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Endocrinology, doi:10.1210/en.2006-1266
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Endocrinology Vol. 148, No. 5 2095-2105
Copyright © 2007 by The Endocrine Society

Coexpression of Somatostatin Receptor Subtype 5 Affects Internalization and Trafficking of Somatostatin Receptor Subtype 2

Nadder Sharif, Louis Gendron, Julia Wowchuk, Philippe Sarret, Jean Mazella, Alain Beaudet and Thomas Stroh

Department of Neurology and Neurosurgery (N.S., L.G., J.W., P.S., A.B., T.S.), McGill University, and the Montreal Neurological Institute, Montréal, Québec, Canada H3A 2B4; and Institut de Pharmacologie Moléculaire et Cellulaire (J.M.), Unité Mixte de Recherche 6097, Université de Nice-Sophia Antipolis, 06560 Valbonne, France

Address all correspondence and requests for reprints to: Thomas Stroh, Ph.D., Montreal Neurological Institute, McGill University, 3801 University Street, Montréal, Québec, Canada H3A 2B6. E-mail: thomas.stroh{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The somatostatin [somatotropin release-inhibiting factor (SRIF)] receptor subtypes sst2A and sst5 are frequently coexpressed in SRIF-responsive cells, including endocrine pituitary cells. We previously demonstrated that sst2A and sst5 exhibit different subcellular localizations and regulation of cell surface expression, although they have similar signaling properties. We investigated here whether sst2A and sst5 functionally interact in cells coexpressing the two receptor subtypes. We stimulated both transfected cells stably expressing sst2A alone (CHO-sst2A) or together with sst5 (CHO-sst2A+5) and the pituitary cell line AtT20, which endogenously expresses the two receptor subtypes, with either the nonselective agonist [D-Trp8]-SRIF-14 or the sst2-selective agonist L-779,976. In CHO-sst2A cells, stimulation with either ligand resulted in the loss of approximately 75% of cell surface SRIF binding sites and massive internalization of sst2A receptors. The cells were desensitized to subsequent stimulation with [D-Trp8]-SRIF-14, which failed to inhibit forskolin-evoked cAMP accumulation. Similarly, in CHO-sst2A+5 and AtT20 cells, [D-Trp8]-SRIF-14 induced the loss of 60–70% of SRIF binding sites as well as massive sst2A endocytosis. By contrast, in cells expressing both sst2A and sst5, selective stimulation of sst2A with L-779,976 resulted in only 20–40% loss of cell surface binding and markedly reduced sst2A internalization. Consequently, whereas CHO-sst2A+5 and AtT20 cells stimulated with [D-Trp8]-SRIF-14 were desensitized to a second stimulation with the same agonist, cells prestimulated with L-779,976 were not desensitized to subsequent [D-Trp8]-SRIF-14 stimulation. These findings indicate that the presence of sst5 in the same cells modulates trafficking and cell surface regulation of sst2A and cellular desensitization to the effects of SRIF.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SOMATOSTATIN, OR SOMATOTROPIN, release-inhibiting factor (SRIF), is a neuropeptide that was originally isolated from ovine hypothalamus and characterized based on its inhibitory action on GH release from the anterior pituitary (1). It is widely distributed throughout the central nervous system and in the periphery (2, 3). SRIF has been implicated in a variety of central functions ranging from neuroendocrine regulation to the control of cognition and locomotion (3, 4), and changes in SRIF levels have been linked to numerous neurological disorders such as Parkinson’s and Alzheimer’s disease (3). In the pituitary gland, in addition to inhibiting GH release from somatotrophs, SRIF has been shown to regulate hormone secretion from almost all the other endocrine cell types of the anterior lobe (2, 5).

The effects of SRIF are mediated by a family of G protein-coupled receptors, designated sst1–sst5, which are coupled negatively to cAMP production via Gi/o proteins. In addition, SRIF receptor subtypes couple to a variety of other second messenger systems (6). Using in situ hybridization histochemistry (7, 8, 9) and immunohistochemistry (10, 11) the presence of all five SRIF receptor subtypes was demonstrated in the pituitary gland. However, the receptor subtypes sst2A and sst5 were found to be the predominant subtypes in anterior pituitary endocrine cells (12, 13, 14). Both were shown to be coexpressed in somatotropic and corticotropic cells and demonstrated to cooperate in the regulation of GH secretion from somatotropes (15) and of ACTH release from corticotropes (16).

In pituitary adenomas, sst2 and sst5 likewise appear to be the dominant receptor subtypes (5, 17). Therefore, patients suffering from neuroendocrine tumors, and notably those exhibiting symptoms of acromegaly, are frequently treated with SRIF analogs such as octreotide or lanreotide, which preferentially bind to sst2 but also to sst5 receptors. Approximately 65% of the patients are responsive to this treatment, and these patients never develop any sign of tolerance to the effects of the drugs, suggesting that their receptor targets are not down-regulated (5, 18).

In both ectopically and endogenously expressing cells, sst2A and sst5 exhibit a strikingly different distribution at rest and regulation after stimulation. All studies agree that sst2A is largely located at the cell surface before stimulation (19, 20, 21, 22). After exposure to an agonist, the receptor-ligand complexes are internalized rapidly and efficiently (19, 20, 21, 22, 23, 24, 25, 26). By contrast, sst5 appears to be predominantly, but not exclusively, located in intracellular compartments in agonist-naive cells (19, 21, 27). However, in response to stimulation, a large number of receptors are recruited to the plasma membrane within minutes of the onset of the stimulus (19, 27). We recently demonstrated that these patterns of cellular distribution and post-stimulation regulation of sst2A and sst5 are also found in an endogenously expressing mouse corticotropic adenoma cell line, AtT20 (21).

The question arises as to whether the opposing cell surface targeting and regulation properties of sst2A and sst5 are responsible for the clinical observation that patients under prolonged SRIF analog treatment do not develop tolerance, i.e. whether in cells expressing both sst2A and sst5, the two receptor subtypes may interact functionally with each other to maintain high densities of cell surface SRIF receptors under continuous exposure to agonists. To investigate this possibility, we monitored sst2A trafficking in cells either ectopically or endogenously expressing both receptor subtypes and determined whether the presence of sst5 in the same cells influenced the cell surface availability and responsiveness of sst2A receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid construction
To construct N-terminally c-Myc-tagged sst2A and HA-tagged sst5 receptors, mouse sst2A and sst5 cDNAs were amplified from plasmid pCMV-6b by PCR using 5'- and 3'-oligonucleotide primers containing NotI-BamHI and EcoRI-NotI restriction site sequences, respectively. PCR products were gel-purified, digested, and directly inserted between the corresponding sites of plasmids pIRES-neo (cMyc-sst2A) and pIRES-puro (HA-sst5) (Clontech; Palo Alto, CA). Constructs were verified by sequencing.

Culture and transfection of cells
CHO-K1 cells (American Type Culture Collection, Rockville, MD) were maintained in Ham’s F12 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin/streptomycin, and 250 g/ml Fungizone (Invitrogen, Burlington, Ontario, Canada). To establish stable cell lines expressing c-Myc-sst2A and/or HA-sst5 receptors, approximately 2.6 x 106 CHO-K1 cells were transfected with 8 µg c-Myc-sst2A pIRES-neo or HA-sst5 pIRES-puro plasmids, using a Lipofectamine transfection reagent (DAC30; Eurogentec, San Diego, CA) according to the manufacturer’s instructions. Individual clones of transfected cells were selected in the presence of 750 µg/ml geneticin G418 (Invitrogen) or 25 µg/ml puromycin (BD Clontech, Mountain View, CA), respectively, and tested for their capacity to bind [125I]Tyr0-[D-Trp8]SRIF-14 in a radioligand binding assay. A clone expressing 600 fmol/mg sst2A receptor (CHO-sst2A) was used to generate CHO-K1 cells stably expressing both receptor subtypes (CHO-sst2A+5) using the same protocol. From the transfected cells, a clone exhibiting 750 fmol/mg protein of SRIF binding sites was selected.

Mouse AtT-20/D16-16 tumor cells were grown in DMEM with high glucose supplemented with 10% fetal bovine serum, 10% horse serum, 100 U/ml penicillin/streptomycin, and 250 g/ml Fungizone. All cells were cultured in 100-mm dishes, maintained in a humidified atmosphere of 95% air and 5% CO2, and passed when the monolayer reached 90% confluence. These cells were previously shown to express approximately 300 fmol/mg SRIF binding sites (20).

Competition binding of [D-Trp8]-SRIF-14 or L-779,976 using [125I]Tyr0-[D-Trp8]-SRIF-14
To determine the binding affinities of [D-Trp8]-SRIF-14, a non-subtype-selective somatostatin peptide analog that is more resistant to metabolic degradation than native SRIF-14 and is six to eight times more potent than the natural ligand (25, 28, 29), and of L-779,976, a nonpeptidic somatostatin analog that shows high affinity and selectivity for sst2A (30, 31) (kindly supplied by Merck, Rahway, NJ), competition binding experiments were performed on whole live CHO-sst2A, CHO-sst5, CHO-sst2A+5, and AtT-20 cells. Cells were washed once and equilibrated for 10 min at 37 C with Earle’s buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.9 mM MgCl2, and 25 mM HEPES, pH 7.4), containing 2% BSA. Next, 0.3 nM [125I]Tyr0-[D-Trp8]-SRIF-14 (1800–2000 Ci/mmol; J. Mazella, Valbonne, France) was added to the equilibrium mixture in the presence of increasing concentrations (from 10–12 to 10–6 M) of either unlabeled [D-Trp8]-SRIF-14 or L-779,976 for 30 min. Nonspecific binding was measured in the presence of 1 µM unlabeled [D-Trp8]-SRIF-14. Subsequently, cells were washed twice with Earle’s buffer and detached using 0.1 M NaOH. The radioactivity of each sample was counted in a {gamma}-counter. Competition binding experiments were done in triplicate and repeated at least twice. IC50 values were calculated using nonlinear regression analysis in SigmaPlot version 7.0 (SPSS Inc., Point Richmond, CA).

[125I]Tyr0-[D-Trp8]-SRIF-14 cell surface binding
Radioactive ligand binding was performed on live CHO-sst2A, CHO-sst2A+5, and AtT-20 cells at 37 C to quantify cell surface binding sites after stimulation with [D-Trp8]-SRIF-14 or L-779,976. At least 12 h before each experiment, approximately 300,000 cells per well were plated on 24-well tissue culture plates (Falcon, Franklin Lakes, NJ). Before stimulation, cells were equilibrated for 10 min at 37 C in Earle’s buffer (pH 7.4) containing 2% BSA and 0.1% D-glucose. The equilibrium mixture was replaced by 300 µl Earle’s buffer containing 0.8 mM 1,10-phenanthroline, and 100 nM of [D-Trp8]-SRIF-14, L-779,976, or Earle’s buffer alone for 0–40 min. After agonist stimulation, cells were washed three times with ice-cold Earle’s buffer, twice with a hypertonic acid solution (Earle’s buffer containing 0.2 M acetic acid and 0.5 M NaCl, pH 4) to wash away surface-bound ligand, and three times again with Earle’s buffer. Next, to quantify the remaining surface binding sites, cells were incubated with 250 µl Earle’s buffer containing 2% BSA, 0.3 nM [125I]Tyr0-[D-Trp8]-SRIF-14 (1800–2000 Ci/mmol; Phoenix Pharmaceuticals, Belmont, CA) for 30 min at 37 C. To inhibit receptor internalization during radioactive labeling, 10 µM phenylarsine oxide (PAO; a well-documented internalization blocker) (32, 33) was added to the above mixture. We repeated the competition binding experiments described earlier in the presence of PAO to ensure that this compound did not affect binding affinity. In addition, we repeated the present experiments in the absence of PAO and incubated cells with [125I]Tyr0-[D-Trp8]-SRIF-14 on ice. Nonspecific binding was measured in the presence of 10 µM unlabeled [D-Trp8]-SRIF-14 and represented less than 10% of the total binding. Total specific surface binding in stimulated cells was normalized to the total specific binding in unstimulated cells (100%). In CHO cells, the data for five separate experiments (each performed in triplicate) were pooled, and statistical significance was verified using a one-way ANOVA followed by Bonferroni’s multiple comparison test. In AtT-20 cells, the data for four separate experiments (each performed in triplicate) were pooled, and statistical significance was verified using an unpaired t test with Welch’s correction.

Immunocytochemistry
To monitor the trafficking of sst2A after stimulation with 100 nM [D-Trp8]-SRIF-14 or L-779,976, we conducted immunocytochemical analysis on CHO-sst2A, CHO-sst2A+5, and AtT-20 cells. Approximately 100,000 cells per well were plated on poly-L-lysine-coated glass coverslips in four-well tissue culture plates (Nunc, Roskilde, Denmark) at least 12 h before each experiment. After agonist stimulation for 0–40 min (as described above), cells were fixed with 4% paraformaldehyde (Polysciences, Warrington, PA) for 20 min and preincubated with a blocking solution for 15 min containing 5% normal goat serum (NGS), 2% BSA, and 0.1% Triton X-100 in Tris-buffered saline (TBS). Immunostaining was performed by incubating cells overnight at 4 C with a rabbit-anti-c-Myc antibody (Sigma Chemical Co., St. Louis, MO) diluted 1:500 in TBS containing 0.05% Triton X-100 and 1% NGS (Jackson ImmunoResearch, West Grove, PA). Subsequently, the cells were thoroughly rinsed in TBS and incubated for 40 min at room temperature with Alexa 488-conjugated goat antirabbit antibodies (Molecular Probes, Eugene, OR) diluted 1:500 in TBS. They were then rinsed again and mounted onto glass object slides. Untransfected CHO-K1 cells were used as a control to ensure antibody specificity. For double labeling of c-Myc-sst2A and HA-sst5 in CHO-sst2A+5 cells, a mouse-anti-HA antibody (1:500; Roche Diagnostics, Indianapolis, IN) was included in the primary antibody incubation and Alexa 594-conjugated goat antimouse in the secondary antibody mixture.

In AtT-20 cells, sst2A was detected using a rabbit antibody (Gramsch Laboratories, Schwabhausen, Germany) directed against the C-terminal segment of the mouse sst2A receptor (34) at a dilution of 1:500. Bound antibodies were revealed using Alexa 488-conjugated goat antirabbit secondary antibodies (Molecular Probes) diluted 1:500. Immunocytochemistry experiments were all performed in duplicate and repeated at least three times.

Immunolabeled cells were observed on a Zeiss laser scanning microscope 510 equipped with Argon2 (488 nm) and He/Ne1 (543 nm) lasers. Single-labeled cells were analyzed using the 488-nm laser line to excite Alexa 488, whereas images of double-labeled cells were acquired by using both lasers simultaneously in multitrack mode. Images were acquired as single optical sections through the center of the cells at the level of the nucleus and processed with Adobe Photoshop 6.0.

Quantification of cell surface sst2A immunofluorescence
We quantified cell surface c-Myc-sst2A after 40 min of stimulation with 100 nM [D-Trp8]-SRIF-14 or L-779,976 to characterize the agonist-induced reduction in cell surface sst2A receptor density. In the case of CHO-sst2A and CHO-sst2A+5 cells, approximately 300,000 cells per well were plated on 24-well tissue culture plates (Falcon) at least 12 h before each experiment. After 40 min of agonist stimulation (as described above), cells were rinsed with ice-cold Earle’s buffer and incubated with a solution containing rabbit anti-c-Myc (1:500; Sigma), 2% BSA, and 1% NGS for 90 min at 4 C (to inhibit receptor endocytosis). They were then fixed with 4% PFA for 20 min at room temperature, incubated for 1 h with a goat antirabbit Alexa 488-conjugated secondary antibody (1:500; Molecular Probes), and fluorescence intensity was measured on a FL600 fluorescence immunoplate reader (FLIPR; Fischer Scientific, Montreal, Quebec, Canada). Means for each condition were calculated and subtracted from background fluorescence (wells not treated with primary antibody). Surface fluorescence intensity in stimulated cells was normalized to fluorescence intensity in unstimulated cells (100%). The data for three separate experiments (each done in quadruplicate) were pooled, and statistical significance was verified using a one-way ANOVA followed by Bonferroni’s multiple comparison test.

Because commercially available antibodies for sst2A receptors are directed against the intracellular C terminus, immunocytochemical detection of endogenously expressed sst2A receptors required permeabilization of the cells. This procedure renders FLIPR techniques unapplicable for the detection of cell surface immunofluorescence. Therefore, to quantify peripheral sst2A immunofluorescence in AtT-20 cells, we resorted to densitometric analysis of confocal images. Images were converted to grayscale using Photoshop 6.0 (Adobe) and imported into ImageJ (http://rsb.info.nih.gov/ij/) for gray-level intensity analysis. Five images for each condition were used for analysis, and each was divided into quadrants. Four to six cells from each quadrant were selected at random; peripheral (corresponding largely to cell surface) fluorescence labeling was traced by hand, and the mean gray level of each traced area was calculated. Peripheral fluorescence intensity of stimulated cells was normalized to the peripheral fluorescence intensity of unstimulated cells (100%). The data for three separate experiments (each performed in duplicate) were pooled, and statistical significance was verified using a one-way ANOVA followed by Bonferroni’s multiple comparison test.

Reappearance of cell surface sst2A
To investigate whether the reappearance of surface sst2A involved receptor recycling, we used monensin, an ionophore that has been demonstrated to prevent endosome acidification (19, 35, 36), in CHO-sst2A and CHO-sst2A+5 cells. For confocal microscopy, cells were plated onto poly-L-lysine-coated glass coverslips in four-well plates (Nunc). For quantification of cell surface immunoreactivity, approximately 300,000 cells per well were plated on 24-well tissue culture plates (Falcon) at least 12 h before each experiment. Fifteen minutes before the start of the experiment, medium was replaced or not by F12 medium containing 25 µM monensin. Cells were equilibrated at 37 C for 10 min in either Earle’s buffer containing 2% BSA and 1% glucose or Earle’s buffer supplemented with 25 µM monensin followed by 40 min of stimulation with 100 nM of either [D-Trp8]-SRIF-14 or L-779,976 with or without 25 µM monensin in Earle’s buffer. Subsequent to rinsing with hypertonic acid buffer, to wash away surface-bound ligand, cells were incubated at 37 C in the absence of ligand for 0–40 min in Earle’s buffer with or without 25 µM monensin to allow for reappearance of receptors at the cell surface. After the recovery period, immunostaining was carried out without permeabilization (see above), and cell surface sst2A immunofluorescence was visualized on a confocal microscope. Quantification of reappearing cell surface immunofluorescence was achieved using FLIPR as described. Surface fluorescence intensity in stimulated cells was normalized to fluorescence intensity in unstimulated cells (100%). The data for three separate experiments (each done in quadruplicate) were pooled, and statistical significance was verified using a one-way ANOVA followed by Bonferroni’s multiple comparison test.

Inhibition of stimulated cAMP production
To investigate the responsiveness of CHO-sst2A, CHO-sst2A+5, and AtT-20 cells prestimulated with [D-Trp8]-SRIF-14 or L-779,976, we measured the ability of [D-Trp8]-SRIF-14 to inhibit forskolin-stimulated cAMP production in a second stimulation. Approximately 200,000 cells per well were plated on 24-well tissue culture plates (Falcon) at least 12 h before each experiment. Cells were equilibrated and prestimulated with 100 nM [D-Trp8]-SRIF-14 or L-779,976 for 40 min. This was followed by another 40-min stimulation with either 100 nM forskolin (Sigma) and 1 mM isobutylmethylxanthine (Sigma) or a mixture containing 100 nM [D-Trp8]-SRIF-14 or L-779,976, 100 nM forskolin, and 1 mM isobutylmethylxanthine. The experiment was terminated by adding lysis reagent (0.5% dodecyltrimethylammonium bromide) to each well for 20 min. Total adenylate cyclase inhibition was measured with an enzyme immunoassay kit (Amersham Biosciences, Little Chalfont, UK), according to the manufacturer’s suggestions. Briefly, cellular extracts were transferred to a 96-well plate (precoated with donkey antirabbit IgG), and rabbit antiserum against cAMP was added to each well at 4 C for 2 h. Next, cAMP conjugated to horseradish peroxidase was added for 60 min to initiate competition, which was terminated by extensive washes with 0.01 M phosphate buffer (pH 7.4), containing 0.05% (vol/vol) Tween 20 (EMD Chemicals, Gibbstown, NJ). Enzyme substrates were added to each well where a blue color was allowed to develop for 1 h; the plate was then read at 630 nm on a µQuant microplate reader (Bio-Tek Instruments Inc., Winooski, VT). Cells receiving no pretreatment followed by forskolin alone were used to calculate maximal cAMP production. The effect of SRIF analogs on forskolin-stimulated cAMP production was expressed as a percentage of the effect of forskolin alone. The data for four separate experiments (each done in triplicate) were pooled, and statistical significance was verified using a one-way ANOVA followed by Tukey’s multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Agonist binding affinities
To establish the binding affinities of [D-Trp8]-SRIF-14 and L-779,976 in live cells, we performed competition binding experiments using [125I]Tyr0-[D-Trp8]-SRIF-14 in CHO-K1 cells stably expressing sst2A (CHO-sst2A), sst5 (CHO-sst5), or both receptor subtypes (CHO-sst2A+5) as well as in AtT20 cells, which endogenously express both receptor subtypes (37). [D-Trp8]-SRIF-14 bound with subnanomolar affinity to CHO cells expressing sst2A either alone or together with sst5, with nanomolar affinity to AtT20 cells, and with markedly lower affinity to cells expressing sst5 alone (61 nM; Table 1Go), in keeping with the reportedly lower affinity of SRIF-14 and [D-Trp8]-SRIF-14 for sst5 compared with other receptor subtypes (38, 39, 40, 41). L-779,976 exhibited lower binding affinities than [D-Trp8]-SRIF-14 in AtT20, CHO-sst2A, and CHO-sst2A+5 cells with IC50 of 5.7, 32, and 50 nM, respectively (Table 1Go). However, in agreement with earlier findings (30), it did not bind to CHO-sst5 cells at concentrations of up to 100 nM, confirming that it was selective for sst2 over sst5.


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  		TABLE 1. Somatostatin analog binding affinities

 
Binding of [125I]Tyr0-[D-Trp8]-SRIF-14 to whole cells
To investigate whether coexpression of sst5 in the same cells had an effect on the trafficking of sst2A, we compared cell surface binding in CHO cells expressing sst2A alone or in combination with sst5, after stimulation with [D-Trp8]-SRIF-14 or L-779,976 for 0–40 min at 37 C. To this end, stimulated cells were rinsed with hypertonic acid buffer to strip off any remaining surface-bound ligand and were incubated at 37 C with [125I]Tyr0-[D-Trp8]-SRIF-14 in the presence of the endocytosis inhibitor PAO to block receptor internalization. In CHO-sst2A cells, [125I]Tyr0-[D-Trp8]-SRIF-14 cell surface binding was reduced to 25% of that recorded in nonstimulated cells after 10 min of exposure to either [D-Trp8]-SRIF-14 or L-779,976 (Fig. 1AGo). This level of remaining cell surface sst2A binding sites was the same after exposure to the ligand for up to 40 min, suggesting that both ligands induced efficient sequestration of sst2A receptors. The same results were obtained in CHO-sst2A+5 cells after [D-Trp8]-SRIF-14 stimulation but not after incubation with L-779,976, which proved significantly less efficient than [D-Trp8]-SRIF-14 at down-regulating cell surface [125I]Tyr0-[D-Trp8]-SRIF-14 binding (60% of cell surface binding remaining after 10–40 min of ligand exposure; P ≤ 0.01, as compared with [D-Trp8]-SRIF-14 stimulation in the same cell line; P ≤ 0.01–0.001, as compared with L-779,976 stimulation in CHO-sst2A cells; n = 5; Fig. 1AGo).


Figure 1
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  		FIG. 1. Cell surface [125I]Tyr0-[D-Trp8]-SRIF-14 binding to CHO-sst2A and CHO-sst2A+5 (A) or AtT20 cells (B) after agonist exposure. Cells were stimulated with 100 nM [D-Trp8]-SRIF-14 (SRIF-14) or L-779,976 for 0–40 min at 37 C, rinsed with hypertonic acid buffer, and incubated with [125I]Tyr0-[D-Trp8]-SRIF-14 in the presence of PAO to quantify remaining cell surface binding sites. A, After stimulation with L-779,976, more surface binding remains in CHO-sst2A+5 ({circ}) as compared with the same type of cells exposed to [D-Trp8]-SRIF-14 (bullet; ##, P ≤ 0.01; one-way ANOVA with Bonferroni’s multiple comparison test) or to CHO-sst2A cells stimulated with L-779,976 ({square}; **, P ≤ 0.01; ***, P ≤ 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). B, In AtT-20 cells, more surface binding remains after stimulation with L-779,976 ({triangleup}; **, P ≤ 0.01; ***, P ≤ 0.001; unpaired t test with Welch’s correction) as compared with [D-Trp8]-SRIF-14 stimulation ({blacktriangleup}). Data represent means ± SEM of three independent experiments done in triplicate.

 
In AtT20 cells, as in CHO-sst2A+5 cells, [D-Trp8]-SRIF-14 induced a pronounced loss of cell surface binding, leaving approximately 40% of binding activity after 10–40 min of stimulation (Fig. 1BGo). Here again, stimulation with L-779,976 was less efficient than that with [D-Trp8]-SRIF-14, leaving 75% of cell surface binding activity after 40 min (P ≤ 0.01–0.001, as compared with [D-Trp8]-SRIF-14; n = 4; Fig. 1BGo).

Sst2A trafficking in stably transfected CHO-K1 cells
To visualize the internalization and trafficking of sst2A after stimulation with 100 nM of either [D-Trp8]-SRIF-14 or L-779,976, CHO-sst2A and CHO-sst2A+5 cells were stimulated for 0, 5, 20, or 40 min, fixed, and stained using an antibody against the N-terminal c-Myc tag of the recombinant sst2A receptor. In both cell types, before stimulation (0 min; Fig. 2Go, A, B, I, and J), sst2A immunoreactivity formed a brightly fluorescent ring outlining the shape of the cell, presumably at the cell surface. In CHO-sst2A cells (Fig. 2Go, A–H), 5 min of stimulation with either agonist led to a disappearance of this peripheral labeling and to a redistribution of sst2A immunolabeling within small, fluorescent vesicles, distributed throughout the cytoplasm (Fig. 2Go, C and D). These vesicles progressively concentrated with time and coalesced into intensely fluorescent agglomerations in the vicinity of the nucleus (Fig. 2Go, E–H). In CHO-sst2A+5 cells, stimulation with [D-Trp8]-SRIF-14 induced the same sequence of sst2A trafficking events (Fig. 2Go, I, K, M, and O). By contrast, stimulation with the sst2-selective agonist L-779,976 did not promote sst2A internalization as efficiently, as demonstrated by the persistence of immunofluorescence in the periphery of the cells at all time points (Fig. 2Go, J, L, N, and P). After 20 min of stimulation, a few intracellular, sst2A-positive vesicles were evident, but much less so than in either the same cell line stimulated with [D-Trp8]-SRIF-14 or in CHO-sst2A cells (Fig. 2NGo). After 40 min, the distribution of sst2A was reminiscent of the labeling observed in nonstimulated cells (Fig. 2PGo).


Figure 2
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  		FIG. 2. Agonist-induced trafficking of c-Myc-sst2A in CHO-sst2A and CHO-sst2A+5 cells. Cells were stimulated with [D-Trp8]-SRIF-14 or L-779,976 for the indicated periods of time, fixed, and immunostained using a rabbit-anti-c-Myc antibody to visualize sst2A. A, B, I, and J, Before stimulation, sst2A immunofluorescence forms a ring outlining the cell surface; A–H, in CHO-sst2A cells, both agonists induce rapid and efficient internalization of sst2A; I–P, in CHO-sst2A+5 cells, stimulation with [D-Trp8]-SRIF-14 also induces massive sst2A internalization. In contrast, after stimulation with L-779,976, clusters of sst2A immunofluorescence are evident at the cell surface (L and N; arrows) but only limited receptor sequestration within the cytoplasm (N; arrowheads). Scale bar, 5 µm.

 
To quantify the apparent differential loss of cell surface sst2A receptors in CHO-sst2A and CHO-sst2A+5 cells after stimulation with either [D-Trp8]-SRIF-14 or L-779,976, cells were stimulated as above, placed on ice, and immunostained in the absence of detergent before fixation to prevent permeabilization and to stain the cell surface only. They were then either visualized under a confocal microscope (Fig. 3Go, A–F) or quantified using a FLIPR (Fig. 3GGo). In both CHO-sst2A (Fig. 3Go, A–C) and CHO-sst2A+5 cells (Fig. 3Go, D–F), there was robust sst2A immunofluorescence at the cell surface before stimulation (Fig. 3Go, A and D). In CHO-sst2A cells, stimulation for 40 min with either [D-Trp8]-SRIF-14 (Fig. 3BGo) or L-779,976 (Fig. 3CGo) induced an almost complete loss of that cell surface labeling as determined by confocal microscopy. However, in CHO-sst2A+5 cells, stimulation with L-779,976 resulted in a much smaller reduction in cell surface fluorescence (Fig. 3FGo) than stimulation with [D-Trp8]-SRIF-14 (Fig. 3EGo). Quantification of cell surface fluorescence by FLIPR revealed that in CHO-sst2A cells, 25–30% of the original sst2A immunolabeling remained on the cell surface after stimulation with either agonist (Fig. 3GGo). Similarly, in CHO-sst2A+5 cells, stimulation with [D-Trp8]-SRIF-14 for 40 min reduced cell surface sst2A immunolabeling to 20% of that measured in nonstimulated cells (Fig. 3GGo). By contrast, in CHO-sst2A+5 cells, stimulation with L-779–976 left more than 40% of cell surface immunofluorescence (P ≤ 0.001, as compared with CHO-sst2A+5 cells stimulated with [D-Trp8]-SRIF-14; n = 3; Fig. 3GGo). This value was also significantly different from that measured in CHO-sst2A cells stimulated with the same ligand (P ≤ 0.001; n = 3; Fig. 3GGo; two-way ANOVA with Bonferroni’s post hoc test: F (interaction) = 3.548; F (treatment) = 393.5; F (cell type) = 3.377).


Figure 3
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  		FIG. 3. Effect of stimulation on cell surface sst2A immunofluorescence. Cells were stimulated for 40 min at 37 C with [D-Trp8]-SRIF-14 or L-779,976, incubated with rabbit anti-c-Myc antibody at 4 C and then fixed and fluorescently stained. A–F, Confocal images acquired as single optical sections through the center of the cells; A and D, before stimulation, intense cell surface immunofluorescence is apparent in both CHO-sst2A and CHO-sst2A+5 cells; B and E, stimulation with [D-Trp8]-SRIF-14 induces a loss of cell surface fluorescence in both cell types; C and F, stimulation with L-779,976 induces loss of cell surface immunofluorescence in CHO-sst2A (C) but not in CHO-sst2A+5 (F) cells. Scale bar, 5 µm. G, Quantification of cell surface labeling by FLIPR. In CHO-sst2A+5 cells stimulated with L-779,976, the ligand-induced loss of cell surface sst2A immunoreactivity is attenuated as compared with both [D-Trp8]-SRIF-14 stimulation in the same cell line (***, P ≤ 0.001) and L-779,976 stimulation in CHO-sst2A cells (***, P ≤ 0.001; two-way ANOVA with Bonferroni’s multiple comparison test).

 
Sst2A trafficking in AtT20 cells
To follow the internalization and trafficking of sst2A in cells endogenously expressing both sst2A and sst5, we stimulated the mouse corticotropic adenoma-derived cell line AtT20 as described. Because in these experiments we were investigating native, endogenously expressed receptors, we used a subtype-specific antibody recognizing the C-terminal domain of sst2A (34). In keeping with previous findings in this cell line (21), in the absence of stimulation, sst2A was predominantly located at the periphery of the cells (Fig. 4Go, A and B). After stimulation with [D-Trp8]-SRIF-14, cell surface fluorescence diminished, whereas more intracellular fluorescent vesicles appeared (Fig. 4CGo) and the fluorescence in the juxtanuclear compartment increased (Fig. 4Go, C and E). After 40 min, the bulk of sst2A immunolabeling was concentrated in a hot spot next to the nucleus (Fig. 4GGo). Stimulation of AtT20 cells with L-779,976 did not induce a sequestration of sst2A immunoreactivity similar to that observed after [D-Trp8]-SRIF-14 exposure. In fact, the outline of the cells was always clearly delineated by sst2A immunofluorescence up to 40 min of stimulation with L-779,976 (Fig. 4Go, B, D, F, and H). Concomitantly, the concentration of sst2A in the juxtanuclear compartment was much less pronounced at all times as compared with [D-Trp8]-SRIF-14 stimulation.


Figure 4
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  		FIG. 4. Ligand-induced trafficking of sst2A in AtT20 cells. Cells were stimulated with [D-Trp8]-SRIF-14 or L-779,976 for the indicated periods of time, fixed, and immunostained using a rabbit anti-sst2A antibody. A, C, E, and G, [D-Trp8]-SRIF-14 induces rapid and efficient internalization of sst2A, as evident by the progressive clusterization of sst2A immunofluorescence in the pericentriolar endosome (also see Ref. 21 ); B, D, F, and H, after stimulation with L-779,976, intracellular sequestration of sst2A immunofluorescence is markedly reduced. Scale bar, 10 µm. I, Densitometric analysis of cell surface fluorescence after 40-min stimulation with either [D-Trp8]-SRIF-14 (gray bar) or L-779,976 (black bar). Data correspond to the means ± SEM of three independent experiments carried out in quadruplicate and expressed as a percentage of the peripheral fluorescence in nonstimulated control cells (white bar = 100%). ***, P ≤ 0.001; one-way ANOVA with Bonferroni’s multiple comparison test.

 
Because our sst2A-specific antibody was directed against the intracellular C terminus of the receptor protein, we could not employ the described live-cell immunolabeling technique for cell surface fluorescence quantification by FLIPR. Alternatively, we quantified the intensity of peripheral immunolabeling from the confocal images by densitometry. This approach revealed a highly significant difference in the loss of cell surface sst2A after 40 min between [D-Trp8]-SRIF-14-stimulated cells, in which approximately 35% of peripheral (presumably cell surface) labeling remained, and cells exposed to the selective agonist L-779,976, which retained 55% of their cell surface sst2A receptor population (Fig. 4IGo; P ≤ 0.001; n = 3).

Reappearance of cell surface sst2A
To investigate whether monensin-sensitive recycling mechanisms contributed to the maintenance of cell surface sst2A receptors in cells coexpressing sst5 and whether [D-Trp8]-SRIF-14 and L-779,976 had the same effect on recycling, we stimulated CHO-sst2A and CHO-sst2A+5 cells for 40 min with either one of these analogs in the presence or absence of monensin. After stripping off any remaining surface-bound ligand, the cells were allowed recovery periods of 5–40 min at 37 C in buffer without agonist before being subjected to live cell immunolabeling and confocal microscopic analysis or FLIPR quantification of cell surface fluorescence as described.

In CHO-sst2A cells stimulated with [D-Trp8]-SRIF-14, confocal microscopy revealed a gradual reappearance of immunoreactive sst2A receptors at the cell surface (Fig. 5AGo). Quantification of cell surface fluorescence in this cell line revealed that after 5 min of recovery, cell surface fluorescence levels corresponded to approximately 25% of baseline values after stimulation with [D-Trp8]-SRIF-14 (white bars) or L-779,976 (light gray bars; Fig. 5BGo). After 40 min of recovery, these values rose to approximately 60 and 50% of baseline, respectively. Coincubation with the recycling inhibitor monensin reduced the reappearance of sst2A after both [D-Trp8]-SRIF-14 (black bars) and L-779,976 (dark gray bars) by approximately 50% (P ≤ 0.05; n = 3; Fig. 5BGo).


Figure 5
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  		FIG. 5. Recycling of sst2A after internalization in CHO-sst2A and CHO-sst2A+5 cells. Cells were stimulated for 40 min with [D-Trp8]-SRIF-14 or L-779,976 at 37 C, incubated for the indicated periods of time with fresh buffer without agonist and with or without 25 µM of the recycling inhibitor monensin at 37 C. Immunostaining with a rabbit-anti-c-Myc antibody was performed before fixation to selectively label cell surface sst2A receptors. A, Confocal images of [D-Trp8]-SRIF-14-stimulated CHO-sst2A cells after 0, 5, 20, and 40 min of recovery illustrate the gradual reappearance of cell surface c-Myc-sst2A immunoreactivity. Images were acquired as single optical sections through the center of the cells. Scale bar, 5 µm. B and C, Quantification of cell surface c-myc-sst2A reappearance by FLIPR in CHO-sst2A (B) and CHO-sst2A+5 cells (C) after stimulation with [D-Trp8]-SRIF-14 (white and black bars) or L-779,976 (light and dark gray bars) in the presence (black bars) or not (dark gray bars) of monensin. Data represent the means ± SEM of three independent experiments carried out in quadruplicate *, P ≤ 0.05; one-way ANOVA with Bonferroni’s multiple comparison test.

 
In CHO-sst2A+5 cells, the same kinetics and monensin sensitivity of sst2A reappearance were observed after stimulation with [D-Trp8]-SRIF-14 (P ≤ 0.05; n = 3; Fig. 5CGo). By contrast, after stimulation with L-779,976 and 5 min of recovery, cell surface fluorescence was at approximately 30–35% of baseline levels, irrespective of the presence of monensin. This level of surface fluorescence rose only slightly, but not statistically significantly, to 40–45% toward the end of the experiment (40 min).

Double staining for sst2A and sst5
To determine whether sst2A and sst5 colocalize at any time during agonist stimulation, we stimulated CHO-sst2A+5 cells for 40 min with 100 nM of either [D-Trp8]-SRIF-14 or L-779,976 and immunostained for both receptor subtypes. The results revealed that the subcellular distribution of sst2A and sst5 was largely separate before stimulation. Although sst2A was predominantly located at the cell periphery, the bulk of sst5 immunoreactivity resided in a hot spot next to the nucleus (Fig. 6Go, A–C), in keeping with previous studies in other cell types (19, 21, 27), stimulation with [D-Trp8]-SRIF-14 for 40 min induced internalization of sst2A and concentration of the immunolabel in a juxtanuclear compartment (Fig. 6DGo). At the same time, sst5 receptors were mobilized in small vesicles distributed throughout the cytoplasm (core and periphery) of the cell (Fig. 6EGo). As evident in merged confocal images (Fig. 6FGo), these two vesicular populations were largely distinct from one another. By contrast, after 40 min of stimulation with L-779,976, the subcellular distributions of sst2A and sst5 were indistinguishable from that before stimulation (Fig. 6Go, G–I).


Figure 6
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  		FIG. 6. Dual immunocytochemical labeling of sst2A and sst5. CHO-sst2A+5 cells were stimulated or not with [D-Trp8]-SRIF-14 or L-779,976 for 40 min and stained with primary antibodies directed against the c-Myc and HA epitope tags. A–C, In nonstimulated cells, sst2A (A) is located at the periphery of the cells and sst5 (B) is distributed throughout the cytoplasm. Merging of images (C) reveals no overlap between sst2A and sst5 immunolabeling. D–F, After stimulation with [D-Trp8]-SRIF-14, sst2A becomes highly concentrated in a juxtanuclear compartment (D), and sst5-positive vesicles (E) are distributed throughout the cytoplasm of the cell. Merging of images (F) shows that these vesicles are largely distinct. G–I, After stimulation with L-779,976, sst2A (G) remains at the cell periphery whereas sst5-positive puncti (H) are largely distributed throughout the cytoplasm. In the merged image (I), there is no overlap between sst2A and sst5 immunostaining. Scale bar, 10 µm.

 
Inhibition of cAMP accumulation after prestimulation with [D-Trp8]-SRIF-14 or L-779,976
To determine whether prestimulation with [D-Trp8]-SRIF-14 or L-779,976 desensitized the cells toward a second stimulation, i.e. to see whether remaining cell surface receptors were functional, we incubated CHO-sst2A, CHO-sst2A+5, and AtT20 cells for 40 min with 100 nM of either [D-Trp8]-SRIF-14 or L-779,976 or in the absence of any agonist, followed by a second stimulation with either forskolin to induce cAMP production or forskolin plus [D-Trp8]-SRIF-14. Intracellular cAMP concentrations were determined by enzyme immunoassay. Cells without previous stimulation by SRIF agonists responded to forskolin with a 20- to 25-fold increase in intracellular cAMP (data not shown). [D-Trp8]-SRIF-14 given together with forskolin in non-prestimulated cells inhibited the forskolin effect by 40% in all cell types (Fig. 7Go, white bars). In cells prestimulated with [D-Trp8]-SRIF-14, a second exposure to the same agonist inhibited the forskolin-induced accumulation of cAMP by only 10% (Fig. 7Go, black bars). The same was observed in CHO-sst2A cells after prestimulation with L-779,976. However, in CHO-sst2A+5 and AtT20 cells prestimulated with the sst2-selective agonist, a subsequent exposure to [D-Trp8]-SRIF-14 had the same inhibitory effect on forskolin-induced cAMP production as in non-prestimulated control cells (Fig. 7Go, light gray bars), suggesting that these cells were not desensitized to the effects of SRIF.


Figure 7
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  		FIG. 7. Effect of agonist exposure on desensitization of CHO-sst2A, CHO-sst2A+5, and AtT20 cells. Cells were not stimulated (white bars) or prestimulated for 40 min with 100 nM [D-Trp8]-SRIF-14 (black bars) or L-779,976 (gray bars) at 37 C, and the inhibition of forskolin-stimulated cAMP production by a second stimulation with 100 nM [D-Trp8]-SRIF-14 was determined. Data are expressed as a percentage of the effect of forskolin alone in the second stimulation on cAMP production as compared with control cells without prestimulation. They represent the means ± SEM of four independent experiments done in quadruplicate. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; one-way ANOVA with Tukey’s multiple comparison test. 0, Non-prestimulated control; FSK, forskolin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that ectopic or endogenous coexpression of the SRIF receptor subtype sst5 in epithelial-like and neuroendocrine cells affects the regulation of cell surface availability of the SRIF receptor subtype sst2A. Stimulation with an sst2-selective agonist in cells expressing both receptor subtypes results in maintenance of immunoreactive sst2A at the cell surface as compared with cells expressing sst2A alone and thereby reduces cellular desensitization to the effects of SRIF.

To compare the effects of nonselective vs. selective receptor stimulation in cells expressing sst2A and/or sst5, the selectivity and affinity of L-779,976 and [D-Trp8]-SRIF-14 binding was assessed in live cells expressing these receptors ectopically or endogenously. As previously observed in transiently transfected cells (25, 27), the binding affinities of the nonselective SRIF receptor agonist [D-Trp8]-SRIF-14 for the rodent sst2A and sst5 were in the sub-nanomolar range in both CHO-sst2A and CHO-sst2A+5 cells and in the nanomolar range in AtT20 cells. L-779,976 was found here to exhibit a lower affinity for mouse sst2A in CHO cells than previously reported toward the human sst2A receptor in membrane preparations (30). However, it still showed IC50 values well less than 10–7 M in both CHO-sst2A and CHO-sst2A+5 cells and in the nanomolar range in AtT20 cells, in keeping with the recent report that L-779,976 and native SRIF-14 inhibited forskolin-induced cAMP accumulation in AtT20 cells with the same EC50 (42). Also, L-779,976 did not displace [125I]Tyr0-[D-Trp8]-SRIF from CHO-K1 cells expressing only sst5, confirming that it was highly selective for sst2 over sst5 (30). These data demonstrate that [D-Trp8]-SRIF and L-779,976 have comparable affinities for sst2 irrespective of whether this receptor is expressed alone or together with sst5 and that differences in the effects of receptor stimulation between cells expressing sst2 or sst2/sst5 cannot be ascribed to different affinities of the agonists.

Radioactive binding assays after stimulation with [D-Trp8]-SRIF-14 revealed that this nonselective agonist promoted a rapid loss of approximately 60–70% of cell surface SRIF binding sites in all cell types investigated. Stimulation with the sst2-selective L-779,976 induced a similar reduction in cell surface SRIF binding sites in CHO-sst2A cells. However, in both CHO-sst2A+5 and AtT20 cells, only about 20–30% of cell surface binding sites were down-regulated, suggesting that coexpression of other SRIF receptor subtypes, and namely of sst5, contributed to the maintenance of SRIF receptors on the cell surface.

To determine whether the relative maintenance of SRIF binding sites in cells expressing both sst2A and sst5 after stimulation with L-779,976 was due to compensatory membrane recruitment of sst5 receptors or to increased resistance of sst2A to down-regulation, we monitored intracellular trafficking of sst2A immunoreactivity after stimulation with either [D-Trp8]-SRIF-14 or L-779,976. In keeping with earlier reports, immunoreactive sst2A receptors were predominantly located at the cell surface before stimulation in all cell types examined, and exposure to [D-Trp8]-SRIF-14 led to their efficient internalization (19, 21, 22, 23, 25, 26, 43), resulting in a reduction of cell surface sst2A immunofluorescence by approximately 70–80% after 40 min. L-779,976 induced the same effect only in CHO-sst2A cells. By contrast, in CHO-sst2A+5 and AtT20 cells, stimulation with L-779,976 induced a loss of cell surface sst2A immunofluorescence of only 60 and 45%, respectively. These data indicate that the maintenance of SRIF binding sites recorded in cells expressing both receptor subtypes using [125I]Tyr0-[D-Trp8]-SRIF binding assays not only reflects compensatory recruitment of sst5 receptors to the plasma membrane (27) but is also due to the maintenance of cell surface sst2A receptors.

Such maintenance of sst2A cell surface receptors in the presence of an agonist might be explained by the agonistic properties of the nonpeptidic L-779,976 as opposed to the peptide agonist [D-Trp8]-SRIF-14. Indeed, a recent report indicated that L-779,976 may be less potent than native SRIF in inducing sst2A internalization in transfected HEK 293 cells (44). However, the fact that in the present study sst2A internalization in CHO-sst2A cells, as assessed using either radioactive ligand binding or immunofluorescence experiments, was the same irrespective of the agonist, argues against this notion. Rather, our observations suggest that in the presence of sst5, stimulation of the latter receptor subtype in addition to that of sst2A is needed to provide efficient down-regulation of sst2A by ligand-induced endocytosis.

To determine whether the relative maintenance of cell surface sst2A receptors may at least in part be due to rapid recycling of internalized receptors as suggested by recent observations (22 , but see also Ref. 45), we stimulated CHO-sst2A and CHO-sst2A+5 cells with either [D-Trp8]-SRIF-14 or L-779–976 in the presence of the recycling inhibitor monensin. The results demonstrate that irrespective of the cell model and the ligand used, sst2A receptors reappear to some extent at the cell surface. This process was inhibited by monensin in CHO-sst2A and CHO-sst2A+5 cells after [D-Trp8]-SRIF-14 stimulation but only in CHO-sst2A cells subsequent to L-779,976 exposure. Given the attenuated loss of cell surface sst2A receptors in response to L-779,976 in CHO-sst2A+5 cells, it is possible that the elevated levels of cell surface immunofluorescence at the beginning of the recovery period precluded the detection of a small effect of monensin. Nonetheless, these experiments suggest that rapid sst2A recycling is unlikely to contribute to the maintenance of cell surface SRIF receptors in cells expressing both sst2A and sst5.

Taken together, the present results therefore suggest that the reduced down-regulation of sst2A receptors observed after stimulation with L-779,976 in cells expressing both sst2A and sst5 results from impaired ligand-induced internalization of sst2A. The mechanism by which sst5 expression affects sst2A internalization (unless sst5 is itself stimulated) is unclear. Receptor heterodimerization has been invoked to explain changes in receptor trafficking upon coexpression of multiple receptor subtypes. For instance, coexpression of the NTS1 with the NTS2 neurotensin receptor subtype markedly reduces cell surface targeting of NTS1. Moreover, NTS1 is less sensitive to down-regulation in response to prolonged stimulation in cells coexpressing NTS1 and NTS2 (46). However, there has so far been no evidence for the formation of sst2A/sst5 heterodimers, although both sst2A and sst5 have been shown to form homodimers as well as heterodimers with other SRIF receptor subtypes and nonrelated G-protein-coupled receptors (47, 48, 49, 50). Furthermore, in the present study, sst2A and sst5 were never found to colocalize by confocal microscopy after stimulation with either [D-Trp8]-SRIF-14 or L-779,976. Other mechanisms must therefore be invoked to account for the relative preservation of sst2A receptor density in CHO-sst2A+5 and AtT20 cells stimulated with L-779,976. Recent studies in AtT20 cells using the subtype-selective compounds BIM 23120 (sst2) and BIM 23206 (sst5) to investigate intracellular Ca2+ homeostasis and receptor internalization demonstrated that sst5 acts as a modulator of sst2A, suggesting that a signaling pathway downstream of sst5 may regulate sst2A trafficking (51). Alternatively, sst5 might sequester an interaction partner in the agonist-naive state and liberate it when stimulated by [D-Trp8]-SRIF-14. This hypothetical chaperone may subsequently interact with sst2A and reinforce sst2A internalization in response to [D-Trp8]-SRIF-14 stimulation. This model would explain why the efficiency of sst2A internalization is reduced in cells that coexpress sst5 but not in CHO-sst2A cells after subtype-selective stimulation with L-779,976.

To ensure that sst2A receptors maintained at the cell surface were functional, we monitored desensitization by measuring the inhibitory effects of [D-Trp8]-SRIF-14 on forskolin-induced cAMP accumulation after prestimulation of the cells. After prestimulation with [D-Trp8]-SRIF-14, all cell types investigated were desensitized to the effects of a second [D-Trp8]-SRIF-14 exposure. By contrast, after prestimulation with L-779,976, only CHO-sst2A cells were desensitized to subsequent [D-Trp8]-SRIF-14 exposure, whereas CHO-sst2A+5 and AtT20 cells were almost fully responsive to the second [D-Trp8]-SRIF-14 stimulation. Although the subpopulation of sst5 receptors at the plasma membrane (27) may contribute to this lack of cellular desensitization to the effects of SRIF in cells expressing both receptor subtypes, these findings suggest that the reduced down-regulation of cell surface sst2A receptors observed in radioligand binding and immunohistochemical studies translates into a preserved cellular response to SRIF.

The inhibitory effects of SRIF on hormone release from both rodent and human anterior pituitary endocrine cells have been shown to be mainly mediated by sst2A and sst5 (12, 13, 14, 15, 16). Synthetic octapeptide SRIF analogs such as octreotide and lanreotide have proven very successful in the therapy of hormone-secreting pituitary adenomas, especially in GH-secreting adenomas causing acromegaly, and other neuroendocrine tumors (5, 17). Patients under long-term octreotide treatment never develop tolerance, i.e. a reduction in the potency of the drug with the need to increase the dose over time (5, 18). Octreotide has a sub-nanomolar binding affinity for sst2 and an approximately 10- to 100-fold lower affinity for sst5 (38, 39, 41). In light of the findings of the present study, it is tempting to speculate that the lack of tolerance observed after chronic treatment with octreotide is due to the pharmacological profile of the drug.

In conclusion, the present study shows that the sst5 SRIF receptor subtype modulates the trafficking of sst2A receptors in cells that either ectopically or endogenously express both receptor proteins. This modulation protects sst2A from desensitization through maintenance of cell surface receptors after stimulation with a sst2-selective agonist. These findings might explain the lack of tolerance toward the effects of long-term treatment with sst2-preferring SRIF analogs in patients with neuroendocrine tumors.


   Acknowledgments
 
We thank Mariette Lavallée and Nicole Kasischke for expert technical assistance and Naomi Takeda for administrative assistance in the preparation of the manuscript.


   Footnotes
 
This work was supported by Fonds de la Recherche en Santé du Québec, Canadian Institutes of Health Research (Operating Grant MOP 7366 to A.B.) and Institut National de la Santé et de la Recherche Médicale (France). N.S. and L.G. were funded by fellowships from the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research (MFE-63497), respectively.

Present address for L.G. and P.S.: Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada.

Author Disclosure Summary: N.S., L.G., J.W., P.S., J.M., A.B., and T.S. have nothing to declare.

First Published Online February 1, 2007

Abbreviations: FLIPR, Fluorescence immunoplate reader; NGS, normal goat serum; PAO, phenylarsine oxide; SRIF, somatotropin release-inhibiting factor; TBS, Tris-buffered saline.

Received September 19, 2006.

Accepted for publication January 25, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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