help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nouel, D.
Right arrow Articles by Beaudet, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nouel, D.
Right arrow Articles by Beaudet, A.
Endocrinology Vol. 138, No. 1 296-306
Copyright © 1997 by The Endocrine Society

ARTICLES

Differential Internalization of Somatostatin in COS-7 Cells Transfected with SST1 and SST2 Receptor Subtypes: A Confocal Microscopic Study Using Novel Fluorescent Somatostatin Derivatives1

Dominique Nouel, Georges Gaudriault, Mariette Houle, Terry Reisine, Jean-Pierre Vincent, Jean Mazella and Alain Beaudet

Montreal Neurological Institute, McGill University (D.N., M.H., A.B.), Montreal, Quebec, Canada H3A 2B4; Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice-Sophia Antipolis (G.G, J.-P.V., J.M.), Valbonne, France; and the Department of Pharmacology, University of Pennsylvania School of Medicine (T.R.), Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Dr. Alain Beaudet, Department of Neurobiology, McGill University, Montreal Neurological Institute, 3801 University Street, Montreal, Quebec, Canada H3A 2B4. E-mail: MCIN{at}MUSICA.MCGILL.CA


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A growing body of evidence suggests that neuropeptide binding to G protein-linked receptors may result in internalization of receptor-ligand complexes, followed by intracellular mobilization and degradation of the ligand into its target cells. Because of discrepant results in the literature concerning the occurrence of such a mechanism for the tetradecapeptide somatostatin (SRIF), we have reinvestigated this question by comparing the binding and internalization of iodinated and fluorescent derivatives of the metabolically stable analog of SRIF, [D-Trp8]SRIF, in COS-7 cells transfected with complementary DNA encoding the sst1 or sst2A receptor subtype. A series of fluoresceinyl and Bodipy fluorescent derivatives of [D-Trp8]SRIF-14 was purified by HPLC, analyzed for purity by mass spectrometry, and tested for biological activity in a membrane binding assay. Of the six compounds tested, fluoresceinyl and Bodipy derivatives labeled in position {alpha} (fluo-SRIF) retained high affinity for SRIF receptors. COS-7 cells transfected with complementary DNA encoding either sst1 or sst2A receptors both displayed specific, high affinity binding of iodinated and fluo-SRIF. At 4 C, the labeling was confined to the cell surface in both cell types, as indicated by the fact that it was entirely removable by a hypertonic acid wash and assumed a pericellular distribution in the confocal microscope. At 37 C, the fate of specifically bound ligand varied markedly according to the type of receptor transfected. In cells encoding the sst1 receptor, approximately 20% of specifically bound ligand was recovered in the acid-resistant (i.e. intracellular) fraction. This fraction remained clustered at the periphery of the cell, suggesting that it was being sequestered either within or immediately beneath the plasma membrane. By contrast, in cells transfected with sst2A receptors, up to 75% of specifically bound ligand was recovered inside the cells, where it clustered into small endosome-like particles. These particles increased in size and moved toward the nucleus with time, suggestive of receptor-ligand complexes proceeding down the endocytic pathway. These results demonstrate that neuropeptides may be processed differently depending on the subtype of receptor expressed in their target cells and suggest that these different processing patterns may reflect different modes of sensitization/desensitization and recycling of the receptors, and thereby of transmembrane signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW clearly established that both forms of the GH inhibitory peptide somatostatin, the tetradecapeptide somatostatin-14 (SRIF) and its N-terminally extended form SRIF-28, exert their central and peripheral effects through G protein-coupled receptors. These receptors have been shown to be linked to a variety of effector systems including adenylate cyclase, Ca2+ and K+ channels, Na+-H+ exchanger, and tyrosine phosphatases (for reviews, see Refs. 1–3).

Recent molecular biological studies have demonstrated the existence of five different SRIF receptor subtypes with distinct central and peripheral distributions. These cloned receptors have been termed sst1 (4), sst2 (4), sst3 (5, 6), sst4 (7), and sst5 (8, 9) according to the order in which they were isolated. The sst2 receptor comprises two isoforms generated from the same gene; sst2A, the unspliced form, and sst2B, a 23-amino acid shorter splice variant (10, 11). Sequence and hydropathy analyses have confirmed that all of these SRIF receptor subtypes contain the seven putative transmembrane domains characteristic of other G protein-coupled receptors. The cloned SRIF receptors have a high degree of amino acid and structural homology among themselves; however, it is believed that they may represent a unique neurosecretagogue receptor subfamily, as their sequences differ from those of any other known receptors (12, 13). Pharmacological studies in cells transfected with complementary DNA (cDNA) encoding the various SRIF receptor subtypes have demonstrated that the sst2A/B, sst3, and sst5 receptors correspond to the so-called type 1 subclass previously defined as displaying a relatively high affinity for the biologically stable synthetic SRIF agonists MK678 and octreotide (14, 15). These receptors are desensitizable and are sensitive to GTP and divalent cations (14, 5). The sst1 and sst4 receptors comprise the type 2 subclass, which displays relatively lower affinity for MK678 and octreotide and is not desensitizable (14, 15).

Data from studies in cell culture systems and in tissue preparations have demonstrated, both biochemically and morphologically, that various ligands interacting with G protein-coupled receptors rapidly enter their target cells after binding to cell surface receptors. This process, referred to as receptor-mediated internalization, is believed to involve local clustering of receptors followed by their endocytosis through clathrin-coated pits (for review, see Ref.16). Ligand and/or receptor internalization has been described for a variety of neuropeptides, including gonadotropin- (17), corticotropin- (18), and thyrotropin-releasing hormones (19), as well as substance P (20, 21, 22), cholecystokinin (23, 24), vasopressin (25), angiotensin II (26), vasoactive intestinal peptide (27) glucagon-like peptide-2 (28), gastrin-releasing peptide (29), and neurotensin (30, 31, 32, 33) in cell cultures in vitro and in whole organs in vivo.

Whether SRIF is internalized in a comparable manner after ligand-receptor interaction has been a matter of debate. Several reports indicate that SRIF is internalized in pancreatic (34, 35), hypophyseal (36, 37, 38), and human carcinoid tumor (39) cells after application of exogenous SRIF ligands in vitro. In vivo peptide scintigraphic data also argue in favor of SRIF internalization (40). Other reports, to the contrary, indicate that SRIF is not internalized to any appreciable extent in either GH4C1 pituitary cells (41) or RINm5F insulinoma cells (42). A possible interpretation for these discrepancies is that there exists a differential capacity of the various SRIF receptor subtypes to internalize SRIF and that the observed tissue variations merely reflect differences in the expression of these subtypes. To test this hypothesis, we compared the binding and internalization of iodinated and fluorescent analogs of the metabolically stable SRIF agonist [D-Trp8]SRIF-14 in COS-7 cells transfected with sst1 and sst2A receptor subtypes, taken as representatives of SRIF-2 and SRIF-1 classes of receptors, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthesis and purification of fluorescent [D-Trp8]SRIF derivatives
A battery of new fluorescent derivatives (fluo-SRIF) of the metabolically stable analog of somatostatin-14, [D-Trp8]SRIF, were synthesized and tested in the present experiments. The first series was prepared by incubating [D-Trp8]SRIF (0.6 µmol; Neosystem, Strasbourg, France) with N-hydroxy-succinimide (NHS)-fluorescein (1 µmol diluted in 200 µl acetonitrile; Pierce, Toronto, Canada) in a final volume of 1 ml borate (50 mM)-phosphate (50 mM), pH 6.5, for 3 h at 4 C. Labeled derivatives were purified by HPLC on a C18 column (10 x 250 mm; Ultrosphere, ODS, Beckman, Mississauga, Canada) and eluted in 0.1% trifluoroacetic acid with a linear gradient of acetonitrile from 20–70% for 100 min. Peaks of elution were then submitted to Edman degradation to determine the position of the labeling.

A second series of fluorescent conjugates was prepared as described above, except that NHS-fluorescein was replaced by the NHS ester of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-3-propionic acid (Bodipy FL C3-SE, Molecular Probes, Eugene, OR). Here again, labeled derivatives were purified by HPLC, and elution peaks were subjected to Edman degradation. The amino acid composition was further verified by amino acid analysis, and the mol wt was measured by laser desorption mass spectrometry. A red fluorescent analog of SRIF was also prepared and purified as described above by reacting [D-Trp8]SRIF with the NHS ester of Bodipy 576/589 C3. The binding properties of this analog were undistinguishable from those of the green fluorescent Bodipy FC C3 (results not shown).

Cell culture
COS-7 cells were grown in DMEM containing glutamine and supplemented with 44 mM NaHCO3, 10% FCS, and 50 mg/liter gentamicin. Twenty-four hours before transfection, the cells were plated in 100-mm diameter petri dishes at a density of 106 cells/dish. When semiconfluent, cells were transfected with cDNA encoding either the sst1 or the sst2A receptor subtype. For this purpose, the PstI-XmnI cDNA fragment (1.5 kilobases) bearing the coding region for the human SSTR1 was inserted into the BamHI site of the pCMV-6b expression vector after addition of BglII linkers. The XbaI cDNA fragment encoding the mouse SSTR2a was subcloned into the corresponding site of the pCMV-6c expression vector. Transfections were carried out by incubating the cells for 30 min at room temperature with 1 ml Tris-buffered saline containing 1 µg recombinant plasmid and 0.5 mg/ml diethylaminoethyl-dextran. At the end of the incubation, the buffer was replaced by culture medium containing 100 µM chloroquine, and the cells were further incubated at 37 C for at least 3 h. They were then rinsed in TBS, covered with culture medium, and grown at 37 C for approximately 60 h.

Binding properties of fluo-SRIF derivatives
The capacity of the different HPLC eluates of fluorescent SRIF to compete with iodinated SRIF binding was tested on membranes prepared from adult rat cerebral cortex. For membrane preparation, rats were killed by decapitation, and the brains were rapidly removed. Cerebral cortex was dissected on ice and placed in a 10-fold volume of 5 mM Tris-HCl supplemented with 2 mM EDTA, homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) for 20 sec, and centrifuged at 15,000 x g for 15 min at 4 C. The pellet was then resuspended in the same volume of buffer, recentrifuged for 15 min at 15,000 x g, and frozen until use.

Binding experiments were carried out by incubating 50 µg membranes with 0.3 nM 125I-labeled Tyr0-[D-Trp8]SRIF (2000 Ci/mmol) in the presence of increasing concentrations of fluorescent peptide (10-12–10-6 M). Tyr0-[D-Trp8]SRIF was iodinated by the chloramine-T method as previously described (43). Monoiodo-[125I]Tyr0-[D-Trp8]SRIF ([125I]SRIF) was purified by HPLC on a C18 column (Lichrosorb, Merck, Clevenot, France) to exclude any possible contamination resulting from chloramine-T-induced destruction of the tryptophan side-chain. The iodinated peptide was eluted in 0.05% triethylamine-0.1% trifluoroacetic acid with a linear gradient of acetonitrile from 10–50% for 56 min. (elution time, 37 min). Binding was carried out for 45 min at room temperature in 250 µl 50 mM Tris-HCl (pH 7.5) containing 2 mM MgCl2, 0.1% BSA, and 1 mM 1,10-phenanthroline to prevent peptide degradation. Experiments were terminated by the addition of 2 ml ice-cold buffer followed by filtration through glass microfiber filters preincubated in 0.5% polyethylenimine (GF/C, Whatman, Clifton, NJ). Filters were then washed twice with ice-cold buffer, and the radioactivity retained in them was counted with a {gamma}-counter. Non specific binding was measured in the presence of 1 µM nonradioactive [D-Trp8]SRIF.

Binding of [125I]SRIF to transfected COS-7 cells
To compare the affinity and efficiency of [125I]SRIF binding to COS-7 cells transfected with the sst1 and sst2A subtypes, the first set of binding assays was carried out on membranes freshly prepared from transfected COS-7 cells. For this purpose, the cells were scraped off the culture dishes with PBS and centrifuged at 5000 x g for 5 min, and the pellets were homogenized by incubation in a hypotonic 5 mM Tris-HCl buffer (pH 7.5) containing 5 µg/ml deoxyribonuclease I for 30 min at 0 C. Membrane homogenates were then recovered by centrifugation at 15,000 x g for 30 min at 4 C. Binding assays were carried out as described above by incubating 10 µg membranes with concentrations of [125I]SRIF ranging between 20 pM and 1 nM for 30 min at room temperature.

To demonstrate [125I]SRIF internalization, a second set of binding experiments was carried out on whole cells at 37 C. The culture medium was discarded from 12-mm diameter dishes containing 2 x 105 transfected COS-7 cells, and the 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, and 25 mM HEPES, pH 7.4) supplemented with 0.1% glucose and 1% BSA in the presence or absence of 10 µM of the endocytosis inhibitor, phenylarsine oxide (PAO). Equilibration buffer was then replaced by 250 µl binding buffer containing 0.3 nM [125I]SRIF in the presence of 1 mM 1,10-phenanthroline for intervals ranging between 3–60 min. At the end of the incubation, the cells were washed twice for 2 min each time with 1 ml pure equilibration buffer with a hypertonic acid (pH 4) buffer consisting of 0.2 M acetic acid and 0.5 M NaCl in Earle-Tris-HEPES buffer to strip off surface-bound radioactivity (30). Cells were harvested with 1 ml 0.1 M NaOH, and associated radioactivity was counted in a {gamma}-counter. Nonspecific binding was measured in the presence of 1 µM unlabeled [D-Trp8]SRIF.

Binding of fluo-SRIF to transfected COS-7 cells
Dissociated COS-7 cells transfected with cDNA encoding sst1 or sst2A receptors, nontransfected cells, and cells transfected with neurotensin in lieu of sst receptor cDNA, were preincubated for 10 min at 37 C in Earle’s buffer containing 0.2% BSA and 0.1% glucose in the presence or absence of 10 µM PAO. They were then incubated for 45 min at 37 C with 5 nM of either {alpha}-fluoresceinyl-[D-Trp8]SRIF-14 (FTC-SRIF) or {alpha}-Bodipy-[D-Trp8]SRIF-14 (Bodipy-SRIF), both generically referred to hereafter as fluo-SRIF, in the same buffer, again supplemented or not with PAO. For determination of nonspecific labeling, 1 µM [D-Trp8]SRIF was added to the incubation medium. To examine temperature dependence, additional cells were incubated in parallel at 4 C. Incubation was terminated by centrifugation at 500 x g for 10 min, followed by a 2-min rinse in Earle’s buffer at 4 C. Cells were then recentrifuged for 5 min at 500 x g, and the pellets were suspended in 20 µl fresh buffer, deposited on glass microscope slides, air-dried, and examined under the confocal microscope.

To selectively visualize internalized fluorescent molecules, transfected COS-7 cells were grown as a monolayer on 12-mm polylysine-treated glass coverslips to 80% confluence in 18-mm petri dishes, incubated with fluo-SRIF as described above, but washed for 2 min with hypertonic acid buffer, before air drying and confocal microscopic examination.

Confocal microscopy
Labeled COS-7 were examined under a Leica confocal laser scanning microscope (CLSM) configured with a Leica Diaplan inverted microscope equipped with an argon/krypton laser with an output power of 2–50 mV and a VME bus MC 68020/68881 computer system coupled to an optical disc for image storage (Leica, St. Laurent, Canada). All image-generating and processing operations were performed with the Leica CLSM software package. Micrographs were taken from the image monitor using a Focus Imagecorder (Foster City, CA). Images were acquired as single optical sections taken through the middle of the cells and averaged over 32 scans/frame. For all types of acquisitions, the gain and black levels were set manually to optimize the dynamic range of the image while ensuring that no region was completely suppressed (intensity = 0) or completely saturated (intensity = 255).

Quantitative data on the number of intracellular fluorescent particles, the area of particles, and the mean distance of each particle from the center of the cell were obtained using the Leica CLSM software. Results were expressed as the mean ± SEM of measurements in 5–11 labeled cells from 3 different experiments. Statistical analyses were performed using a one-way ANOVA, followed by a regression curve analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Molecular characterization of fluorescent [D-Trp8]SRIF derivatives
Purification of fluoresceinyl-[D-Trp8]SRIF conjugates by HPLC yielded four distinct peaks eluting at 35, 45, 47, and 50 min, respectively (Fig. 1Go). The first of these peaks (peak N; elution time, 35 min) was found by Edman degradation to correspond to native [D-Trp8]SRIF. The second fraction (peak 1; elution time, 45 min) was resistant to phenylisothiocyanate and was thereby ascribed to {alpha}-fluoresceinyl-[D-Trp8]SRIF. The third fraction (peak 2, elution time, 47 min) had the expected sequence of [D-Trp8]SRIF, except for the fourth phenylthiohydanthoin amino acid, which corresponded to none of the 20 natural amino acids. This derivative was, therefore, taken to correspond to {epsilon}4-fluoresceinyl-[D-Trp8]SRIF. Similarly, the fourth fraction (peak 3; elution time, 50 min) was identified as {epsilon}9-fluoresceinyl-[D-Trp8]SRIF. The three fluoreceinyl peptides were further characterized by amino acid analysis and mass spectroscopy, which confirmed that the {alpha}-, {epsilon}4-, {epsilon}9-fluoresceinyl derivatives were isomers (same mass and same amino acid content) and that each fluorescent peptide contained a single fluoresceinyl group per mol peptide.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Purification of fluoresceinyl analogs of [D-Trp8]SRIF by reverse phase HPLC. Incubation and elutions were carried out as described in Materials and Methods. Elution was followed by automatic recording of optical density at 230 nm. Fractions (3 ml) were collected, lyophilized, and reconstituted in 100 µl dimethylsulfoxide for further analysis. Fluorescent peptides were protected from photobleaching by working either in the dark or under dim light. Fractions corresponding to peaks N, 1, 2, and 3 were identified as native [D-Trp8]SRIF and {alpha}-, {epsilon}4-, and {epsilon}9-fluoresceinyl-[D-Trp8]SRIF, respectively. Fluorescent peptides were stored in a dry state at -20 C.

 
The {alpha}-, {epsilon}4- and {epsilon}9-Bodipy derivatives of [D-Trp8]SRIF were purified by HPLC and chemically characterized as described above for the corresponding fluorescein-labeled analogs. Complete purification of the three Bodipy-labeled peptides was easily achieved as the observed elution times of each HPLC fractions were clearly different (35, 44, 58, and 63 min for native SRIF and its {alpha}-, {epsilon}4-, and {epsilon}9-Bodipy analogs, respectively).

Pharmacological characterization of fluorescent [D-Trp8]SRIF derivatives
When tested for its ability to compete with specific binding of [125I]SRIF to membranes of rat cerebral cortex, {alpha}-fluoresceinyl-[D-Trp8]SRIF was 16 times less potent than unlabeled [D-Trp8]SRIF (Fig. 2AGo and Table 1Go). {epsilon}4-Fluoresceinyl-[D-Trp8]SRIF also competed with specific [125I]SRIF binding, but with an apparent affinity 2–3 times lower than that of the {alpha}-fluoresceinyl peptide (Fig. 2AGo and Table 1Go). By contrast, the {epsilon}9-analog competed extremely poorly with [125I]SRIF binding (Fig. 2AGo and Table 1Go).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Competition of fluoresceinyl-[D-Trp8]SRIF (A) and Bodipy-[D-Trp8]SRIF (B) derivatives with [125I]SRIF-specific binding to rat brain homogenates. Rat brain homogenates (50 µg protein) were incubated with 0.3 nM [125I]SRIF (2000 Ci/mmol) and the indicated concentrations of unlabeled peptide for 45 min at 25 C in a total volume of 0.25 ml Tris-HCl, pH 7.5, containing 0.2% BSA and 2 mM MgCl2. The incubation medium was then diluted in 4 ml ice-cold incubation buffer and filtered under reduced pressure on GF/C filters (Whatman) preincubated in 0.5% polyethylenimine. Filters were rinsed twice with 4 ml ice-cold buffer and counted in a {gamma}-counter. Data from one representative experiment are shown. A: •, [D-Trp8]SRIF; {square}, {alpha}-fluoresceinyl [D-Trp8]SRIF; {triangleup}, {epsilon}4-fluoresceinyl-[D-Trp8]SRIF; {triangledown}, {epsilon}9-fluoresceinyl-[D-Trp8]SRIF. B: •, [D-Trp8]SRIF; {square}, {alpha}-Bodipy-[D-Trp8]SRIF; {triangleup}, {epsilon}1-[D-Trp8]SRIF; {triangledown}, {epsilon}2-[D-Trp8]SRIF.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Comparative potency of SRIF fluorescent derivatives in competing for the binding of 125I-labeled [D-Trp8]SRIF-14 in rat cortical homogenates

 
{alpha}-Bodipy-[D-Trp8]SRIF competed for [125I]SRIF binding with the highest affinity of all of the fluorescent ligands tested (Fig. 2BGo and Table 1Go). This affinity, however, was still 4 times lower than that of the unlabeled peptide. The {epsilon}4-Bodipy derivative competed for [125I]SRIF binding with an affinity comparable to that of the {alpha}-fluoresceinyl compound. Its solubility, however, was considerably less than that of the {alpha}-fluoresceinated derivative, which precluded its use in biological assays. Finally, as with fluorescein, Bodipy labeling of [D-Trp8]SRIF in {epsilon}9 completely abolished the capacity of the peptide to compete with [125I]SRIF binding (Fig. 2BGo and Table 1Go). {alpha}-Fluoresceinyl-[D-Trp8]SRIF and {alpha}-Bodipy-[D-Trp8]SRIF were used interchangeably in the imaging studies described below and are referred to in the text under the generic term fluo-SRIF.

Binding of [125I]SRIF to COS-7 cells transfected with cDNA encoding sst1 or sst2A receptor subtypes
[125I]SRIF bound specifically to membrane homogenates from COS-7 cells transfected with cDNA encoding either sst1 or sst2A receptor subtypes, with Kd values of 0.68 and 0.28 nM, respectively. In both types of cells, the overall maximal binding capacity was on the order of 1 pmol/mg whole cell protein, which, based on an average protein content of 50 µg for 2 x 105 cells, corresponds to 1.5 x 105 sites/cell.

[125I]SRIF also bound specifically to whole sst1-expressing cells incubated at 37 C. As shown in Fig. 3AGo, this specific binding reached a plateau between 40–60 min. Hypertonic acid washes of the bound radioactivity at various time points indicated that the bulk of specifically bound radioactivity remained extracellular (i.e. acid washable) at all times (Fig. 3AGo). Experiments in which the cells were pretreated with the endocytosis inhibitor PAO before the incubation with the radioactive ligand yielded a saturation curve comparable to that of the acid-washable fraction, confirming that the greatest proportion of the ligand had remained surface bound (Fig. 3AGo). Accordingly, the amount of the intracellular (i.e. acid-resistant) fraction remained low throughout, accounting for less than 20% of the specifically bound radioactivity after 45 min of incubation (Fig. 4Go). The radioactivity associated with cells preincubated with PAO was entirely acid washable, confirming the efficacy of PAO in preventing internalization of [125I]SRIF into the cells (Fig. 3AGo).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Binding of [125I]SRIF to COS-7 cells transfected with sst1 (A) or sst2A (B) receptor subtype: cell surface binding profiles. Transfected cells were incubated with 0.3 nM [125I]SRIF at 37 C in the presence or absence of the endocytosis inhibitor PAO. At the indicated time, cells were washed twice with binding buffer, incubated for 2 min at 37 C with an acid-NaCl buffer, and washed twice with binding buffer. The radioactivity that remained bound to the cells was recovered with 0.1 M NaOH and counted in a {gamma}-counter. Nonspecific [125I]SRIF binding was determined in parallel at each time by incubation in the presence of 1 µM nonradioactive [D-Trp8]SRIF and accounted for less than 25% of total. Values are the mean ± SEM of three different experiments. Maximal specific binding at saturation, 26,000 cpm. •, Specific binding; {triangleup}, acid NaCl-washable binding; {blacksquare}, binding in the presence of PAO; {square}, acid NaCl-resistant binding in the presence of PAO.

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 4. Binding of [125I]SRIF to COS-7 cells transfected with sst1 ({diamond}) or sst2A ({diamondsuit}) receptor subtypes: internalization profiles. Experiments were the same as described in Fig. 3Go. The acid NaCl-resistant fraction is expressed as a percentage of specific binding. Note that the efficiency of [125I]SRIF internalization is almost 4 times greater in cells transfected with the sst2A than the sst1 receptor.

 
Binding of [125I]SRIF to COS-7 cells transfected with cDNA encoding the sst2A receptor was also specific and saturable (Fig. 3BGo). However, in contrast to what was observed in sst1-expressing cells, an important fraction of the specifically bound radioactivity remained acid resistant throughout. This fraction of internalized radioactivity increased rapidly with time (t1/2 = 8–10 min) to reach a plateau at 40 min, at which time it accounted for almost 75% of the total amount of radioactivity specifically associated with the cells (Fig. 4Go). Consequently, the acid-washable fraction of the bound radioactivity followed a reverse trend, as did the saturation profile observed in the presence of PAO (Fig. 3BGo). Finally, virtually all the radioactivity specifically bound in the presence of PAO was acid wash-sensitive, confirming the efficacy of PAO in blocking the internalization of [125I]SRIF (Fig. 3BGo).

Confocal microscopic visualization of fluo-SRIF binding to and internalization in transfected COS-7 cells
After incubation of COS-7 cells transfected with cDNA encoding either sst1 or sst2A receptor subtype in the presence of 5 nM of fluo-SRIF for 45 min at 4 or 37 C, 25–30% of the cells exhibited intense fluorescent labeling (Fig. 5Go). This labeling was specific, in that it was no longer apparent when the incubation was carried out in the presence of 1 µM unlabeled [D-Trp8]SRIF (Fig. 6Go, C and D) or in cells either nontransfected or transfected with an irrelevant receptor (not shown). After incubation at 4 C, the labeling was confined to the cell surface, as demonstrated by confocal imaging of midcellular planes (Fig. 6Go, A and B) and confirmed by its total abolition after hypertonic acid wash (Fig. 6Go, E and F).



View larger version (110K):
[in this window]
[in a new window]
  		Figure 5. Binding of Bodipy-SRIF to COS-7 cells transfected with sst1 (A and A') or sst2A (B and B') receptor subtype. Cells were incubated for 45 min at 37 C with 5 nM fluorescent ligand. A and B, Nomarsky optics. A' and B', Epifluorescence. Note that only a subpopulation (~30%) of the cells have internalized the fluorescent ligand (arrows) in conformity with reported transfection yields in this cell line (64). Scale bar = 20 µm.

 


View larger version (81K):
[in this window]
[in a new window]
  		Figure 6. Confocal microscopic imaging of FTC-SRIF binding to COS-7 cells transfected with cDNA encoding sst1 (A, C, and E) or sst2A (B, D, and F) receptor subtypes at 4 C. Images were acquired as single midcellular optical sections at 32 scans/frame. In both types of transfected cells, the labeling is confined to the cell surface, along which it forms a more or less continuous ring (A and B). This labeling is no longer apparent in cells incubated in the presence of an excess of nonfluorescent ligand (C and D) or subjected to hypertonic acid wash to strip the cells from surface binding (E and F). Scale bar = 10 µm.

 
COS-7 cells transfected with cDNA encoding the sst1 receptor and incubated with fluo-SRIF for 1–60 min at 37 C exhibited intense pericellular labeling at all times examined (Fig. 7AGo). This pericellular ring was no longer apparent in cells incubated in the presence of an excess of nonfluorescent ligand or in cells transfected with an irrelevant receptor (not shown). At short time intervals (1–5 min), the labeling was almost completely abolished by a hypertonic acid wash, indicating that the bulk of the labeling was extracellular (Fig. 7BGo). The remaining bound fluorescent molecules were typically clustered at the periphery of the cells (Fig. 7BGo). At longer time intervals (10–60 min), a greater proportion of the original labeling was still detected after acid wash (Fig. 7CGo). However, it remained confined to the periphery of the cells, in a pattern undistinguishable from that observed before acid wash in cells incubated at 4 C (Fig. 6AGo) or in which receptor internalization was prevented by treatment with PAO (Fig. 7DGo).



View larger version (128K):
[in this window]
[in a new window]
  		Figure 7. Confocal microscopic imaging of specific binding of FTC-SRIF to COS-7 cells transfected with cDNA encoding the sst1 receptor at 37 C. Images were acquired as single midcellular optical sections at 32 scans/frame. Total binding (A) is confined to the periphery of the cells. Accordingly, both intracellular (i.e. acid-resistant; B and C) and cell-surface bound molecules, as observed in the presence of PAO (D), also form eccentric pericellular rings. Note that the labeling is more intense after 45 min (C and D) than after 5 min (A and B) of incubation. Scale bar = 10 µm.

 
COS-7 cells transfected with cDNA encoding the sst2A receptor and incubated for 1–60 min at 37 C exhibited at all time intervals heterogeneous, punctate labeling (Fig. 8AGo). This labeling was not observed in cells incubated in the presence of an excess of nonfluorescent [D-Trp8]SRIF (Fig. 8BGo) or in COS-7 cells either nontransfected or transfected with a plasmid encoding the neurotensin receptor in lieu of the somatostatin receptor (not shown). Stripping of cell surface labeling with a hypertonic acid wash variably reduced (depending on the duration of incubation), but never abolished, the fluorescent labeling, indicating that part of the labeling was intracellular (Fig. 8CGo). Accordingly, in cells incubated in the presence of PAO, bound fluo-SRIF molecules remained clustered on the surface of the cells (Fig. 8DGo) in a pattern comparable to that seen after incubation at 4 C or in cells transfected with cDNA encoding the sst1 receptor.



View larger version (135K):
[in this window]
[in a new window]
  		Figure 8. Confocal microscopic imaging of FTC-SRIF binding to COS-7 cells transfected with cDNA encoding the sst2A receptor at 37 C. Images were acquired as single midcellular optical sections at 32 scans/frame. A, Total binding. After 45 min of incubation with 5 nM fluo-SRIF, bound fluorescent molecules are apparent both at the surface and in the cytoplasm of the cell. B, Nonspecific binding. The labeling is completely abolished when the incubation is carried out in the presence of a thousandfold excess of nonfluorescent SRIF. C, Acid wash. Stripping of cell surface binding with a hypertonic acid wash reveals a predominantly intracellular labeling in the form of small endosome-like particles (arrows). D, Treatment with PAO. When internalization is prevented by treatment with the endocytosis inhibitor PAO, bound fluorescent molecules remain clustered at the periphery of the cells. Scale bar = 10 µm.

 
Serial confocal microscopic optical sectioning of sst2A-expressing cells subjected to hypertonic acid washes indicated that the intracytoplasmic labeling took the form of small endosome-like particles (Fig. 9Go). The size and intracytoplasmic distribution of these fluorescent particles varied significantly with the duration of the incubation (Fig. 9Go and Table 2Go). At short incubation times (1–5 min), the particles were small (1.16 ± 0.24 µm2 in area) and sparsely distributed at the periphery of the cells (Fig. 9AGo). Their mean surface progressively increased with time [F(1, 31) = 15.8; t = 2.88; P < 0.01] to reach 2.74 ± 0.44 µm2 at 45 min (Table 2Go). Conversely, their mean distance significantly decreased with time [F(1, 31) = 15.1; t = 4.28; P < 0.001] as they invaded the entire cytoplasm and converged toward the nucleus (Table 2Go and Fig. 9Go, B and C). There also was a trend toward a time-dependent decrease in the mean number of intracytoplasmic fluorescent particles, which did not reach statistical significance (Table 2Go). The nucleus remained devoid of labeling at all times examined.



View larger version (70K):
[in this window]
[in a new window]
  		Figure 9. Confocal microscopic imaging of internalized Bodipy-SRIF in COS-7 cells expressing the sst2A receptor. Cells were incubated at 37 C, and surface-bound molecules were stripped by hypertonic acid wash. After 3 min of incubation with 5 nM fluo-SRIF (A), internalized label is mainly apparent at the periphery of the cell (arrows). At 10 min (B), the label is segregated within small endosome-like particles that are still eccentrically distributed (arrowheads). At 45 min (C), intracellular particles are larger and distributed throughout the cytoplasm of the cells (arrows). Scale bar = 10 µm.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Morphometric analysis of internalized fluorescent particles in COS-7 cells transfected with cDNA encoding the sst2A receptor and incubated for 5–45 min with 5 nM fluo-SRIF at 37 °C

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study confirms the occurrence of receptor-mediated internalization of SRIF in mammalian cells and demonstrates that this internalization is receptor subtype specific.

To achieve confocal microscopic visualization of SRIF-receptor interactions, we resorted to a series of fluorescent derivatives of the SRIF analog [D-Trp8]SRIF-14. This analog was chosen for its resistance to metabolic degradation and high affinity for all SRIF receptor subtypes (15, 44). Furthermore, this ligand had been used successfully in iodinated form for autoradiographic detection of SRIF receptors (45), suggesting that it might prove equally useful for their confocal receptor imaging. We originally synthesized six different fluorescent compounds, each differing in the type of fluorophore (fluorescein or Bodipy) and the position of the fluorescent group on one of the three amine functions of the peptide sequence. Structure-function activity studies and conformation analysis of SRIF analogs had previously indicated that the sequence required for the biological activity of SRIF consists in the ß-turn fragment Phe-Trp-Lys-Thr corresponding to the residue 7–10 of SRIF-14 (46). In agreement with these data, we found that acetylation of the amine function on the side-chain of Lys9 with either fluorescein or Bodipy lead to fluorescent [D-Trp8]SRIF analogs that were almost completely devoid of binding activity in rat cortical homogenates. By contrast, fluorescent analogs substituted on the {alpha}-amino group or on the side-chain of Lys4 retained their capacity to specifically bind to brain SRIF receptors, in conformity with previous data which had shown that deletion of Ala1 and Lys4 in the SRIF sequence did not abolish SRIF’s biological activity (47, 48).

Admittedly, the affinity of both {alpha}-fluoresceinyl and {alpha}-Bodipy compounds was somewhat reduced compared to that of native [D-Trp8]SRIF-14, which may presumably be accounted for by steric hindrance of the fluorophors. Nonetheless, both of the compounds (heretofore referred to as fluo-SRIF) retained sufficient biological activity for confocal imaging of sst1 and sst2 receptors in transfected cells.

Membrane binding assays on COS-7 cells transfected with cDNA coding for sst1 or sst2A receptor subtypes confirmed the expression of specific, high affinity 125I-labeled [D-Trp8]SRIF-binding sites by both types of cells. As previously documented in stably transfected cells, COS-7 cells transfected with the sst2A subtype exhibited a greater affinity for the D-Trp8 derivative than those transfected with the sst1, although the differential observed in our study was not as high as that reported by others (15, 44).

After incubation of whole sst1- and sst2A-transfected cells at 4 C, specific [125I]SRIF or fluo-SRIF binding remained confined to the cell surface, as indicated by the fact that it was entirely acid washable and assumed a pericellular distribution in the confocal microscope. By contrast, after incubation at 37 C, a fraction of the bound radioactive/fluorescent ligand became resistant to hypertonic acid washes. However, both the amount and intracellular distribution of sequestered radioactive/fluorescent ligand were distinct for each cell type.

In cells transfected with cDNA encoding the sst1 receptor, the maximal amount of acid-resistant radioactivity ranged between 20–25% of the specifically bound [125I]SRIF. Comparable effects of hypertonic acid washes had previously been observed for [125I]SRIF binding to RINm5F insulinoma cells (42), pancreatic acinar cells (35), and GH4C1 pituitary cells (41). However, in the case of both RINm5F and GH4C1 cells, this acid-resistant fraction was temperature independent, which led the researchers to conclude against the occurrence of [125I]SRIF internalization in these cells. By contrast, in both the present study and that of Viguerie et al. (35), the build up of the acid-resistant fraction was temperature dependent, suggesting that it might reflect internalization of the radioactivity within the cells. In keeping with this interpretation, pretreatment of the transfected COS-7 cells with the endocytosis inhibitor PAO prevented the formation of an acid-resistant compartment. However, in contrast to the subcellular fractionation results of Viguerie et al. (35), which showed an accumulation of internalized [125I]SRIF into microsomal and nuclear fractions of pancreatic acinar cells, our confocal microscopic observations on sst1-transfected cells suggest that the acid-resistant fraction remains sequestered into or immediately beneath the plasma membrane. This distribution is reminiscent of the insulation effect described for fluorescent CCK on the surface of pancreatic acinar cells, where the ligand was shown to become immobilized and, hence, acid wash resistant, in a time and temperature-dependent manner within the cell membrane (23). Possible mechanisms for this sequestration include the trapping of bound fluorescent molecules within caveolae, a process previously invoked to account for signal transduction of endothelin in transfected COS-7 cells (49) as well as for part of the endocytic processing of fluorescent cholecystokinin in Chinese hamster ovary cells (24).

COS-7 cells transfected with cDNA encoding the sst2A receptor and incubated at 37 C with [125I]SRIF exhibited a rapid, time-dependent increase in the acid-resistant fraction of specifically bound radioactivity. This increase was prevented by coincubation with PAO, indicating that it reflected progressive endocytosis of surface-bound ligand. Both the t1/2 of the internalization process (10 min) and the maximal proportion of bound radioactivity internalized (75%) are concordant with figures reported for other sst2 receptor agonists (39) as well as for other peptide ligands acting through the neurotensin (51, 52), NK1 (20), gastrin (29), cholecystokinin (23, 24), or TRH (19) receptors.

Confocal microscopy confirmed that specifically bound SRIF ligand was taken up inside sst2-transfected cells and demonstrated that the internalization proceeded through the formation of small intracytoplasmic organelles. These endosome-like particles increased in size with time, presumably due to their conglomeration and/or fusion given the trend toward their concurrent decrease in number. They also proceeded toward the center of the cell, where they showed a tendency to cluster along the nuclear membrane. This pattern of internalization is congruent with what has now been described for a variety of peptide ligands both in vitro and in vivo and shown to correspond, at least in some systems, to sequestration of receptor-ligand complexes into clathrin-coated pits, leading to their transfer into early endosomes, late endosomes, multivesicular bodies, and lysosomes (16). It is also consistent with the results of subcellular fractionation studies, which demonstrated sequestration of [125I]SRIF into coated vesicles (36) as well as with electron microscopic data that showed labeling of vesicular as well as lysosomal elements after in vivo or in vitro labeling of pituitary cells with [125I]SRIF (37, 38) or gold-conjugated SRIF (35, 52). Whether, as demonstrated for other peptides (19, 29, 53), this internalization process results in a recycling of the receptors and/or provides for their down-regulation remains to be established.

Previous immunohistochemical (54, 55) and autoradiographic (56, 57) studies have demonstrated striking differences in the steady state subcellular distribution of different peptide receptor subtypes expressed by the same type of cells. The present findings further indicate that there may be major differences in the way peptide receptor subtypes are sorted. Subtype-specific differences in receptor regulation profiles in response to agonist exposure have previously been reported for {alpha}- and ß-adrenergic receptors (58, 59), and it is, therefore, possible that subtype-specific sorting constitutes a general feature of the G protein-coupled receptor superfamily. The precise difference in the amino acid sequence between the sst1 and sst2A receptors that underlies their different pattern of ligand internalization is unknown. It is likely, however, to be imparted by variations in amino acid sequences between their respective C terminals, which differ by more than 51%. This hypothesis appears all the more likely because C terminal amino acid sequences have been reported to be critical for ligand internalization mediated by neurotensin (60) TRH (61), gastrin-releasing peptide (62), and ß2-adrenergic (63) receptors. Additional molecular biological studies are needed to substantiate this hypothesis.

The subtype-specific differences in the kinetic and distributional profiles of SRIF internalization observed here in COS-7 cells may explain the differences in the efficiency of SRIF internalization previously reported among various cell lines. They may also account for the difference in the progression of endocytosed [125I]SRIF from endocytic vesicles to downstream vesicular structures observed between A/B- and D-type pancreatic islet cells (34). The physiological significance of this differential internalization pattern remains a matter of speculation. The low efficiency, intra- or subplasmalemmal sequestration of SRIF mediated by the sst1 receptor may reflect rapid recycling of the receptor and maintenance of cell surface receptor availability. By contrast, the high efficiency internalization of SRIF mediated by the sst2A receptor may underlie rapid receptor down-regulation and, consequently, high receptor turnover. It is interesting in this context that the sst1 receptor, which internalizes poorly, does not desensitize (14), whereas the sst2 receptor, which exhibits a high efficiency internalization, desensitizes rapidly (14, 15). Clearly, elucidation of the mechanisms underlying these subtype-specific differences will prove critical for understanding their role in the regulation of SRIF signal transduction.


   Footnotes
 
1 This work was supported by Grant MA-7366 (to A.B.) from the Medical Research Council of Canada, the Centre National de la Recherche Scientifique (G.G., J.-P.V.), a fellowship (to D.N.) and a Visiting Scientist Award (to J.M.) from the Fonds de la Recherche en Santé du Québec, and Advanced Bioconcept, Inc. Back

Received May 14, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Raynor K, Reisine T 1992 Somatostatin receptors. Crit Rev Neurobiol 6:273–279[Medline]
  2. Rens-Domiano S, Reisine T 1992 Biochemical and functional properties of somatostatin receptors. J Neurochem 58:87–93
  3. Epelbaum J, Dournaud P, Fodor M, Viollet C 1994 The neurobiology of somatostatin. Crit Rev Neurobiol 8:25–44[Medline]
  4. Yamada Y, Post SR, Wang K, Tager HS, Bell GI, Seino S 1992 Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney. Proc Natl Acad Sci USA 89:251–255[Abstract/Free Full Text]
  5. Yasuda K, Rens-Domiano S, Breder CD, Law SF, Saper CB, Reisine T, Bell GI 1992 Cloning of a novel somatostatin receptor, SSTR3, coupled to adenylyl cyclase. J Biol Chem 267:20422–20428[Abstract/Free Full Text]
  6. Kluxen FW, Bruns C, Lubbert H 1992 Expression cloning of a rat brain somatostatin receptor cDNA. Proc Natl Acad Sci USA 89:4618–4623[Abstract/Free Full Text]
  7. Bruno JF, Xu Y, Song J, Berelowitz M 1992 Molecular cloning and functional expression of a brain-specific somatostatin receptor. Proc Natl Acad Sci USA 89:11151–11155[Abstract/Free Full Text]
  8. O’Carroll A-M, Lolait SJ, Konig M, Mahan LC 1992 Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28. Mol Pharmacol 42:939–946[Abstract]
  9. Panetta R, Greenwood MT, Warszynska A, Demchyshyn LL, Day R, Niznik HB, Srikant CB, Patel YC 1994 Molecular cloning, functional characterization, and chromosomal localization of a human somatostatin receptor (somatostatin receptor type 5) with preferential affinity for somatostatin-28. Mol Pharmacol 45:417–427[Abstract]
  10. Vanetti M, Kouba M, Wang X, Vogt G, Hollt V 1992 Cloning and expression of a novel mouse somatostatin receptor (SSTR2B). FEBS Lett 311:290–294[CrossRef][Medline]
  11. Vanetti M, Vogt G, Hollt V 1993 The two isoforms of the mouse somatostatin receptor (mSSTR2A and mSSTR2B) differ in coupling efficiency to adenylate cyclase and in agonist induced receptor desensitization. FEBS Lett 331:260–266[CrossRef][Medline]
  12. Reisine T, Bell GI 1995 Molecular biology of somatostatin receptors. Endocr Rev 16:427–442[CrossRef][Medline]
  13. Patel YC, Greenwood MT, Panetta R, Demchyshyn L, Niznik H, Srikant CB 1995 The somatostatin receptor family. Life Sci 57:1249–1265[CrossRef][Medline]
  14. Rens-Domiano S, Law SF, Yamada Y, Seino S, Bell GI, Reisine T 1992 Pharmacological properties of two cloned somatostatin receptors. Mol Pharmacol 42:28–34[Abstract]
  15. Raynor K, Murphy WA, Coy DH, Taylor JE, Moreau J-P, Yasuda K, Bell GI, Reisine T 1993 Cloned somatostatin receptors: identification of subtype-selective peptides and demonstration of high affinity binding of linear peptides. Mol Pharmacol 43:838–844[Abstract]
  16. Goldstein JL, Brown MS, Anderson RG, Russell DW, Schneider WJ 1985 Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol 1:1–39[CrossRef]
  17. Hazum E, Cuatrecasas P, Marian J, Conn PM 1980 Receptor-mediated internalization of fluorescent gonadotropin-releasing hormone by pituitary gonadotropes. Proc Natl Acad Sci USA 77:6692–6695[Abstract/Free Full Text]
  18. Leroux P, Pelletier G 1984 Radioautographic study of binding and internalization of corticotropin-releasing factor by rat anterior pituitary corticotrophs. Endocrinology 114:14–21[Abstract]
  19. Ashworth R, Yu R, Nelson EJ, Dermer S, Gershengorn MC Hinkle PM 1995 Visualization of the thyrotropin-releasing hormone receptor and its ligand during endocytosis and recycling. Proc Natl Acad Sci USA 92:512–516[Abstract/Free Full Text]
  20. Garland AM, Grady EF, Payan DG, Vigna SR, Bunnett NW 1994 Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells. Biochem J 303:177–186
  21. Garland AM, Grady EF, Lovett M, Vigna SR, Frucht MM, Krause JE, Bunnett NW 1996 Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors. Mol Pharmacol 49:438–446[Abstract]
  22. Mantyh PW, Allen CJ, Ghilardi JR, Rogers SD, Mantyh CR, Liu H, Basbaum AI, Vigna SR, Maggio JE 1995 Rapid endocytosis of a G protein-coupled receptor: substance P-evoked internalization of its receptor in the rat striatum in vivo. Proc Natl Acad Sci USA 92:2622–2626[Abstract/Free Full Text]
  23. Roettger BF, Rentsch RU, Hadac EM, Hellen EH, Burghardt TP, Miller LJ 1995 Insulation of a G-protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cells. J Cell Biol 130:579–590[Abstract/Free Full Text]
  24. Roettger BF, Rentsch RU, Pinon D, Holicky E, Hadac E, Larkin JM, Miller LJ 1995 Dual pathways of internalization of the cholecystokinin receptor. J Cell Biol 128:1029–1041[Abstract/Free Full Text]
  25. Lutz W, Sanders M, Salisbury J, Kumar R 1990 Internalization of vasopressin analogs in kidney and smooth muscle cells: evidence for receptor-mediated endocytosis in cells with V2 or V1 receptors. Proc Natl Acad Sci USA 87:6507–6511[Abstract/Free Full Text]
  26. Husain A, DeSilva P, Speth RC, Bumpus FM 1987 Regulation of angiotensin II in rat adrenal gland. Circ Res 60:640–648[Abstract/Free Full Text]
  27. Svoboda M, De Neef P, Tastenoy M, Christophe J 1988 Molecular characteristics and evidence for internalization of vasoactive intestinal peptide (VIP) receptors in the tumoral rat pancreatic acinar cell line AR 4–2J. Eur J Biochem 76:707–713
  28. Widmann C, Dolci W, Thorens B 1995 Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas. Biochem J 310:203–214
  29. Grady EF, Slice LW, Brant WO, Walsh JH, Payan DG, Bunnett NW 1995 Direct observation of endocytosis of gastrin releasing peptide and its receptor. J Biol Chem 270:4603–4611[Abstract/Free Full Text]
  30. Mazella J, Leonard K, Chabry J, Kitabgi P, Vincent JP, Beaudet A 1991 Binding and internalization of iodinated neurotensin in neuronal cultures from embryonic mouse brain. Brain Res 564:249–255[CrossRef][Medline]
  31. Castel MN, Malgouris C, Blanchard JC, Laduron PM 1990 Retrograde axonal transport of neurotensin in the dopaminergic nigrostriatal pathway in the rat. Neuroscience 36:425–430[CrossRef][Medline]
  32. Castel MN, Woulfe J, Wang X, Laduron PM, Beaudet A 1992 Light and electron microscopic localization of retrogradely transported neurotensin in rat nigrostrial dopaminergic neurons. Neuroscience 50:269–282[CrossRef][Medline]
  33. Faure MP, Alonso A, Nouel D, Gaudriault G, Dennis M, Vincent JP, Beaudet A 1995 Somatodendritic internalization and perinuclear targeting of neurotensin in the mammalian brain. J Neurosci 15:4140–4147[Abstract]
  34. Amherdt M, Patel YC, Orci L 1989 Binding and internalization of somatostatin, insulin, and glucagon by cultured rat islet cells. J Clin Invest 84:412–417
  35. Viguerie N, Estève JP, Susini C, Vaysse N, Ribet A 1987 Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini. Am J Physiol 252:G535–G542
  36. Draznin B, Sherman N, Sussman K, Dahl R, Vatter A 1985 Internalization and cellular processing of somatostatin in primary culture of rat anterior pituitary cells. Endocrinology 117:960–966[Abstract]
  37. Morel G, Leroux P, Pelletier G 1985 Ultrastructural autoradiographic localization of somatostatin-28 in the rat anterior pituitary gland. Endocrinology 116:1615–1619[Abstract]
  38. Morel G, Pelletier G, Heisler S 1986 Internalization and subcellular distribution of radiolabeled somatostatin-28 in mouse anterior pituitary tumor cells. Endocrinology 119:1972–1979[Abstract]
  39. Hofland LJ, van Koetsveld PM, Waaijers M, Zuyderwijk J, Breeman WAP, Lamberts SWJ 1995 Internalization of the radioiodinated somatostatin analog [125I-Tyr3]octreotide by mouse and human pituitary tumor cells: increase by unlabeled octreotide. Endocrinology 136:3698–3706[Abstract]
  40. Breeman W, Kwekkeboom DJ, Kooij P, Bakker WH, Hofland LJ, Visser TJ, Ensing GJ, Lamberts S, Krenning EP 1995 Effect of dose and specific activity on tissue distribution of indium-111-pentetreotide in rats. J Nucl Med 36:623–627[Abstract/Free Full Text]
  41. Presky DH, Schonbrunn A 1986 Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells. J Cell Biol 102:878–888[Abstract/Free Full Text]
  42. Sullivan SJ, Schonbrunn A 1986 The processing of receptor-bound [125I-Tyr11]somatostatin by RINm5F insulinoma cells. J Biol Chem 261:3571–3577[Abstract/Free Full Text]
  43. Sadoul JL, Mazella J, Amar S, Kitabgi P, Vincent JP 1984 Preparation of neurotensin selectively iodinated on the tyrosine 3 residue. Biological activity and binding properties on mammalian neurotensin receptors. Biochem Biophys Res Commun 120:812–819[CrossRef][Medline]
  44. Patel YC, Srikant BS 1994 Subtype selectivity of peptide analogs for all five cloned human somatostatin receptors (hsstr 1–5). Endocrinology 135:2814–2817[Abstract]
  45. Epelbaum J, Moyse E, Tannenbaum GS, Kordon C, Beaudet A 1989 Combined autoradiographic and immunohistochemical evidence for an association of somatostatin binding sites with growth hormone-releasing factor containing nerve cell bodies in the rat arcuate nucleus. J Neuroendocrinol 1:109–115
  46. Veber DF, Holly FW, Nutt RF, Bergstrand SJ, Brady SF, Hirschmann R, Glitzer MS, Saperstein R 1979 Highly active cyclic and bicyclic somatostatin analogs of reduced ring size. Nature 280:512–514[CrossRef][Medline]
  47. Sarantakis D, McKinley WA, Jaunakais I, Clark D, Grant NH 1976 Structure activity studies on somatostatin. Clin Endocrinol (Oxf) [Suppl] 5:275S–278S
  48. Brown M, Rivier J, Vale W 1979 Somatostatin: central nervous system actions on glucoregulation. Endocrinology 104:1709–1915[Abstract]
  49. Chun M, Liyanage UK, Lisanti MP, Lodish HF 1994 Signal transduction of a G protein-coupled receptor in caveolae: colocalization of endothelin and its receptor with caveolin. Proc Natl Acad Sci USA 91:11728–11732[Abstract/Free Full Text]
  50. Chabry J, Gaudriault G, Vincent JP, Mazella J 1993 Implication of various forms of neurotensin receptors in the mechanism of internalization of neurotensin in cerebral neurons. J Biol Chem 268:17138–17144[Abstract/Free Full Text]
  51. Faure MP, Labbé-Jullié C, Cashman N, Kitabgi P, Beaudet A 1995 Binding and internalization of neurotensin in hybrid cells derived from septal cholinergic neurons. Synapse 20:106–116[CrossRef][Medline]
  52. Mentlein R, Buchholz C, Krisch B 1989 Binding and internalization of gold-conjugated somatostatin and growth hormone-releasing hormone in cultured rat somatotropes. Cell Tissue Res 258:309–317[Medline]
  53. Petrou CP, Tashjian AH Jr 1995 Evidence that the thyrotropin-releasing hormone receptor and its ligand are recycled dissociated from each other. Biochem J 306:107–113
  54. Arvidsson U, Dado RJ, Riedl M, Lee JH, Law PY, Loh HH, Elde R, Wessendorf MW 1995 Delta-opioid receptor immunoreactivity: distribution in brainstem and spinal cord, and relationship to biogenic amines and enkephalin. J Neurosci 15:1215–1235[Abstract]
  55. Arvidsson U, Riedl M, Chakrabarti S, Lee JH, Nakano AH, Dado RJ, Loh HH, Law PY, Wessendorf MW, Elde R 1995 Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. J Neurosci 15:3328–3341[Abstract]
  56. Hamel E, Beaudet A 1984 Electron microscopic autoradiographic localization of opioid receptors in rat neostriatum. Nature 312:155–157[Medline]
  57. Pasquini F, Bochet P, Garbay-Jaureguiberry C, Roques BP, Rossier J, Beaudet A 1992 Electron microscopic localization of photoaffinity-labelled delta opioid receptors in the neostriatum of the rat. J Comp Neurol 326:229–244[CrossRef][Medline]
  58. von Zastrow M, Link R, Daunt D, Barsh G, Kobilka B 1993 Subtype-specific differences in the intracellular sorting of G protein-coupled receptors. J Biol Chem 268:763–766[Abstract/Free Full Text]
  59. Suzuki T, Nguyen CT, Nantel F, Bonin H, Valiquette M, Frielle T, Bouvier M 1991 Distinct regulation of ß1- and ß2-adrenergic receptors in chinese hamster fibroblasts. Mol Pharmacol 41:542–548[Abstract]
  60. Chabry J, Botto JM, Nouel D, Beaudet A, Vincent JP, Mazella J 1995 Thr-422 and Tyr-424 residues in the carboxyl terminus are critical for the internalization of the rat neurotensin receptor. J Biol Chem 270:2439–2442[Abstract/Free Full Text]
  61. Nussenzveig DR, Heinflink M, Gershengorn MC 1993 Agonist-stimulated internalization of the thyrotropin-releasing hormone receptor is dependent on two domains in the receptor carboxyl terminus. J Biol Chem 268:2389–2392[Abstract/Free Full Text]
  62. Benya RV, Fathi Z, Battey JF, Jensen RT 1993 Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization. J Biol Chem 268:20285–20290[Abstract/Free Full Text]
  63. Barak LS, Tiberi M, Freedman NJ, Kwatra MM, Lefkowitz RJ, Caron MG 1994 A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated beta 2-adrenergic receptor sequestration. J Biol Chem 269:2790–2795[Abstract/Free Full Text]
  64. Pollard JW, Luqmani Y, Bateson A, Chotai K 1984 DNA transformation of mammalian cells. In: Walker JM (ed) Methods in Molecular Biology. Humana Press, Clifton, vol 2:321–332



This article has been cited by other articles:


Home page
 Eur J EndocrinolHome page
A. Fusco, G. Gunz, P. Jaquet, H. Dufour, A. L. Germanetti, M. D Culler, A. Barlier, and A. Saveanu
Somatostatinergic ligands in dopamine-sensitive and -resistant prolactinomas
Eur. J. Endocrinol., May 1, 2008; 158(5): 595 - 603.
[Abstract] [Full Text] [PDF]

Home page