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Intracellular degradation of somatostatin-14 following somatostatin-receptor 3-mediated endocytosis in rat insulinoma cells Dirk Roosterman1, Nicole E I Brune2, Oliver J Kreuzer2, Micha Feld1, Sylvia Pauser1, Kim Zarse2, Martin Steinhoff1 and Wolfgang Meyerhof2 Department of Dermatology, IZKF Munster and Ludwig Boltzmann Institute for Cell and Immunobiology of the Skin, Germany ¨ Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Keywords endocytosis; G-protein-coupled receptor; neuropeptide; proteolysis; somatostatin Correspondence D Roosterman, Department of Dermatology and IZKF Munster, Von-Esmarch-Strasse 58, ă D-48148 Munster, Germany ă 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 differentially 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 endosome-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 internalized SST-14 and the regulation of SSTR3 markedly differ from the events mediated by the other SSTR subtypes 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 biological effects, including inhibition of secretion of growth hormone, insulin, glucagon and gastrin as well as other hormones secreted from the pituitary, skin and gastrointestinal 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 therefore 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 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 4728 FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS D Roosterman et al success of SRS is based on specific interactions of stable 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 diagnostics 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 reinternalization 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 internalized 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 critically dependent on phosphorylation of the C-terminal tail [15] As the phosphorylation sites not correspond to consensus sequences for second messengerregulated protein kinases, protein kinase A or protein kinase C, it was suggested that specific G-proteincoupled receptor kinases were involved [16] Moreover, colocalization studies and the use of dominant-negative mutants of arrestin-2 demonstrated that internalization 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 Intracellular degradation of somatostatin 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 degradation, whereas octreotide endocytosed with SSTR3 but was released as an intact peptide from the cells Moreover, chronic stimulation of SSTR3 with SST14 induced time-dependent downregulation of the receptor Our results demonstrate that SSTR3 trafficking 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 (Fig 1A) Incubation was stopped by placing cells on ice, and surface binding sites were determined Stimulation 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 Recovery of cell surface binding sites Next, we determined whether or not cell surface binding recovered following removal of the stimulus RINSSTR3 cells were first stimulated with SST-14 for 120 Cell surface-bound ligand was washed off, and cells were incubated for another 0–120 The recovery of surface binding sites was determined as described above Interestingly, during the first 15 of incubation, surface binding recovered to 76% However, incubation of the cells for a period of up to 120 did not lead to recovery of surface binding beyond this value (Fig 1B) This result indicates that prolonged incubation of SSTR3-expressing cells with SST-14 induced marked downregulation of the receptor We therefore determined the time dependence of SSTR3 downregulation by chronic stimulation Therefore, RIN-SSTR3 cells were stimulated for 0–1300 with SST-14 at 37 °C Subsequently, the agonist peptide was washed off, and cells were incubated for recovery of surface binding sites (Fig 1C) Chronic stimulation resulted in FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS 4729 Intracellular degradation of somatostatin D Roosterman et al A B C D E F Fig Loss and recovery of SST-14 binding sites (A) SST-14-mediated reduction of cell surface binding sites RIN-SSTR3 cells were stimulated with SST-14 at 37 °C for the indicated times Cells were washed with acidic buffer, and surface binding sites were determined by incubation with [125I]SST-14 at °C (B) Recovery of cell surface binding sites RIN-SSTR3 cells were stimulated for 120 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 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 saponin 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 Then, the peptide was removed and cells were allowed to recover for 90 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) time-dependent downregulation of the receptor After a period of 1300 of stimulation and 120 of recovery, cell surface binding was reduced to 43 ± 5% The time-dependent loss of surface binding sites during stimulation with SST-14 suggests that the receptor was internalized We determined the ratio between cell surface-located receptors and internalized receptors after h of incubation with SST-14 (Fig 1D) by measuring cell surface binding and total cellular binding in the presence of saponin [17] Incubation of untreated cells with saponin did not significantly change the 4730 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 stimulation, approximately 40% of the SSTR3 receptors were localized in intracellular compartments, and the data clearly indicate that SSTR3 was internalized during stimulation These data are in line with the fluorescence microscopy quantification of intracellularly located SSTR3 [18] The loss of cell surface binding sites during chronic stimulation suggests that the receptor is sequestered FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS D Roosterman et al Intracellular degradation of somatostatin within the cells or that it is downregulated by degradation To distinguish between the two possibilities, we determined the localization of SSTR3 after 1300 of stimulation with SST-14 and 90 of recovery In untreated cells, SSTR3 showed a bright immunofluorescence signal at the cell membrane (Fig 1E, arrows) In cells chronically stimulated with SST-14, the fluorescence signal was weaker than the signal seen in untreated cells Moreover, in all of these cells, the SSTR3 immunofluorescence signal was locally concentrated 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 [125I]Tyr1-octreotide in SSTR3-expressing rat insulinoma cells To examine the fate of SST-14 cointernalized with SSTR3, we measured the uptake of [125I]Tyr11-SST14 in RIN-SSTR3 cells in the absence (Fig 2A, diamonds) or presence (Fig 2A, triangles) of ammonium chloride Treatment of the cells with A B C D E F Fig SSTR3-mediated uptake of 125I-labeled peptides (A) SSTR3-mediated uptake of [125I]Tyr11-SST-14 RIN-SSTR3 cells were incubated with [125I]SST-14 in the presence (triangle) or absence (diamonds) of ammonium chloride Cell surface [125I]Tyr11-SST-14 was washed off, and the amount of cell-associated radioactivity was determined (B) SSTR3-mediated uptake of [125I]Tyr1-octreotide RIN-SSTR3 cells were incubated with [125I]Tyr1-octreotide Cell surface [125I]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 [125I]Tyr11-SST-14 at 37 °C for 30 in the presence of ammonium chloride, washed with acidic buffer, and analyzed by HPLC (D) HPLC separation of the cell supernatant incubated for 30 with [125I]Tyr11-SST-14 (E) Time course of SSTR3-mediated uptake of [125I]Tyr11-SST-14 RIN- SSTR3 cells were stimulated for 0–15 with [125I]Tyr11-SST-14, washed with acidic buffer, and incubated for 0–90 Cell-associated (triangles) and released (diamonds) radioactivity was determined by HPLC (F) Time course of SSTR3-mediated uptake of [125I]Tyr1-octreotide RIN- SSTR3 cells were incubated for 0–15 with [125I]Tyr1octreotide, washed with acidic buffer, and incubated for 0–90 at 37 °C Cell-associated radioactivity and radioactivity from the supernatants was determined by HPLC The data are expressed as mean ± SEM values from three independent experiments FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS 4731 Intracellular degradation of somatostatin D Roosterman et al ammonium chloride neutralized acidic cellular compartments [19] Within 10–15 at 37 °C, the cellular uptake of [125I]Tyr11-SST-14 reached maximal levels in the absence of ammonium chloride corresponding to  80% of the amount of the cell surface-bound peptide The amount of intracellular radioactivity then quickly declined over a period of  15 to very low levels corresponding to about 30% of the amount of cell surface-bound peptide These levels slowly decreased over the next 90 to < 20% of the initial value These observations are best explained by assuming that specific intracellular 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 probably reflects the steady-state level of [125I]Tyr11-SST-14 determined by receptor-mediated uptake and degradation The receptor population engaged in agonist uptake appears to be largely diminished at these times, due to receptor internalization and desensitization [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 ammonium chloride (Fig 2A, triangles), radioactivity accumulated with a similar kinetic during the first 10 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 continuous uptake of the ligand During the first 30 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 h of incubation, the amount of internalized radioactivity was equivalent to 256% of cell surfacebound octreotide In order to distinguish between radioactivity corresponding 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 of stimulation with [125I]SST-14 in the presence of ammonium chloride This treatment blocked the 4732 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 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 radioactive 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 (diamonds) in the extracellular medium, as assayed by HPLC analyses of the fractions During the first 15 of stimulation, [125I]Tyr11-SST-14 accumulated within the cells After removal of the peptide agonist by washing and further incubation at 37 °C, the amount of cellassociated [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 accordance with Fig 2B,F (filled circles) shows that [125I]Tyr1-octreotide was rapidly internalized by RINSSTR3 cells during the 15 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 Interestingly, HPLC analysis of the cell lysate demonstrated 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 ligandspecific processing Whereas SST-14 is sorted into an ammonium-sensitive degradation pathway, octreotide FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS D Roosterman et al bypasses degradation, accumulates in the cell and is released as intact ligand from the cells in the surrounding medium Intracellular degradation of somatostatin A 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 °C After removal of unbound peptide, a temperature shift to 37 °C induced internalization of cell surface-bound agonist for 2, 30 or 60 At the beginning of the observation period at min, fluorescence signals of FITC–SST-14 were barely visible However, 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 of stimulation, most of the receptors were recycled to the plasma membrane (Fig 3A, red arrows, bottom panels), whereas the fluorescence signal of the ligand was still observed within intracellular vesicular structures Traces of SSTR3–HSV were also detected within intracellular 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 lm SST-14 or octreotide for 15 (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 B Fig Agonist-induced internalization of SSTR3–HSV (A) SSTR3mediated uptake of FITC–SST-14 RIN-SSTR3 cells were incubated for 60 with FITC–SST-14 at °C Cells were washed and incubated for 2, 30 and 60 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) Agonistinduced mobilization of arrestin-2–EGFP RIN-SSTR3 cells were transiently transfected with arrestin-2–EGFP Cells were stimulated with SST-14 (1 lM) or octreotide (1 lM) for 15 at 37 °C SSTR3–HSV was localized using an antibody against HSV and arrestin-2-EGFP by EGFP The experiment was performed three times, with similar results 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 after stimulation, arrestin-2 was only partially associated with internalized SSTR3–HSV, indicating that arrestin-2 FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS 4733 Intracellular degradation of somatostatin D Roosterman et al dissociated 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, octreotide, 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 colocalization of rhodamine-B–SST-14 with cathepsin D, a lysosomal protease [23], were performed (Fig 4) Therefore, we incubated RIN-SSTR3 cells with rhodamine–SST-14 at °C Under these conditions, the fluorescence signals of the peptide were predominantly observed as a punctate pattern at the cell surface, whereas the fluorescence signals of the lysosomal protease appeared to be scattered within the cytoplasm (Fig 4, top panels) Warming the cells to 37 °C induced the internalization of rhodamine–SST-14 and Fig Rhodamine-B–SST-14 is transported to lysosomes RINSSTR3 cells were incubated for h with rhodamine-B–SST-14 at °C, washed, and incubated for 0, 30 and 60 at 37 °C Rhodamine-B–SST-14 (red) was detected using rhodamine-B fluorescence; lysosomes (green) were detected using an antibody against cathepsin D The experiment was performed three times, with similar results 4734 aggregation of lysosomes (Fig 4, middle panels) After 30 of stimulation, clear colocalization of cathepsin D and rhodamine-B–SST-14 was observed, indicating 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 internalized SST-14 in RIN-SSTR3 cells Discussion Recent studies provided clear evidence that the SSTR subtypes (SSTR1, SSTR2, SSTR3 and SSTR5) internalize 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 transiently with arrestin-2, and internalized SSTR2A formed a stable complex with arrestin-2 [11,16,25] The differences between the receptor subtypes in their interaction with arrestins indicate the existence of internalization and trafficking pathways that are specific for the SSTR subtypes Determining the fate of the internalized ligand revealed three different pathways of receptor trafficking and agonist processing SSTR1 mediates accumulation and release of intact SST-14 This phenomenon was accomplished by a dynamic process of internalization, recycling and reinternalization of the peptide, a pathway consistent with the role of SSTR1 as an autoreceptor [11] In contrast, SSTR2A induced sequestration of the receptor–ligand complex within early endosomes SSTR2A did not recycle within a period of 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 sequestration 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 [125I]Tyr11SST-14 and [125I]Tyr1-octreotide Our data show that SSTR3 transiently interacts with arrestins and FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS D Roosterman et al directs 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 demonstrated 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 fluorescent dye-labeled SST-14 showed strong colocalization of the fluorescence signal with cathepsin D, a specific marker protein for lysosomes The persistence of the fluorescence signal within lysosomes as compared to the rapid degradation of the radioligand within 30 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 chloride [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 Interestingly, 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 subtype, 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 h of chronic stimulation, 256% of surface-bound octreotide was observed to be cell-associated, indicating that SSTR3 was continuously recycled to the cell membrane 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] Intracellular degradation of somatostatin Interestingly, chronic stimulation of SSTR3 with SST-14 induced time-dependent downregulation of SSTR3 One hundred and twenty minutes after stimulation, SSTR3 was recycled up to 75%, whereas it was recycled up to only 43% after 1300 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, immunofluorescence 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 responsible 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 degradation, 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 internalization is a complex process depending on the receptor subtype and the nature of the stimulating agonist Besides the above-described differences in the regulation of receptor internalization, trafficking and recycling, 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 endocytosis 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 trafficking They also indicate that the considerable knowledge about the interaction of octreotide with SSTR2A FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS 4735 Intracellular degradation of somatostatin D Roosterman et al cannot be generalized to other SSTR subtypes and ligands without experimental proof The receptor subtype-specific transport of SSTR2A and the ligandspecific 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 desensitization 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 The rhodamine ⁄ ByBop ⁄ N-methylmorpholine ratio was : 0.9 : in mL of dimethylformamide The coupling reaction was performed two times for 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 RhodamineB–SST-14 has a 10-fold lower affinity for SSTR3 than unlabeled SST-14 [24] Experimental procedures Reduction of cell surface binding Materials SST-14 and octreotide were obtained from Bachem (Weil am Rhein, Germany), [125I]Tyr11-SST-14 (2000 CiỈmmol)1) was from Amersham (Braunschweig, Germany), and [125I]Tyr1-octreotide was from Anawa (Wangen, Switzerland) 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 purchased from Sigma (Taufkirchen, Germany) The polyclonal antiserum against cathepsin D was a generous gift from A Hille-Rehfeld (Goettingen, Germany), and has been described in detail elsewhere [23] Recovery of cell surface binding 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 glycoprotein 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 · 104 cells 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 activated 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 4736 Cells grown in 24-well dishes were stimulated with lm SST-14 in RPMI-1640 (0.1% BSA) for 0–120 at 37 °C 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 °C for 90 Bound [125I]Tyr11SST-14 was collected after lysing of the cells in mL of m NaOH and determined in a c-counter (Canberra Packard, 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] Cells grown in 24-well dishes were stimulated with SST-14 (1 lm) in RPMI-1640 (0.1% BSA) for 120 at 37 °C 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 °C for 90 Bound [125I]Tyr11-SST-14 was collected after lysing of the cells in mL of 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 37 °C for 60 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 FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS D Roosterman et al Surface 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 °C for 90 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 °C for 90 Bound [125I]Tyr11-SST-14 was collected after lysing of the cells in mL of m NaOH and determined 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 37 °C Surface-bound SST-14 was removed by three acidic washes with HBS (acetic acid, pH 4.8) and incubated for 120 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 °C for 90 Bound [125I]Tyr11-SST-14 was collected after lysing of the cells in mL of 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% confluence The culture medium was replaced by serum-free medium containing [125I]Tyr11-SST-14 or [125I]Tyr1-octreotide (100 000 c.p.m., 2000 CiỈmmol)1) and 0.1% BSA prewarmed to 37 °C, 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-associated radioactivity was determined in a c-counter (LKB Wallac, Ontario, Canada) following lysis of the cells in m NaOH In parallel experiments, cell surface binding was determined at °C [20] Cell-associated radioactivity was expressed as percentage of total cell surface-bound radioactivity 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 with [125I]Tyr11-SST-14 or [125I]Tyr1-octreotide (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 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 subjected to HPLC separation Cell-associated radioactivity Intracellular degradation of somatostatin 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 reversephase C-18 column (2 · 25 mm) A separating gradient of 0–40% acetonitrile ⁄ 0.08% trifluoroacetic acid for 25 at a flow rate of mLỈmin)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 of incubation with 100 000 c.p.m.ỈmL)1 radioactivity [11] Microscopy and immunofluorescence Cells were incubated with SST-14 (1 lm) for 1300 min, washed, and incubated for 90 at 37 °C The cells were fixed with paraformaldehyde 4%, washed, and incubated for 30 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 antimouse IgG (1 : 200) In other experiments, cells were incubated 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 37 °C, washed with HBS ⁄ acetic acid (pH 4.75) at °C, fixed, and permeabilized for 30 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 °C) FITC-conjugated or Texas Red-conjugated goat anti-(mouse IgG) or goat anti-(rabbit IgG) were used as secondary antibodies (1 : 200, 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 Munster (RO 120611) (to D Roosterman) ă References Olias G, Viollet C, Kusserow H, Epelbaum J & Meyerhof W (2004) Regulation and function of somatostatin receptors J Neurochem 89, 1057–1091 Csaba Z & Dournaud P (2001) Cellular biology of somatostatin receptors Neuropeptides 35, 1–23 FEBS Journal 275 (2008) 4728–4739 ª 2008 The Authors Journal compilation ª 2008 FEBS 4737 Intracellular degradation of somatostatin D Roosterman et al Reubi JC & Laissue JA (1995) Multiple actions of somatostatin in neoplastic disease Trends Pharmacol Sci 16, 110–115 de Herder WW, Hofland LJ, van der Lely AJ & Lamberts SW (2003) Somatostatin receptors in gastroentero-pancreatic neuroendocrine tumours Endocr Relat Cancer 10, 451–458 Resmini E, Dadati P, Ravetti JL, Zona G, Spaziante R, Saveanu A, Jaquet P, Culler 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ARTICLE Mammalian xanthine oxidoreductase – mechanism of transition from xanthine dehydrogenase to xanthine oxidase Tomoko Nishino 1 , Ken Okamoto 1 , Bryan. mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase. J Biochem (Tokyo) 141, 52 5–5 34. 29 Nishino T, Okamoto K, Kawaguchi