-Regulated Internalization of the Opioid Receptor-Like 1: A Confocal Study

Maithé Corbani, Christophe Gonindard and Jean-Claude Meunier

Institut de Pharmacologie et de Biologie Structurale (M.C., J.-C.M.), Centre National de la Recherche Scientifique, 31017 Toulouse, and the Institut Européen de Biologie Cellulaire (C.G.), 31520 Ramonville-Sainte-Agne, France

Address all correspondence and requests for reprints to: Maithé Corbani, Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique-Unité Mixte de Recherche 5089, 205 route de Narbonne 31017 Toulouse cedex 4, France. E-mail: maithe.corbani{at}ipbs.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To better understand opioid receptor-like 1 (ORL1) internalization, we fused the C terminus of ORL1, the nociceptin (noc) receptor, to the N terminus of a green fluorescent protein and used the fusion protein to characterize receptor endocytosis in live human embryonic kidney cells. The fusion altered neither the affinity of the receptor for noc or other ORL1 receptor ligands nor the ability of the receptor to mediate agonist-induced binding of GTP{gamma}35S, i.e. coupling with heterotrimeric G protein. Confocal microscopy showed that the fluorescent receptor was mostly associated (>75%) with the periplasmic membrane. In the presence of 0.1 µM noc, approximately 80% of receptors were internalized, and half-maximum internalization was reached in approximately 12 min at 22 C and approximately 6 min at 37 C. After washing, a normal receptor level was recovered within 70 min at 22 C. The lack of internalization in the presence of 0.45 M sucrose suggests that noc-induced receptor endocytosis mainly occurred via clathrin-coated pits. Coincubation of the recombinant cells with noc and tetramethylrhodamine-transferrin showed that ORL1 was mainly internalized through the endosome compartment. Lofentanil and Ro64-6198 ([(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one]) promoted endocytosis of the fluorescent receptor as efficiently as noc. Among the two ORL1 receptor antagonists, J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one), but not III-BTD, blocked the noc-induced internalization of the fluorescent receptor. Two partial agonists were dramatically less efficient than noc to promote ORL1 internalization. They recruited very little (the pseudopeptide [Phe1{psi}(CH2-NH)Gly2]-noc-(1–13)NH2) or no (the hexapeptide Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH2) G protein receptor kinase type 2 coupled to red fluorescent protein 1 at the membrane, suggesting that subsequent receptor phosphorylation necessary for internalization via coated pits is altered. Thus, partial agonists that induce a prolonged cell response without causing substantial receptor internalization may be good tools for further clinical treatments.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OPIOID RECEPTOR-LIKE 1 (ORL1) receptor belongs to the G protein-coupled receptor (GPCR) family and was identified from a human cDNA library on the basis of homology with the µ, {delta}, and {kappa} opioid receptors (1). However, it exhibits low affinity for opioid agonists (reviewed in Ref. 2). At the cellular level, the activation of ORL1 by the natural ligand nociceptin (3), also called orphaninFQ (4), causes inhibition of cAMP and of voltage-gated calcium channels (5). Nociceptin also stimulates the inwardly rectifying potassium conductance (6). Both opioid receptors and ORL1 are involved in modulating the MAPK activity (7).

In rodents, nociceptin modulates nociception and, unlike opioids, appears to be free of abuse potential but also displays multiple functions. Nociceptin stimulates prolactin and GH release in male and female rats (8) and was recently shown to decrease plasma corticosterone elicited in mice after intracerebroventricular injection (9). In the same respect, the first nonpeptide agonist Ro64-6198 ([(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one]), synthesized for the purpose of reducing anxiety in humans, shows anxiolytic properties in rats (10). The µ-opioid receptors and ORL1 distribution overlaps with the distribution of estrogen and progesterone receptors in the hypothalamus and the limbic systems, and nociceptin regulates sexual receptivity in female rats (11). The nociceptin/ORL1 system is also involved in food intake, and nociceptin displays orexigenic behaviors in rats (12). Nociceptin also participates in the water reabsorption process and activates the inwardly rectifying conductance in vasopressin-containing neurons (13).

The identification of the natural ORL1 ligand (3, 4) was followed by the synthesis, on a structural basis, of several peptide (14) or nonpeptide (15) agonists, the discovery of short peptide agonists after library screening (16), and antagonists (17, 18, 19) providing new tools for in vivo and in vitro studies (20, 21).

Repeated or sustained activation of the ORL1 receptor by nociceptin results in a rapid decrease in cell response (desensitization) in a variety of cellular contexts such as in locus coeruleus and hippocampal neurons, in SK-N-BE and SK-N-SH neuroblastoma cells, in NG 108-15 neuroblastoma x glioma cells, and in the ORL1 receptor-transfected nonneuronal Chinese hamster ovary and human embryonic kidney (HEK) cell lines (7). Likewise, rodents have been shown to develop tolerance to the antinociceptive (22) and locomotor-inhibiting (23) effects of nociceptin. Although the mechanisms involved in the development of these nociceptin-induced adaptive processes are largely unknown, it is possible that receptor endocytosis is involved.

The rate and amplitude of receptor endocytosis vary considerably in function of receptor type and of agonist used. For example, the three types of opioid receptor, µ, {delta}, and {kappa}, all undergo endocytosis. However, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO, an enkephalin derivative) and etorphine, but not morphine, trigger both the phosphorylation and efficient internalization of µ-opioid receptors in HEK293 cells (24, 25). In fact, DAMGO-driven endocytosis of the µ-opioid receptor reduces the development of tolerance to the analgesic effects of morphine (26). On the contrary, morphine fails to induce the µ-opioid receptor phosphorylation necessary for ß-arrestin recruitment and association with partners such as clathrin and adaptor protein type 2 (26, 27).

In the last few years, several receptors have been successfully labeled by fusion with a green fluorescence protein (GFP) molecule (review in Refs. 28 ,29). To determine in what condition the ORL1 receptor, the fourth member of the opioid receptor family, undergoes endocytosis, we constructed and stably expressed, in HEK293 cells, ORL1 carrying at the C terminus one molecule of enhanced GFP (EGFP). After pharmacological characterization, we conducted a detailed pharmacology of ORL1 internalization using confocal microscopy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of the eukaryotic expression vector encoding the ORL1-EGFP fusion protein
The human ORL1 cDNA was cloned into the pEGFP-N3 vector (Clontech, Palo Alto, CA) encoding the P64L/S65T mutant (EGFP) of the natural jellyfish (Aequoria victoria) GFP. The HindIII/KpnI-digested human ORL1 (hORL1) cDNA, the double-stranded oligodeoxynucleotide 5'-CGGCGGCCCGCA-3' (Genome Express, Grenoble, France) encoding the C-terminal tetrapeptide (Pro-Arg-Pro-Ala) of the receptor and containing the 5'end of the BamH1 site, and the HindIII/BamH1-digested pEGFP-N3 vector were ligated together, and the ligation product was introduced into Escherichia coli (DH5{alpha}) and used to prepare DNA. The fusion product was submitted to inverse PCR using oligo 1 (5'-ATGGTGAGCAAGGGCGAGGAG-3') and oligo 2 (5'-TGCGGGCCGCGGTACCGTC-3') as forward and reverse primers, respectively. Oligo 1 corresponded to the N-terminal part of the EGFP without the Kozak sequence, to prevent any reinitiation of transcription and the undesirable production of soluble EGFP. Oligo 2 corresponded to the complementary strand of the ORL1 C-terminal sequence and included a (silent) mutation of the EagI restriction site. After digestion of the parental plasmid with DpnI (New England Biolabs, Beverly, MA), ligation of the PCR product, and transformation, positive colonies were identified by lack of BamH1 and EagI sites. The recombinant DNA thus encoded the full-size hORL1 fused at the C terminus with the N terminus of EGFP. The final construction was checked by complete DNA sequencing (Millegen, Toulouse, France).

Stable expression of hORL1-EGFP in HEK-293 cells
HEK cells (HEK293) were stably transfected with the recombinant vector using polybrene and grown in DMEM/Nut Mix F12 medium (Life Technologies, Gaithersburg, MD) containing 7% (vol/vol) fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, 200 mM glutamine, and 0.4 mg/ml G418 (Life Technologies). Positive clones were selected on the basis of intense plasma membrane fluorescence, as assessed by confocal microscopy (see below). One clone was subcloned by limit dilution and amplified.

Transitory expression of G protein receptor kinase type 2 coupled to red fluorescent protein 1 (GRK2-DsRed)
A stable clone expressing 3.6 pmol/mg protein of ORL1-EGFP was transiently transfected by incubating 30 µg/ml bovine GRK2-DsRed plasmid and 20 µl/ml lipofectamine 2000 (Invitrogen, Groningen, The Netherlands) in 1 ml of serum-free medium for 3 h. The cells were resuspended, evenly distributed in Labtek II chambers, and cultured for 48 h. Each Labtek chamber was treated with the relevant ligand, submitted to laser scanning confocal microscopy, or fixed with 3.8% paraformaldehyde in PBS before further observation.

Membrane preparation
Recombinant cells were harvested in ice-cold PBS, collected by centrifugation, and frozen at –70 C. The cell pellet was homogenized in 50 mM Tris-HCl, 1 mM EDTA (pH 7.4) in a Potter-Elvehjem tissue grinder. The nuclear pellet was discarded after centrifugation at 1000 x g, and the supernatant was recentrifuged at 105,000 x g for 35 min at 4 C. The membrane pellet was diluted in 10 mM Tris, 5 mM EDTA (pH 7.5) to a final protein concentration of 5 mg/ml as estimated by the method of Lowry, divided into aliquots and stored at –70 C.

Ligand binding studies
Ligand binding studies were all performed at 25 C for 1 h in 0.5 ml Tris-HCl buffer (50 mM, pH 7.4), supplemented with 0.1 mg/ml of proteinase-free BSA (fraction V, Sigma Chemical Co., St Louis, MO), in propylene tubes to minimize tube wall adsorption of the radioligand. For saturation binding experiments, cell membranes (5–30 µg protein) were incubated in triplicate with increasing concentrations (0.05–3 nM) of [3H]nociceptin (23 Ci/mmol; Amersham, Little Chalfont, UK) in the absence (total binding) and in the presence (nonspecific binding) of 1 µM unlabeled nociceptin. For competition experiments, cell membranes (5–30 µg protein) were incubated in triplicate with 1 nM [3H]nociceptin and the unlabeled ligand at the desired concentration in the absence (total binding) and in the presence (nonspecific binding) of 1 µM unlabeled nociceptin. The bound radioligand was collected by filtration on polyethyleneimine-treated glass fiber filters (GF/B; Whatman, Clifton, NJ), and radioactivity was counted in a Packard model 2100TR liquid scintillation analyzer (Packard, Meriden, CT). The data were fitted using the Prism program (GraphPad Software, San Diego, CA). IC50 values, the concentrations of inhibitor that halve specific radioligand binding, were converted to Ki values by applying the Cheng and Prussof equation: Ki = IC50/([1+([L]/Kd)], where [L] and Kd are the concentration and dissociation constants of radioligand, respectively.

GTP{gamma}35S binding assay
Five to 10 µg of cell membrane preparation were incubated in 0.2 ml (final volume) of 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.1 mg/ml BSA, 40 µM GDP (Sigma), 0.1 nM stabilized GTP{gamma}35S (1117 Ci/mmol, Amersham), and the ligand at the desired concentration. Nonspecific GTP{gamma}35S binding was measured in the presence of 10 µM cold GTP{gamma}S (Sigma). After a 1-h incubation at 30 C, the samples were filtered on glass fiber disks (GF/B; Whatman) that had been presoaked in 50 mM Tris-HCl (pH 7.4), supplemented with 50 mM NaCl, 5 mM MgCl2, and 1 mM EDTA. After four washes with 5 ml of ice-cold soaking buffer, the radioactivity of the filters was counted using a Packard model 2100TR liquid scintillation analyzer (Packard). The percentage of stimulation was calculated as [{Delta}stim/{Delta}basal] x 100, where {Delta}stim and {Delta}basal represent total minus nonspecifically bound GTP{gamma}35S in the presence and absence of ligand, respectively. Experimental data were fitted to a sigmoid dose-response curve with variable slope parameter using the Prism software (GraphPad).

Cell labeling with tetramethylrhodamine (TMR)-transferrin
HEK cells stably expressing ORL1-EGFP were incubated for 5, 15, or 30 min at 22 C with 40 µg/ml TMR-transferrin (Molecular Probes, Leiden, The Netherlands) in the absence or presence of 0.1 µM nociceptin. After two washes, the cells were fixed in paraformaldehyde (3.8% vol/vol) in PBS and stored at 4 C for further observation.

Internalization of the fluorescent receptor
The recombinant HEK293 cells were harvested using the nonproteolytic agent Splittix (Biomedia, Boussens, France). Between 30,000 and 50,000 cells were seeded in Labtek I flasks in a 2-ml volume. The next day, the cells were incubated in 1 ml of DMEM/Nut Mix F12 culture medium containing 0.1 mg BSA, 25 mM HEPES (pH 7.4) and the ligand at the desired concentration. Sequential laser scanning under the confocal microscope followed internalization in living cells in real time at the desired temperature (4, 22, or 37 C). When necessary, the cells were fixed with 3.8% paraformaldehyde in PBS and stored at 4 C.

Laser scanning confocal microscopy
Confocal images were recorded by means of an inverted LSM 510 laser scanning confocal microscope (Carl Zeiss Inc., Oberkochen, Germany). The 488-nm argon-ion laser was used to detect EGFP, with a 505- to 530-nm band-pass filter. TMR was excited with the helium/neon laser at 543 nm, and emission was detected through a 560-nm long-pass filter in multitrack mode, providing a very distinct noncontaminated image. The inverted Axiovert 100 M microscope was equipped for phase transmission, Nomarski, and fluorescence observation, and a x40 oil-immersion objective (1.3 numerical aperture, Carl Zeiss) was used. All acquisitions were performed under a once-forever set mode to prevent any variation due to setting conditions and to allow the use of the full nonsaturated dynamic scale of fluorescence. The images were stored on CD-ROM and analyzed with Zeiss LSM software.

Statistical analysis
Data were analyzed using the Student’s t test or one-way ANOVA followed by the Dunnet’s test (Prism GraphPad Software). P values lower than 0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pharmacological characterization of the ORL1-EGFP fluorescent receptor expressed in HEK cells
We first tested the ability of the ORL1-EGFP receptor to bind to selected ORL1 receptor ligands. The selected ligands included pure agonists, i.e. nociceptin (3, 4), Ro64-6198 (15), and lofentanil (30); partial agonists, i.e. the hexapeptide Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH2 (16) and the pseudopeptide nociceptin analog [Phe1{psi}(CH2-NH)Gly2]nociceptin-(1–13)-NH2 (14, 31); and the antagonists III-BTD (17) and J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) (18). The affinity of the fluorescent receptor for [3H]nociceptin (Kd ~0.24 nM) was similar to that of the wild-type (wt) receptor (Kd ~0.2 nM), with a Bmax value approximately 3.6 pmol/mg membrane protein (Table 1Go). Likewise, the fluorescent receptor-bound lofentanil with a Ki approximately 24.2 nM was similar to the wt receptor (24 nM) (32). Similarly, Ro64-6198 (~0.48 nM), the hexapeptide (~1.28 nM), the pseudopeptide (~0.76 nM), III-BTD (~12.4 nM), and J-113397 (~2.12 nM) were bound equally well by ORL1-EGFP and wt ORL1.


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  		TABLE 1. Pharmacological characteristics of the ORL1-EGFP expressed in HEK293 cells

 
We then evaluated the functionality of the fluorescent receptor, i.e. its ability to mediate agonist-induced binding of GTP{gamma}35S (Table 1Go and Fig. 1Go). Nociceptin stimulated the fluorescent receptor to promote binding of GTP{gamma}35S with an EC50 of approximately 4.7 nM, identical with the wt receptor (Ki = 5 nM) (33). Likewise, lofentanil and Ro64-6198 were equally as efficient in inducing the binding of GTP{gamma}35S via the fluorescent as via the wt receptor (34). They displayed similar potencies compared with nociceptin (~100%). However, the hexapeptide and the pseudopeptide elicited, respectively, at most 76 and 65% of the maximal response elicited by nociceptin, lofentanil, and Ro64-6198 (Fig. 1Go), consistent with their previously reported partial agonist nature (16, 31, 35). The two ORL1 receptor antagonists, III-BTD and J-113397, blocked nociceptin-induced, fluorescent receptor-mediated binding of GTP{gamma}35S, the EC50 values being approximately 64 and 6.3 nM, respectively, close to the values reported for the wt receptor (17, 18).



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  		FIG. 1. Stimulation of GTP{gamma}35S binding by nociceptin, Ro64-6198, lofentanil, hexapeptide, or pseudopeptide (1 2 3 4 5 6 7 8 9 10 11 12 13 ) on membrane fractions from HEK cells expressing the ORL1-EGFP receptor. Specific GTP{gamma}35S binding was measured after 1 h at 30 C in the presence of the unlabeled ligand at the indicated concentration. Nociceptin (Noc), lofentanil, and Ro64-6198 stimulated the binding of the GTP analog to the same maximum extent (2.6 times the basal binding). Lofentanil was less efficient. The maximum stimulation by the hexapeptide or pseudopeptide (1 2 3 4 5 6 7 8 9 10 11 12 13 ) was significantly lower (1.7-fold). Each data point is the mean (± SEM) of three values from independent determinations.

 
Thus, the fusion of EGFP to the C terminus of ORL1 did not seem to alter the binding properties and the G protein coupling of the receptor that we measured.

Internalization of the ORL1-EGFP fluorescent receptor
Confocal imaging of the recombinant HEK cells expressing the ORL1-EGFP fusion protein revealed that the green fluorescence was largely confined to the plasma membrane (Fig. 2Go, left). This was confirmed by colocalization with TMR-concanavalin A, a marker of the glycocalyx at the cell surface (data not shown). Indeed, according to the Zeiss LSM software, the colocalization of the two fluorophores was estimated to amount to approximately 75%.



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  		FIG. 2. Nociceptin-induced internalization of the ORL1-EGFP receptor. Membrane-associated fluorescence was quantified before (0 min, left) and after 60 min exposure (right) of the recombinant HEK cells to 0.1 µM nociceptin at 22 C. The quantitative fluorescence intensity vs. distance profiles (lower row) were obtained by fluorometric scanning along the straight line shown on the confocal images (upper row), using the Zeiss LSM software. The plasma membrane (pm) boundaries, indicated by arrows, were located by simultaneous examination of the cells in light transmission mode (gray line profile) and in fluorescent mode (black line profile). Before exposure to nociceptin, the fluorescence is mainly seen as sharp peaks corresponding to the plasma membrane (fluorometric profile on the left). After exposure to nociceptin, the main fluorescence pattern is no longer coincidental with the plasma membrane but localizes to intracellular compartments indicating a new receptor distribution (fluorometric profile on the right). In this particular experiment, representative of many, nociceptin induced a 78% decrease in plasma membrane-associated fluorescence. Control cells (not shown) incubated in parallel without nociceptin showed constant membrane fluorescent levels between time 0 (202 ± 18 AU) and 60 min (207 ± 16 AU). The 512 x 512-pixel image was obtained using a x40 oil immersion objective and magnified with a x3 zoom.

 
Because these cells were repeatedly laser scanned for dynamic studies, they were tested for bleaching of the EGFP fluorophore. In fact, bleaching was very low, amounting to only 2.4% when the cells were scanned every 2.5 min for 1 h with an argon laser set at 6% Wmax.

Exposure of the recombinant HEK(ORL1-EGFP) cells to 0.1 µM nociceptin for 60 min at 22 C resulted in a dramatic (over 75%) decrease in plasma membrane-associated fluorescence and a concomitant and transitory increase in cytoplasmic fluorescence, indicative of a robust internalization of the fluorescent receptor (Fig. 2Go, top and bottom rows). Fluorometric scanning across the confocal image represented by the profile measurement (Fig. 2Go, bottom row) was used to quantify, using Zeiss LSM software, the membrane fluorescence of the cells. To take into account possible cell movements, the limits of the plasma membrane are detected by superposition of the confocal image (black line profile) with the corresponding light transmission image (gray line profile), and the fluorescence remaining at the membrane after agonist treatment was compared with control values. At least 40 individual cells were quantified for each set of conditions, and each experiment was repeated three times. This mode of quantification was applied to all experiments additionally reported.

No internalization of the fluorescent receptor was detected at 4 C, even after 1 h (Fig. 3Go). The membrane fluorescence values were 152 ± 12 arbitrary units (AU) at time 0 and 155 ± 8 AU after 60 min of incubation, very similar to control cells incubated at 22 C (158 ± 17 AU) or at 37 C (150 ± 11 AU). At 22 C, nociceptin promoted the internalization of up to 80% of the fluorescent receptor, half-maximal internalization being reached after approximately 12 min. As expected, the process was even more rapid at 37 C, half-maximal depletion occurring after only approximately 6 min of exposure to 0.1 µM nociceptin (Fig. 3Go).



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  		FIG. 3. Time course of internalization of the ORL1-EGFP receptor at 4, 22, and 37 C. The recombinant HEK cells were incubated with 0.1 µM nociceptin for the indicated periods of time. For each time point, internalization was quantified as the mean percentage, relative to control values (time 0), of loss of plasma membrane-associated fluorescence in 30–40 nociceptin-treated cells. Each value represents the mean (± SEM) of three independent experiments. Nociceptin promoted rapid and extensive (>75%) internalization of the fluorescent receptor at 22 C ({blacktriangleup}) and 37 C ({blacksquare}) but not at 4 C ({blacktriangledown}). Half-maximal internalization was reached after approximately 12 min at 22 C and after approximately 6 min at 37 C.

 
Internalization pathway
To characterize the internalization of the fluorescent receptor further, we tested the effect of hypertonic sucrose, which selectively inhibits clathrin-mediated endocytosis (36). When the recombinant cells were first incubated for 30 min in 0.45 M sucrose, then exposed to 10 nM nociceptin for another 30 min at 22 C, considerably less of the fluorescent receptor was internalized (8 vs. 50%) than in control, non-sucrose-treated cells (Fig. 4Go). This suggests that the nociceptin-induced receptor endocytosis mainly occurs via clathrin-coated pits.



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  		FIG. 4. Blockade of nociceptin-induced internalization of the ORL1-EGFP receptor by hypertonic sucrose. Left panel, Control recombinant cells. Middle panel, Recombinant HEK cells were preincubated in ice-cold culture medium for 30 min and then exposed to 10 nM nociceptin for 30 min at 22 C. In this case, membrane-associated fluorescence intensity decreased to approximately 50% of control values. Right panel, Same as middle panel, except that the cells were preincubated in ice-cold medium containing hypertonic (0.45 M) sucrose. Hardly any internalization was detected in this case, the fluorescence intensity being decreased by only 8% compared with control values.

 
Moreover, the nociceptin-activated fluorescent receptor appeared to translocate to intracellular vesicles within minutes. Experiments using TMR-transferrin showed that these vesicles were the endosomal compartment (Fig. 5Go). When the recombinant HEK cells were incubated with TMR-transferrin alone, the green and red fluorescence signals were mostly associated with the periplasmic membrane (Fig. 5AGo for ORL1-EGFP alone, 5B for TMR-transferrin alone, and 5C for overlapping in a single cell), although a few tiny vesicles were spread throughout the cytosol. Almost no colocalization (<=10%) was observed in the absence of the agonist, indicating that spontaneous endocytosis is very limited in recombinant HEK cells. The addition of nociceptin (0.1 µM) for 15 min resulted in the massive internalization of both the fluorescent receptor (Fig. 5DGo) and TMR-transferrin (Fig. 5EGo), which colocalized in yellowish vesicles in the cytoplasm (Fig. 5FGo).



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  		FIG. 5. Endosomal pathway for nociceptin-induced internalization of the ORL1-EGFP receptor. The recombinant HEK cells were incubated in the presence of 40 µg/ml TMR-transferrin (control, A–C) or 40 µg/ml TMR-transferrin and 0.1 µM nociceptin (nociceptin-treated, D–F) for 5, 15 (results shown here), or 30 min at 22 C. A and D, ORL1-EGFP channel; B and E, TMR-transferrin channel; C and F, EGFP/TMR overlap indicating ORL1-EGFP and TMR-transferrin colocalization in yellow. In the absence of nociceptin, TMR-transferrin strongly labeled the outer membrane in red (B) in addition to the green labeling due to ORL1-EFGP receptors (A). Very few internal red or green and very rarely yellow vesicles were seen inside the cells (C), indicating a slow constitutive internalization of both transferrin and ORL1 receptors. After nociceptin addition, numerous yellowish endosomal vesicles appeared within 5 min, peaking at 15 min, where a population of large vesicles merged (F), indicating that ORL1-EGFP receptors were internalized via the endosomal compartment.

 
Receptor resensitization
We evaluated the recovery of the receptor at the cell surface after chronic nociceptin treatment by incubating the recombinant HEK(ORL1-EGFP) cells for 1 h with 0.1 µM nociceptin, washing three times with medium and then examining the cells cultured in medium every 10 min for 90 min by confocal microscopy (Fig. 6Go). We also evaluated photobleaching in control cells incubated in parallel to correct the test values (data not shown). The green fluorescence of ORL1-EGFP appeared gradually at the cell surface, indicating progressive fusion of recycling or newly synthesized vesicles with the membrane, as shown on pictures taken at time zero (Fig. 6AGo) and after 30, 50, or 90 min of recovery (Fig. 6Go, B, C, and D, respectively). A normal receptor level was recovered after 70 min and remained stable until 90 min (Fig. 6Go, curve).



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  		FIG. 6. Membrane fluorescence recovery of ORL1-EGFP after chronic treatment with nociceptin. The timing of receptor resensitization was evaluated on cells incubated first with 0.1 µM nociceptin for 1 h at 22 C, washed three times with medium, and then incubated again in complete medium without nociceptin for another 90 min. Membrane fluorescence levels were evaluated every 10 min (lower panel). When nociceptin stimulation was removed, ORL1-EGFP gradually reconstituted the green labeling of the plasma membrane, and normal fluorescence was recovered after 70 min. A, Time 0 corresponding to nociceptin treatment for 1 h; B, 30 min of receptor recovery; C, 50 min of recovery; D, 90 min of recovery.

 
Effects of agonists and antagonists on internalization of the ORL1-EGFP fluorescent receptor
Next, we examined the effects of various ORL1 receptor ligands on internalization of the fluorescent receptor (Fig. 7Go). The recombinant cells were exposed to ligands for 60 min at 22 C, and internalization was measured. Internalization is expressed as the percentage of residual vs. initial levels of plasma membrane-associated fluorescence. As shown in Fig. 7AGo, 0.1 µM nociceptin decreased membrane fluorescence down to 21% of control values. Lofentanil (1 µM) and Ro64-6198 (1 µM) were as efficient as nociceptin in promoting the internalization of the fluorescent receptor, residual membrane-associated fluorescence amounting to less than 20 and 26% of control values, respectively. The two ORL1 receptor antagonists, III-BTD, and J-113397, each used at 10 µM, had no effect. However, as expected, J-113397 totally prevented nociceptin from promoting the internalization of the fluorescent receptor; the membrane fluorescence of cells that had been exposed to a mixture of J-113397 (10 µM) and nociceptin (10 nM) for 60 min was more than 90% of that of control cells, compared with less than 30% in cells treated with nociceptin alone. J-113397 also completely blocked lofentanil-induced internalization of the fluorescent receptor, restoring residual membrane fluorescence levels from 16% (lofentanil alone) up to more than 90% of control (data not shown). However, III-BTD (10 µM) did not block nociceptin-induced internalization, residual fluorescence amounting to only 24% of control, similar to the value (29%) seen in cells treated with nociceptin alone.



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  		FIG. 7. Ability of various agonists and antagonists to promote internalization of the ORL1-EGFP receptor. Recombinant HEK cells expressing fluorescent receptors were exposed to ligands at the final concentrations indicated (A). After 1 h at 22 C, the percentage of remaining membrane fluorescence, compared with control cells, was determined for each ligand as described in the legends to Figs. 3Go and 4Go and represented as histogram bars. The pure ORL1 receptor agonists (nociceptin, lofentanil, and Ro64-6198) induced strong internalization, whereas the partial agonists hexapeptide (HP5, Ac-Arg-Tyr-Tyr-Lys-Trp-Arg-NH2) and pseudopeptide (1 2 3 4 5 6 7 8 9 10 11 12 13 ) promoted only an approximately 30% loss of membrane fluorescence. This was confirmed by the dose-dependence curves (B) obtained by treating the cells with increasing concentrations of nociceptin ({blacktriangleup}), Ro64-6198 ({blacktriangledown}), pseudopeptide (1 2 3 4 5 6 7 8 9 10 11 12 13 ) ({diamondsuit}), or hexapeptide ({blacksquare}) ranging from 1 nM to 1 µM for 1 h. In comparison with nociceptin and Ro64-6198, both hexapeptide and pseudopeptide (1 2 3 4 5 6 7 8 9 10 11 12 13 ) were able to promote only partial (28 and 35%, respectively, vs. 78% for nociceptin) depletion of plasma membrane-associated fluorescence. When administered alone, the nonpeptide antagonists III-BTD and J-113397 did not show any effect (A), but J-113397 significantly blocked the nociceptin-induced ORL1 internalization. The µ-opioid-specific agonist DAMGO did not induce any ORL1-EGFP internalization. Each data point is the mean (± SEM) of three values from independent determinations.

 
The hexapeptide (0.1 µM) induced only a partial internalization of the fluorescent receptor (Fig. 7AGo). Indeed, a maximum 28% internalization was seen after 60 min at 22 C in the presence of 0.1 or 1 µM hexapeptide, the highest concentration tested, whereas under identical conditions, nociceptin and Ro64-6198 induced up to 75% internalization (Fig. 7BGo). Likewise, the pseudopeptide induced only a maximum 35% internalization even at the highest concentration tested (1 µM). The µ-opioid-specific ligand DAMGO (1 µM) did not induce any significant internalization (Fig. 7AGo).

Trafficking of GRK2-DsRed in recombinant HEK(ORL1-EGFP+) cells
Cells were treated with different ligands for 2, 4, 6, or 8 min at 22 C, and GRK2-DsRed translocation was evaluated as the accumulation of red fluorescence at the cell membrane during the time period compared with control cells. As shown in Fig. 8Go, nociceptin (0.1 µM) triggered the distribution of red fluorescence at the membrane in a time-dependent manner. The maximum (255%) was reached after 4 min, and then the red fluorescence signal at the membrane started to decrease, indicating the beginning of the receptor endocytosis process. The pseudopeptide (0.1 µM) induced GRK2-DsRed redistribution to a lesser extent with a transitory increase at 181% after 2 min, followed by a rapid decrease. On the contrary, the hexapeptide (0.1 µM) and the antagonist J-113397 showed no effect, exhibiting red fluorescence levels at the membrane similar to control values all during the treatment. Images depicting this phenomenon after 4 min of treatment can be seen in Fig. 9Go. Control (Fig. 9AGo) and hexapeptide-treated (Fig. 9CGo) cells exhibited a cytoplasmic distribution of GRK2-DsRed, with almost no membrane labeling. By contrast, in nociceptin-treated cells, a strong membrane colabeling (yellow) resulting in green receptor and red GRK2-DsRed colocalization was observed (Fig. 9BGo).



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  		FIG. 8. Kinetics of GRK2-DsRed trafficking in ORL1-EGFP-expressing cells. Cells stably expressing ORL1-EGFP were transiently transfected with cDNAs encoding GRK2-DsRed. After 48 h, the cells were treated for up to 8 min with 0.1 µM agonists such as nociceptin (noc) ({blacksquare}), pseudopeptide ({blacktriangledown}), or hexapeptide ({blacktriangleup}) or with 1 µM antagonist J-113397 ({diamondsuit}) and compared with cells incubated in control medium ({bullet}). The recruitment of GRK2-DsRed at the membrane was evaluated by confocal imaging and quantified. The values are expressed as a percentage of red fluorescence at the membrane compared with control values. The data are the means (± SEM) of three independent experiments.

 


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  		FIG. 9. Confocal imaging of GRK2-DsRed trafficking in ORL1-EGFP-expressing cells. Confocal microscopy was performed on cells stably expressing ORL1-EGFP and transiently transfected with GRK2-DsRred for 48 h. The cells were treated for 4 min, the optimal time for maximal effect as established in the kinetics shown in Fig. 8Go. There was no change between control cells (A) and cells treated with 0.1 µM hexapeptide (C), whereas 0.1 µM nociceptin induces a strong translocation of GRK2-DsRed at the membrane, as attested by the yellowish labeling indicating ORL1 and GRK2 colocalization (B). Images were obtained in multitracking mode to prevent any channel contamination.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because GFP-tagged receptors have been used successfully to demonstrate and to monitor the endocytosis of different GPCRs (for review see Ref. 28), we fused the ORL1 receptor to the GFP (EGFP) to demonstrate and to characterize receptor endocytosis in real time in live HEK (HEK293) cells. Fusion of the N terminus of EGFP to the C terminus of ORL1 did not alter the affinity of the receptor for nociceptin and other ORL1 receptor ligands, nor did it affect the ability of the receptor to mediate agonist-induced binding of GTP{gamma}35S, i.e. to couple with the heterotrimeric G protein. Thus, the chimeric fluorescent ORL1 receptor appeared to be functional. Most of the authors found that GFP-tagged receptors behave in internalization processes very similarly compared with wt receptors, evaluated with antireceptor antibodies. The only reported difference seems to be in kinetics, the GFP-tagged receptor internalization and trafficking being sometimes slower (37). In HEK293 cells, the fluorescent receptor was primarily localized at the periplasmic membrane, as confirmed by TMR-concanavalin A labeling, and was efficiently internalized when activated by nociceptin, the natural agonist of the ORL1 receptor.

In the absence of ligand, the colocalization between ORL1-EGFP and TMR-transferrin was mainly observed at the membrane (~90%), indicating a poor spontaneous endocytosis in HEK cells. After 15 min at 22 C in the presence of nociceptin, there was a massive increase in internalized colabeling. The internalized receptors appeared as yellow vesicles, indicating the simultaneous presence of ORL1-EGFP and TMR-transferrin. These data show that ORL1 endocytosis takes place through regular endosomes under nociceptin stimulation, as is the case for other receptors such as cholecystokinin (38) or neuropeptide Y receptors (29).

In cells preincubated with hypertonic sucrose, nociceptin treatment did not significantly decrease plasma membrane fluorescence. This indicates that the nociceptin-induced internalization of ORL1 proceeds mainly through clathrin-coated pits, similarly to what has been described for other GPCRs such as the cholecystokinin receptor type A (38, 39), the ß2-adrenergic receptor (40), and opioid receptors (41). The ORL1-EGFP receptor was rapidly (t1/2 ~12 min at 22 C and ~6 min at 37 C) and strongly (up to 75–80% of the initial membrane-bound fluorescence) internalized.

In the absence of any convenient cellular labeling of ORL1 (fluorescent ligand or antibodies against ORL1), other studies have demonstrated the down-regulation of the ORL1 receptors by challenging ORL1 binding after nociceptin administration in neuroblastomas or other cell lines stably transfected with ORL1 (15, 42, 43). Spampinato et al. (43) reported that nociceptin promoted ORL1 receptor endocytosis in NK-S-BE, a human neuroblastoma cell line that expresses the receptor naturally, albeit at very low levels of receptors (38 fmol/mg membrane protein). In their study, receptor internalization was evaluated as a decrease in cell surface [3H]nociceptin binding sites after washing the nociceptin-exposed cells to remove the bound unlabeled ligand. At 37 C, the decrease was extremely rapid (t1/2 <= 2 min) yet only partial, reaching only 20% of internalization for 0.1 µM nociceptin, and was not maximal before 5 µM. This is quite different from what we observed in HEK293 cells, where 0.1 µM nociceptin induced the internalization of approximately 75% of the ORL1 receptor at t1/2 = 6 min. The presence of multiple other families of receptors such as opioids, somatostatin, or adrenergic in SK-N-BE may also modulate each other’s desensitization. Thus, Jordan et al. (44) showed that the coexpression of the {kappa}-opioid receptor, but not µ- or {delta}-opioid receptors, together with the ß2-adrenergic receptor prevents ß2-adrenergic receptor internalization. In fact, the rate and amplitude of GPCR endocytosis may differ depending on the cellular context. The HEK293 cells were preferred as a model (15, 29) because they are equipped, unlike COS cells, with most of the cellular partners necessary for receptor phosphorylation and internalization. They are now extensively used for studies involving cellular trafficking of receptors. Because HEK293 cells do not contain any of the opioid-related receptors, this cell line offers a model of choice to explore receptor crosstalk by transfecting sequentially different receptor cDNAs.

Using a method similar to that used by Spampinato et al. (43), Dautzenberg et al. (15) recently found that nociceptin does not induce the internalization of the ORL1 receptor in the same cellular context as in our study, i.e. HEK293 cells. They found that the nociceptin-binding sites at the cell surface were fully recovered after an acid wash of nociceptin-treated (desensitized), but not of Ro64-6198-desensitized, HEK cells. They concluded that, unlike Ro64-6198, nociceptin does not promote receptor endocytosis in these recombinant cells. This is in marked contrast with our observation that nociceptin and Ro64-6198 are equally efficient in eliciting the strong endocytosis of the fluorescent ORL1 receptor.

In terms of receptor resensitization, after 60 min of incubation with nociceptin, the time necessary for complete receptor recovery of the membrane after ligand washing was 70–80 min at 22 C, a value close to what has been published for the ß2-adrenergic receptor in HEK293 cells (40). This also indicates that EGFP-tagged receptors can recycle as native ones.

The quantitative study of various ligands led to two major observations. First, the two antagonists tested did not show an equivalent capacity to block nociceptin-induced internalization. J-113397 completely blocked this process, whereas an equal concentration of III-BTD (10 µM) failed to do so, although they both show a relatively good affinity (Ki = 2.1 nM for J-113397 and 12 nM for III-BTD). We do not have clear explanations for this feature. It is possible that the kinetics of association of III-BTD to ORL1 in the cell medium is much lower than the association of nociceptin to ORL1, the former finally failing to prevent the initiation of internalization by the agonist, even in the presence of 1000 times excess antagonist. This may be reflected by the relatively low affinity (Ki 12.4 nM) of III-BTD for ORL1 compared with nociceptin (0.24 nM). However, based on the formulae (17, 18), it is likely that the hairpin-shaped III-BTD and the 4-anilino-piperidin nucleus of J-113397 do not interact similarly with the ORL1 receptor.

Our second observation is that, whereas nociceptin and lofentanil both induced a 75% maximal internalization rate, the partial agonists hexapeptide and the pseudopeptide failed to induce the internalization of more than 28 and 35%, respectively, even at the very high dose of 1 µM. One hypothesis could be that nociceptin- and hexapeptide-induced internalization does not occur through the same pathways, as is the case for cholecystokinin (39). As confirmed by the sucrose inhibition assay, nociceptin operates via clathrin-coated pits. However, as 0.1 µM of hexapeptide induced only 18% of receptor internalization after 30 min, we could not significantly determine whether or not the sucrose hyperosmolarity was inhibiting ORL1 internalization. We propose to use a negative dominant of Eps15, deleted in EH2 and EH3 domains (45), which prevents clathrin-coated pit formation and would help us to clarify this point.

Schulz et al. (46) reported that the activation of {delta}-opioid receptor triggers a rapid translocation of cytoplasmic GRK2-DsRed toward the cell membrane, which, in turn, releases vesicles carrying both green and red fluorescence. We have used the same technique based on cell imaging. In our hands, the confocal microscopy on living cells was an outstanding tool for following red (GRK2) and green (ORL1) fluorescence distribution in individual cells as a function of time. We could see that nociceptin did indeed mobilize GRK2-DsRed in a time-dependent manner. Conversely, the hexapeptide failed to do so, suggesting that it could not induce the receptor phosphorylation necessary for the assembly of additional partners. We provide here the first evidence that partial agonists differently affect ORL1 interaction with internalization partners compared with full agonists such as nociceptin or Ro64-6198. In that respect, as morphine for the µ-opioid receptor, the hexapeptide and, to a lesser extend, the pseudopeptide might induce G protein coupling to ORL1 and subsequent signal transduction without causing significant receptor desensitization. This is of primary importance in the design of molecules for clinical treatments, and several laboratories are currently developing the synthesis of hexapeptide derivatives or analogs (47) that could structurally interact with ORL1 in a different way compared with nociceptin, as we have recently shown using photoaffinity labeling (48). Thus, we demonstrated that hexapeptides of the Houghten family are suspected to interact superficially with extracellular portions of the receptor such as the C terminus of transmembrane helix II of ORL1 residues (amino acid residues 107–113), whereas nociceptin, and probably the pseudopeptide 1–13, will be localized in an opioid-like binding pocket of ORL1 (residues 1–6 of nociceptin, with residues 7–13 staying outside), according to the model we proposed (49). Additional studies should improve our understanding of the ligand discrepancies observed here.


   Acknowledgments
 
We thank Dr. Giro Calo (University of Ferrara, Italy) for the pseudopeptide (1–13), Dr. Satoshi Ozaki (Banyu Tsukuba Research Institute, Tsukuba, Japan) for J-113397, Dr. François Jenck and Dr. Jurgen Wichmann (F. Hoffmann-La Roche AG, Basel, Switzerland) for Ro64-6198, and Dr. Rudiger Schulz for the GRK2-DsRed plasmid (Institute of Pharmacology, Toxicology, and Pharmacy, Munich, Germany). We thank Jean-Michel Lago (Carl Zeiss, LePecq, France) for training us on confocal microscopy and Zeiss software.


   Footnotes
 
This work was supported in part by grants from the Association pour la Recherche sur le Cancer (ARC 9428) and the European Commission (BMH4 CT97 2317).

Abbreviations: AU, Arbitrary unit; DAMGO, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin; DsRed, red fluorescent protein 1; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; GRK2, G protein receptor kinase type 2; HEK, human embryonic kidney; hORL1, human opioid receptor-like 1; TMR, tetramethylrhodamine; wt, wild-type.

Received January 20, 2004.

Accepted for publication March 3, 2004.


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 Introduction
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