Our data show that SST-14 was cointernalized with SSTR3, was uncoupled from the receptor, and was sorted into an endocytic degradation pathway, whereas octreotide was recycled as an inta
Trang 1somatostatin-receptor 3-mediated endocytosis in rat
insulinoma cells
Dirk Roosterman1, Nicole E I Brune2, Oliver J Kreuzer2, Micha Feld1, Sylvia Pauser1, Kim Zarse2, Martin Steinhoff1and Wolfgang Meyerhof2
1 Department of Dermatology, IZKF Mu¨nster and Ludwig Boltzmann Institute for Cell and Immunobiology of the Skin, Germany
2 Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany
Somatostatin is a cyclic peptide that is widely
expressed throughout the central nervous system,
endocrine tissue, skin and gastrointestinal tract [1]
Somatostatin exerts a wide range of important
biolo-gical effects, including inhibition of secretion of growth
hormone, insulin, glucagon and gastrin as well as other
hormones secreted from the pituitary, skin and
gastro-intestinal tract [2]
Among other actions, somatostatin elicits strong antiproliferative effects in in vivo as well as in vitro models of cancer [3–5] Somatostatin analogs are there-fore being used in the diagnosis and therapy of various tumors, in particular neuroendocrine tumors, which express somatostatin receptors (SSTRs) [3–5] SSTR scintigraphy (SRS), a widely used imaging technique,
is employed to detect and localize such tumors The
Keywords
endocytosis; G-protein-coupled receptor;
neuropeptide; proteolysis; somatostatin
Correspondence
D Roosterman, Department of Dermatology
and IZKF Mu¨nster, Von-Esmarch-Strasse 58,
D-48148 Mu¨nster, Germany
Fax: +49 0251 8357452
Tel: +49 0251 8352932
E-mail: roosterman@gmx.net
(Received 27 March 2008, revised 20 June
2008, accepted 23 July 2008)
doi:10.1111/j.1742-4658.2008.06606.x
Somatostatin receptor (SSTR) endocytosis influences cellular responsiveness
to agonist stimulation and somatostatin receptor scintigraphy, a common diagnostic imaging technique Recently, we have shown that SSTR1 is dif-ferentially regulated in the endocytic and recycling pathway of pancreatic cells after agonist stimulation Additionally, SSTR1 accumulates and releases internalized somatostatin-14 (SST-14) as an intact and biologically active ligand We also demonstrated that SSTR2A was sequestered into early endosomes, whereas internalized SST-14 was degraded by endosomal peptidases and not routed into lysosomal degradation Here, we examined the fate of peptide agonists in rat insulinoma cells expressing SSTR3 by biochemical methods and confocal laser scanning microscopy We found that [125I]Tyr11-SST-14 rapidly accumulated in intracellular vesicles, where
it was degraded in an ammonium chloride-sensitive manner In contrast, [125I]Tyr1-octreotide accumulated and was released as an intact peptide Rhodamine-B-labeled SST-14, however, was rapidly internalized into endo-some-like vesicles, and fluorescence signals colocalized with the lysosomal marker protein cathepsin D Our data show that SST-14 was cointernalized with SSTR3, was uncoupled from the receptor, and was sorted into an endocytic degradation pathway, whereas octreotide was recycled as an intact peptide Chronic stimulation of SSTR3 also induced time-dependent downregulation of the receptor Thus, the intracellular processing of inter-nalized SST-14 and the regulation of SSTR3 markedly differ from the events mediated by the other SSTR subtypes
Abbreviations
EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; HSV, herpes simplex virus glycoprotein D; RIN, rat insulinoma; SRS, somatostatin receptor scintigraphy; SST-14, somatostatin-14; SSTR, somatostatin receptor.
Trang 2success of SRS is based on specific interactions of
sta-ble radiolabeled somatostatin analogs injected into
patients, causing them to bind to SSTRs expressed by
tumor cells [4,6] These interactions are not restricted
to the binding of the peptide agonist to its cognate
receptor, but also lead to agonists accumulating in the
tumor cell after internalization of the receptor–agonist
complex [7–9] Understanding the internalization of
somatostatin–receptor complexes and their intracellular
fate is therefore of considerable interest in tumor
diag-nostics and therapy as well as neuroinflammation
Somatostatin binds to and activates six different
G-protein-coupled SSTR subtypes: SSTR1, SSTR2A,
SSTR2B, SSTR3, SSTR4 and SSTR5 The various
SSTR subtypes show distinct internalization pathways
[10,11] In human embryonic kidney 293 cells and
neuroendocrine pancreatic b-cells, rat SSTR1,
SSTR2A, SSTR3 and SSTR5 but not SSTR4 were
internalized upon stimulation by somatostatin Further
investigation of SSTR1 and SSTR2A clearly indicated
that the fate of internalized somatostatin-14 (SST-14)
strongly depends on the receptor subtype, although the
particular pathways are not yet fully explored We
recently demonstrated that chronic stimulation of
SSTR1 induced accumulation of SST-14 in cells via a
dynamic process of internalization, recycling and
rein-ternalization of the ligand [11] In contrast, stimulation
of SSTR2A with SST-14 or its stable analog octreotide
induced prolonged sequestration of the receptor–ligand
complex into early endosomes that was dependent on
arrestins [12,13] Subsequently, the endosomal
peptidase endothelin-converting enzyme-1 cleaves
inter-nalized SST-14 between positions Asn5-Phe6 and
Thr10-Phe11, leading to release of internalized SST-14
as SST-14(6–10) (FFWKT) and SST-14(1–5)⁄ (11–14)
(AGCLN⁄ FTSC) [13]
Further analysis of SSTR3, an SSTR subtype of
particular importance in human thymoma [14], shows
that agonist-mediated internalization of SSTR3 is
criti-cally dependent on phosphorylation of the C-terminal
tail [15] As the phosphorylation sites do not
corre-spond to consensus sequences for second
messenger-regulated protein kinases, protein kinase A or protein
kinase C, it was suggested that specific
G-protein-coupled receptor kinases were involved [16] Moreover,
colocalization studies and the use of
dominant-nega-tive mutants of arrestin-2 demonstrated that
internali-zation of SSTR3 involves arrestin-2, the adaptor
protein-2 complex, and proceeds via clathrin-coated
pits and vesicles [16] In contrast to the trafficking
process of the receptor, the fate of the peptide agonist
has not thus far been analyzed after cointernalization
with SSTR3
Therefore, we examined the fate of SST-14 and octreotide cointernalized with SSTR3 in transfected rat insulinoma (RIN) cells by biochemical methods and confocal laser scanning microscopy We show that SST-14 endocytosed with SSTR3, uncoupled from the receptor and proceeded to lysosomal degra-dation, whereas octreotide endocytosed with SSTR3 but was released as an intact peptide from the cells Moreover, chronic stimulation of SSTR3 with
SST-14 induced time-dependent downregulation of the receptor Our results demonstrate that SSTR3 traf-ficking and ligand processing differ markedly from the mechanisms observed for either SSTR1 or SSTR2A
Results
Reduction of cell surface binding sites
To examine the time course of SST-14-induced loss of SSTR3-specified cell surface binding sites, RIN-SSTR3 cells were stimulated with SST-14 for 0–120 min (Fig 1A) Incubation was stopped by placing cells on ice, and surface binding sites were determined Stimu-lation of the SSTR3-expressing cells with SST-14 induced a relatively slow reduction in the number of surface binding sites as compared to SSTR1 [11] Sixty minutes after chronic stimulation with SST-14, the number of binding sites was decreased to 50% of the original density, and it remained at this level for another 60 min
Recovery of cell surface binding sites Next, we determined whether or not cell surface binding recovered following removal of the stimulus RIN-SSTR3 cells were first stimulated with SST-14 for
120 min Cell surface-bound ligand was washed off, and cells were incubated for another 0–120 min The recov-ery of surface binding sites was determined as described above Interestingly, during the first 15 min of incuba-tion, surface binding recovered to 76% However, incu-bation of the cells for a period of up to 120 min did not lead to recovery of surface binding beyond this value (Fig 1B) This result indicates that prolonged incuba-tion of SSTR3-expressing cells with SST-14 induced marked downregulation of the receptor We therefore determined the time dependence of SSTR3 downregula-tion by chronic stimuladownregula-tion Therefore, RIN-SSTR3 cells were stimulated for 0–1300 min with SST-14 at
37C Subsequently, the agonist peptide was washed off, and cells were incubated for recovery of surface binding sites (Fig 1C) Chronic stimulation resulted in
Trang 3time-dependent downregulation of the receptor After a
period of 1300 min of stimulation and 120 min of
recov-ery, cell surface binding was reduced to 43 ± 5%
The time-dependent loss of surface binding sites
during stimulation with SST-14 suggests that the
recep-tor was internalized We determined the ratio between
cell surface-located receptors and internalized receptors
after 1 h of incubation with SST-14 (Fig 1D) by
mea-suring cell surface binding and total cellular binding in
the presence of saponin [17] Incubation of untreated
cells with saponin did not significantly change the
number of binding sites After stimulation with SST-14, cell surface binding decreased to 52 ± 3% of that of untreated cells, whereas total binding remained at 91 ± 4% of that of untreated cells Thus, after peptide stimu-lation, approximately 40% of the SSTR3 receptors were localized in intracellular compartments, and the data clearly indicate that SSTR3 was internalized during stimu-lation These data are in line with the fluorescence micros-copy quantification of intracellularly located SSTR3 [18] The loss of cell surface binding sites during chronic stimulation suggests that the receptor is sequestered
Fig 1 Loss and recovery of SST-14 binding sites (A) SST-14-mediated reduction of cell surface binding sites RIN-SSTR3 cells were stimu-lated with SST-14 at 37 C for the indicated times Cells were washed with acidic buffer, and surface binding sites were determined by incu-bation with [ 125 I]SST-14 at 4 C (B) Recovery of cell surface binding sites RIN-SSTR3 cells were stimulated for 120 min with SST-14 at
37 C Cells were washed with acidic buffer and incubated for the indicated times, and cell surface binding sites were determined as described above (C) Downregulation of surface binding sites RIN-SSTR3 cells were stimulated with SST-14 for the indicated times, washed with acidic buffer, and incubated for 120 min at 37 C Surface binding sites were determined as described above (D) Determination of cell surface and total binding after stimulation with SST-14 RIN-SSTR3 cells were stimulated with SST-14 in the absence or presence of sapo-nin Binding was measured as described above The data are expressed at mean ± SEM values from three independent experiments (E, F) Distribution of SSTR3–HSV in control cells and under conditions of receptor downregulation RIN cells expressing SSTR3–HSV were stimulated (F) or not stimulated (E) with SST-14 for 1300 min Then, the peptide was removed and cells were allowed to recover for 90 min Thereafter, the epitope-tagged SSTR3 was visualized by indirect immunfluorescence (F) Localization of SSTR3 after chronic stimulation with SST-14 Chronic stimulation of SSTR3 with SST-14 mediates downregulation of SSTR3 The immunofluorescence signal of SSTR3 is concentrated in one area of the cell and not equally distributed in the cell membrane (arrows).
Trang 4within the cells or that it is downregulated by
degrada-tion To distinguish between the two possibilities, we
determined the localization of SSTR3 after 1300 min
of stimulation with SST-14 and 90 min of recovery In
untreated cells, SSTR3 showed a bright
immunofluo-rescence signal at the cell membrane (Fig 1E, arrows)
In cells chronically stimulated with SST-14, the
fluores-cence signal was weaker than the signal seen in
untreated cells Moreover, in all of these cells, the
SSTR3 immunofluorescence signal was locally
concen-trated in only one area of the cell surface and not
evenly distributed over the plasma membrane (Fig 1F,
arrows)
Together, the results indicate that stimulation of SSTR3 with SST-14 induced internalization of the receptor Chronic stimulation with SST-14 mediated downregulation of SSTR3
Uptake of [125I]Tyr11-SST-14 and [125 I]Tyr1-octre-otide in SSTR3-expressing rat insulinoma cells
To examine the fate of SST-14 cointernalized with SSTR3, we measured the uptake of [125
I]Tyr11-SST-14 in RIN-SSTR3 cells in the absence (Fig 2A, diamonds) or presence (Fig 2A, triangles) of ammo-nium chloride Treatment of the cells with
Fig 2 SSTR3-mediated uptake of 125 I-labeled peptides (A) SSTR3-mediated uptake of [ 125 I]Tyr11-SST-14 RIN-SSTR3 cells were incubated with [ 125 I]SST-14 in the presence (triangle) or absence (diamonds) of ammonium chloride Cell surface [ 125 I]Tyr11-SST-14 was washed off, and the amount of cell-associated radioactivity was determined (B) SSTR3-mediated uptake of [ 125 I]Tyr1-octreotide RIN-SSTR3 cells were incubated with [ 125 I]Tyr1-octreotide Cell surface [ 125 I]Tyr1-octreotide was washed off, and the amount of cell-associated radioactivity was determined (C) HPLC separation of cell-associated, agonist-bound internalized radioactivity in the presence of ammonium chloride Cells were pretreated with ammonium chloride, incubated with [ 125 I]Tyr11-SST-14 at 37 C for 30 min in the presence of ammonium chloride, washed with acidic buffer, and analyzed by HPLC (D) HPLC separation of the cell supernatant incubated for 30 min with [ 125 I]Tyr11-SST-14 (E) Time course of SSTR3-mediated uptake of [125I]Tyr11-SST-14 RIN- SSTR3 cells were stimulated for 0–15 min with [125I]Tyr11-SST-14, washed with acidic buffer, and incubated for 0–90 min Cell-associated (triangles) and released (diamonds) radioactivity was determined by HPLC (F) Time course of SSTR3-mediated uptake of [ 125 I]Tyr1-octreotide RIN- SSTR3 cells were incubated for 0–15 min with [ 125 I]Tyr1-octreotide, washed with acidic buffer, and incubated for 0–90 min at 37 C Cell-associated radioactivity and radioactivity from the superna-tants was determined by HPLC The data are expressed as mean ± SEM values from three independent experiments.
Trang 5ammonium chloride neutralized acidic cellular
com-partments [19] Within 10–15 min at 37C, the
cellu-lar uptake of [125I]Tyr11-SST-14 reached maximal
levels in the absence of ammonium chloride
corre-sponding to 80% of the amount of the cell
sur-face-bound peptide The amount of intracellular
radioactivity then quickly declined over a period of
15 min to very low levels corresponding to about
30% of the amount of cell surface-bound peptide
These levels slowly decreased over the next 90 min
to < 20% of the initial value These observations
are best explained by assuming that specific
intracel-lular degradation destroys the radiolabeled peptide,
and the degradation products are then released from
the cells The low level of radioactivity observed to
be cell-associated between 60 and 120 min probably
reflects the steady-state level of [125I]Tyr11-SST-14
determined by receptor-mediated uptake and
degra-dation The receptor population engaged in agonist
uptake appears to be largely diminished at these
times, due to receptor internalization and
desensitiza-tion [20] Notably, the amount of the internalized
peptide corresponds to 80% of the cell surface-bound
peptide, suggesting that most of the agonist-occupied
receptors were engaged in endocytosis in the presence
of subnanomolar concentrations of agonist When the
experiment was carried out in the presence of
ammo-nium chloride (Fig 2A, triangles), radioactivity
accu-mulated with a similar kinetic during the first 10 min of
incubation but reached a plateau corresponding to
almost 100% of cell surface-bound [125I]Tyr11-SST-14
Thus, under conditions in which the vesicular pH is
neutral [19], almost all cell surface-bound radioactivity
accumulated and remained in the cells
Next, we determined the SSTR3-mediated uptake of
[125I]octreotide (Fig 2B) Chronic stimulation of
SSTR3-expressing cells with octreotide induced
contin-uous uptake of the ligand During the first 30 min of
incubation, 118% of surface-bound octreotide was
found to be cell-associated Further incubation induced
a linear accumulation of radioactivity within the cells
After 4 h of incubation, the amount of internalized
radioactivity was equivalent to 256% of cell
surface-bound octreotide
In order to distinguish between radioactivity
corre-sponding to degraded or intact peptide, we examined
the cell-associated radioactivity by HPLC We found
that [125I]Tyr eluted in fractions 1–5, degraded peptide
fragments in fractions 9–11, and intact [125I]SST-14
in fractions 14–17 (Fig 2C,D) Figure 2C shows a
radiogram of the cell-associated radioactivity after
30 min of stimulation with [125I]SST-14 in the presence
of ammonium chloride This treatment blocked the
degradation of [125I]SST-14 For instance, more than 95% of the cell-associated radioactivity eluted as intact [125I]SST-14 in fraction 16 Minor amounts of peptide fragments of [125I]SST-14 were observed in fractions 9–11, suggesting modest degradation of [125I]SST-14 by peptidases The degradation of [125I]SST-14 to [125I]Tyr was completely blocked Together, these results suggest that internalized [125I]Tyr11-SST-14 was targeted in a degradation pathway that is sensitive to ammonium chloride
In contrast, a representative HPLC chromatogram of the supernatant collected 30 min after stimulation of the cells incubated with [125I]Tyr11-SST-14 shows that 97%
of the radioactivity was [125I]Tyr (Fig 2D) [21,22] This result suggests that SST-14 was completely degraded to its amino acids, which were subsequently released into the supernatant Similar HPLC analyses of the radio-active degradation products found in the supernatant of stimulated SSTR1 cells have demonstrated that SST-14
is relatively stable in the supernatant and is only slowly degraded by phosphoamidon-sensitive peptidase [19] Thus, we conclude that [125I]SST-14 is processed to amino acids within the cells
Figure 2E shows the time courses of association of [125I]Tyr11-SST-14 (triangles) with the cells and the accumulation of its degradation product [125I]Tyr (dia-monds) in the extracellular medium, as assayed by HPLC analyses of the fractions During the first 15 min
of stimulation, [125I]Tyr11-SST-14 accumulated within the cells After removal of the peptide agonist by wash-ing and further incubation at 37C, the amount of cell-associated [125I]Tyr11-SST-14 rapidly decreased Ninety minutes after stimulation, the amount of cell-associated [125I]Tyr11-SST-14 was reduced to 6% This decay was paralleled by accumulation of [125I]Tyr (Fig 2E, diamonds) in the medium Thus, all of the internalized peptide was degraded to its amino acids
Next, we analyzed whether or not octreotide was degraded during the internalization process In accor-dance with Fig 2B,F (filled circles) shows that [125I]Tyr1-octreotide was rapidly internalized by RIN-SSTR3 cells during the 15 min stimulation period, i.e as long as the peptide was present However, when the stimulus was removed, the cells released the endocytosed peptide into the supernatatant Inter-estingly, HPLC analysis of the cell lysate demon-strated that octreotide was not degraded (data not shown) Both the radioactivity determined in the supernatant and the cell-associated radioactivity eluted with a retention time identical to that of [125I]Tyr1-octreotide Thus, SSTR3 mediates ligand-specific processing Whereas SST-14 is sorted into an ammonium-sensitive degradation pathway, octreotide
Trang 6bypasses degradation, accumulates in the cell and is
released as intact ligand from the cells in the
sur-rounding medium
SSTR3-mediated uptake of fluorescein
isothiocyanate (FITC)–SST-14
To directly visualize the receptor-mediated uptake of
the peptide agonist, internalization of FITC-labeled
SST-14 was examined in RIN-SSTR3 cells by confocal
laser scanning microscopy The fluorescent peptide was
colocalized with herpes simplex virus glycoprotein D
tagged SSTR3 (SSTR3–HSV), as detected by indirect
immunofluorescence Cells were incubated with FITC–
SST-14 at 4C After removal of unbound peptide, a
temperature shift to 37C induced internalization of
cell surface-bound agonist for 2, 30 or 60 min At the
beginning of the observation period at 2 min,
fluores-cence signals of FITC–SST-14 were barely visible
How-ever, a few discrete zones were labeled at the cell surface
(Fig 3A, green arrows, top left) that colocalized with
SSTR3–HSV (Fig 3A, red arrows, top middle panel,
yellow arrows in the overlay) After 30 min, internalized
FITC–SST-14 and SSTR3–HSV frequently colocalized
in intracellular vesicles (Fig 3A, middle panels, yellow
arrows) However, vesicles that appear only in red or
green suggest that some FITC–SST-14 (Fig 3A, green
arrow) dissociated from SSTR3–HSV (Fig 3A, red
arrow) and that SSTR3 and the agonist peptide were
sorted into different cell pathways After 60 min of
stimulation, most of the receptors were recycled to the
plasma membrane (Fig 3A, red arrows, bottom
pan-els), whereas the fluorescence signal of the ligand was
still observed within intracellular vesicular structures
Traces of SSTR3–HSV were also detected within
intra-cellular vesicular compartments, where it colocalized
with SST-14 (Fig 3A, yellow arrowheads)
Agonist-induced mobilization of arrestin-2–
enhanced green fluorescent protein (EGFP)
HPLC analysis of internalized SST-14 and octreotide
demonstrated that SST-14 but not octreotide was
metabolized after internalization We reasoned that the
integrity of the ligand could influence the association
of arrestins with the internalized receptor Therefore,
we analyzed the localization of arrestin-2–EGFP and
SSTR3–HSV after stimulation with 1 lm SST-14 or
octreotide for 15 min (Fig 3B) In unstimulated cells,
arrestin-2–EGFP was diffusely located within the cells
and SSTR3–HSV was primarily located at the plasma
membrane (Fig 3B, top panels) Stimulation with
either of the two agonists induced internalization of
the receptor (Fig 3B, red arrows, middle panel) Accordingly, arrestin-2 was mobilized and transported from the cytosol to the cell membrane (Fig 3B, green arrows, middle panel) Interestingly, 15 min after stim-ulation, arrestin-2 was only partially associated with internalized SSTR3–HSV, indicating that arrestin-2
A
B
Fig 3 Agonist-induced internalization of SSTR3–HSV (A) SSTR3-mediated uptake of FITC–SST-14 RIN-SSTR3 cells were incubated for 60 min with FITC–SST-14 at 0 C Cells were washed and incu-bated for 2, 30 and 60 min at 37 C FITC–SST-14 was detected using the FITC label (shown in green) SSTR3 was detected using antibody directed against the HSV tag (shown in red) (B) Agonist-induced mobilization of arrestin-2–EGFP RIN-SSTR3 cells were transiently transfected with arrestin-2–EGFP Cells were stimulated with SST-14 (1 l M ) or octreotide (1 l M ) for 15 min at 37 C SSTR3–HSV was localized using an antibody against HSV and arres-tin-2-EGFP by EGFP The experiment was performed three times, with similar results.
Trang 7dissociated from the internalized receptor at or close
to the plasma membrane Virtually no differences
could be determined in the localization of arrestin-2–
EGFP after stimulation with SST-14 or octreotide
Thus, our data indicate that the stable ligand,
octreo-tide, did not induce stronger association of arrestin-2–
EGFP with the internalized SSTR3 than did SST-14
Internalized SST-14 is transported to lysosomes
The complete intracellular degradation of internalized
SST-14 suggested that the ligand was processed into
the lysosomal degradation pathway in RIN-SSTR3
cells To examine whether fluorescent dye-labeled
SST-14 was sorted to lysosomes, experiments on
colo-calization of rhodamine-B–SST-14 with cathepsin D, a
lysosomal protease [23], were performed (Fig 4)
Therefore, we incubated RIN-SSTR3 cells with
rhoda-mine–SST-14 at 4C Under these conditions, the
fluo-rescence signals of the peptide were predominantly
observed as a punctate pattern at the cell surface,
whereas the fluorescence signals of the lysosomal
pro-tease appeared to be scattered within the cytoplasm
(Fig 4, top panels) Warming the cells to 37C
induced the internalization of rhodamine–SST-14 and
aggregation of lysosomes (Fig 4, middle panels) After
30 min of stimulation, clear colocalization of cathe-psin D and rhodamine-B–SST-14 was observed, indi-cating that the peptide was routed to a lysosomal degradation pathway (Fig 4, middle panel, yellow arrowhead) After 60 min, both fluorescence signals still colocalized within these compartments (Fig 4, middle panel, yellow arrowhead) The observation that endocytosed SST-14 colocalized with cathepsin D agrees with our data obtained using biochemical assays, demonstrating complete degradation of inter-nalized SST-14 in RIN-SSTR3 cells
Discussion
Recent studies provided clear evidence that the SSTR subtypes (SSTR1, SSTR2, SSTR3 and SSTR5) inter-nalize to similar extents after stimulation with SST-14, somatostatin-28, and synthetic agonists [16,18,20, 24,25] However, detailed analyses of the endocytic processes and the pathways of trafficking of the SSTR subtypes revealed explicit differences
For example, SSTR1 did not mobilize arrestin-2 during internalization, whereas SSTR3 interacted tran-siently with arrestin-2, and internalized SSTR2A formed a stable complex with arrestin-2 [11,16,25] The differences between the receptor subtypes in their inter-action with arrestins indicate the existence of internali-zation and trafficking pathways that are specific for the SSTR subtypes
Determining the fate of the internalized ligand revealed three different pathways of receptor traffic-king and agonist processing SSTR1 mediates accumu-lation and release of intact SST-14 This phenomenon was accomplished by a dynamic process of internaliza-tion, recycling and reinternalization of the peptide, a pathway consistent with the role of SSTR1 as an auto-receptor [11] In contrast, SSTR2A induced sequestra-tion of the receptor–ligand complex within early endosomes SSTR2A did not recycle within a period of
2 h after agonist stimulation SST-14, endocytosed with SSTR2A, was degraded by endothelin-converting enzyme-1 and other peptidases and was not routed into lysosomal degradation This strong association of arrestins with the internalized receptor and the seques-tration of the receptor in early endosomes is indicative
of a class B receptor [13,26] Stimulation of SSTR2A with octreotide induced long-lasting sequestration of the intact ligand into early endosomes
Here, we investigated the intracellular trafficking of SSTR3 and the processing of internalized [125 I]Tyr11-SST-14 and [125I]Tyr1-octreotide Our data show that SSTR3 transiently interacts with arrestins and
Fig 4 Rhodamine-B–SST-14 is transported to lysosomes
RIN-SSTR3 cells were incubated for 2 h with rhodamine-B–SST-14 at
4 C, washed, and incubated for 0, 30 and 60 min at 37 C
Rhoda-mine-B–SST-14 (red) was detected using rhodamine-B
fluores-cence; lysosomes (green) were detected using an antibody against
cathepsin D The experiment was performed three times, with
sim-ilar results.
Trang 8directs SST-14 to lysosomal degradation This
transient interaction of the receptor with arrestins is
indicative of a class A receptor, and our data are in
line with data in [16] and [25]
When we analyzed the fate of the ligand, we
demon-strated that SSTR3 routed internalized SST-14 towards
a lysosomal degradation pathway Internalized
[125I]Tyr11-SST-14 was rapidly degraded, and [125I]Tyr
was released into the cell supernatant Confocal laser
scanning microscopic analyses of internalized
fluores-cent dye-labeled SST-14 showed strong colocalization of
the fluorescence signal with cathepsin D, a specific
mar-ker protein for lysosomes The persistence of the
fluores-cence signal within lysosomes as compared to the rapid
degradation of the radioligand within 30 min most
likely reflects the stability of the fluorophore and not
that of the peptide moiety It is known that internalized
ligands that are routed to lysosomes are degraded by
acidic proteases [27] To address the question of whether
somatostatin is also degraded by acidic proteases, we
interfered with the acidification of endocytic vesicles by
incubating the cells in the presence of ammonium
chlo-ride [19] Under these conditions, the degradation of the
internalized radioligand and the continued endocytosis
of the peptide ligand were markedly blocked, suggesting
that receptor trafficking proceeds via acidic vesicles and
that the degradation of somatostatin is accomplished by
acidic proteases when endocytosed with SSTR3
Inter-estingly, the endosomal degradation of internalized
SST-14, observed after internalization through
SSTR2A, was partially inhibited by neutralization of
acidic cell compartments [13], suggesting that different
peptidases are involved in the degradation process of
SST-14, depending on the coendocytosed SSTR
sub-type, i.e either SSTR2A or SSTR3
We also analyzed the intracellular processing of
octreotide in SSTR3-expressing cells Octreotide is a
synthetic SST-14 analog that binds to SSTR2A as well
as SSTR3 Octreotide is resistant to degradation by
endosomal peptidases [13] Interestingly, octreotide
was also stable when it was internalized via SSTR3,
suggesting that the synthetic agonist is also resistant to
lysosomal degradation or, alternatively, that it was not
routed to the lysosomes Chronic stimulation of the
cells with octreotide induced continuous accumulation
of the intact peptide within these cells After 4 h of
chronic stimulation, 256% of surface-bound octreotide
was observed to be cell-associated, indicating that
SSTR3 was continuously recycled to the cell
mem-brane and reinternalized during chronic stimulation,
thereby mediating the accumulation of octreotide in
the cells A similar observation was described for the
SSTR1-mediated accumulation of SST-14 [11]
Interestingly, chronic stimulation of SSTR3 with SST-14 induced time-dependent downregulation of SSTR3 One hundred and twenty minutes after stimu-lation, SSTR3 was recycled up to 75%, whereas it was recycled up to only 43% after 1300 min In contrast, SSTR1, which does not direct SST-14 to lysosomal degradation, recovered up to 100% under the same conditions [11,20] This observation underlines our finding that SSTR3 continuously recycles and is re-endocytosed under chronic stimulation One hour after stimulation of the cells with SST-14, immunofluo-rescence signals of SSTR3–HSV still colocalized with the fluorescence signal of the internalized ligand At this time point, the ligand was simultaneously detected within lysosomes in SSTR3-expressing cells The data suggest that lysosomal targeting of SSTR3 is responsi-ble for the downregulation of the receptor
Taken together, our results show that: (a) SSTR3 continuously internalizes, recycles and reinternalizes under chronic agonist stimulation; (b) the internalized SST-14 is routed to lysosomal degradation, where internalized [125I]Tyr11-SST-14 is degraded to [125I]Tyr; (c) internalized octreotide is resistant to deg-radation, but is accumulated within cells as an intact ligand; and (d) chronic stimulation of SSTR3 with SST-14 induces time-dependent downregulation of the receptor, probably through lysosomal degradation of SSTR3
At least two conclusions may be drawn from these observations First, agonist-induced SSTR internaliza-tion is a complex process depending on the receptor subtype and the nature of the stimulating agonist Besides the above-described differences in the regula-tion of receptor internalizaregula-tion, trafficking and recy-cling, further functional differences among SSTR subtypes may be postulated through interactions with distinct SSTR-binding proteins In fact, such binding proteins that specifically associate with SSTR subtypes have been recently identified [28,29]
Radiolabeled or fluorescent dye-labeled somatostatin analogs accumulating in certain cancer cells are used with the diagnostic method SRS, and conjugates of stable somatostatin analogs with toxic compounds or radioisotopes have been used for chemotherapy in certain tumors [30] Therefore, detailed knowledge of the mechanisms underlying agonist-induced endocyto-sis and trafficking of the SSTR subtypes is of great clinical importance, and cancer patients may benefit from it in the future
Our results indicate that future drugs should be tested for all known aspects of agonist-induced traf-ficking They also indicate that the considerable knowl-edge about the interaction of octreotide with SSTR2A
Trang 9cannot be generalized to other SSTR subtypes and
ligands without experimental proof The receptor
subtype-specific transport of SSTR2A and the
ligand-specific processing of octreotide go well with the use of
octreotide in SSTR2A scintigraphy [13] On the other
hand, these advantages adversely affect the use of
octreotide in tumor treatment, because this peptide
leads to extensive sequestration of SSTR2A and
desen-sitization of the targeted tumor cells for prolonged
periods [13] Our results also show that octreotide
recycled during the SSTR3-mediated transport
Because it did not remain sequestered in the cell when
internalized with SSTR3, labeled octreotide appears
not to be suitable for detecting SSTR3 in receptor
scintigraphy
Experimental procedures
Materials
SST-14 and octreotide were obtained from Bachem (Weil
am Rhein, Germany), [125I]Tyr11-SST-14 (2000 CiÆmmol)1)
was from Amersham (Braunschweig, Germany), and
[125I]Tyr1-octreotide was from Anawa (Wangen,
Switzer-land) FITC–SST-14 was from Advanced Bioconcept
(Derry, NH, USA) FITC-conjugated anti-rabbit IgG,
Texas Red-conjugated anti-mouse IgG, paraformaldehyde,
glycerol⁄ gelatin solution and BSA (fraction IV) were
pur-chased from Sigma (Taufkirchen, Germany) The
poly-clonal antiserum against cathepsin D was a generous gift
from A Hille-Rehfeld (Goettingen, Germany), and has
been described in detail elsewhere [23]
Generation of cDNA constructs and cell line
The construct with arrestin-2 tagged with EGFP has been
described previously [31] Generation of the neuroendocrine
RIN 1046-38 cell line stably expressing the C-terminal HSV
epitope-tagged rat SSTR3 tagged with the HSV
glycopro-tein D epitope at the C-terminus (SSTR3–HSV) has been
described previously, and it has been demonstrated to
possess a maximal binding capacity of 1660 (± 350) fmol
per 2· 104cells for [125I]Tyr11-SST-14 [20]
Synthesis of rhodamine-B-labeled SST-14
SST-14 was generated on a LIPS vario multiple peptide
synthesizer using the robot’s standard protocol following
the Fmoc strategy (peptides&elephants, Potsdam, Germany)
The rhodamine-B label was attached by deprotection of the
N-terminal a-amino function The rhodamine-B was
acti-vated using ByBop (Novabiochem, Darmstadt, Germany)
and N-methylmorpholine as a base Rhodamine-B was
added in a four-fold surplus to the a-amino function
The rhodamine⁄ ByBop ⁄ N-methylmorpholine ratio was
1 : 0.9 : 2 in 1 mL of dimethylformamide The coupling reaction was performed two times for 3 h The resin was washed with dimethylformamide until the washing solution was colorless After this, the resin was washed with dichloromethane and dried overnight The next day, the peptide was cleaved and deprotected using reagent K [tri-isopropylsilan (5%), water (2.5%), trifluoroacetic acid (92.5%)] The cleavage was performed for 2.5 h at room temperature After this, the peptide was precipitated with diethyl ether and washed three times with ice-cold diethyl ether Cyclization was performed following the protocol of Bodansky and Bodansky [32]
The peptide was further purified by HPLC, and the identity was confirmed by MALDI-TOF MS Rhodamine-B–SST-14 has a 10-fold lower affinity for SSTR3 than unlabeled SST-14 [24]
Reduction of cell surface binding Cells grown in 24-well dishes were stimulated with 1 lm SST-14 in RPMI-1640 (0.1% BSA) for 0–120 min at 37C Cells were placed on ice, washed three times with chilled acidic buffer, and incubated with 100 000 c.p.m per 0.3 mL of [125I]Tyr11-SST-14, 0.01 nm SST-14, and 0.1% BSA in RPMI-1640, at 4C for 90 min Bound [125
I]Tyr11-SST-14 was collected after lysing of the cells in 1 mL of
1 m NaOH and determined in a c-counter (Canberra Pack-ard, Dreieich, Germany) Calculations and graphical presentations were carried out using ms-excel and adobe photoshop Unspecific binding was determined in the presence of 0.1 mm SST-14 [20]
Recovery of cell surface binding Cells grown in 24-well dishes were stimulated with SST-14 (1 lm) in RPMI-1640 (0.1% BSA) for 120 min at 37C Surface-bound SST-14 was removed by three acidic washes with Hank’s buffered saline (HBS) (acetic acid, pH 4.8) and incubated for the indicated times in RPMI-1640 (0.1% BSA) The cells were placed on ice and incubated with
100 000 c.p.m per 0.3 mL of [125I]Tyr11-SST-14, 0.01 nm SST-14, and 0.1% BSA in RPMI-1640, at 4C for 90 min Bound [125I]Tyr11-SST-14 was collected after lysing of the cells in 1 mL of 1 m NaOH and determined in a c-counter (Canberra Packard) [20]
Determination of cell surface and total binding RIN-SSTR3 cells grown in 24-well dishes were stimulated with SST-14 (1 lm) in RPMI-1640 (0.1% BSA) at 37C for 60 min Cells were washed three times with HBS (acetic acid, pH 4.8) in the presence or absence of 0.1% saponin The cells were washed with RPMI-1640 to adjust the pH
Trang 10Surface binding sites were determined by incubation with
100 000 c.p.m per 0.3 mL of [125I]Tyr11-SST-14, 0.01 nm
SST-14, and 0.1% BSA in RPMI-1640, at 4C for 90 min
Total binding was determined by incubation with
100 000 c.p.m per 0.3 mL of [125I]Tyr11-SST-14, 0.01 nm
SST-14, 0.1% BSA and 0.1% saponin in RPMI-1640, at
4C for 90 min Bound [125I]Tyr11-SST-14 was collected
after lysing of the cells in 1 mL of 1 m NaOH and
deter-mined in a c-counter (Canberra Packard) [20]
Downregulation of cell surface binding sites
RIN-SSTR3 cells grown in 24-well dishes were stimulated
with SST-14 (1 lm) in RPMI-1640 (0.1% BSA) for the
indicated times at 37C Surface-bound SST-14 was
removed by three acidic washes with HBS (acetic acid,
pH 4.8) and incubated for 120 min in RPMI-1640 (0.1%
BSA) The cells were placed on ice and incubated with
100 000 c.p.m per 0.3 mL of [125I]Tyr11-SST-14, 0.01 nm
SST-14, and 0.1% BSA in RPMI-1640, at 4C for 90 min
Bound [125I]Tyr11-SST-14 was collected after lysing of the
cells in 1 mL of 1 m NaOH and determined in a c-counter
(Canberra Packard) [20]
Uptake of [125I]Tyr-labeled ligand
RIN 1046-38 cells transfected with SSTR3–HSV cDNA
were seeded in 24-well microplates and grown to 75%
con-fluence The culture medium was replaced by serum-free
medium containing [125I]Tyr11-SST-14 or [125
I]Tyr1-octreo-tide (100 000 c.p.m., 2000 CiÆmmol)1) and 0.1% BSA
pre-warmed to 37C, and incubated at this temperature for the
indicated times Cells were then washed at acidic pH to
remove all cell surface-bound peptide [33], and
cell-associ-ated radioactivity was determined in a c-counter (LKB
Wallac, Ontario, Canada) following lysis of the cells in 1 m
NaOH In parallel experiments, cell surface binding was
determined at 0C [20] Cell-associated radioactivity was
expressed as percentage of total cell surface-bound
radio-activity In addition, the experiment was carried out in the
presence of 40 mm NH4Cl [11]
HPLC analysis of internalized and released
[125I]Tyr-labeled ligand
RIN-SSTR3 cells grown in 24-well dishes were stimulated
for 0–30 min with [125I]Tyr11-SST-14 or [125
I]Tyr1-octreo-tide (100 000 c.p.m.) in RPMI-1640 (0.1% BSA) in the
presence or absence of 40 mm NH4Cl The cells were
washed in acidic buffer and incubated for 0–90 min in
RPMI-1640 (0.1% BSA) The supernatants were collected,
and acidified by adding 10 lL of trifluoroacetic acid The
supernatants were centrifuged (5 min, 13 000 g) and
sub-jected to HPLC separation Cell-associated radioactivity
was determined by adding 0.5 mL of HPLC buffer A Lysed cells were centrifuged (5 min, 13 000 g) and subjected
to HPLC separation HPLC was performed on a reverse-phase C-18 column (2· 25 mm) A separating gradient of 0–40% acetonitrile⁄ 0.08% trifluoroacetic acid for 25 min at
a flow rate of 1 mLÆmin)1 was used with an HPLC-Akta (General Healthcare, Munich, Germany) The HPLC gradient was fractionated every minute, and the eluted radioactivity was determined in a c-counter (LKB Wallac) The radioactivity of each fraction was divided by the initial amount of cell-associated radioactivity determined after
15 min of incubation with 100 000 c.p.m.ÆmL)1 radioacti-vity [11]
Microscopy and immunofluorescence Cells were incubated with SST-14 (1 lm) for 1300 min, washed, and incubated for 90 min at 37C The cells were fixed with paraformaldehyde 4%, washed, and incubated for
30 min in NaCl⁄ Pi(0.05% saponin, 5% normal goat serum) SSTR3–HSV was detected using mouse antibody against glycoprotein D (1 : 10 000) and Texas Red-conjugated anti-mouse IgG (1 : 200) In other experiments, cells were incu-bated with FITC–SST-14 or rhodamime-B–SST-14 on ice in RPMI-1640 and 0.1% BSA Unbound ligand was washed off, and the cells were incubated for the indicated times at
37C, washed with HBS ⁄ acetic acid (pH 4.75) at 4 C, fixed, and permeabilized for 30 min in HBS, 5% normal goat serum, and 0.05% saponin SST-14 was detected using the fluorescence dye, cathepsin D was detected using polyclonal antiserum against cathepsin D, and SSTR3–HSV was detected by using mouse antibody against glycoprotein D (1 : 10 000, overnight incubation at 4C) FITC-conjugated
or Texas Red-conjugated goat anti-(mouse IgG) or goat anti-(rabbit IgG) were used as secondary antibodies (1 : 200,
1 h, room temperature) Cells were embedded in Vectashield mounting medium (Vector, Burlingame, CA, USA) and observed with confocal microscopy [20,32]
Acknowledgements
This work was supported by grants from IZKF (STEI2⁄ 076 ⁄ 06), SFB 293 (A14), SFB 492 (B13), DFG STE 1014⁄ 2-2 (to M Steinhoff), the Rosacea Foundation (to M Steinhoff and D Roosterman) and IMF Mu¨nster (RO 120611) (to D Roosterman)
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