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Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor
Molecular and Cellular Endocrinology 387 (2014) 44–51
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Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor q Andreas Lehmann 1, Andrea Kliewer 1, Dagmar Schütz, Falko Nagel, Ralf Stumm, Stefan Schulz ⇑ Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University, 07747 Jena, Germany
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Article history: Received 5 November 2012 Received in revised form 16 February 2014 Accepted 16 February 2014 Available online 22 February 2014 Keywords: Somatostatin Octreotide Pasireotide Somatostatin receptor Internalization Desensitization
a b s t r a c t The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven b-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and b-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The somatostatin receptor sst2 is highly expressed at the plasma membrane of human tumors including pancreatic, gastrointestinal and pulmonary neuroendocrine tumors, pituitary adenomas, breast carcinomas, meningiomas, neuroblastomas, medulloblastomas, pheochromocytomas, and paragangliomas. This is the molecular basis for clinical application of stable somatostatin analogs for tumor imaging and tumor therapy. A number of metabolically stable somatostatin analogs have been synthesized two of which, octreotide and lanreotide, were approved for clinical use. Octreotide and lanreotide bind with high sub-nanomolar affinity Abbreviations: CHO, chinese hamster ovarian cells; GH, growth hormone; GPCR, G-protein coupled receptor; HEK293, human embryonic kidney 293 cells; SS-14, somatostatin-14; sst, somatostatin receptor. q Phosphorylation of multiple carboxyl-terminal sites within the including S341, S343, T353, T354, T356 and T359 directly regulates agonist-driven internalization and desensitization of the human sst2 somatostatin receptor. ⇑ Corresponding author. Tel.: +49 3641 9325650; fax: +49 3641 9325652. E-mail address: [email protected] (S. Schulz). 1 These authors contributed equally.
http://dx.doi.org/10.1016/j.mce.2014.02.009 0303-7207/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
to sst2 only, have moderate affinity to sst3 and sst5 and show very low or absent binding to sst1 and sst4 (Colao et al., 2010). In clinical practice, octreotide and lanreotide are used as first choice medical treatment of neuroendocrine tumors such as GHsecreting adenomas and carcinoids (Donangelo and Melmed, 2005; Oberg et al., 2010). Octreotide initially controls symptoms caused by hormonal overproduction in about 90% of carcinoid patients. After 1 year of treatment, however, some 50% of patients show an escape of response (Oberg, 2005; Asnacios et al., 2008). In contrast, octreotide can normalize GH levels for prolonged periods in 65% of acromegalic patients (Donangelo and Melmed, 2005). Recently, the novel multireceptor somatostatin analog, pasireotide (SOM230), has been synthesized (Bruns et al., 2002). Pasireotide is a cyclohexapeptide, which binds with high affinity to all somatostatin receptors except to sst4 (Lewis et al., 2003). We have recently uncovered that octreotide and pasireotide stimulate distinct agonist-selective patterns of sst2 somatostatin receptor phosphorylation and internalization (Poll et al., 2010; Lesche et al., 2009; Kliewer et al., 2012). Like somatostatin, octreotide promotes the phosphorylation of at least six carboxylterminal serine and threonine residues namely S341, S343, T353,
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T354, T356 and T359, which in turn leads to a robust sst2 receptor endocytosis (Nagel et al., 2011; Kao et al., 2011). Unlike somatostatin, pasireotide fails to induce a substantial phosphorylation or internalization of the sst2 receptor (Nagel et al., 2011). These findings suggest that agonist-driven phosphorylation may facilitate sst2 receptor endocytosis. However, earlier studies have failed to establish a causal relationship between phosphorylation and internalization for the rat sst2 receptor (Liu et al., 2008). Here, we have constructed a series of phosphorylation-deficient mutants and examined the contribution of individual phosphorylation events to agonist-dependent regulation of the human sst2 receptor. 2. Materials and methods 2.1. Reagents, plasmids and antibodies Pasireotide and octreotide were kindly provided by Dr. Herbert Schmid (Novartis, Basel, Switzerland). SS-14 was obtained from Bachem (Weil am Rhein, Germany). DNA for HA-tagged 19S/T-A, 14S/ T-A, 6S/T-A, 4T-A and 2S-A mutants of the human sst2 receptor was generated via artificial gene synthesis and cloned into pcDNA3.1 by imaGenes (Berlin, Germany). The human HA-tagged sst2 receptor was obtained from UMR cDNA Resource Center (Rolla, MO). The phosphorylation-independent rabbit monoclonal anti-sst2 antibody {UMB-1} (Epitomics, Burlingame, CA) and the phosphositespecific sst2 antibodies anti-pS341/pS343 {3157}, anti-pT353/ pT354 {0521}, anti-pT356/pT359 {0522} and the rabbit polyclonal anti-HA antibody were generated and extensively characterized as previously described (Poll et al., 2010; Fischer et al., 2008). Antibodies {0521} and {0522} were generated against a peptide containing pT353, pT354, pT356 and pT359 and subsequently affinity purified against peptides containing either pT353 and pT354 {0521} or pT356 and pT359 {0522} (Poll et al., 2010, 2011; Nagel et al., 2011). 2.2. Cell culture and transfection Human embryonic kidney HEK293 cells were obtained from the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany). HEK293 cells were grown in DMEM supplemented with 10% fetal calf serum. Cells were transfected with plasmids encoding for wild-type or mutant sst2 receptors using Lipofectamine 2000 according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Stable transfectants were selected in the presence of 400 lg/ml G418. Stable cells were characterized using radioligand-binding assays, Western blot analysis, surface ELISA assay and immunocytochemistry as described previously (Tulipano et al., 2004; Pfeiffer et al., 2001). All mutants tested were present at the cell surface, expressed similar amounts of receptor protein and had similar affinities for SS-14 as the wild-type receptor. The level of receptor expression was 800 fmol/mg membrane protein for all experiments using stably transfected cells. The level of receptor expression was between 1500 and 2000 fmol/mg membrane protein for all experiments using transiently transfected cells. 2.3. Analysis of receptor internalization by confocal microscopy HEK293 cells satbly expressing HA-tagged human sst2 receptors were grown on poly-L-lysine-coated coverslips overnight. When indicated cells were transiently transfected with 1 lg GRK2 plasmid DNA per well containing 100,000 cells using TurboFect™ (Fermentas) according to the instructions of the manufacturer. After the appropriate treatment with either 1 lM SS-14, 1 lM Octreotide or 10 lM Pasireotide at 37 °C, cells were fixed with 4%
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paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 30 min at room temperature and washed several times. Specimens were permeabilized and then incubated with anti-sst2 {UMB-1} antibody followed by Alexa488-conjugated secondary antibody (Amersham, Braunschweig, Germany). Specimens were mounted and examined using a Zeiss LSM510 META laser scanning confocal microscope (Lesche et al., 2009). 2.4. Quantification of receptor internalization by ELISA Receptor internalization was quantified using a linear surface receptor ELISA that has been characterized extensively (Poll et al., 2010; Lesche et al., 2009; Nagel et al., 2011; Pfeiffer et al., 2001). Equal numbers of stably transfected HEK293 cells expressing HA-tagged human sst2 receptors were seeded onto poly-L-lysine-treated 24-well plates (200.000 cells per well). The next day, cells were preincubated with 1 lg/ml anti-HA antibody for 2 h at 4 °C. After the appropriate treatment with SS-14 (1 lM) at 37 °C, cells were fixed and incubated with peroxidase-conjugated antirabbit antibody overnight. After washing, plates were developed with ABTS solution and analyzed at 405 nm using a microplate reader. When indicated cells were transiently transfected with 1 lg GRK2 plasmid DNA per well using TurboFect™ (Fermentas) 24 h later, cells were preincubated with 1 lg/ml anti-HA antibody and treated as described above. Statistical analysis was carried out with unpaired t-test. p-Values of <0.05 were considered statistically significant. 2.5. Western blot analysis HEK293 cells stably expressing HA-tagged human sst2 receptors were plated onto 60-mm dishes and grown to 80% confluence. After treatment with SS-14 at 37 °C, cells were lysed in detergent buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) in the presence of protease and phosphatase inhibitors Complete mini and PhosSTOP (Roche Diagnostics, Mannheim, Germany) and centrifuged at 16,000g for 20 min at 4 °C. Glycosylated proteins were partially enriched using wheat germ lectin-agarose beads as described (Schulz et al., 2000; Mundschenk et al., 2003; Plockinger et al., 2008). Proteins were eluted from the beads using SDS-sample buffer for 20 min at 65 °C and then resolved on 8% SDS–polyacrylamide gels. After electroblotting, membranes were incubated with phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} or anti-pT356/pT359 {0522} at a concentration of 0.1 lg/ml followed by detection using enhanced chemiluminescence (Amersham). Blots were subsequently stripped and reprobed with anti-sst2 antibody {UMB-1} to confirm equal loading of the gels. 2.6. b-Arrestin-EGFP mobilization assay Untransfected HEK293 cells were seeded onto 35-mm glassbottom culture dishes (Mattek, Ashland, MA). The next day, cells were transiently cotransfected with 0.2 lg b-arrestin-2-EGFP and 2 lg human sst2 receptor or with a mixture of 0.2 lg b-arrestin2-EGFP, 0.8 lg GRK2 and 1.2 lg human sst2 receptor per dish containing 200,000 cells using TurboFect™. After 24 h, cells were transferred onto a temperature-controlled microscope stage set at 37 °C of a Zeiss LSM510 META laser scanning confocal microscope. Images were collected sequentially using single line excitation at 488 nm with 515–540-nm band pass emission filters. Saturating concentrations of SS-14 (1 lM) were applied directly into the culture medium immediately after the initial image was taken.
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2.7. [Ca2+]i assay HEK293 cells stably expressing HA-tagged human sst2 receptors were transiently transfected with G protein chimera qi5 (0.25 lg plasmid DNA per well containing 38,000 cells) consisting of murine Gaq with the 5 carboxyl-terminal amino acids changed from Gaq to Gai2. We were also able to trigger calcium responses of the sst2 receptor in the absence of qi5. Given that transfection with qi5 considerably improves the signal-to-noise ratio, we preferred this approach. G protein signaling was examined 24 h after plating by measuring increases in [Ca2+]i using the FlexStation3 microplate reader as described (Hoffmann et al., 2012). Baseline values were recorded for 30 s before the maximal possible response to 100 nM SS-14 was determined. In desensitization experiments, a prestimulus with 10 nM SS-14 was applied before stimulating with 100 nM SS-14. Desensitization of a sst2 mutant, was calculated as the difference between the maximal possible response and the response to 100 nM SS-14 with prestimulus and expressed in percent of the maximal possible response of the mutant. 2.8. Data analysis Data were analyzed using GraphPad Prism 4.0 software. Statistical analysis was carried out with unpaired t-test, or one-way ANOVA followed by the Dunnett’s post-test. p-Values of <0.05 were considered statistically significant. 3. Results The human sst2 receptor contains many potential phosphate acceptor sites in its third intracellular loop and carboxyl-terminal tail (Fig. 1A). Although phosphosite-specific antibodies have been successfully generated against some of these sites, a clear functional role has not yet been assigned to individual phosphorylation events. We therefore constructed a series of phosphorylation-deficient mutations including 2S-A, 4T-A, 6S/T-A, 14S/T-A and 19S/T-A (Fig. 1B). In initial Western blot studies, we confirmed that all of these mutants expressed similar receptor levels (Fig. 1C, bottom
panels). In addition, we tested whether these receptors were present at the plasma membrane using surface ELISA (Supplemental Fig. S1). We also confirmed that all mutant receptors exhibited binding and signaling properties similar to the wild-type sst2 receptor (Supplemental Fig. S3). Next, we examined agonist-induced phosphorylation of the wild-type sst2 receptor and its mutants using phosphosite-specific antibodies for pS341/343, pT353/354 and pT356/359 (Fig. 1C). Phosphorylation at the three sites was detectable within 5 min of SS-14 exposure in the wildtype sst2 receptor. SS-14 promoted phosphorylation at T353/354 and T356/359 in the 2S-A receptor as efficient as in wild-type sst2. The 2S-A receptor carries alanine mutations of S341 and S343. Conversely, phosphorylation of S341/S343 was still detectable in the 4T-A receptor, in which T353, T354, T356 and T359 were converted into alanines. As expected, phosphorylation at the three sites was no longer detectable in the 6S/T-A receptor, which carries alanine mutations in the S341/S343 site and in the T353/T354/T356/T359 cluster. Similar results were obtained with the 19S/T-A receptor, in which all potential phosphate acceptor sites in the third intracellular loop and the carboxyl-terminal tail have been mutated, and the 14S/T-A receptor with mutation of all potential phosphate acceptor sites in the carboxyl-terminal tail. These findings suggest that SS-14-promoted phosphorylation at T353/T354 and T356/359 occurs independent from phosphorylation at S341/343 and that phosphorylation at S341/343 occurs independent from phosphorylation at threonine cluster T353/ 354/356/359. Quantitative and qualitative analysis of receptor sequestration using ELISA and immunocytochemistry revealed that treatment of the wild-type human sst2 receptor with SS-14 produced a 80% loss of surface receptors within 30 min of agonist exposure (Fig. 2A, B). SS-14-induced internalization was reduced but clearly detectable in the 2S-A mutant. In the 4T-A and 6S/T-A mutants, SS14-induced internalization was strongly inhibited, i.e. after 30 min SS-14 exposure there was a loss of surface receptors of less than 10% (Fig. 2A and B). In fact, replacement of all fourteen carboxylterminal serine and threonine residues by alanine (14S/T-A) was required to completely abrogate sst2 receptor internalization in
Fig. 1. Construction of phosphorylation-deficient sst2 receptor mutants. (A) Schematic representation of the human sst2 receptor indicating all potential phosphate acceptor sites (depicted in gray or black) within the third intracellular loop and carboxyl-terminal tail. Epitopes of the phosphosite-specific anti-pS341/pS343, anti-pT353/pT354 and anti-pT356/pT359 antibodies (depicted in black) as well as the epitope of the phosphorylation-independent anti-sst2 antibody (UMB-1) are indicated. (B) Depiction of amino acid sequences of the third intracellular loop (237–250) and carboxyl-terminal tail (304–369) of the human sst2 receptor. Phosphate acceptor sites mutated to alanine in the mutants are depicted in black. (C) HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A or 19S/T-A were treated with 1 lM SS-14 for 5 min. The levels of phosphorylated sst2 receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} or anti-pT356/pT359 {0522}. Blots were subsequently stripped and reprobed with the phosphorylation-independent anti-sst2A antibody {UMB-1} to confirm equal loading of the gels (hsst2). Blots shown are representative of three independent experiments each. The positions of molecular mass markers are indicated on the left (in kDa).
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Fig. 2. Phosphorylation of the 353TTETQRT359 motif is required for sst2 internalization. (A) HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A or 19S/T-A were treated for 30 min with 1 lM SS-14 (A) or vehicle (not shown). Receptor sequestration was measured by ELISA. Data represent per cent loss of cell-surface receptors in agonist-treated cells as compared to sister cultures receiving 30 min vehicle (100%). Data are presented as mean ± SEM from at least four independent experiments performed in quadruplicate. Results were analyzed by unpaired t-test vs. wild type receiving 30 min vehicle (p < 0.05). (B) HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/ T-A or 19S/T-A were treated with 1 lM SS-14 for 15 or 30 min. Cells were then fixed, stained with the anti-sst2 antibody {UMB-1} and examined by confocal microscopy. Shown are representative images from one of at least three independent experiments performed in duplicate. Scale bar, 15 lm. (C) HEK293 cells stably expressing human WT or mutants were transfected with GRK2 and next day treated for 30 min with 1 lM SS-14 (A) or vehicle (not shown). Receptor sequestration was measured by ELISA. Data represent per cent loss of cell-surface receptors in agonist-treated cells as compared to sister cultures receiving 30 min vehicle (100%). Data are presented as mean ± SEM from at least four independent experiments performed in quadruplicate. Results were analyzed by unpaired t-test vs. wild type receiving 30 min vehicle (p < 0.05). (D) HEK293 cells stably expressing wild-type hsst2 or mutants were transfected with GRK2 and next day treated with 1 lM SS-14 for 15 or 30 min. Cells were then fixed, stained with the anti-sst2 antibody {UMB-1} and examined by confocal microscopy. Shown are representative images from one of at least three independent experiments performed in duplicate. Scale bar, 15 lm.
HEK293 cells (Fig. 2A and B). Additional mutation of all five serine residues within the third intracellular loop (19S/T-A) produced similar effects. We have recently shown that agonist-driven sst2 phosphorylation is mediated in part via GRK2 (Poll et al., 2010).
Consequently, we evaluated whether GRK2 overexpression would enhance internalization of wild-type or mutant sst2 receptors. Under these conditions, however, the extent of receptor internalization detected by ELISA as loss of surface receptors after
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the 30 min SS-14 exposure was very similar to that observed without GRK2 overexpression (Fig. 2C). Nevertheless, overexpression of GRK2 accelerated internalization of the 2S-A mutant at earlier time points (15 min) as detected by confocal microscopy (Fig. 2D). We then asked whether overexpression of GRK2 and b-arrestin-2 would drive internalization of the 14S/T-A receptor. As depicted in Supplemental Fig. S2, the loss of surface receptors was less than 5% under these conditions. Collectively, these results suggest that GRK-mediated multi-site phosphorylation of the sst2 carboxyl-terminal tail is a major requirement for its agonist-induced internalization. Octreotide and pasireotide stimulate distinct patterns of sst2 receptor phosphorylation (Lesche et al., 2009). Like somatostatin, octreotide promotes the phosphorylation of at least six carboxylterminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343. We therefore tested the effect of different mutations on octreotide- and pasireotide-induced internalization. Similar to that observed with SS-14, octreotide-driven internalization was strongly reduced in the 4T-A, 6S/TA and 14S/T-A mutants, while internalization of the 2S-A receptor was still clearly detectable (Fig. 3, left panel). In contrast, exchange of S341 and S343 by alanine (2S-A) was sufficient to prevent pasireotide-driven internalization (Fig. 3, right panel). We then addressed the functional role of carboxyl-terminal sst2 receptor phosphorylation. First, we employed functional b-arrestin-2 conjugated to enhanced green fluorescent protein (EGFP) to visualize the patterns of sst2-stimulated b-arrestin mobilization in live HEK293 cells under conditions with or without GRK2 overexpression. In the absence of agonist, b-arrestin-2-EGFP was
uniformly distributed throughout the cytoplasm (Fig. 4). The addition of saturating concentrations of SS-14 to the wild-type sst2 receptor induced a redistribution of b-arrestin-2 from the cytoplasm to the plasma membrane within 1 min resulting in fluorescence outlining of the cell shape (Fig. 4). After extended time periods (30 min), a redistribution of b-arrestin-2-EGFP into the cytosol was noted in cells transfected with the wild-type human sst2 receptor (Fig. 4). A rapid redistribution (<1 min) of b-arrestin-2 to the plasma membrane was also observed in the 2S-A mutant (Fig. 4, left panels). Interestingly, b-arrestin-2 mobilization was much less pronounced and required more extended time periods (10–30 min) in the 4T-A and 6S/T-A mutants (Fig. 4, left panels). In contrast, no b-arrestin-2 redistribution was observed in cells expressing the 14S/T-A or the 19S/T-A mutant under otherwise identical conditions (Fig. 4, left panels). GRK2 overexpression facilitated b-arrestin-2 mobilization in the 4T-A and 6S/T-A receptors but was not able to promote a similar redistribution in cells expressing the 14S/T-A or the 19S/T-A mutant (Fig. 4, right panels). These findings suggest that carboxyl-terminal multi-site phosphorylation of the sst2 receptor is a major determinant for agonist-induced b-arrestin mobilization. The residual b-arrestin-2 translocation observed in the 6S/T-A mutants suggests that carboxyl-terminal phosphate acceptor sites other than S341, S343, T353, T354, T356 and T359 may undergo agonist-induced phosphorylation. Moreover, this residual and delayed b-arrestin-2 recruitment may be responsible for the small amount of receptor internalization (<10%) seen in the 14S/T-A mutant after 30 min SS-14 exposure. We then used a calcium mobilization assay to determine the effect of the mutations on sst2 desensitization. Stably expressing
Fig. 3. Differential effects of sst2 mutations on octreotide- and pasireotide-induced internalization. HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A or 14S/T-A were treated with 1 lM Octreotide or 10 lM Pasireotide for 15 or 30 min. Cells were then fixed, stained with the anti-sst2 antibody {UMB-1} and examined by confocal microscopy. Shown are representative images from one of at least two independent experiments performed in duplicate. Scale bar, 15 lm.
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Fig. 4. Phosphorylation-dependent b-arrestin mobilization. (A) HEK293 cells were transiently transfected with either hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A or 19S/T-A and barrestin-2-EGFP. The distribution of b-arrestin-2 was visualized sequentially in the same live cells before (0 min) and after (1 to 30 min) the addition of 1 lM SS-14 to the culture medium. Shown are representative images from one of four independent experiments performed in duplicate. Scale bar, 15 lm. (B) HEK293 cells were transiently cotransfected with receptor, GRK2 and b-arrestin-2-EGFP. The distribution of b-arrestin-2 was visualized sequentially in the same live cells before (0 min) and after (1– 30 min) the addition of 1 lM SS-14 to the culture medium. Shown are representative images from one of four independent experiments performed in duplicate. Scale bar, 15 lm.
cells were transfected with a qi5 G protein chimera that was engineered to connect Gi-coupled receptors to the calcium pathway (Hoffmann et al., 2012). Under these conditions, application of 100 nM SS-14 evoked a similar strong calcium response in all mutants tested (Supplemental Fig. S3). Sequential stimulation was then used to determine homologous sst2 desensitization. In cells expressing human wild-type sst2, 100 nM SS-14 evoked a strongly reduced calcium response when administered 180 s after a precedent 10 nM SS-14 stimulus as compared with sister cultures experiencing 100 nM SS-14 without pre-stimulus (Fig. 5A). In the 14S/ T-A mutant, the pre-stimulus with 10 nM SS-14 provoked a modest reduction of the 100 nM stimulus as compared with sister cultures experiencing no SS-14 pre-stimulus (Fig. 5B). We then calculated desensitization for each mutant. To this end, we determined the differences in the height of the 100 nM peaks without and with pre-stimulus and expressed it as percent of the 100 nM peak without pre-stimulus. This showed that the 10 nM SS-14 pre-stimulus caused more than 80% desensitization of G protein signaling of the wild-type sst2 receptor as well as in the 2S-A and 4T-A mutants but only 60% and 50% desensitization of the 6S/T-A and 14S/T-A mutants, respectively (Fig. 5C). This loss of signal largely reflects homologous receptor desensitization and was not due to an exhaustion of the Ca response because a subsequent third pulse with the muscarinic receptor agonist carbachol evoked similar strong responses in all tested mutant and wild-type sst2 receptors (Supplemental Fig. S4). By comparing the different mutants to wild-type sst2, it became apparent that desensitization was unchanged in the 2S-A and 4T-A mutants, suggesting that both the S341/S343 and the 353TTETQRT359 motif are sufficient to mediate
full homologous sst2 receptor desensitization. Combined elimination of the S341/S343 and 353TTETQRT359 motifs was required to significantly diminish sst2 desensitization. Enhanced desensitization of the 14S/T-A mutant as compared with the 6S/T-A mutant indicates that phosphoacceptor sites other than S341/S343 and T353/T354/T356/T359 may additionally contribute to sst2 receptor desensitization.
4. Discussion The overexpression of the sst2 receptor in human tumors is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analog octreotide. In gastroenteropancreatic neuroendocrine tumors, octreotide initially controls symptoms caused by hormonal overproduction in about 90% of patients. After 1 year of treatment, however, some 50% of patients show an escape of response (Oberg, 2005; Asnacios et al., 2008). Consequently, the elucidation of regulatory mechanisms of the sst2 somatostatin receptor has attracted much attention. Thus, direct evidence using phosphosite-specific antibodies has been presented for agonistdependent phosphorylation of at least six carboxyl-terminal serine and threonine residues in vivo and in vitro (Poll et al., 2010; Lesche et al., 2009; Kliewer et al., 2012; Nagel et al., 2011; Liu et al., 2009; Waser et al., 2012). For many GPCRs a direct correlation between phosphorylation and internalization has been observed. Nevertheless, an earlier study analyzed the rat sst2 somatostatin receptor expressed on CHO cells with mutations of either all serine or all threonine residues as well as a combined mutation of all serine
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multiple sites including S341, S343, T353, T354, T356 and T359 contributes to agonist-driven internalization and desensitization of the human sst2 somatostatin receptor. In fact, we have previously established that rapid agonist-induced phosphorylation of all of these sites occurs via GRK2/3-dependent mechanisms (Poll et al., 2010; Nagel et al., 2011). In a manner similar to previous studies (Liu et al., 2008; Tulipano et al., 2004), we also show that multi-site phosphorylation is also a precondition for b-arrestin mobilization indicating that the agonist-induced conformational change of a phosphorylation-deficient sst2 receptor by itself was not sufficient for b-arrestin binding. Our observation that carboxyl-terminal phosphorylation is the initial step determining the rate of internalization and desensitization of the human sst2 receptor, may have important implications for the clinical utility of octreotide and pasireotide. Recent evidence indicates that octreotide and pasireotide display functional selectivity (Kliewer et al., 2012). In fact, pasireotide and octreotide are equally active in inducing classical G protein-dependent signaling at the sst2 receptor (Lesche et al., 2009; Nagel et al., 2011). Yet, they promote strikingly different patterns of sst2 phosphorylation, internalization and b-arrestin trafficking (Poll et al., 2010). Similar to that observed with SS-14, octreotide promotes the phosphorylation of multiple sites including S341, S343, T353, T354, T356 and T359, which is followed by a robust internalization, stable b-arrestin interaction and slow recycling. In contrast, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor, which is followed by a partial receptor sequestration, instable b-arrestin interaction but fast recycling (Poll et al., 2010; Lesche et al., 2009; Nagel et al., 2011). Nevertheless, we have to await additional clinical data to see inasmuch these differences in acute internalization and desensitization may regulate longterm responses to octreotide and pasireotide. It is also possible that during long-term treatment additional yet unknown mechanisms of receptor downregulation contribute to the regulation of tumor responsiveness. In conclusion, we show that the human sst2 receptor is regulated like a prototypical GPCR. Multi-site phosphorylation of clusters of carboxyl-terminal serine and threonine residues within the middle portion of the receptor’s cytoplasmic tail is a critical initial event regulating its internalization, b-arrestin trafficking and desensitization. Disclosure statement The authors have nothing to disclose. Fig. 5. Multi-site phosphorylation regulates sst2 receptor desensitization. HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A were transfected with qi5 chimeric G protein and changes in [Ca2+]i were monitored. Averaged traces with SEM showing two subsequent stimulations with 10 nM SS-14 or vehicle and 100 nM SS-14 for wild-type hsst2 (A) and 14S/T-A (B). Traces with the mock prestimulus (vehicle) are gray and traces with 10 nM SS-14 pre-stimulus are black. For each receptor, data were normalized to peak height of the 100 nM SS-14 stimulus without pre-stimulus (maximal stimulation). (C) For each receptor, homologous desensitization was calculated as the difference between the two peaks after 100 nM SS-14 and expressed in percent of the 100 nM peak without pre-stimulus. Data are presented as mean ± SEM from quadruplicate determinations. Two independent experiments gave similar results. Results were analyzed by one-way ANOVA followed by the Dunnett’s posttest (p < 0.05).
and threonine residues (Liu et al., 2008). Although sst2 internalization was found to be partially inhibited by mutation of the threonine residues, none of the mutations analyzed resulted in a complete block of receptor internalization (Liu et al., 2008). The present study established for the first time a causal relationship between carboxyl-terminal phosphorylation and internalization for the human sst2 receptor. Indeed phosphorylation of
Acknowledgements We thank Heidrun Guder and Heike Stadler for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft grant SCHU924/10-3 and the Deutsche Krebshilfe Grant 109952. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mce.2014.02.009. References Asnacios, A., Courbon, F., Rochaix, P., Bauvin, E., Cances-Lauwers, V., Susini, C., Schulz, S., Boneu, A., Guimbaud, R., Buscail, L., 2008. Indium-111-pentetreotide scintigraphy and somatostatin receptor subtype 2 expression: new prognostic factors for malignant well-differentiated endocrine tumors. J. Clin. Oncol. 26, 963–970. Bruns, C., Lewis, I., Briner, U., Meno-Tetang, G., Weckbecker, G., 2002. SOM230: a novel somatostatin peptidomimetic with broad somatotropin release inhibiting
A. Lehmann et al. / Molecular and Cellular Endocrinology 387 (2014) 44–51 factor (SRIF) receptor binding and a unique antisecretory profile. Eur. J. Endocrinol. 146, 707–716. Colao, A., Faggiano, A., Pivonello, R., 2010. Somatostatin analogues: treatment of pituitary and neuroendocrine tumors. Prog. Brain Res. 182, 281–294. Donangelo, I., Melmed, S., 2005. Treatment of acromegaly: future. Endocrine 28, 123–128. Fischer, T., Doll, C., Jacobs, S., Kolodziej, A., Stumm, R., Schulz, S., 2008. Reassessment of sst2 somatostatin receptor expression in human normal and neoplastic tissues using the novel rabbit monoclonal antibody UMB-1. J. Clin. Endocrinol. Metab. 93, 4519–4524. Hoffmann, F., Müller, W., Schütz, D., Penfold, M.E., Wong, Y.H., Schulz, S., Stumm, R., 2012. Rapid uptake and degradation of CXCL12 depend on CXCR7 carboxylterminal serine/threonine residues. J. Biol. Chem. 287, 28362–28377. Kao, Y.J., Ghosh, M., Schonbrunn, A., 2011. Ligand-dependent mechanisms of sst2A receptor trafficking: role of site-specific phosphorylation and receptor activation in the actions of biased somatostatin agonists. Mol. Endocrinol. 25, 1040–1054. Kliewer, A., Mann, A., Petrich, A., Poll, F., Schulz, S., 2012. A transplantable phosphorylation probe for direct assessment of g protein-coupled receptor activation. PLoS One 7, e39458. Lesche, S., Lehmann, D., Nagel, F., Schmid, H.A., Schulz, S., 2009. Differential effects of octreotide and pasireotide on somatostatin receptor internalization and trafficking in vitro. J. Clin. Endocrinol. Metab. 94, 654–661. Lewis, I., Bauer, W., Albert, R., Chandramouli, N., Pless, J., Weckbecker, G., Bruns, C., 2003. A novel somatostatin mimic with broad somatotropin release inhibitory factor receptor binding and superior therapeutic potential. J. Med. Chem. 46, 2334–2344. Liu, Q., Dewi, D.A., Liu, W., Bee, M.S., Schonbrunn, A., 2008. Distinct phosphorylation sites in the SST2A somatostatin receptor control internalization, desensitization, and arrestin binding. Mol. Pharmacol. 73, 292–304. Liu, Q., Bee, M.S., Schonbrunn, A., 2009. Site specificity of agonist and second messenger-activated kinases for somatostatin receptor subtype 2A (Sst2A) phosphorylation. Mol. Pharmacol. 76, 68–80. Mundschenk, J., Unger, N., Schulz, S., Hollt, V., Schulz, S., Steinke, R., Lehnert, H., 2003. Somatostatin receptor subtypes in human pheochromocytoma:
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subcellular expression pattern and functional relevance for octreotide scintigraphy. J. Clin. Endocrinol. Metab. 88, 5150–5157. Nagel, F., Doll, C., Poll, F., Kliewer, A., Schroder, H., Schulz, S., 2011. Structural determinants of agonist-selective signaling at the sst(2A) somatostatin receptor. Mol. Endocrinol. 25, 859–866. Oberg, K., 2005. Neuroendocrine tumors of the gastrointestinal tract: recent advances in molecular genetics, diagnosis, and treatment. Curr. Opin. Oncol. 17, 386–391. Oberg, K.E., Reubi, J.C., Kwekkeboom, D.J., Krenning, E.P., 2010. Role of somatostatins in gastroenteropancreatic neuroendocrine tumor development and therapy. Gastroenterology 139, 742–753 (753 e741). Pfeiffer, M., Koch, T., Schroder, H., Klutzny, M., Kirscht, S., Kreienkamp, H.J., Hollt, V., Schulz, S., 2001. Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A). J. Biol. Chem. 276, 14027–14036. Plockinger, U., Albrecht, S., Mawrin, C., Saeger, W., Buchfelder, M., Petersenn, S., Schulz, S., 2008. Selective loss of somatostatin receptor 2 in octreotide-resistant growth hormone-secreting adenomas. J. Clin. Endocrinol. Metab. 93, 1203– 1210. Poll, F., Lehmann, D., Illing, S., Ginj, M., Jacobs, S., Lupp, A., Stumm, R., Schulz, S., 2010. Pasireotide and octreotide stimulate distinct patterns of sst2A somatostatin receptor phosphorylation. Mol. Endocrinol. 24, 436–446. Poll, F., Doll, C., Schulz, S., 2011. Rapid dephosphorylation of G protein-coupled receptors by protein phosphatase 1beta is required for termination of betaarrestin-dependent signaling. J. Biol. Chem. 286, 32931–32936. Schulz, S., Pauli, S.U., Schulz, S., Handel, M., Dietzmann, K., Firsching, R., Hollt, V., 2000. Immunohistochemical determination of five somatostatin receptors in meningioma reveals frequent overexpression of somatostatin receptor subtype sst2A. Clin. Cancer Res. 6, 1865–1874. Tulipano, G., Stumm, R., Pfeiffer, M., Kreienkamp, H.J., Hollt, V., Schulz, S., 2004. Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes. J. Biol. Chem. 279, 21374–21382. Waser, B., Cescato, R., Liu, Q., Kao, Y.J., Korner, M., Christ, E., Schonbrunn, A., Reubi, J.C., 2012. Phosphorylation of sst2 receptors in neuroendocrine tumors after octreotide treatment of patients. Am. J. Pathol. 180, 1942–1949.
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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Carboxyl-terminal multi-site phosphorylation regulates internalization and desensitization of the human sst2 somatostatin receptor q Andreas Lehmann 1, Andrea Kliewer 1, Dagmar Schütz, Falko Nagel, Ralf Stumm, Stefan Schulz ⇑ Institute of Pharmacology and Toxicology, Jena University Hospital, Friedrich-Schiller-University, 07747 Jena, Germany
a r t i c l e
i n f o
Article history: Received 5 November 2012 Received in revised form 16 February 2014 Accepted 16 February 2014 Available online 22 February 2014 Keywords: Somatostatin Octreotide Pasireotide Somatostatin receptor Internalization Desensitization
a b s t r a c t The somatostatin receptor 2 (sst2) is the pharmacological target of somatostatin analogs that are widely used in the diagnosis and treatment of human neuroendocrine tumors. We have recently shown that the stable somatostatin analogs octreotide and pasireotide (SOM230) stimulate distinct patterns of sst2 receptor phosphorylation and internalization. Like somatostatin, octreotide promotes the phosphorylation of at least six carboxyl-terminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359, which in turn leads to a robust receptor endocytosis. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor followed by a partial receptor internalization. Here, we show that exchange of S341 and S343 by alanine is sufficient to block pasireotide-driven internalization, whereas mutation of T353, T354, T356 and T359 to alanine is required to strongly inhibited both octreotide- and somatostatin-induced internalization. Yet, combined mutation of T353, T354, T356 and T359 is not sufficient to prevent somatostatin-driven b-arrestin mobilization and receptor desensitization. Replacement of all fourteen carboxyl-terminal serine and threonine residues by alanine completely abrogates sst2 receptor internalization and b-arrestin mobilization in HEK293 cells. Together, our findings demonstrate for the first time that agonist-selective sst2 receptor internalization is regulated by multi-site phosphorylation of its carboxyl-terminal tail. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The somatostatin receptor sst2 is highly expressed at the plasma membrane of human tumors including pancreatic, gastrointestinal and pulmonary neuroendocrine tumors, pituitary adenomas, breast carcinomas, meningiomas, neuroblastomas, medulloblastomas, pheochromocytomas, and paragangliomas. This is the molecular basis for clinical application of stable somatostatin analogs for tumor imaging and tumor therapy. A number of metabolically stable somatostatin analogs have been synthesized two of which, octreotide and lanreotide, were approved for clinical use. Octreotide and lanreotide bind with high sub-nanomolar affinity Abbreviations: CHO, chinese hamster ovarian cells; GH, growth hormone; GPCR, G-protein coupled receptor; HEK293, human embryonic kidney 293 cells; SS-14, somatostatin-14; sst, somatostatin receptor. q Phosphorylation of multiple carboxyl-terminal sites within the including S341, S343, T353, T354, T356 and T359 directly regulates agonist-driven internalization and desensitization of the human sst2 somatostatin receptor. ⇑ Corresponding author. Tel.: +49 3641 9325650; fax: +49 3641 9325652. E-mail address: [email protected] (S. Schulz). 1 These authors contributed equally.
http://dx.doi.org/10.1016/j.mce.2014.02.009 0303-7207/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
to sst2 only, have moderate affinity to sst3 and sst5 and show very low or absent binding to sst1 and sst4 (Colao et al., 2010). In clinical practice, octreotide and lanreotide are used as first choice medical treatment of neuroendocrine tumors such as GHsecreting adenomas and carcinoids (Donangelo and Melmed, 2005; Oberg et al., 2010). Octreotide initially controls symptoms caused by hormonal overproduction in about 90% of carcinoid patients. After 1 year of treatment, however, some 50% of patients show an escape of response (Oberg, 2005; Asnacios et al., 2008). In contrast, octreotide can normalize GH levels for prolonged periods in 65% of acromegalic patients (Donangelo and Melmed, 2005). Recently, the novel multireceptor somatostatin analog, pasireotide (SOM230), has been synthesized (Bruns et al., 2002). Pasireotide is a cyclohexapeptide, which binds with high affinity to all somatostatin receptors except to sst4 (Lewis et al., 2003). We have recently uncovered that octreotide and pasireotide stimulate distinct agonist-selective patterns of sst2 somatostatin receptor phosphorylation and internalization (Poll et al., 2010; Lesche et al., 2009; Kliewer et al., 2012). Like somatostatin, octreotide promotes the phosphorylation of at least six carboxylterminal serine and threonine residues namely S341, S343, T353,
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T354, T356 and T359, which in turn leads to a robust sst2 receptor endocytosis (Nagel et al., 2011; Kao et al., 2011). Unlike somatostatin, pasireotide fails to induce a substantial phosphorylation or internalization of the sst2 receptor (Nagel et al., 2011). These findings suggest that agonist-driven phosphorylation may facilitate sst2 receptor endocytosis. However, earlier studies have failed to establish a causal relationship between phosphorylation and internalization for the rat sst2 receptor (Liu et al., 2008). Here, we have constructed a series of phosphorylation-deficient mutants and examined the contribution of individual phosphorylation events to agonist-dependent regulation of the human sst2 receptor. 2. Materials and methods 2.1. Reagents, plasmids and antibodies Pasireotide and octreotide were kindly provided by Dr. Herbert Schmid (Novartis, Basel, Switzerland). SS-14 was obtained from Bachem (Weil am Rhein, Germany). DNA for HA-tagged 19S/T-A, 14S/ T-A, 6S/T-A, 4T-A and 2S-A mutants of the human sst2 receptor was generated via artificial gene synthesis and cloned into pcDNA3.1 by imaGenes (Berlin, Germany). The human HA-tagged sst2 receptor was obtained from UMR cDNA Resource Center (Rolla, MO). The phosphorylation-independent rabbit monoclonal anti-sst2 antibody {UMB-1} (Epitomics, Burlingame, CA) and the phosphositespecific sst2 antibodies anti-pS341/pS343 {3157}, anti-pT353/ pT354 {0521}, anti-pT356/pT359 {0522} and the rabbit polyclonal anti-HA antibody were generated and extensively characterized as previously described (Poll et al., 2010; Fischer et al., 2008). Antibodies {0521} and {0522} were generated against a peptide containing pT353, pT354, pT356 and pT359 and subsequently affinity purified against peptides containing either pT353 and pT354 {0521} or pT356 and pT359 {0522} (Poll et al., 2010, 2011; Nagel et al., 2011). 2.2. Cell culture and transfection Human embryonic kidney HEK293 cells were obtained from the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany). HEK293 cells were grown in DMEM supplemented with 10% fetal calf serum. Cells were transfected with plasmids encoding for wild-type or mutant sst2 receptors using Lipofectamine 2000 according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Stable transfectants were selected in the presence of 400 lg/ml G418. Stable cells were characterized using radioligand-binding assays, Western blot analysis, surface ELISA assay and immunocytochemistry as described previously (Tulipano et al., 2004; Pfeiffer et al., 2001). All mutants tested were present at the cell surface, expressed similar amounts of receptor protein and had similar affinities for SS-14 as the wild-type receptor. The level of receptor expression was 800 fmol/mg membrane protein for all experiments using stably transfected cells. The level of receptor expression was between 1500 and 2000 fmol/mg membrane protein for all experiments using transiently transfected cells. 2.3. Analysis of receptor internalization by confocal microscopy HEK293 cells satbly expressing HA-tagged human sst2 receptors were grown on poly-L-lysine-coated coverslips overnight. When indicated cells were transiently transfected with 1 lg GRK2 plasmid DNA per well containing 100,000 cells using TurboFect™ (Fermentas) according to the instructions of the manufacturer. After the appropriate treatment with either 1 lM SS-14, 1 lM Octreotide or 10 lM Pasireotide at 37 °C, cells were fixed with 4%
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paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 30 min at room temperature and washed several times. Specimens were permeabilized and then incubated with anti-sst2 {UMB-1} antibody followed by Alexa488-conjugated secondary antibody (Amersham, Braunschweig, Germany). Specimens were mounted and examined using a Zeiss LSM510 META laser scanning confocal microscope (Lesche et al., 2009). 2.4. Quantification of receptor internalization by ELISA Receptor internalization was quantified using a linear surface receptor ELISA that has been characterized extensively (Poll et al., 2010; Lesche et al., 2009; Nagel et al., 2011; Pfeiffer et al., 2001). Equal numbers of stably transfected HEK293 cells expressing HA-tagged human sst2 receptors were seeded onto poly-L-lysine-treated 24-well plates (200.000 cells per well). The next day, cells were preincubated with 1 lg/ml anti-HA antibody for 2 h at 4 °C. After the appropriate treatment with SS-14 (1 lM) at 37 °C, cells were fixed and incubated with peroxidase-conjugated antirabbit antibody overnight. After washing, plates were developed with ABTS solution and analyzed at 405 nm using a microplate reader. When indicated cells were transiently transfected with 1 lg GRK2 plasmid DNA per well using TurboFect™ (Fermentas) 24 h later, cells were preincubated with 1 lg/ml anti-HA antibody and treated as described above. Statistical analysis was carried out with unpaired t-test. p-Values of <0.05 were considered statistically significant. 2.5. Western blot analysis HEK293 cells stably expressing HA-tagged human sst2 receptors were plated onto 60-mm dishes and grown to 80% confluence. After treatment with SS-14 at 37 °C, cells were lysed in detergent buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) in the presence of protease and phosphatase inhibitors Complete mini and PhosSTOP (Roche Diagnostics, Mannheim, Germany) and centrifuged at 16,000g for 20 min at 4 °C. Glycosylated proteins were partially enriched using wheat germ lectin-agarose beads as described (Schulz et al., 2000; Mundschenk et al., 2003; Plockinger et al., 2008). Proteins were eluted from the beads using SDS-sample buffer for 20 min at 65 °C and then resolved on 8% SDS–polyacrylamide gels. After electroblotting, membranes were incubated with phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} or anti-pT356/pT359 {0522} at a concentration of 0.1 lg/ml followed by detection using enhanced chemiluminescence (Amersham). Blots were subsequently stripped and reprobed with anti-sst2 antibody {UMB-1} to confirm equal loading of the gels. 2.6. b-Arrestin-EGFP mobilization assay Untransfected HEK293 cells were seeded onto 35-mm glassbottom culture dishes (Mattek, Ashland, MA). The next day, cells were transiently cotransfected with 0.2 lg b-arrestin-2-EGFP and 2 lg human sst2 receptor or with a mixture of 0.2 lg b-arrestin2-EGFP, 0.8 lg GRK2 and 1.2 lg human sst2 receptor per dish containing 200,000 cells using TurboFect™. After 24 h, cells were transferred onto a temperature-controlled microscope stage set at 37 °C of a Zeiss LSM510 META laser scanning confocal microscope. Images were collected sequentially using single line excitation at 488 nm with 515–540-nm band pass emission filters. Saturating concentrations of SS-14 (1 lM) were applied directly into the culture medium immediately after the initial image was taken.
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2.7. [Ca2+]i assay HEK293 cells stably expressing HA-tagged human sst2 receptors were transiently transfected with G protein chimera qi5 (0.25 lg plasmid DNA per well containing 38,000 cells) consisting of murine Gaq with the 5 carboxyl-terminal amino acids changed from Gaq to Gai2. We were also able to trigger calcium responses of the sst2 receptor in the absence of qi5. Given that transfection with qi5 considerably improves the signal-to-noise ratio, we preferred this approach. G protein signaling was examined 24 h after plating by measuring increases in [Ca2+]i using the FlexStation3 microplate reader as described (Hoffmann et al., 2012). Baseline values were recorded for 30 s before the maximal possible response to 100 nM SS-14 was determined. In desensitization experiments, a prestimulus with 10 nM SS-14 was applied before stimulating with 100 nM SS-14. Desensitization of a sst2 mutant, was calculated as the difference between the maximal possible response and the response to 100 nM SS-14 with prestimulus and expressed in percent of the maximal possible response of the mutant. 2.8. Data analysis Data were analyzed using GraphPad Prism 4.0 software. Statistical analysis was carried out with unpaired t-test, or one-way ANOVA followed by the Dunnett’s post-test. p-Values of <0.05 were considered statistically significant. 3. Results The human sst2 receptor contains many potential phosphate acceptor sites in its third intracellular loop and carboxyl-terminal tail (Fig. 1A). Although phosphosite-specific antibodies have been successfully generated against some of these sites, a clear functional role has not yet been assigned to individual phosphorylation events. We therefore constructed a series of phosphorylation-deficient mutations including 2S-A, 4T-A, 6S/T-A, 14S/T-A and 19S/T-A (Fig. 1B). In initial Western blot studies, we confirmed that all of these mutants expressed similar receptor levels (Fig. 1C, bottom
panels). In addition, we tested whether these receptors were present at the plasma membrane using surface ELISA (Supplemental Fig. S1). We also confirmed that all mutant receptors exhibited binding and signaling properties similar to the wild-type sst2 receptor (Supplemental Fig. S3). Next, we examined agonist-induced phosphorylation of the wild-type sst2 receptor and its mutants using phosphosite-specific antibodies for pS341/343, pT353/354 and pT356/359 (Fig. 1C). Phosphorylation at the three sites was detectable within 5 min of SS-14 exposure in the wildtype sst2 receptor. SS-14 promoted phosphorylation at T353/354 and T356/359 in the 2S-A receptor as efficient as in wild-type sst2. The 2S-A receptor carries alanine mutations of S341 and S343. Conversely, phosphorylation of S341/S343 was still detectable in the 4T-A receptor, in which T353, T354, T356 and T359 were converted into alanines. As expected, phosphorylation at the three sites was no longer detectable in the 6S/T-A receptor, which carries alanine mutations in the S341/S343 site and in the T353/T354/T356/T359 cluster. Similar results were obtained with the 19S/T-A receptor, in which all potential phosphate acceptor sites in the third intracellular loop and the carboxyl-terminal tail have been mutated, and the 14S/T-A receptor with mutation of all potential phosphate acceptor sites in the carboxyl-terminal tail. These findings suggest that SS-14-promoted phosphorylation at T353/T354 and T356/359 occurs independent from phosphorylation at S341/343 and that phosphorylation at S341/343 occurs independent from phosphorylation at threonine cluster T353/ 354/356/359. Quantitative and qualitative analysis of receptor sequestration using ELISA and immunocytochemistry revealed that treatment of the wild-type human sst2 receptor with SS-14 produced a 80% loss of surface receptors within 30 min of agonist exposure (Fig. 2A, B). SS-14-induced internalization was reduced but clearly detectable in the 2S-A mutant. In the 4T-A and 6S/T-A mutants, SS14-induced internalization was strongly inhibited, i.e. after 30 min SS-14 exposure there was a loss of surface receptors of less than 10% (Fig. 2A and B). In fact, replacement of all fourteen carboxylterminal serine and threonine residues by alanine (14S/T-A) was required to completely abrogate sst2 receptor internalization in
Fig. 1. Construction of phosphorylation-deficient sst2 receptor mutants. (A) Schematic representation of the human sst2 receptor indicating all potential phosphate acceptor sites (depicted in gray or black) within the third intracellular loop and carboxyl-terminal tail. Epitopes of the phosphosite-specific anti-pS341/pS343, anti-pT353/pT354 and anti-pT356/pT359 antibodies (depicted in black) as well as the epitope of the phosphorylation-independent anti-sst2 antibody (UMB-1) are indicated. (B) Depiction of amino acid sequences of the third intracellular loop (237–250) and carboxyl-terminal tail (304–369) of the human sst2 receptor. Phosphate acceptor sites mutated to alanine in the mutants are depicted in black. (C) HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A or 19S/T-A were treated with 1 lM SS-14 for 5 min. The levels of phosphorylated sst2 receptors were then determined using the phosphosite-specific antibodies anti-pS341/pS343 {3157}, anti-pT353/pT354 {0521} or anti-pT356/pT359 {0522}. Blots were subsequently stripped and reprobed with the phosphorylation-independent anti-sst2A antibody {UMB-1} to confirm equal loading of the gels (hsst2). Blots shown are representative of three independent experiments each. The positions of molecular mass markers are indicated on the left (in kDa).
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Fig. 2. Phosphorylation of the 353TTETQRT359 motif is required for sst2 internalization. (A) HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A or 19S/T-A were treated for 30 min with 1 lM SS-14 (A) or vehicle (not shown). Receptor sequestration was measured by ELISA. Data represent per cent loss of cell-surface receptors in agonist-treated cells as compared to sister cultures receiving 30 min vehicle (100%). Data are presented as mean ± SEM from at least four independent experiments performed in quadruplicate. Results were analyzed by unpaired t-test vs. wild type receiving 30 min vehicle (p < 0.05). (B) HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/ T-A or 19S/T-A were treated with 1 lM SS-14 for 15 or 30 min. Cells were then fixed, stained with the anti-sst2 antibody {UMB-1} and examined by confocal microscopy. Shown are representative images from one of at least three independent experiments performed in duplicate. Scale bar, 15 lm. (C) HEK293 cells stably expressing human WT or mutants were transfected with GRK2 and next day treated for 30 min with 1 lM SS-14 (A) or vehicle (not shown). Receptor sequestration was measured by ELISA. Data represent per cent loss of cell-surface receptors in agonist-treated cells as compared to sister cultures receiving 30 min vehicle (100%). Data are presented as mean ± SEM from at least four independent experiments performed in quadruplicate. Results were analyzed by unpaired t-test vs. wild type receiving 30 min vehicle (p < 0.05). (D) HEK293 cells stably expressing wild-type hsst2 or mutants were transfected with GRK2 and next day treated with 1 lM SS-14 for 15 or 30 min. Cells were then fixed, stained with the anti-sst2 antibody {UMB-1} and examined by confocal microscopy. Shown are representative images from one of at least three independent experiments performed in duplicate. Scale bar, 15 lm.
HEK293 cells (Fig. 2A and B). Additional mutation of all five serine residues within the third intracellular loop (19S/T-A) produced similar effects. We have recently shown that agonist-driven sst2 phosphorylation is mediated in part via GRK2 (Poll et al., 2010).
Consequently, we evaluated whether GRK2 overexpression would enhance internalization of wild-type or mutant sst2 receptors. Under these conditions, however, the extent of receptor internalization detected by ELISA as loss of surface receptors after
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the 30 min SS-14 exposure was very similar to that observed without GRK2 overexpression (Fig. 2C). Nevertheless, overexpression of GRK2 accelerated internalization of the 2S-A mutant at earlier time points (15 min) as detected by confocal microscopy (Fig. 2D). We then asked whether overexpression of GRK2 and b-arrestin-2 would drive internalization of the 14S/T-A receptor. As depicted in Supplemental Fig. S2, the loss of surface receptors was less than 5% under these conditions. Collectively, these results suggest that GRK-mediated multi-site phosphorylation of the sst2 carboxyl-terminal tail is a major requirement for its agonist-induced internalization. Octreotide and pasireotide stimulate distinct patterns of sst2 receptor phosphorylation (Lesche et al., 2009). Like somatostatin, octreotide promotes the phosphorylation of at least six carboxylterminal serine and threonine residues namely S341, S343, T353, T354, T356 and T359. Unlike somatostatin, pasireotide stimulates a selective phosphorylation of S341 and S343. We therefore tested the effect of different mutations on octreotide- and pasireotide-induced internalization. Similar to that observed with SS-14, octreotide-driven internalization was strongly reduced in the 4T-A, 6S/TA and 14S/T-A mutants, while internalization of the 2S-A receptor was still clearly detectable (Fig. 3, left panel). In contrast, exchange of S341 and S343 by alanine (2S-A) was sufficient to prevent pasireotide-driven internalization (Fig. 3, right panel). We then addressed the functional role of carboxyl-terminal sst2 receptor phosphorylation. First, we employed functional b-arrestin-2 conjugated to enhanced green fluorescent protein (EGFP) to visualize the patterns of sst2-stimulated b-arrestin mobilization in live HEK293 cells under conditions with or without GRK2 overexpression. In the absence of agonist, b-arrestin-2-EGFP was
uniformly distributed throughout the cytoplasm (Fig. 4). The addition of saturating concentrations of SS-14 to the wild-type sst2 receptor induced a redistribution of b-arrestin-2 from the cytoplasm to the plasma membrane within 1 min resulting in fluorescence outlining of the cell shape (Fig. 4). After extended time periods (30 min), a redistribution of b-arrestin-2-EGFP into the cytosol was noted in cells transfected with the wild-type human sst2 receptor (Fig. 4). A rapid redistribution (<1 min) of b-arrestin-2 to the plasma membrane was also observed in the 2S-A mutant (Fig. 4, left panels). Interestingly, b-arrestin-2 mobilization was much less pronounced and required more extended time periods (10–30 min) in the 4T-A and 6S/T-A mutants (Fig. 4, left panels). In contrast, no b-arrestin-2 redistribution was observed in cells expressing the 14S/T-A or the 19S/T-A mutant under otherwise identical conditions (Fig. 4, left panels). GRK2 overexpression facilitated b-arrestin-2 mobilization in the 4T-A and 6S/T-A receptors but was not able to promote a similar redistribution in cells expressing the 14S/T-A or the 19S/T-A mutant (Fig. 4, right panels). These findings suggest that carboxyl-terminal multi-site phosphorylation of the sst2 receptor is a major determinant for agonist-induced b-arrestin mobilization. The residual b-arrestin-2 translocation observed in the 6S/T-A mutants suggests that carboxyl-terminal phosphate acceptor sites other than S341, S343, T353, T354, T356 and T359 may undergo agonist-induced phosphorylation. Moreover, this residual and delayed b-arrestin-2 recruitment may be responsible for the small amount of receptor internalization (<10%) seen in the 14S/T-A mutant after 30 min SS-14 exposure. We then used a calcium mobilization assay to determine the effect of the mutations on sst2 desensitization. Stably expressing
Fig. 3. Differential effects of sst2 mutations on octreotide- and pasireotide-induced internalization. HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A or 14S/T-A were treated with 1 lM Octreotide or 10 lM Pasireotide for 15 or 30 min. Cells were then fixed, stained with the anti-sst2 antibody {UMB-1} and examined by confocal microscopy. Shown are representative images from one of at least two independent experiments performed in duplicate. Scale bar, 15 lm.
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Fig. 4. Phosphorylation-dependent b-arrestin mobilization. (A) HEK293 cells were transiently transfected with either hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A or 19S/T-A and barrestin-2-EGFP. The distribution of b-arrestin-2 was visualized sequentially in the same live cells before (0 min) and after (1 to 30 min) the addition of 1 lM SS-14 to the culture medium. Shown are representative images from one of four independent experiments performed in duplicate. Scale bar, 15 lm. (B) HEK293 cells were transiently cotransfected with receptor, GRK2 and b-arrestin-2-EGFP. The distribution of b-arrestin-2 was visualized sequentially in the same live cells before (0 min) and after (1– 30 min) the addition of 1 lM SS-14 to the culture medium. Shown are representative images from one of four independent experiments performed in duplicate. Scale bar, 15 lm.
cells were transfected with a qi5 G protein chimera that was engineered to connect Gi-coupled receptors to the calcium pathway (Hoffmann et al., 2012). Under these conditions, application of 100 nM SS-14 evoked a similar strong calcium response in all mutants tested (Supplemental Fig. S3). Sequential stimulation was then used to determine homologous sst2 desensitization. In cells expressing human wild-type sst2, 100 nM SS-14 evoked a strongly reduced calcium response when administered 180 s after a precedent 10 nM SS-14 stimulus as compared with sister cultures experiencing 100 nM SS-14 without pre-stimulus (Fig. 5A). In the 14S/ T-A mutant, the pre-stimulus with 10 nM SS-14 provoked a modest reduction of the 100 nM stimulus as compared with sister cultures experiencing no SS-14 pre-stimulus (Fig. 5B). We then calculated desensitization for each mutant. To this end, we determined the differences in the height of the 100 nM peaks without and with pre-stimulus and expressed it as percent of the 100 nM peak without pre-stimulus. This showed that the 10 nM SS-14 pre-stimulus caused more than 80% desensitization of G protein signaling of the wild-type sst2 receptor as well as in the 2S-A and 4T-A mutants but only 60% and 50% desensitization of the 6S/T-A and 14S/T-A mutants, respectively (Fig. 5C). This loss of signal largely reflects homologous receptor desensitization and was not due to an exhaustion of the Ca response because a subsequent third pulse with the muscarinic receptor agonist carbachol evoked similar strong responses in all tested mutant and wild-type sst2 receptors (Supplemental Fig. S4). By comparing the different mutants to wild-type sst2, it became apparent that desensitization was unchanged in the 2S-A and 4T-A mutants, suggesting that both the S341/S343 and the 353TTETQRT359 motif are sufficient to mediate
full homologous sst2 receptor desensitization. Combined elimination of the S341/S343 and 353TTETQRT359 motifs was required to significantly diminish sst2 desensitization. Enhanced desensitization of the 14S/T-A mutant as compared with the 6S/T-A mutant indicates that phosphoacceptor sites other than S341/S343 and T353/T354/T356/T359 may additionally contribute to sst2 receptor desensitization.
4. Discussion The overexpression of the sst2 receptor in human tumors is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analog octreotide. In gastroenteropancreatic neuroendocrine tumors, octreotide initially controls symptoms caused by hormonal overproduction in about 90% of patients. After 1 year of treatment, however, some 50% of patients show an escape of response (Oberg, 2005; Asnacios et al., 2008). Consequently, the elucidation of regulatory mechanisms of the sst2 somatostatin receptor has attracted much attention. Thus, direct evidence using phosphosite-specific antibodies has been presented for agonistdependent phosphorylation of at least six carboxyl-terminal serine and threonine residues in vivo and in vitro (Poll et al., 2010; Lesche et al., 2009; Kliewer et al., 2012; Nagel et al., 2011; Liu et al., 2009; Waser et al., 2012). For many GPCRs a direct correlation between phosphorylation and internalization has been observed. Nevertheless, an earlier study analyzed the rat sst2 somatostatin receptor expressed on CHO cells with mutations of either all serine or all threonine residues as well as a combined mutation of all serine
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multiple sites including S341, S343, T353, T354, T356 and T359 contributes to agonist-driven internalization and desensitization of the human sst2 somatostatin receptor. In fact, we have previously established that rapid agonist-induced phosphorylation of all of these sites occurs via GRK2/3-dependent mechanisms (Poll et al., 2010; Nagel et al., 2011). In a manner similar to previous studies (Liu et al., 2008; Tulipano et al., 2004), we also show that multi-site phosphorylation is also a precondition for b-arrestin mobilization indicating that the agonist-induced conformational change of a phosphorylation-deficient sst2 receptor by itself was not sufficient for b-arrestin binding. Our observation that carboxyl-terminal phosphorylation is the initial step determining the rate of internalization and desensitization of the human sst2 receptor, may have important implications for the clinical utility of octreotide and pasireotide. Recent evidence indicates that octreotide and pasireotide display functional selectivity (Kliewer et al., 2012). In fact, pasireotide and octreotide are equally active in inducing classical G protein-dependent signaling at the sst2 receptor (Lesche et al., 2009; Nagel et al., 2011). Yet, they promote strikingly different patterns of sst2 phosphorylation, internalization and b-arrestin trafficking (Poll et al., 2010). Similar to that observed with SS-14, octreotide promotes the phosphorylation of multiple sites including S341, S343, T353, T354, T356 and T359, which is followed by a robust internalization, stable b-arrestin interaction and slow recycling. In contrast, pasireotide stimulates a selective phosphorylation of S341 and S343 of the human sst2 receptor, which is followed by a partial receptor sequestration, instable b-arrestin interaction but fast recycling (Poll et al., 2010; Lesche et al., 2009; Nagel et al., 2011). Nevertheless, we have to await additional clinical data to see inasmuch these differences in acute internalization and desensitization may regulate longterm responses to octreotide and pasireotide. It is also possible that during long-term treatment additional yet unknown mechanisms of receptor downregulation contribute to the regulation of tumor responsiveness. In conclusion, we show that the human sst2 receptor is regulated like a prototypical GPCR. Multi-site phosphorylation of clusters of carboxyl-terminal serine and threonine residues within the middle portion of the receptor’s cytoplasmic tail is a critical initial event regulating its internalization, b-arrestin trafficking and desensitization. Disclosure statement The authors have nothing to disclose. Fig. 5. Multi-site phosphorylation regulates sst2 receptor desensitization. HEK293 cells stably expressing hsst2, 2S-A, 4T-A, 6S/T-A, 14S/T-A were transfected with qi5 chimeric G protein and changes in [Ca2+]i were monitored. Averaged traces with SEM showing two subsequent stimulations with 10 nM SS-14 or vehicle and 100 nM SS-14 for wild-type hsst2 (A) and 14S/T-A (B). Traces with the mock prestimulus (vehicle) are gray and traces with 10 nM SS-14 pre-stimulus are black. For each receptor, data were normalized to peak height of the 100 nM SS-14 stimulus without pre-stimulus (maximal stimulation). (C) For each receptor, homologous desensitization was calculated as the difference between the two peaks after 100 nM SS-14 and expressed in percent of the 100 nM peak without pre-stimulus. Data are presented as mean ± SEM from quadruplicate determinations. Two independent experiments gave similar results. Results were analyzed by one-way ANOVA followed by the Dunnett’s posttest (p < 0.05).
and threonine residues (Liu et al., 2008). Although sst2 internalization was found to be partially inhibited by mutation of the threonine residues, none of the mutations analyzed resulted in a complete block of receptor internalization (Liu et al., 2008). The present study established for the first time a causal relationship between carboxyl-terminal phosphorylation and internalization for the human sst2 receptor. Indeed phosphorylation of
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