JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M313522200 on March 4, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21374-21382, May 14, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/20/21374    most recent
M313522200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tulipano, G.
Right arrow Articles by Schulz, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tulipano, G.
Right arrow Articles by Schulz, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Differential {beta}-Arrestin Trafficking and Endosomal Sorting of Somatostatin Receptor Subtypes*

Giovanni Tulipano, Ralf Stumm, Manuela Pfeiffer, Hans-Jürgen Kreienkamp{ddagger}, Volker Höllt, and Stefan Schulz§

From the Institut für Pharmakologie und Toxikologie, Otto-von-Guericke-Universität, 39120 Magdeburg, Germany and {ddagger}Institut für Zellbiochemie und klinische Neurobiologie, Universitätskrankenhaus Hamburg-Eppendorf, Universität Hamburg, 20246 Hamburg, Germany

Received for publication, December 10, 2003 , and in revised form, February 25, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst1-sst5) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of {beta}-arrestin mobilization and endosomal sorting. The {beta}-arrestin-dependent trafficking of the sst2A somatostatin receptor resembled that of a class B receptor in that upon receptor activation, {beta}-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst2A receptor. Unlike other class B receptors, however, the sst2A receptor was rapidly resensitized and recycled to the plasma membrane. The {beta}-arrestin mobilization of the sst3 and the sst5 somatostatin receptors resembled that of a class A receptor in that upon receptor activation, {beta}-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, {beta}-arrestin was excluded from sst3-containing vesicles. Unlike other class A receptors, a large proportion of sst3 receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst4 somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and {beta}-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Somatostatin (SS-14)1 is an important regulator of neurotransmission in the brain as well as of hormone secretion from the anterior pituitary gland, the pancreas, and the gastrointestinal tract. Five genes encoding six different somatostatin receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, sst5) have been cloned. These receptors are widely expressed in the central nervous system and periphery, and multiple somatostatin receptor subtypes often coexist in the same cell (1, 2). The high density of somatostatin receptors on human neuroendocrine tumors (3-5) has been used clinically to treat the symptoms of hormonal hypersecretion in patients with growth hormone- and thyrotropin-secreting pituitary adenomas and patients harboring islet cell or carcinoid tumors (6-8). Moreover, it has allowed the development of somatostatin receptor scintigraphy for tumor imaging as well as somatostatin receptor-targeted radiotherapy (9, 10). The fact that naturally occurring somatostatin peptides have only short half-lives has necessitated the development of stable somatostatin peptide analogs including octreotide and lanreotide. Although SS-14 binds with high affinity to all five somatostatin receptors, octreotide and lanreotide bind only to sst2 with high affinity and to sst3 and sst5 with moderate affinity (1, 11).

It is well known that the physiological responses to SS-14 are diminished with continued exposure (1). However, important differences have been observed in the response of neuroendocrine tumors to the long-term application of stable somatostatin analogs. In patients harboring somatostatin receptor-expressing growth hormone-secreting adenoma, the inhibitory effects of somatostatin analogs on hormone secretion persist for many years during long-term treatment, and only few thyrotropin-secreting tumors escape from octreotide therapy. In contrast, islet cell tumors and carcinoids are very likely to undergo desensitization within weeks to months of octreotide exposure (12). Although studies in transfected host cells have examined agonist-induced internalization of the various somatostatin receptor subtypes (2), the molecular basis for the distinct long-term responsiveness of individual target cells has not been established. So far only species-related differences in agonist-mediated endocytosis of somatostatin receptors have been observed, e.g. the rat sst4 receptor appears to be largely resistant to agonist-induced internalization and desensitization (13), whereas the human sst4 receptor has been reported to undergo a very slow but clearly detectable endocytosis. Other studies have shown that the human sst1 receptor but not the rat sst1 receptor failed to internalize in an agonist-dependent manner (14, 15).

The aim of the present study was to characterize early molecular events after agonist activation that lead to functional desensitization and sequestration of the distinct somatostatin receptor subtypes. Moreover, we wanted to delineate the molecular determinants that dictate the differential trafficking and endosomal sorting of somatostatin receptors. All five somatostatin receptors belong to the superfamily of G protein-coupled receptors (GPCRs). After agonist binding and activation of GPCRs, the signaling is turned off by phosphorylation of intracellular receptor domains and subsequent recruitment of cytoplasmic proteins termed arrestins that interrupt coupling between the receptor and its cognate heterotrimeric G protein (16, 17). {beta}-Arrestins also function as docking proteins that link the receptor to components of the endocytic machinery such as AP-2 and clathrin and as scaffolding proteins to turn on signaling to mitogen-activated protein kinase (MAPK) cascades (18). Within the endosomal compartment, {beta}-arrestins regulate the rate at which internalized receptors are dephosphorylated and recycled to the plasma membrane (19, 20). Based on their binding properties to different isoforms of {beta}-arrestin, GPCRs have been categorized into two classes. Class A receptors (e.g. µ-opioid, {beta}2 and {alpha}1B adrenergic, endothelin A, and dopamine D1A receptors) do not bind visual arrestin and have a higher affinity for nonvisual {beta}-arrestin-2 than {beta}-arrestin-1. Class B receptors (e.g. substance P, angiotensin AT1a, neurotensin 1, and vasopressin 2 receptors) bind visual arrestin and have similar affinities for {beta}-arrestin-1 and {beta}-arrestin-2 (16). Class A and class B receptors also differ in the fate of the {beta}-arrestin-receptor complex. For class A receptors, {beta}-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors, {beta}-arrestin forms stable complexes with the receptors, such that the receptor-{beta}-arrestin complex internalizes as a unit into early endosomes (17).

Here we identify the sst3 and sst5 somatostatin receptors as class A receptors and the sst2A somatostatin receptor as class B receptor. The trafficking of the sst1 and sst4 somatostatin receptors did not appear to depend on {beta}-arrestins. We demonstrate that the formation of stable complexes between {beta}-arrestin and sst2A requires G protein-coupled receptor kinase 2 (GRK2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst2A receptor. We also show that sst2A and sst3 underwent differential endosomal sorting. In contrast to what would be expected from their {beta}-arrestin mobilization patterns, the sst2A receptor was rapidly recycled to the plasma membrane whereas the sst3 receptor was subject to ubiquitination-dependent lysosomal degradation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Monoclonal mouse anti-ubiquitin antibody (UbP4D1) was from Santa Cruz Biotechnology. Mouse monoclonal anti-T7 antibody and mouse monoclonal anti-T7 antibody covalently coupled to Sepharose beads were obtained from Novagen (Madison, WI). The rabbit anti-sst2A antibody was generated to the peptide ETQRTLLNGDLQTSI, which corresponds to residues 355-369 of the carboxyl-terminal tail of rat/mouse/human sst2A. The anti-sst3 antibody was generated to the peptide TAGDKASTLSHL, which corresponds to residues 417-428 of the carboxyl-terminal tail of the rat/mouse sst3. Both antibodies have been characterized extensively (21). All polyclonal rabbit antisera were purified by affinity chromatography using the Sulfo-Link coupling gel coupled to the appropriate immunizing peptide according to the instructions of the manufacturer (Pierce). Plasmid constructs of the rat sst1, sst2A, sst3, sst4, and sst5 containing the amino-terminal T7 epitope tag sequence MASMTGGQQMG in pcDNA3 have been described previously (13, 15, 21). Truncated sst2A mutants were created by PCR using primers that introduce stop codons at suitable positions (22). Point mutations were introduced into sst2A by PCR replacing threonine 353, 355, and 356 with alanine. The sequences of all constructs were verified by dideoxynucleotide sequencing. The plasmid encoding {beta}-arrestin-1-enhanced green fluorescence protein (EGFP) was kindly provided by N. W. Bunnett (University of California, San Francisco). The construct encoding the {beta}-arrestin-2-EGFP fusion protein was obtained from Biosignal (Montreal, Canada). Transferrin-Alexa Fluor 488 was purchased from Molecular Probes (Leiden, The Netherlands).

Cell Culture and Transfection—Human embryonic kidney (HEK) 293 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 10% CO2. Transfections were performed using LipofectAMINE 2000 according to the instructions of the manufacturer (Invitrogen). Stable transfectants were selected in the presence of 500 µg/ml G418 (Invitrogen). HEK 293 cells stably expressing the T7-tagged sst2A (Bmax, 820 ± 19 fmol/mg membrane protein) or T7-tagged sst3 receptors (Bmax, 1,182 ± 31 fmol/mg membrane protein) were characterized using radioligand binding assays, Western blot analysis, and immunocytochemistry as described previously (21). The level of somatostatin receptor expression was between 1,500 and 2,000 fmol/mg membrane protein for all experiments using transiently transfected HEK 293 cells.

Confocal Microscopy—One day after transfection, cells were seeded into 35-mm glass-bottom culture dishes (Mattek, Ashland, MA). The next day, cells were incubated for 2 h in serum-free OPTIMEM-1 medium (Invitrogen) containing 10 mM Hepes (pH 7.4). Cells were then transferred onto a temperature-controlled microscope stage set at 37 °C. Confocal microscopy was performed using a Leica TCS NT laser scanning confocal microscope (Heidelberg, Germany). Analysis of {beta}-arrestin translocation was performed in cells transiently cotransfected with 1.5 µg of either {beta}-arrestin-1-EGFP or {beta}-arrestin-2-EGFP and 6 µg of the various somatostatin receptor subtypes. When indicated, 4 µg of GRK2 or 4 µg of empty vector (MOCK) were included in the transfections. 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 µM) were applied directly into the culture medium immediately after the initial image was taken.

Immunocytochemistry—Cells were grown on poly-L-lysine-coated coverslips overnight. After the appropriate treatment with SS-14, cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (pH 6.9) for 40 min at room temperature and washed several times in TPBS (10 mM Tris-HCl, pH 7.4, 10 mM phosphate buffer, 137 mM NaCl, and 0.05% thimerosal). Specimens were then incubated for 3 min in 50% methanol and 3 min in 100% methanol, washed in TPBS, and preincubated with TPBS supplemented with 3% normal goat serum for 1 h at room temperature. Cells were then incubated with affinity-purified anti-T7, anti-sst2A, or anti-sst3 antibodies at a concentration of 1 µg/ml in TPBS supplemented with 1% normal goat serum overnight. After washing with TPBS, bound primary antibody was detected with cyanine 3.18-conjugated secondary antibody (Jackson Immunoresearch, West Grove, PA). Cells were then dehydrated, cleared in xylol, and permanently mounted in DPX (Fluka, Deisenhofen, Germany). For {beta}-arrestin-2-EGFP/sst2A colocalization studies, cells were mounted directly in Vectashield Hard Set mounting medium (Vector, Burlingame, CA). Specimens were examined using a Leica TCS NT laser scanning microscope. Cyanine 3.18 was imaged with 568 nm excitation and 570-630-nm band pass emission filters.

Transferrin Trafficking—A previously described pulse-chase assay was used to estimate the degree to which a "pulse" of internalized sst2A or sst3 was accessible to a subsequent "chase" of endocytosed transferrin (23). HEK 293 cells stably expressing T7-tagged sst2A or sst3 were grown on glass coverslips, and receptors were surface-labeled with 1 µg/ml T7 antibody. In the first set of experiments, cells were simultaneously incubated with 1 µM SS-14 and 5 µg/ml Alexa Fluor 488-conjugated transferrin for 30 min at 37 °C. In a second set of experiments, cells were first incubated with 1 µM SS-14 for 30 min at 37 °C to drive endocytosis of antibody-labeled receptors. Next, cells were chilled on ice, rinsed with EDTA-supplemented phosphate-buffered saline, rewarmed for 20 min in medium lacking agonist but containing 5 µg/ml Alexa Fluor 488-conjugated transferrin. Cells were fixed and permeabilized, and antibody-labeled sst2A or sst3 receptors were detected using cyanine 3.18-conjugated secondary antibodies. Specimens were examined as described above.

Whole Cell Phosphorylation Assay—HEK 293 cells were plated at a density of 106 cells/dish onto 100-mm dishes. The next day, cells were transfected with the appropriate plasmids. Two days later, cells were washed with serum- and phosphate-free medium and then labeled with 200 µCi/ml carrier-free 32P-orthophosphate (285 Ci/mg Pi, ICN, Eschwege, Germany) for 60 min at 37 °C. Labeled cells were then exposed to 1 µM SS-14 for 20 min. Subsequently, cells were placed on ice and washed with ice-cold phosphate-buffered saline. Cells were then scraped into 1 ml of radioimmune precipitation 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, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, and 10 µg/ml bacitracin). Cells were solubilized on a rotating platform for 60 min at 4 °C. After centrifugation at 30,000 x g for 60 min at 4 °C, receptor proteins were immunoprecipitated using T7 affinity beads. Receptors were eluted from the beads using SDS-sample buffer for 20 min at 60 °C. The samples were subjected to 8% SDS-PAGE followed by autoradiography. The extent of receptor phosphorylation was quantitated using a Fuji phosphoimaging system and BAS 1000 software. Loading of equal amounts of receptor proteins in each lane was confirmed by Western blot analysis. Receptor phosphorylation was expressed as a percent of wild-type sst2A. Means ± S.E. of three independent experiments are reported.

Western Blot Analysis—Stably transfected HEK 293 cells were plated onto poly-L-lysine-coated 100-mm dishes and grown to 80% confluence. After treatment with SS-14 in serum-free medium, cells were lysed in radioimmune precipitation buffer as described above. Glycosylated proteins were partially enriched using wheat germ-lectin-agarose beads (Amersham Biosciences). Proteins were eluted from the beads using SDS-sample buffer for 20 min at 60 °C and then resolved on 8% SDS-polyacrylamide gels. After electroblotting, the membranes were incubated with 1 µg/ml anti-sst2A or anti-sst3 antibody for 12 h at 4 °C followed by detection using an enhanced chemiluminescence detection system (Amersham Biosciences). For ubiquitination studies, 200 µM chloroquine (Sigma) and 25 µM MG132 (Calbiochem) were added to the culture medium 30 min before and during agonist treatment. Cells were then lysed in radioimmune precipitation buffer containing 200 µM chloroquine, 25 µM MG132, and 10 mM N-ethylmaleimide (Calbiochem). Receptor proteins were precipitated with T7 affinity beads. Ubiquitinated receptors were detected using anti-ubiquitin antibodies (2 µg/ml). NIH Image 1.62 software was used to desensitize and quantify protein bands detected on Western blots. Statistical analysis was carried out with the Student's t test using GraphPad Prism 3.0 software. p-values of <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Differential {beta}-Arrestin Mobilization of Somatostatin Receptors—We employed functional {beta}-arrestin-1 and {beta}-arrestin-2 conjugated to enhanced green fluorescent protein to visualize the translocation of {beta}-arrestins to the plasma membrane in live HEK 293 cells transfected with distinct somatostatin receptor subtypes. In the absence of the agonist, both {beta}-arrestin-EGFP isoforms were uniformly distributed throughout the cytoplasm of the cells (Fig. 1, 0 min). {beta}-Arrestin-1 was also detectable inside the cell nucleus. The addition of saturating concentrations of SS-14 induced a rapid redistribution of {beta}-arrestin-1 as well as {beta}-arrestin-2 from the cytoplasm to the plasma membrane in sst2A- and sst3-transfected cells. In sst3-expressing cells, fluorescence outlining the shape of the cells was less marked in cells cotransfected with {beta}-arrestin-1 as compared with cells cotransfected with {beta}-arrestin-2. In sst5-transfected cells, only translocation of {beta}-arrestin-2 but not {beta}-arrestin-1 was observed. Translocation of {beta}-arrestin-1 or {beta}-arrestin-2 to the plasma membrane was not detectable in cells transfected with either sst1 or sst4. After extended agonist exposure, internalization of {beta}-arrestin-EGFP into endocytotic vesicles was observed only in sst2A-expressing cells (Fig. 1, 50 min). Dual color confocal microscopy revealed that {beta}-arrestin-2-EGFP and sst2A were extensively colocalized in early endosomes (Fig. 2). In both sst3- and sst5-expressing cells, {beta}-arrestin-EGFP ultimately redistributed to the cytoplasm after prolonged SS-14 exposure (Fig. 1, 50 min).



View larger version (102K):
[in this window]
[in a new window]
  		FIG. 1.
Differential {beta}-arrestin mobilization of somatostatin receptors. Left panel, HEK 293 cells were transiently transfected with 1.5 µg of {beta}-arrestin-1-EGFP, 4 µg of GRK2, and 6 µg of one of the five somatostatin receptor subtypes (sst1-5). Right panel, HEK 293 cells were transiently transfected with 1.5 µg of {beta}-arrestin-2-EGFP, 4 µg of GRK2, and 6 µg of one of the five somatostatin receptor subtypes. The distribution of {beta}-arrestin was visualized sequentially in the same live cells before (0 min) and after (5 and 50 min) the addition of SS-14 to the culture medium. Receptor expression levels were between 1,500 and 2,000 fmol/mg membrane protein. Shown are representative images from one of four independent experiments performed in duplicate. Arrowheads, translocation of {beta}-arrestin-EGFP to the plasma membrane; arrows, trafficking of {beta}-arrestin-2-EGFP into early endosomes. Scale bar, 20 µm.

 



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 2.
Cotrafficking of {beta}-arrestin and sst2A into early endosomes. HEK 293 cells-expressing sst2A were transiently transfected with 1.5 µgof {beta}-arrestin-2-EGFP and 4 µg of GRK2. Cells were exposed to SS-14 for 0, 1, or 30 min. Cells were then fixed, processed for dual immunofluorescence, and examined by confocal microscopy. Receptor expression level was ~800 fmol/mg membrane protein. Shown are representative images from one of three independent experiments performed in duplicate. Scale bar, 10 µm.

 
Agonist-induced Receptor Phosphorylation and {beta}-Arrestin Trafficking—Formation of stable complexes between {beta}-arrestin and GPCRs strongly depends on the presence of clusters of phosphate acceptor sites (defined as serine/threonine residues occupying three consecutive positions or three of four positions) within the carboxyl-terminal tail of the receptor (16, 17). These clusters are remarkably conserved in their position within the carboxyl-terminal domain and serve as primary sites of agonist-dependent receptor phosphorylation. The carboxyl-terminal tails of the sst2A, sst3, and sst4 receptors contain such clusters of phosphate acceptor sites; however, they occupy different positions with regard to the conserved NPILY motif, which marks the end of the seventh transmembrane domain and the beginning of the cytoplasmic tail (Fig. 3A). To delineate a mechanistic basis for the observed differences in the trafficking patterns of these three receptors, we assessed whole cell receptor phosphorylation and {beta}-arrestin-dependent trafficking in the presence and absence of GRK2. Overexpression of GRK2 promoted a marked increase in SS-14-dependent phosphorylation of the sst2A receptor (Fig. 3C, left panel) and facilitated the recruitment of {beta}-arrestin-2 to the plasma membrane as well as its trafficking with the receptor into the endocytotic vesicles (Fig. 3B, upper panel), indicating that these processes strongly depend on the cellular complement of GRK2. In sst3-expressing cells, agonist-induced receptor phosphorylation (Fig. 3C, middle panel) and {beta}-arrestin-2 trafficking (Fig. 3B, lower panel) were largely independent of the presence of overexpressed GRK2. Although the sst4 receptor contains several putative phosphorylation sites in its cytoplasmic tail (Fig. 3A), we were not able to detect any agonist-induced receptor phosphorylation (Fig. 3C, right panel) or {beta}-arrestin-2 mobilization (not shown) in the presence or absence of GRK2.



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 3.
Agonist-induced receptor phosphorylation and {beta}-arrestin mobilization. A, carboxyl-terminal amino acid sequences of sst2A, sst3, and sst4 are depicted beginning with the conserved NPILY motif, which marks the end of the seventh transmembrane domain and the beginning of the cytoplasmic tail. Potential phosphate acceptor sites are underlined. B, HEK 293 cells were transiently transfected with 1.5 µg of {beta}-arrestin-2-EGFP, 6 µg of sst2A, or 6 µg of sst3 and either 4 µg of GRK2 or 4 µg of empty vector. The distribution of {beta}-arrestin was visualized sequentially in the same live cells before (0 min) and after (3, 5, 10, and 30 min) the addition of SS-14 to the culture medium. Receptor expression levels were between 1,500 and 2,000 fmol/mg membrane protein. Shown are representative images from one of three independent experiments performed in duplicate. Arrowheads, translocation of {beta}-arrestin-2-EGFP to the plasma membrane; arrows, trafficking of {beta}-arrestin-2-EGFP into early endosomes. Scale bar, 20 µm. C, HEK 293 cells were transfected with sst2A, sst3, or sst4 and either GRK2 or empty vector. Two days later, cells were either not exposed or exposed to 1 µM SS-14 for 20 min, and whole cell receptor phosphorylation was determined as described under "Experimental Procedures." Upper panel, autoradiographs from representative experiments are shown. Lower panel, aliquots of the immunoprecipitates (IP) were immunoblotted with T7 antibody (IB, immunoblot) to confirm equal loading of the gels. Two additional experiments gave similar results. Note that overexpression of GRK2 strongly increased agonist-induced phosphorylation of sst2A (179 ± 25%, p < 0.05) compared with agonist-induced phosphorylation in the absence of overexpressed GRK2, whereas agonist-induced phosphorylation of sst3 (113 ± 18%) was not significantly increased in the presence of overexpressed GRK2. The position of molecular mass markers are indicated on the left (in kDa).

 
Molecular Determinants Underlying the Cotrafficking of {beta}-Arrestin and sst2A into Endocytotic Vesicles—To determine the sites required for GRK2-mediated phosphorylation and {beta}-arrestin mobilization of the sst2A receptor, serial truncations were constructed deleting either 10 (359X), 15 (354X), or 20 (349X) amino acids of its carboxyl-terminal tail (Fig. 4A). As depicted in Fig. 4, B and C, deletion of 10 amino acids of the sst2A receptor tail (359X mutant), which contain serine-368 and threonine-367 as potential phosphorylation sites, reduced GRK2-mediated phosphorylation by ~10% (residual phosphorylation, 89% ± 4%; n = 3) (Fig. 4C). Analysis of {beta}-arrestin mobilization of the 359X mutant revealed that the receptor is still phosphorylated sufficiently enough to induce a translocation of {beta}-arrestin-2-EGFP to the plasma membrane (Fig. 4B). However, analysis of the 359X receptor also showed that phosphorylation of serine 368 and/or threonine 367 is required for formation of stable {beta}-arrestin-sst2A complexes (Fig. 4B). Deletion of 15 amino acids of the sst2A receptor tail (354X mutant), which contain two more potential phosphorylation sites, namely threonine 359 and threonine 356, reduced GRK2-mediated phosphorylation by ~40% (residual phosphorylation, 62% ± 14%; n = 3) (Fig. 4C). Analysis of {beta}-arrestin trafficking of the 356X mutant revealed that the residual receptor phosphorylation was not sufficient to induce translocation of {beta}-arrestin-2-EGFP to the plasma membrane (Fig. 4B). Deletion of 20 amino acids of the sst2A receptor tail (349X mutant), which contain two additional potential phosphorylation sites, namely threonine 354 and threonine 353, strongly reduced GRK2-mediated phosphorylation (residual phosphorylation, 19% ± 4%; n = 3) and {beta}-arrestin binding (Fig. 4, B and C). In the AAEA mutant, the cluster of threonine 353, 355, and 356 was replaced by alanine residues. The level of agonist-induced phosphorylation was reduced by 55% as compared with the wild-type sst2A (residual phosphorylation, 44% ± 2%; n = 3) (Fig. 4C). In cells expressing the AAEA mutant, agonist-induced {beta}-arrestin-2 translocation was strongly inhibited, and fluorescence outlining the cell shape was hardly detectable. Immunocytochemical staining of nonpermeabilized cells using anti-T7 antibodies revealed that all sst2A mutants were targeted to the plasma membrane (not shown). These data suggest that many if not all of the potential phosphate acceptor sites contained within the 20 carboxyl-terminal amino acids are involved in GRK2-mediated phosphorylation of the sst2A receptor and that phosphorylation of these sites is required for formation of stable {beta}-arrestin-sst2A complexes. These data also suggest that the cluster of threonine 353, 355, and 356 may represent the primary site for GRK2-mediated phosphorylation and {beta}-arrestin binding.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 4.
{beta}-Arrestin mobilization and agonist-dependent phosphorylation of truncated sst2A receptors. A, carboxyl-terminal amino acid sequences of wild-type sst2A and serial truncation mutants of the sst2A receptor. The positions where stop codons were introduced are indicated by X. Potential phosphate acceptor sites are underlined. B, HEK 293 cells were transiently transfected with 1.5 µgof {beta}-arrestin-2-EGFP, 4 µg of GRK2, and 6 µg of sst2A, 359X, 354X, 349X, or AAEA mutant. The distribution of {beta}-arrestin was visualized sequentially in the same live cells before (0 min) and after (5 and 40 min) the addition of SS-14 to the culture medium. Receptor expression levels were between 1,500 and 2,000 fmol/mg membrane protein. Shown are representative images from one of three independent experiments performed in duplicate. Arrowheads, translocation of {beta}-arrestin-2-EGFP to the plasma membrane; arrows, trafficking of {beta}-arrestin-2-EGFP into early endosomes. Scale bar, 20 µm. C, HEK 293 cells were transfected with GRK2 and sst2A, 359X, 354X, 349X, or AAEA mutant. Two days later, cells were either not exposed or exposed to 1 µM SS-14 for 20 min, and whole cell receptor phosphorylation was determined as described under "Experimental Procedures." Autoradiographs from representative experiments are shown. Two additional experiments gave similar results. The positions of molecular mass markers are indicated on the left (in kDa).

 
Fate of sst2A and sst3 after Agonist-induced Internalization—The pattern of {beta}-arrestin trafficking is believed to dictate the rate of receptor recycling and resensitization. In fact, many class A receptors have been shown to recycle rapidly, whereas class B receptor-like trafficking patterns are often observed for slowly recycling receptors. We therefore examined the redistribution of sst2A and sst3 after agonist-induced internalization. Cells were treated for 30 min with SS-14, washed extensively, and subjected to an additional incubation in the absence of agonist for 10, 20, or 40 min. As depicted in Fig. 5, sst2A and sst3 were almost exclusively confined to the plasma membrane in untreated cells. After 30 min of SS-14 exposure, both receptors were extensively sequestered into intracellular vesicles. In the absence of agonist it became apparent that nearly the entire pool of internalized sst2A receptors recycled to the plasma membrane during the 40-min SS-14-free interval. In contrast, only part of the internalized sst3 receptors were redistributed to the plasma membrane, whereas a large proportion of sst3 receptors was sequestered into intracellular clusters of relatively large vesicles (Fig. 5, 20 and 40 min). To examine whether sst2A and sst3 receptors were internalized into the same pool of endosomes, we used an immunocytochemical pulse-chase assay to estimate the degree to which internalized sst2A or sst3 receptors remain associated with endocytic vesicles that can be labeled with Alexa Fluor 488-labeled transferrin, a well established marker of early and recycling endosomes that mediate rapid recycling (23). T7-tagged sst2A and sst3 receptors present in the plasma membrane were specifically labeled by incubating intact cells with antibody. When these cells were simultaneously incubated with SS-14 and Alexa Fluor 488-conjugated transferrin for 30 min, a high degree of colocalization of both sst2A and sst3 with transferrin was observed, confirming that these receptors were internalized via the same endocytic pathway (Fig. 6, left panel; colocalization is indicated by the numerous yellow structures observed in the overlay panels (bottom row)). However, when these cells were first incubated with SS-14 to initiate a 30-min pulse of endocytosis and then chased for an additional 20 min in the presence of labeled transferrin, the extent of colocalization between the pulse of internalized sst2A or sst3 and the endocytic tracer was dramatically different (Fig. 6, right panel). Most vesicles containing internalized sst2A colocalized with endocytosed transferrin, which is consistent with rapid recycling of internalized sst2A via early and recycling endosomes. In contrast, under the same conditions, sst3 was observed in a large number of endocytic vesicles that did not contain detectable transferrin. These observations support the idea that the failure of internalized sst3 to recycle rapidly is mediated, at least in part, by sorting of internalized receptors to a population of endocytic vesicles distinct from those that constitute the conserved recycling pathway marked by transferrin.



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 5.
Internalization and recycling of sst2A and sst3. HEK 293 cells expressing either sst2A or sst3 were exposed for 30 min to 1 µM SS-14. Cells were washed extensively followed by an agonist-free interval of 0, 10, 20, or 40 min. Cells were subsequently fixed, processed for immunofluorescence, and examined by confocal microscopy. Both receptors were detected using affinity-purified anti-T7 tag antibodies. Representative images from one of three independent experiments performed in duplicate are shown. Scale bar, 20 µm.

 



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 6.
Pulse-chase analysis of internalized sst2A and sst3 relative to endocytosed transferrin. Left panel, confocal microscopy of double-labeled cells stably expressing sst2A or sst3 was used to examine colocalization between antibody-labeled receptors (red) and transferrin (green). Extensive overlap between internalized sst2A or sst3 receptors with transferrin is indicated by numerous yellow vesicular structures visualized in the overlay image. Right panel, confocal microscopy of double-labeled cells stably expressing sst2A or sst3 was used to examine colocalization between a 30-min agonist pulse of antibody-labeled receptors (red) followed by a 20-min chase with transferrin (green) in the absence of SS-14 as described under "Experimental Procedures." The majority of vesicles containing internalized sst2A also contained detectable labeled transferrin. In contrast, in the same experiment conducted using sst3-expressing cells, we observed numerous sst3-enriched vesicles that contained no detectable labeled transferrin (arrows). Shown are representative images from one of three independent experiments performed in duplicate. Scale bar, 15 µm.

 
Differential Down-regulation of sst2A and sst3—Given the differential intracellular trafficking of sst2A and sst3, we next assessed the down-regulation of these receptors during prolonged SS-14 exposure by Western blot analysis. As shown in Fig. 7A, we did not observe any detectable change in the level of cellular sst2A receptors during the 16-h treatment period. In contrast, the sst3 receptor underwent pronounced down-regulation, which became clearly detectable after 3 h of agonist exposure (65 ± 9% of untreated control) (Fig. 7B). The degradation of sst3 was blocked completely by the lysosomal inhibitor chloroquine (Fig. 7C). Surprisingly, the degradation of sst3 was also blocked by the proteasomal inhibitor MG132 (Fig. 7C). Although MG132 is a highly potent inhibitor of a number of proteasome-associated proteases, this compound is not specific for proteasomes and also potently inhibits various cysteine proteases and cathepsins (25). These results suggest that sst2A and sst3 undergo differential endosomal sorting. Although sst2A was sequestered into the endosomal recycling compartment, a huge proportion of endocytosed sst3 receptors was sorted into the degradative lysosomal pathway. To establish a molecular basis for the differential endosomal sorting of the sst2A and the sst3 receptors, we examined the agonist-dependent ubiquitination of these receptors. Cells were exposed to SS-14, receptor proteins were immunoprecipitated, and ubiquitinated receptors were detected using an anti-ubiquitin monoclonal antibody. As depicted in Fig. 8, the sst3 receptor underwent time-dependent ubiquitination, which was apparent as a broad high molecular weight band that represents the receptor linked to multiple ubiquitin monomers. In contrast, SS-14 treatment of sst2A-expressing cells did not result in any detectable change in sst2A receptor ubiquitination.



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 7.
Down-regulation of sst2A and sst3 during prolonged agonist exposure. A, HEK 293 cells expressing sst2A were treated with 1 µM SS-14 for 0, 1, 3, 5, or 16 h. B, HEK 293 cells expressing sst3 were treated with 1 µM SS-14 for 0, 1, 3, 5, or 16 h. C, HEK 293 cells expressing sst3 were pretreated for 30 min with 200 µM chloroquine or 25 µM MG132. Cells were then exposed to SS-14 for 3 h in the continued presence of chloroquine or MG132. The cellular receptor content was then determined by Western blot analysis as described under "Experimental Procedures." Three additional experiments gave similar results. *, significant difference (p < 0.05) compared with untreated cells. The positions of molecular mass markers are indicated on the left (in kDa).

 



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 8.
Agonist-dependent ubiquitination of sst3. HEK 293 cells stably expressing either sst2A or sst3 were pretreated for 30 min with 200 µM chloroquine and 25 µM MG132. Cells were then exposed to SS-14 for 0, 15, 30, or 60 min in the presence of chloroquine and MG132. Cells were lysed, and the receptors were precipitated using T7 affinity beads and Western-blotted using anti-ubiquitin antibodies as described under "Experimental Procedures." Two additional experiments gave similar results. The position of molecular mass markers is indicated on the left (in kDa).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we reveal previously unappreciated differences in {beta}-arrestin mobilization and endosomal sorting of the five somatostatin receptor subtypes. Based on their {beta}-arrestin-dependent trafficking patterns, we identify the sst3 and sst5 somatostatin receptors as class A receptors and the sst2A somatostatin receptor as class B receptor. On the contrary, the trafficking of the sst1 and sst4 somatostatin receptors did not appear to depend on {beta}-arrestins. We also show that sst2A and sst3 underwent differential endosomal sorting. In contrast to what would be expected from their {beta}-arrestin trafficking patterns, the sst2A receptor was rapidly recycled to the plasma membrane, whereas the sst3 receptor was subject to ubiquitination-dependent lysosomal degradation.

Using a series of truncated sst2A receptors, we demonstrate that the molecular determinants for the formation of stable complexes between {beta}-arrestin and sst2A are contained within clusters of phosphate acceptor sites at the end of its cytoplasmic tail. Such clusters of phosphate acceptor sites are defined as serine/threonine residues occupying three consecutive positions or three of four positions (17). For many class B receptors, these clusters are remarkably conserved in their position within the carboxyl-terminal domain and serve as primary sites of agonist-dependent receptor phosphorylation. It should be noted that not only the sst2A receptor but also the sst3 and sst4 receptors contain such clusters of phosphate acceptor sites. However, with regard to the conserved NPILY motif these clusters occupy more downstream positions in the sst3 and sst4 receptors than in the sst2A receptor. We also demonstrate that the phosphorylation of the sst2A receptor but not of the sst3 and sst4 receptors strongly depends on the cellular complement of GRK2.

The sst3 receptor formed complexes with {beta}-arrestin that dissociated at or near the plasma membrane. Neither {beta}-arrestin mobilization nor phosphorylation of the sst3 receptor was changed by overexpression of GRK2. Interestingly, it has previously been shown that replacement of four serine and threonine residues by alanine diminishes agonist-induced phosphorylation and internalization of the sst3 receptor (24). Consistent with the present findings, these residues are localized upstream to the putative GRK2 phosphorylation cluster of sst3.

In contrast to the sst2A and sst3 receptors, the sst1 and sst4 receptors failed to induce {beta}-arrestin translocation to the plasma membrane following agonist treatment. These results imply either peculiar mechanisms of receptor desensitization or resistance to desensitization. In fact, it has previously been shown that the rat sst4 receptor fails to internalize after agonist activation (13). Consistent with the previous findings, we show that sst4 did not undergo any detectable phosphorylation after agonist activation despite the presence of a number of potential phosphate acceptor sites including a cluster of threonine residues occupying four of five positions in its carboxyl-terminal tail (13). In addition, we demonstrate that the sst4 receptor was resistant to agonist-induced phosphorylation and {beta}-arrestin-dependent trafficking even in the presence of GRK2. With regard to the sst1 receptor it should be noted that upon heterologous expression in HEK 293 cells, a large proportion of receptor protein remains in an as yet unidentified intracellular vesicular compartment (4). Thus, the fact that only a fraction of cellular sst1 receptors is targeted to the plasma membrane may explain that agonist exposure of this receptor did not promote efficient {beta}-arrestin translocation under these conditions.

In sst5-transfected HEK 293 cells, SS-14 treatment induced {beta}-arrestin translocation to the plasma membrane. However, this effect was only transient and less pronounced than that observed for the sst2A and sst3 receptors. Given that sst5 has a higher affinity for SS-28 than for SS-14, the limited recruitment of {beta}-arrestin-2 could be because of limited activation of the sst5 receptor by SS-14 (26). We therefore performed {beta}-arrestin mobilization assays using the non-peptide sst5 agonist L-817,818. The fact that very similar results were obtained under these conditions confirms that the sst5 receptor exhibits a class A receptor-like trafficking pattern.

It has been suggested recently that distinct intracellular trafficking patterns of {beta}-arrestin determine the fate of internalized GPCRs (19, 20). Many class A receptors (e.g. µ-opioid, {beta}2 and {alpha} 1B adrenergic, endothelin A, and dopamine D1A receptors) have been shown to recycle rapidly, whereas class B receptor-like trafficking patterns are often observed for slowly recycling receptors (e.g. substance P, angiotensin AT1a, neurotensin-1, and vasopressin-2 receptors) (20). However, recent work shows that not all GPCRs fit into this classification, i.e. the N-formyl peptide receptor requires {beta}-arrestin for its recycling but not for its internalization (27). Here, we show the sst2A receptor was rapidly recycled to the plasma membrane without any detectable loss of cellular sst2A receptors even during prolonged agonist exposure. In contrast, a large proportion of internalized sst3 receptors was ubiquitinated and sorted into the degradative lysosomal pathway, resulting in a rapid down-regulation of cellular sst3 receptors. Thus, our findings suggest that endosomal sorting of sst2A and sst3 was regulated by differential ubiquitination rather than differential {beta}-arrestin binding of these receptors.

In conclusion, we provide evidence for differential {beta}-arrestin-dependent trafficking and endosomal sorting of somatostatin receptor subtypes. The high density of somatostatin receptors on human neuroendocrine tumors has allowed the development of somatostatin receptor scintigraphy for tumor imaging as well as somatostatin receptor-targeted radiotherapy (9, 10). The effectiveness of these diagnostic and therapeutic manipulations strongly depends on receptor internalization and recycling. Thus, a sst2A-expressing tumor would be expected to take up radiolabeled somatostatin analogs more efficiently than tumors that express predominantly sst3 or sst4. In addition, differences have been observed in the response of neuroendocrine tumors to long-term application of stable somatostatin analogs. In patients with growth hormone-secreting pituitary adenoma, the inhibitory effects of somatostatin analogs on hormone secretion persist for many years during long-term treatment. In contrast, carcinoids are very likely to undergo desensitization within weeks to months of octreotide exposure. Although sst2A is most frequently detected, neuroendocrine tumors often express distinct patterns of somatostatin receptor subtypes (3). Thus, the differential intracellular sorting of somatostatin receptors may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant SCHU924/4-4 (to S. S.) and SFB545/B7 (to H.-J. K.) and European Commission Grant QLG3-CT-1999-00908 (to S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie, Otto-von-Guericke-Universität, Leipziger Strasse 44, 39120 Magdeburg, Germany. Tel.: 49-391-671-5881; Fax: 49-391-671-5869; E-mail: Stefan.Schulz{at}Medizin.Uni-Magdeburg.de.

1 The abbreviations used are: SS-14, somatostatin; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HEK, human embryonic kidney; sst, somatostatin receptor; TPBS, Tris/phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Dana Mayer and Anke Reichenauer for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Patel, Y. C. (1999) Front. Neuroendocrinol. 20, 157-198[CrossRef][Medline] [Order article via Infotrieve]
  2. Csaba, Z., and Dournaud, P. (2001) Neuropeptides 35, 1-23[CrossRef][Medline] [Order article via Infotrieve]
  3. Kulaksiz, H., Eissele, R., Rossler, D., Schulz, S., Hollt, V., Cetin, Y., and Arnold, R. (2002) Gut 50, 52-60[Abstract/Free Full Text]
  4. Schulz, S., Pauli, S. U., Schulz, S., Handel, M., Dietzmann, K., Firsching, R., and Hollt, V. (2000) Clin. Cancer Res. 6, 1865-1874[Abstract/Free Full Text]
  5. Shimon, I., Yan, X., Taylor, J. E., Weiss, M. H., Culler, M. D., and Melmed, S. (1997) J. Clin. Investig. 100, 2386-2392[Medline] [Order article via Infotrieve]
  6. Lamberts, S. W., van der Lely, A. J., de Herder, W. W., and Hofland, L. J. (1996) N. Engl. J. Med. 334, 246-254[Free Full Text]
  7. Giustina, A., Zaltieri, G., Negrini, F., and Wehrenberg, W. B. (1996) Pharmacol. Res. 34, 247-268[Medline] [Order article via Infotrieve]
  8. De Herder, W., and Lamberts, S. (2003) Endocrine 20, 285-290[Medline] [Order article via Infotrieve]
  9. Lamberts, S. W., Krenning, E. P., and Reubi, J. C. (1991) Endocr. Rev. 12, 450-482[Medline] [Order article via Infotrieve]
  10. Forster, G. J., Engelbach, M. J., Brockmann, J. J., Reber, H. J., Buchholz, H. G., Macke, H. R., Rosch, F. R., Herzog, H. R., and Bartenstein, P. R. (2001) Eur. J. Nucl. Med. 28, 1743-1750[CrossRef][Medline] [Order article via Infotrieve]
  11. Culler, M. D., Taylor, J. E., and Moreau, J. P. (2002) Ann. Endocrinol. (Paris) 63, 2S5-12[Medline] [Order article via Infotrieve]
  12. Hofland, L. J., and Lamberts, S. W. (2003) Endocr. Rev. 24, 28-47[Abstract/Free Full Text]
  13. Kreienkamp, H. J., Roth, A., and Richter, D. (1998) DNA Cell Biol. 17, 869-878[Medline] [Order article via Infotrieve]
  14. Hukovic, N., Rocheville, M., Kumar, U., Sasi, R., Khare, S., and Patel, Y. C. (1999) J. Biol. Chem. 274, 24550-24558[Abstract/Free Full Text]
  15. Roth, A., Kreienkamp, H. J., Nehring, R. B., Roosterman, D., Meyerhof, W., and Richter, D. (1997) DNA Cell Biol. 16, 111-119[Medline] [Order article via Infotrieve]
  16. Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G., and Barak, L. S. (2000) J. Biol. Chem. 275, 17201-17210[Abstract/Free Full Text]
  17. Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (2001) J. Biol. Chem. 276, 19452-19460[Abstract/Free Full Text]
  18. Pierce, K. L., Luttrell, L. M., and Lefkowitz, R. J. (2001) Oncogene 20, 1532-1539[CrossRef][Medline] [Order article via Infotrieve]
  19. Shenoy, S. K., McDonald, P. H., Kohout, T. A., and Lefkowitz, R. J. (2001) Science 294, 1307-1313[Abstract/Free Full Text]
  20. Shenoy, S. K., and Lefkowitz, R. J. (2003) J. Biol. Chem. 278, 14498-14506[Abstract/Free Full Text]
  21. Pfeiffer, M., Koch, T., Schroder, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., Hollt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036[Abstract/Free Full Text]
  22. Schwartkop, C. P., Kreienkamp, H. J., and Richter, D. (1999) J. Neurochem. 72, 1275-1282[CrossRef][Medline] [Order article via Infotrieve]
  23. Tsao, P. I., and Von Zastrow M. (2000) J. Biol. Chem. 275, 11130-11140[Abstract/Free Full Text]
  24. Roth, A., Kreienkamp, H. J., Meyerhof, W., and Richter, D. (1997) J. Biol. Chem. 272, 23769-23774[Abstract/Free Full Text]
  25. Tanowitz, M., and Von Zastrow, M. (2002) J. Biol. Chem. 277, 50219-50222[Abstract/Free Full Text]
  26. Roosterman, D., Roth, A., Kreienkamp, H. J., Richter, D., and Meyerhof, W. (1997) J. Neuroendocrinol. 9, 741-751[CrossRef][Medline] [Order article via Infotrieve]
  27. Vines, C. M., Revankar, C. M., Maestas, D. C., LaRusch, L. L., Cimino, D. F., Kohout, T. A., Lefkowitz, R. J., and Prossnitz, E. R. (2003) J. Biol. Chem. 278, 41581-41584[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
J. Meijer, J. Ogink, B. Kreike, D. Nuyten, K. E. de Visser, and E. Roos
The Chemokine Receptor CXCR6 and Its Ligand CXCL16 Are Expressed in Carcinomas and Inhibit Proliferation
Cancer Res., June 15, 2008; 68(12): 4701 - 4708.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
D. Roosterman, C. Kempkes, G. S. Cottrell, B. E. Padilla, N. W. Bunnett, C. W. Turck, and M. Steinhoff
Endothelin-Converting Enzyme-1 Degrades Internalized Somatostatin-14
Endocrinology, May 1, 2008; 149(5): 2200 - 2207.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
B. Lelouvier, G. Tamagno, A. M. Kaindl, A. Roland, V. Lelievre, V. Le Verche, C. Loudes, P. Gressens, A. Faivre-Baumann, Z. Lenkei, et al.
Dynamics of Somatostatin Type 2A Receptor Cargoes in Living Hippocampal Neurons
J. Neurosci., April 23, 2008; 28(17): 4336 - 4349.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
A. Kolodziej, S. Schulz, A. Guyon, D.-F. Wu, M. Pfeiffer, V. Odemis, V. Hollt, and R. Stumm
Tonic Activation of CXC Chemokine Receptor 4 in Immature Granule Cells Supports Neurogenesis in the Adult Dentate Gyrus
J. Neurosci., April 23, 2008; 28(17): 4488 - 4500.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
S. L. Fougner, J. Bollerslev, F. Latif, J. K. Hald, T. Lund, J. Ramm-Pettersen, and J. P. Berg
Low Levels of Raf Kinase Inhibitory Protein in Growth Hormone-Secreting Pituitary Adenomas Correlate with Poor Response to Octreotide Treatment
J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1211 - 1216.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Ginj, H. Zhang, K.-P. Eisenwiener, D. Wild, S. Schulz, H. Rink, R. Cescato, J. C. Reubi, and H. R. Maecke
New Pansomatostatin Ligands and Their Chelated Versions: Affinity Profile, Agonist Activity, Internalization, and Tumor Targeting
Clin. Cancer Res., April 1, 2008; 14(7): 2019 - 2027.
[Abstract] [Full Text] [PDF]