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Intracellular Dynamics of sst₅ Receptors in Transfected COS-7 Cells: Maintenance of Cell Surface Receptors during Ligand-Induced Endocytosis¹

Montreal Neurological Institute, McGill University (T.S., A.C.J., A.B.), Montréal, Québec, Canada H3A 2B4; Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis (P.S., C.D.F., J.-P.V., J.M.), 06560 Valbonne, France; and Institut für Zellbiochemie und Klinische Neurobiologie, Universität Hamburg (H.-J.K.), D-20246 Hamburg, Germany

Address all correspondence and requests for reprints to: Alain Beaudet, M.D., Ph.D., Montreal Neurological Institute, McGill University, 3801 University Street, Montréal, Québec, Canada H3A 2B4. E-mail: mcin{at}musica.mcgill.ca

	Abstract

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Internalization of G protein-coupled receptors is crucial for resensitization of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulating cell surface availability of the somatostatin (SRIF) receptor subtype sst₅, we characterized its internalization properties in transfected COS-7 cells using biochemical, confocal microscopic, and electron microscopic techniques. Our results demonstrated rapid and efficient sequestration of specifically bound [¹²⁵I]Tyr⁰-D-Trp⁸-SRIF (up to 45% of bound radioactivity). Combined immunocytochemical detection of sst₅ and visualization of a fluorescent SRIF analog by confocal microscopy revealed that whereas the internalized ligand progressively clustered toward the cell center with time, immunoreactive receptors remained predominantly associated with the plasma membrane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of saturability of [¹²⁵I]Tyr⁰-D-Trp⁸-SRIF binding at 37 C. Binding was rendered saturable by the drug monensin, showing that receptor recycling played a key role in the preservation of cell surface receptors. Electron microscopy demonstrated that in addition to receptor recycling, internalization of receptor-ligand complexes triggered a massive recruitment of sst₅ receptor molecules from intracellular stores to the membrane. This combination of recycling and recruitment of spare receptors may protect sst₅ from long term down-regulation through sequestration and, therefore, facilitate extended SRIF signaling.

	Introduction

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SOMATOSTATIN [somatotropin release-inhibiting factor (SRIF)] is widely distributed throughout the mammalian central nervous system and periphery (for review, see Refs. 1, 2). It exists in one of two forms, derived from a single gene: SRIF-14 and its N-terminally extended form, SRIF-28. Both forms are involved in the regulation of hormone release from the anterior pituitary (3) and from peripheral glands (4, 5). They also act as neurotransmitters and neuromodulators in the central and peripheral nervous systems (1) and as regulators of cell proliferation in peripheral tissues, including SRIF receptor-expressing tumors (6, 7, 8, 9).

The actions of SRIF are mediated through a family of G protein-coupled membrane receptors. The known receptors are encoded by five individual genes, designated sst₁-sst₅ (10, 11, 12, 13, 14, 15). One of the cloned receptor genes, sst₂, gives rise to two splice variants, termed sst_2A and sst_2B, whereas the other receptor genes lack introns. Although these different receptor subtypes have been linked to a variety of signal transduction pathways (for review, see Ref. 16), their individual roles have yet to be fully elucidated. In the pituitary, sst₂ and sst₅ appear to play a major role in mediating SRIF’s regulatory influence on the release of pituitary hormones such as GH (17). In the pancreas, sst₅ is reportedly a key player in the regulation of insulin release by SRIF (5). All five sst receptor subtypes are present in mammalian brain (18, 19, 20). Whereas sst_1–3 appear to be abundantly expressed in brain, the distribution of sst₄ and sst₅ is much more limited (18, 19, 20, 21, 22, 23, 24, 25, 26).

All five SRIF receptors, with the possible exception of sst_4, have been shown to internalize upon ligand activation (27, 28, 29, 30, 31). Internalization of receptor-ligand complexes has been shown to play a predominant role both for resensitization through dephosphorylation (32) and for desensitization through sequestration (33, 34) of G protein-coupled receptors. Furthermore, recent studies on a variety of G protein-coupled receptors, including those of the sst family (35), have suggested that internalization might subserve specific signaling functions either through endosomal signaling (36) or through activation of mitogen-activated protein kinase pathways independent of G protein recruitment (37, 38).

A striking feature of sst receptor internalization is the wide spectrum of maximal capacities and intracellular trafficking patterns reported between the various subtypes (27, 28, 29, 30). For example, in COS-7 cells transfected with the sst₁ receptor, SRIF only poorly internalizes and remains clustered beneath the plasma membrane at all times (28). By contrast, in COS-7 cells transfected with sst_2A, internalization of fluorescent SRIF is extremely efficient and characterized by a rapid down-regulation of cell surface receptors and the formation of small intracytoplasmic particles reminiscent of endosomes (28). Internalization of immunoreactive sst₃ receptors, as studied in transfected HEK-293 (30) and RIN 1046–38 cells (29), is also highly efficient and gives rise to the same type of intracellular labeling pattern as that observed in cells transfected with sst_2A. In the case of sst₄, major species differences have been reported between rat and human clones. Whereas the native rat sst₄ receptor does not internalize following SRIF binding (29, 30), the human sst₄ internalizes well (27). Species differences were also observed in the case of sst₅; rat sst₅ internalizes only in response to stimulation with SRIF-28 and not SRIF-14 (29, 30), whereas human sst₅ does internalize when stimulated with D-Trp⁸-SRIF (39).

The aim of the present study was to further characterize the properties and pattern of SRIF-induced internalization of the sst₅ receptor subtype, in an attempt to gain further insight into the regulation of this receptor. We employed biochemical, confocal, and electron microscopic techniques to track sst₅ and SRIF through the events following SRIF-induced internalization. We show that a constant population of functional sst₅ receptors is maintained at the cell surface at all times through a process of receptor recycling and recruitment of intracellular sst₅ proteins. We propose that this mechanism may protect sst₅ from long term desensitization.

	Materials and Methods

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Cell culture and transfection
The monkey kidney epithelium-derived cell line COS-7 was cultured in DMEM (Life Technologies, Inc., Burlington, Canada) supplemented with 10% FBS (Harlan Bioproducts, Inc., Indianapolis, IN) and 1% penicillin/streptomycin (Life Technologies, Inc.). The cells were transfected with 1 µg/ml of either pcD2 or pcDNA3 eukaryotic expression vectors containing the rat sst₅ receptor complementary DNA (cDNA; provided by Dr. A. M. O’Carroll, NIMH) according to the DEAE-dextran/chloroquine procedure. After 48–72 h, cells were harvested and plated onto poly-L-lysine-coated coverslips in four-well tissue culture plates for light microscopy or directly into the wells for electron microscopy. Cells for radioactive ligand binding assays were grown in 12-mm culture dishes.

Preparation of cell homogenates
Confluent COS-7 cells were washed and scraped into ice-cold Tris-buffered saline (pH 7.5). Subsequently, the cells were centrifuged for 5 min in microfuge tubes at 13,000 x g and resuspended in hypotonic TE buffer (5 mM EDTA and 10 mM Tris-HCl, pH 7.5). Membrane homogenates were then sonicated, recentrifuged at 13,000 x g for 30 min at 4 C, and resuspended in the same buffer.

Binding of ¹²⁵I-Tyr⁰-D-Trp⁸-SRIF to COS-7 cells
To assess the pharmacological properties of sst₅ receptors in transfected COS-7 cells, binding experiments were performed on freshly prepared membranes. Briefly, 25 µg membranes were incubated with concentrations of [¹²⁵I]Tyr⁰-D-Trp⁸-SRIF (2000 Ci/mmol; hereafter referred to as [¹²⁵I]SRIF) ranging from 0.1–100 nM for 30 min at 25 C in 250 µl Tris-HCl (pH 7.5) containing 2 mM MgCl₂, 0.1% BSA (Roche Molecular Biochemicals, Laval, Canada), and 0.8 mM 1,10-phenanthroline (Sigma, St. Louis, MO) to prevent peptide degradation. Binding was terminated by the addition of 2 ml ice-cold buffer followed by filtration through glass-microfiber filters (GF/C, Whatman, Clifton, NJ) preincubated in 0.5% polyethylenimine. After washing twice with 2 ml ice-cold buffer, the radioactivity retained on the filters was counted in a -counter. Nonspecific binding was measured in the presence of 1 µM nonlabeled D-Trp⁸-SRIF (Sigma). The dissociation constant (K_d) and maximal binding capacity (B_max) were derived from Scatchard analysis of the data.

For confocal microscopic tracking of internalized ligand, the pH-insensitive dye Bodipy 576/589 (40) (Molecular Probes, Inc., Eugene, OR) emitting red fluorescence was covalently conjugated to the degradation-resistant SRIF analog D-Trp⁸-SRIF in the position (for more detail on preparation and purification, see Ref. 28). To compare the affinity of this fluorescent SRIF analog (hereafter referred to as fluo-SRIF) toward sst₅ with those of native molecular forms of SRIF and other synthetic SRIF analogs, 25 µg membranes were incubated with 0.3 nM [¹²⁵I]SRIF in the presence of increasing concentrations of SRIF-14, SRIF-28, D-Trp⁸-SRIF, Tyr⁰-D-Trp⁸-SRIF (all from Sigma), or fluo-SRIF ranging from 1 pM to 1 µM. Binding was performed for 30 min at room temperature in 250 µl Tris-HCl buffer, pH 7.5, containing 2 mM MgCl₂, 0.1% BSA, and 0.8 mM 1,10-phenanthroline. The experiment was terminated, and radioactivity was counted as described above.

To characterize the kinetics of sst₅ internalization in transfected COS-7 cells, internalization assays were carried out on whole cells in 12-mm dishes containing 2 x 10⁵ cells. After discarding the culture medium, cells were equilibrated for 10 min at 37 C in Earle’s buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.9 mM MgCl₂·6H₂0, and 25 mM HEPES) containing 0.8 mM phenanthroline, 0.1% D-glucose, and 1% BSA in the presence or absence of 10 µM phenylarsine oxide (PAO; Sigma), a drug that blocks endocytosis (but also many other cellular functions) through chemical reaction with sulfhydryl groups (41), or of 25 µM monensin (Sigma), which inhibits the membrane recycling process by raising the pH within endosomes and thereby preventing the dissociation of receptor and ligand (41). Some experiments were carried out in the presence of 70 µM cycloheximide (Sigma) (41), a protein synthesis inhibitor, in which case the preincubation period was increased to 2 h. Equilibration buffer was then replaced with 250 µl binding buffer containing 0.3 nM [¹²⁵I]SRIF for 3–40 min (time course) or 0.1–100 nM [¹²⁵I]SRIF for 45 min (saturation), both at 37 C. In time-course experiments, cells were then washed twice with pure equilibration buffer or a hypertonic acid buffer (Earle’s buffer containing 0.2 M acetic acid and 0.5 M NaCl, pH 4) to strip off surface-bound radioactivity (41). In saturation experiments, the cells were washed with equilibration buffer. All cells were harvested with 1 ml 0.1 M NaOH, and associated radioactivity was counted in a -counter. Nonspecific binding was measured in the presence of an excess (1 µM) of nonlabeled D-Trp⁸-SRIF.

Internalization of -Bodipy Red-D-Trp⁸-SRIF in sst₅-transfected COS-7 cells
COS-7 cells transfected with cDNA encoding sst₅ were preincubated for 10 min at 37 C in Earle’s buffer containing 0.8 mM phenanthroline, 0.9 g/ml D-glucose, and 0.2% BSA and then incubated for 5–60 min in the presence of 10 nM fluo-SRIF in the same buffer. To determine nonspecific binding, 1 µM nonfluorescent D-Trp⁸-SRIF was added to the incubation medium. To evaluate temperature dependence, additional cells were incubated at 4 C. At the end of the incubation, cells were rinsed twice for 2 min each time with cold Earle’s buffer and air-dried. To selectively observe internalized fluo-SRIF, cells were washed twice with hypertonic acid buffer, pH 4, before rinsing and air-drying. To determine whether fluo-SRIF internalization was clathrin-mediated, cells were preincubated for 30 min in equilibration buffer containing either 20 µM PAO or 0.45 sucrose (two compounds known to inhibit clathrin-mediated endocytosis) (41). They were then incubated with fluo-SRIF in the presence of PAO or sucrose, respectively, and treated as described above. Air-dried cells were examined under a Carl Zeiss laser scanning microscope attached to an Axiovert 100 inverted microscope (Carl Zeiss Canada Ltd., Con Mills, Canada). Images were acquired as single optical sections through the center of the cells at 16 scans/frame in the LUTS mode. After the initial adjustment of laser power and contrast, all images of cells from 1 experiment were acquired using identical settings of the confocal unit. The images were processed using Photoshop version 4.0.1 and Illustrator version 7.0 (both from Adobe Systems, Inc., San Jose, CA) on an IBM-compatible computer.

Pulse-chase experiments: confocal microscopy
To examine the fate of SRIF in parallel with that of sst₅ receptors in the course of ligand-induced internalization, transfected COS-7 cells were preincubated for 90 min at 37 C in DMEM containing 70 µM cycloheximide, a protein synthesis inhibitor, followed by 5- to 10-min equilibration at 4 C in Earle’s buffer containing 70 µM cycloheximide, 0.8 mM phenanthroline, 0.9 g/ml D-glucose, and 0.2% BSA. Cycloheximide was also included in the incubation medium in all following steps before the final rinses. After exposure to 10 nM fluo-SRIF for 30 min at 4 C in supplemented Earle’s buffer, the incubation medium containing the peptide was aspirated and replaced with supplemented Earle’s buffer at 37 C without the ligand, and internalization was allowed to proceed for 0, 5, 10, 20, 40, or 60 min, respectively. Finally, the cells were subjected to hypertonic acid wash followed by air-drying. Additional cells from the same transfection were incubated with 10 nM nonlabeled D-Trp⁸-SRIF in lieu of fluo-SRIF. These cells were incubated for 30 min at 4 C, chased at 37 C as described above, rinsed twice in 0.1 M phosphate buffer, pH 7.4 (PB), and fixed with 4% paraformaldehyde (PFA) in the same buffer at room temperature. Subsequently, the cells were rinsed twice in PB and twice in 0.1 M Tris-buffered saline, pH 7.4 (TBS). They were then incubated overnight at 4 C with 0.2 µg/ml affinity-purified rabbit anti-sst₅ antibody (for details on antibody generation and characterization, see Refs. 26, 30) in TBS containing 0.5% normal goat serum (NGS) and 0.05% Triton X-100. The next day, the cells were rinsed twice in TBS and incubated for 40 min at room temperature with goat antirabbit-Cy3 secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:800 in TBS followed by two final rinses in TBS and mounting in Aquamount (Polyscience, Markham, Canada). As controls, transfected cells were immunostained under the same conditions, but without stimulation by D-Trp⁸-SRIF. All specimens were examined under a confocal microscope as described above.

Pulse-chase experiments: electron microscopy
To follow the fate of internalized sst₅ receptors at the subcellular level, a series of pulse-chase experiments was carried out as described above. However, for the sake of better ultrastructural preservation, cycloheximide was excluded from all incubation buffers, and pulse-labeling with 20 nM D-Trp⁸-SRIF was performed at 10 C, followed by chase periods of 0, 10, and 40 min at 37 C. Subsequent to this pulse-chase protocol, cells were fixed for 20 min in 2% acrolein/2% PFA in 0.1 M PB followed by another 20 min in 2% PFA alone. They were then briefly washed in 0.1 M TBS, exposed for 30 min to a preincubation solution consisting of 3% NGS in 0.1 M TBS, and incubated overnight at 4 C with 0.4 µg/ml sst₅ receptor antibody along with 0.02% Triton X-100 and 0.5% NGS in 0.1 M TBS. After primary antibody incubation, the cells were repeatedly rinsed in 0.01 M PBS, and nonspecific sites were blocked with a mixture of 0.1% gelatin and 0.8% BSA in 0.01 M PBS. Cells were then incubated with a 1:50 dilution of goat antirabbit IgG-gold (AuroProbe One GAR, Amersham Pharmacia Biotech, Arlington Heights, IL) at room temperature for 2 h, fixed with 2% glutaraldehyde in 0.01 M PBS, and rinsed in 0.2 M citrate buffer, after which the reaction was enhanced by incubation with ionic silver (IntenSE M Silver Enhancement Kit, Amersham Pharmacia Biotech). After a rinse in 0.1 M PB, cells were postfixed in a solution of 2% osmium tetroxide in 0.1 M PB in the dark for 10 min, dehydrated in graded concentrations of ethanol, and embedded in Epon. Ultrathin sections were cut and impregnated with uranyl acetate and lead citrate according to standard electron microscopy protocols before examination with a JEOL 100CX transmission electron microscope (JEOL USA, Inc., Peabody, MA).

For quantification, an average of 20 separate cell profiles were obtained from each experimental condition in every individual experiment. The distribution of immunogold particles was then analyzed using computer-assisted morphometry (Biocom, Les Ulis, France). Specifically, the total numbers of gold particles associated with the plasmalemma and located within the intracellular compartment were counted separately and normalized as a function of membrane length and area of cell sampled, respectively. The distance between each intracellular receptor and the plasmalemma was also measured and distributed into bins at increasing distance from the cell surface. Finally, a frequency distribution of intracellular particles and the ratio between intracellular and membrane-associated receptors were calculated for each experimental condition. Statistical significance was verified using Student’s t test, (two-tailed) for distant measurements and Mann-Whitney or Wilcoxon tests (two tailed) for membrane/cell interior ratios. All calculations and statistical analyses were performed using Excel 5.0 (Microsoft Corp., San Francisco, CA).

	Results

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Binding of [¹²⁵I]SRIF to transfected COS-7 cell membranes
[¹²⁵I]SRIF binding to membranes prepared from COS-7 cells transfected with cDNA encoding the rat sst₅ receptor was specific and saturable. Saturation experiments yielded a linear Scatchard plot, demonstrating the presence of a single binding site with a K_d of 0.3 ± 0.1 nM (Fig. 1a). The maximal binding capacity was 92 ± 5 fmol/mg (n = 3; Fig. 1a). Membranes prepared from nontransfected cells were totally devoid of [¹²⁵I]SRIF binding (not shown).

View larger version (21K): [in this window] [in a new window]   Figure 1. Kinetics of [125I]Tyr0-D-Trp8-SRIF binding to membranes of COS-7 cells transfected with cDNA encoding the sst5 receptor. Membranes (25 µg) were incubated with increasing concentrations of (a) or with 0.3 nM (b) [125I]Tyr0-D-Trp8-SRIF for 30 min at 25 C. Specific binding was calculated as total binding minus binding in the presence of 1 µM D-Trp8-SRIF. a, Scatchard representation of saturation data. b, Competitive binding inhibition using -Bodipy-D-Trp8-SRIF (), D-Trp8-SRIF (•), Tyr0-D-Trp8-SRIF (), SRIF-28 (), and SRIF-14 () as competitors. For IC50 values, see Table 1.

View larger version (18K): [in this window] [in a new window]   Figure 2. Kinetics of [125I]Tyr0-D-Trp8-SRIF binding to whole COS-7 cells expressing the rat sst5 receptor. Cells were incubated with 0.3 nM [125I]SRIF for 3–40 min (a) or with increasing concentrations of [125I]SRIF for 45 min (b) at 37 C. a, Time course. Experiments were performed in the presence (circles) or absence (squares) of 10 µM PAO, followed (closed symbols) or not (open symbols) by a hypertonic acid wash to strip off cell surface binding. b, Saturation. Experiments were performed in the absence of any drug (•) or in the presence of 10 µM PAO (), 25 µM monensin (), 70 µM cycloheximide (), or monensin and cycloheximide ().

Internalization of fluo-SRIF in sst₅-transfected COS-7 cells
Incubation of transfected COS-7 cells with 10 nM fluo-SRIF for 40 min at 4 C (data not shown) or 37 C (Figs. 3a and 4) resulted in intense fluorescent labeling of approximately 20% of the cells. This labeling was specific, in that it was no longer apparent in the presence of a 100-fold excess (1 µM) of nonradioactive D-Trp⁸-SRIF (Fig. 3b). When the incubation was carried out at 4 C, or at 37 C in the presence of 20 µM PAO or 0.45 M sucrose, the labeling remained confined to the periphery of the cells (Fig. 3c). This peripheral labeling was completely abolished by acid wash, confirming that it was exclusively surface bound (Fig. 3d).

View larger version (81K): [in this window] [in a new window]   Figure 3. Binding of fluo-SRIF to COS-7 cells transfected with cDNA encoding the sst5 receptor. a, Incubation of transfected cells for 40 min at 37 C in the presence of 10 nM fluo-SRIF results in intense intracytoplasmic labeling of approximately 10–20% of the cells. b, This binding is specific, in that it is abolished in the presence of 1 µM of the nonlabeled agonist, D-Trp8-SRIF. c, When the incubation was carried out in the presence of 20 µM PAO, the labeling remained pericellular as opposed to intracellular. d, This pericellular labeling was completely abolished by the hypertonic acid wash, indicating that it is confined to the cell surface. Single optical sections were taken through the center of the cells. Scale bar, 10 µm.

View larger version (69K): [in this window] [in a new window]   Figure 4. Internalization of fluo-SRIF in sst5-transfected COS-7 cells. Incubation of transfected cells with 10 nM fluo-SRIF for 5–40 min at 37 C gives rise to intense, punctate fluorescent labeling, which increases over time (left column). Hypertonic acid wash reveals that the bulk of this labeling is intracellular (right column). After 5- to 10-min incubation, intracellular labeling was mainly found at the periphery of the cells. After 20 min, intensely fluorescent particles filled most of the cytoplasm, sparing the nucleus. At 40 min, fluo-SRIF had accumulated in the perinuclear zone. Single optical sections were taken through the nuclear plane. Scale bar, 10 µm.

Parallel tracking of fluo-SRIF and sst₅ receptor immunoreactivity in the course of ligand-induced internalization
In pulse-chase experiments (n = 3), in which the cells were preincubated at 4 C with or without 10 nM nonlabeled D-Trp⁸-SRIF or with 10 nM fluo-SRIF followed by sst₅ immunolabeling or air-drying, respectively, the fate of the receptor and the ligand in the course of the internalization process were followed in parallel. These experiments were carried out after 90 min of preincubation with 70 µM cycloheximide and in the presence of cycloheximide throughout to suppress receptor neosynthesis during the course of the experiment. Cells incubated with fluo-SRIF were acid-washed after the labeling period for selective visualization of internalized ligand.

At time zero, i.e. at the end of the 4 C pulse, no intracellular ligand fluorescence was evident (Fig. 5, right column). Immunocytochemistry revealed that at this time point, receptors were present both at the cell surface and in a prominent intracellular pool (Fig. 5, left and middle columns). In cells that were not exposed to D-Trp⁸-SRIF, this pattern of receptor distribution showed little change over time (Fig. 5, left column). By contrast, dramatic movements of both receptor and ligand were observed in cells incubated with SRIF (Fig. 5, middle and right columns). At short time intervals (5 and 10 min), internalized fluo-SRIF was confined to the periphery of the cells. At the same time points, sst₅ immunoreactivity was still visible at the cell surface and within the intracellular core. After 20 min of chase at 37 C, internalized fluo-SRIF was visible throughout the cytoplasm, in the form of small fluorescent vesicles. At the same time point, sst₅ immunofluorescence was still evident at the cell surface. However, the brightness of the central cytoplasmic pool of receptor immunofluorescence was greatly reduced, and the whole cytoplasm was pervaded by punctate receptor immunoreactivity. The most dramatic differences between ligand and receptor labeling patterns were observed after long periods of chase (40 and 60 min). At those time points, internalized fluo-SRIF became progressively more concentrated in the cell center, near the nucleus. By contrast, receptor immunoreactivity was entirely confined to the cell periphery.

View larger version (61K): [in this window] [in a new window]   Figure 5. Parallel tracking of fluo-SRIF and sst5 in the course of internalization. In COS-7 cells fixed immediately after pulse labeling with D-Trp8-SRIF at 4 C, no internalization of the ligand is observed (0', right column). At this time point, immunoreactive receptors are present both at the cell surface and in a prominent cytoplasmic pool (0', left and middle columns). In nonstimulated cells, this pattern of receptor distribution changes only minimally over time (left column). By contrast, in stimulated cells, immunoreactive receptors are first (5–20 min) apparent on the plasma membrane and within the cytoplasm and later (40–60 min) exclusively associated with the plasma membrane (middle column). During the same time, internalized fluo-SRIF (right column) is first confined to the cell periphery (5 and 10 min) and is then progressively mobilized toward the center of the cell (20–60 min). All cells exposed to fluo-SRIF were subjected to hypertonic acid wash to strip off surface-bound ligand before air-drying and imaging. All images were taken in the transnuclear plane and acquired using identical settings of the confocal unit. Scale bar, 10 µm.

View larger version (174K): [in this window] [in a new window]   Figure 6. Electron microscopic detection of sst5 immunoreactive receptors without (a) and with (b) exposure to D-Trp8-SRIF. Transfected COS-7 cells were incubated for 30 min at 10 C with Earle’s buffer containing (b) or not (a) 20 nM D-Trp8-SRIF followed by 10 min of chase at 37 C, allowing for internalization to occur. Subsequently, the cells were immunolabeled using the silver-enhanced immunogold technique. In both stimulated and nonstimulated cells, immunolabeling is detected on the plasma membrane as well as in the subplasmalemmal cytoplasm. In cells exposed to buffer alone, intracytoplasmic gold particles are relatively scarce and predominantly associated with vesicular elements (arrows). By contrast, in cells exposed to D-Trp8-SRIF, immunoreactive sst5 receptors are much more abundant than in controls both in association with the plasma membrane and in the subplasmalemmal zone of the cytoplasm (arrows). Scale bar, 450 nm.

View larger version (18K): [in this window] [in a new window]   Figure 7. Effects of SRIF exposure on membrane-associated/intracellular sst5 receptor ratios. Cells were pulse labeled (black bars) or not (white bars) with 20 µM D-Trp8-SRIF at 10 C, followed by chase periods of 0 (baseline), 10, and 40 min at 37 C. Receptors were visualized by electron microscopic immunocytochemistry using immunogold. Ratios between membrane-associated and intracellular receptors were calculated for each time point and each experimental condition after normalizing the number of gold particles associated with the plasmalemma for membrane length and the number of intracellular gold particles for area of cell sampled. Data are expressed as the mean ± SEM of 20 different cells/condition.

View larger version (142K): [in this window] [in a new window]   Figure 8. Electron microscopic distribution of immunoreactive sst5 receptors after 30 min pulse-labeling with D-Trp8-SRIF at 10 C and 10-min (a–c) or 40-min (d) chase at 37 C. a, After 10 min of chase, prominent sst5 immunolabeling is still evident at the level of the plasma membrane. Note the association of intracellular receptors with tubular endosome-like compartments (arrows). b, Also after 10 min of chase, a single large endosome (arrows) containing at least 10 gold particles is observed deeper in the cytoplasm. (c) At the same time point, a gold particle is observed in association with an invaginating coated pit (arrows). d, After 40 min of chase, a clathrin-coated vesicle containing a gold particle is located next to the plasma membrane, whereas other immunoreactive receptors are associated with an endosome (arrows). Scale bars: a, 400 nm; c, 200 nm; b and d, 185 nm.

View larger version (25K): [in this window] [in a new window]   Figure 9. Intracellular mobilization of sst5 receptors in transfected COS-7 cells after ligand exposure. Electron microscopic distribution of intracellular immunoreactive sst5 receptors in cells pulse-labeled or not with D-Trp8-SRIF. Data from one representative experiment corresponding to the number of gold particles counted within bins of distance from the membrane surface. The inset line graphs are a different representation of the same set of data to underscore the dynamics of intracellular receptor trafficking. Immediately after pulse labeling, the maximum intracellular receptor density is found 2–3 µm from the membrane in both stimulated and nonstimulated cells (0 min). In nonstimulated cells, this pattern of distribution remains virtually unchanged throughout the experiment. By contrast, after 10 min of chase at 37 C, D-Trp8-SRIF-stimulated cells show a significant mobilization of intracellular receptors to the subplasmalemmal zone. After 40 min, two peaks of receptor density are apparent, one deep inside the cytoplasm and one at 0.25 µm from the membrane, indicative of a prolonged up-regulation of receptor numbers in the subplasmalemmal zone.

	Discussion

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The pharmacological characteristics of recombinant rat sst₅ in our experiments conformed to those obtained by others in various transfection systems (42; for review, see Ref. 43). Thus, as in previous studies investigating both rat and human sst₅ in transfected CHO-K1 or COS-7 cells (13, 42, 43), SRIF-28 was approximately 5 times as potent as SRIF-14 in displacing radioiodinated Tyr⁰-D-Trp⁸-SRIF from the sst₅ binding site. The synthetic analogs D-Trp⁸-SRIF and Tyr⁰-D-Trp⁸-SRIF showed identical affinities for the receptor and were almost as potent as SRIF-28 (IC₅₀ = 0.6 nM). In CHO-K1 cells, D-Trp⁸-SRIF exhibited an even higher potency in displacing [¹²⁵I]CGP23996 from the sst₅-binding site and was more potent in doing so than SRIF-28 itself (IC₅₀ = 0.002 nM) (42). Finally, the fluorescent ligand employed in our study, -Bodipy Red-D-Trp⁸-SRIF, was virtually equipotent with SRIF-14 in competing for [¹²⁵I]D-Trp⁸-SRIF binding at the sst₅ site, validating its usefulness as a high affinity marker of sst₅ receptors.

Previous studies have demonstrated that both human (27, 39) and rat (29, 30) sst₅ are internalized in the presence of SRIF in a variety of cell lines. In HEK cells transfected with cDNA encoding rat sst₅ (30) as well as in RIN 1046–38 insulinoma cells transfected with tagged rat sst₅ (29), this process was ligand specific, in that internalization was observed after exposure to SRIF-28, but not to SRIF-14. The present results indicate that in COS-7 cells expressing the rat sst5 receptor, as in CHO-K1 cells expressing the human sst5 receptor (39), D-Trp⁸-SRIF-14 is able to efficiently induce sst₅ internalization. Whether this property is dependent upon the cell lines used or reflects specific properties of this SRIF analog, which could be linked to its near equipotency with SRIF-28 at the sst₅ binding site, remains to be determined.

The internalization process observed in sst₅-transfected COS-7 cells is rapid, reaching a plateau within 20 min (t_1/2 = 10 min). These kinetics are comparable to those of SRIF internalization via the rat sst_2A receptor in the same cell type (28). With 45% of total specifically bound radioactivity, however, internalization via sst₅ does not reach the same capacity as sst_2A-mediated endocytosis (85%; 28). In CHO-K1 cells transfected with individual human sst subtypes, on the other hand, sst₅ internalized more efficiently than sst_2A (66%; 27).

As in the case of sst_2A (28), fluo-SRIF internalization via sst₅ was entirely blocked by incubation with the endocytosis inhibitor PAO or in hypertonic sucrose, indicating that it is dependent on the formation of clathrin-coated pits (41). Immunoelectron microscopic experiments confirmed that this was indeed the case, by demonstrating SRIF-induced mobilization of sst₅ in clathrin-coated pits and vesicles. Molecular studies previously demonstrated the presence of both positive and negative sequence motifs for internalization in the intracellular C-terminal domain of the cloned human and rat sst5 receptors (30, 39), indicating a pivotal role for the C-terminus in mediating internalization. Moreover, a variant of the consensus sequence NPXXY for binding of G protein-coupled receptors to the clathrin-adaptor molecule ß-arrestin (for review, see Refs. 34, 41) is present in position 300–304 (in fact, NPLLY) at the C-terminal end of the cloned rat receptor (13) in exactly the same position as in the human sst5 molecule (39). Although direct evidence is still missing, these data suggest that sst5 might, like other G protein-coupled receptors, interact with clathrin through binding to ß-arrestin.

Combined confocal microscopic tracking of fluo-SRIF and sst₅ receptors in cells pretreated with the protein synthesis inhibitor cycloheximide allowed us to determine the fate of receptor-ligand complexes after ligand-induced internalization. Five to 10 min after transfer of fluo-SRIF-labeled COS-7 cells from 4 to 37 C, internalized ligand was confined to the periphery of the cells in the form of small fluorescent particles. By 20 min after the onset of internalization, these endosome-like particles pervaded most of the cytoplasm. Finally, after longer time intervals (40 and 60 min), the ligand had accumulated inside the cytoplasm and concentrated in the center of the cells in close proximity to the nucleus. This course of events is similar to that observed in the same type of cells transfected with cDNA encoding the rat sst_2A receptor (28). By contrast, cells transfected with the sst₁ receptor barely sequester fluo-SRIF (28). A pattern of endocytic events similar to that seen here was also observed in COS-7 cells transfected with the NT1 neurotensin receptor (44), in CHO cells transfected with the cholecystokinin receptor (45), and in Kirsten murine sarcoma virus-transformed rat kidney (KNRK) cells ectopically expressing the substance P receptor (46). In all of these cell systems, the fluorescent ligand was targeted to a perinuclear compartment after prolonged internalization periods. However, whereas in NT1-transfected cells this juxtanuclear compartment was interpreted as belonging to the trans-Golgi network by virtue of its immunoreactivity for syntaxin-6 (44), in CHO and KNRK cells, it was found to correspond to lysosomes (45, 46). Conceivably, the fate of a metabolically protected ligand such as D-Trp⁸-SRIF, which was used in our experiments, may differ from that of native SRIF, in that the latter may be more readily targeted to lysosomes and degraded than the former. However, recent studies on CHO cells expressing the sst2 receptor subtype have shown iodinated native SRIF-14 to be internalized, recycled to the extracellular medium, and reinternalized several times (47), suggesting that even native peptide may be targeted to recycling compartments. Clearly, further experiments using native ligands are needed to determine the fate of SRIF internalized via the sst5 receptor subtype under physiological conditions.

The pattern of sst₅ internalization, as visualized here by receptor immunofluorescence, differed sharply from that of its fluorescent ligand. At time zero, sst5 immunoreactivity was concentrated at the level of the plasma membrane as well as in a very prominent cytoplasmic pool in the core of the cell. The existence of such a pool of intracellular sst₅, even after prolonged preincubation with cycloheximide, was previously reported by others in different cell lines (29) and presumably largely corresponds to trans-Golgi network/Golgi stores. After exposure to the ligand, there was very little change in the intensity of cell surface labeling. However, the intracellular cytoplasmic pool became progressively less prominent as most of the immunoreactivity accumulated at the periphery of the cell. The lack of an apparent decrease in cell surface labeling was unlike what had previously been reported for either sst_2A (35) or sst₃ (29, 30) receptors expressed either naturally or ectopically in other cell lines. It also differed from the confocal microscopic pattern of ligand-induced internalization reported for other G protein-coupled receptors, such as the neurokinin-1 receptor (46), the ß-adrenergic receptor (48), or the TRH receptor (49), all cases for which a significant decrease in cell surface labeling was observed in the initial stages of internalization. The question arose as to whether the preservation of cell surface receptors observed here in the case of sst₅ could be attributed to receptor recycling and/or to a mobilization of receptors from intracellular stores to the cell surface.

To determine the extent to which receptor recycling contributed to the preservation of cell surface labeling, internalization assays were carried out in the presence and the absence of the recycling inhibitor monensin. In the absence of monensin, [¹²⁵I]SRIF association with sst₅-transfected COS-7 cells did not reach equilibrium, even at concentrations of the ligand as high as 1 µM. This lack of saturability was clearly a consequence of the internalization process, as in the presence of the endocytosis inhibitor PAO, saturation was reached. In the presence of monensin, association of [¹²⁵I]SRIF with whole cells was also saturable and amounted to roughly 40% of the binding observed at equimolar concentrations of the ligand in the absence of the drug. These results demonstrate that receptor recycling accounts for part of the maintenance of cell surface receptors in the course of internalization, but that other mechanisms, such as targeting of receptors from an intracellular reserve pool to the membrane, must also be invoked.

To further investigate this idea, we carried out an immunoelectron microscopic study of sst₅ after pulse-chase stimulation with SRIF. These experiments confirmed that sst₅ receptors were present at the plasma membrane at all times, even after ligand exposure. In fact, they revealed that there was even an overshoot in receptor targeting to the membrane at 10 min of incubation, as reflected by the ratio between cell surface-associated and intracellular receptors, which rose from approximately 0.2 at 0 min to almost 0.9 after 10 min of incubation with the ligand. This effect was accompanied by a redistribution of receptor molecules inside the cytoplasm, which, from a peak distance of 2–3 µm from the plasma membrane at time zero, shifted to a peak distance of 0.25–0.5 µm at 10 min, suggesting a massive recruitment of reserve receptors to the plasmalemma. At 40 min, the intracellular receptor distribution had started to return to normal, as reflected by the presence of two peaks, one still close to the membrane (as seen at 10 min), but the second deeper inside the cell.

These joint recycling and recruitment mechanisms, providing for continuous availability of functional receptors at the cell surface even during massive and prolonged ligand exposure, have profound implications for receptor function, in that they might effectively protect the receptor from long term desensitization. It is interesting to recall, in this context, that the rapidly desensitizing receptor subtype sst_2A (28, 45, 50, 51) acts together with sst₅ to regulate GH release from the anterior pituitary (17). It may be that in cells such as these, and perhaps also in regions of the brain in which sst_2A and sst₅ coexist (21, 22, 26), sst₅ actually provides for a prolongation of SRIF signaling after sst_2A has been down-regulated.

In conclusion, the present study demonstrates that SRIF is rapidly and efficiently internalized via the sst₅ receptor in a heterologous expression system. It shows that this endocytosis is mediated through a clathrin-dependent mechanism. A major finding is that in response to agonist stimulation and endocytosis, internalized sst₅ receptors are rapidly recycled back to the cell surface. At the same time, spare receptors from an intracytoplasmic reserve pool are addressed to the plasma membrane. The combination of these two processes provides for the continuous presence of sst₅ receptors at the cell surface and, therefore, accounts for the lack of saturability of SRIF binding to intact cells expressing sst₅.

	Acknowledgments

 
We thank Dr. A.M. O’Carroll for providing us with the sst5 plasmid and Mariette Houle and Hans-Hinrich Hönck for excellent technical assistance.

	Footnotes

 
¹ This work was supported by grants from the Medical Research Council of Canada (to A.B.; MT-7366) and the Deutsche Forschungsgemeinschaft (to H.-J.K.; Ha 2445/1–1). Travel between A.B.’s and J.M.’s laboratories was supported by an INSERM/FRSQ exchange program. T.S. was funded by a research fellowship from the Deutsche Forschungsgemeinschaft (Str 565/1–1).

Received June 16, 1999.

	References

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