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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 12 6073-6079
Copyright © 2003 by The Endocrine Society

In Vivo Phosphorylation of the Somatostatin 2A Receptor in Human Tumors

Qisheng Liu, Jean-Claude Reubi, Yining Wang, Brian J. Knoll and Agnes Schonbrunn

Department of Integrative Biology and Pharmacology (A.S., Q.L., Y.W.), University of Texas Health Science Center–Houston, Houston, Texas 77225; Department of Pharmacological and Pharmaceutical Sciences (B.J.K.), College of Pharmacy, University of Houston, Houston, Texas 77204; and Division of Cell Biology and Experimental Cancer Research (J.C.R.), Institute of Pathology, University of Berne, CH-3010 Berne, Switzerland

Address all correspondence and requests for reprints to: Dr. Agnes Schonbrunn, Department of Integrative Biology and Pharmacology, University of Texas–Houston, P.O. Box 20708, Houston, Texas 77225. Email: agnes.schonbrunn{at}uth.tmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hormone-stimulated receptor internalization and desensitization occur widely in the G protein-coupled receptor (GPCR) family. A critical first step in both these processes is thought to be receptor phosphorylation, a reaction which has been extensively characterized in cell culture. However, little is known about GPCR phosphorylation in vivo. The somatostatin (SS) receptor subtype (sst)2A is widely distributed in human neuroendocrine tumors, and SS analogs are commonly used to target this receptor for both therapy and diagnosis. In cultured pituitary cells sst2A is rapidly phosphorylated and internalized after hormone binding. The aim of the present study was to go one crucial step further and characterize the phosphorylation state of this receptor in human neuroendocrine tumors using a newly developed gel-shift assay. The receptor from a somatostatinoma was completely phosphorylated. In contrast, only unphosphorylated sst2A was present in human tumors that were not exposed to autocrine stimulation. Both in vivo and in cultured cells, the phosphorylation state of the sst2A receptor was correlated with its subcellular localization: phosphorylated receptor was mostly intracellular, whereas unphosphorylated receptor was localized at the cell surface. These results are the first to demonstrate ligand-stimulated GPCR phosphorylation in human tissue in situ, providing a crucial step toward a better understanding of receptor regulation in vivo. Analysis of sst2A phosphorylation promises to provide a sensitive indicator of the effectiveness of SS analogs in diagnostic and therapeutic situations in tumor patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTIVATION OF SEVEN-TRANSMEMBRANE domain, G protein-coupled receptors (GPCR) by ligand usually leads both to the initiation of signal transduction and to receptor internalization and desensitization (1, 2, 3). The latter two processes are adaptive mechanisms that prevent persistent receptor stimulation from producing detrimental cellular effects. Internalization may also play a role in receptor resensitization. A critical first step in both GPCR internalization and desensitization is believed to be receptor phosphorylation by G protein-receptor kinases and second-messenger-activated kinases. Phosphorylated receptors then bind regulatory proteins called arrestins, which inhibit further signaling by blocking receptor-G protein interaction (1, 2, 3). Arrestins also play a role in receptor internalization and some pathways of signal transduction by serving as adapters to localize essential proteins to the ligand-activated GPCR (4). These molecular events have been extensively characterized in a variety of cell culture model systems and are hypothesized to occur similarly in vivo. However, the only direct evidence to support the conclusion that activation leads to GPCR phosphorylation in intact animals comes from studies of rhodopsin: exposure of mice to light has been shown to stimulate the phosphorylation of retinal rhodopsin (5, 6). To date, no such evidence has been provided with human material or with a ligand-stimulated GPCR. Factors that have made the detection and study of agonist stimulated receptor phosphorylation notoriously difficult in vivo include the extremely low abundance of individual GPCR subtypes in tissues, the difficulty of GPCR purification, the absence of sufficiently sensitive methods for the detection of receptor phosphorylation, and, in the case of human tissues, the difficulty in getting tissue samples with and without exposure to an appropriate agonist.

The peptide somatostatin (SS) exerts its physiological effects by interacting with a family of GPCRs, encoded by five genes, named sst1–sst5 (7, 8, 9). The receptor subtype (sst)2A is widely distributed in tissues, including brain, pituitary, pancreas, and gastrointestinal tract, and is also present in a large variety of human neuroendocrine tumors (7, 8, 9, 10). In fact, stable SS analogs are routinely used clinically for the treatment of sst2A-receptor-containing neoplasms (11, 12, 13). In addition, taking advantage of receptor-mediated ligand internalization, radiolabeled analogs of SS are used in receptor scintigraphy for sensitive and specific visualization of sst2A-receptor-containing tumors and for their radiotherapy (14, 15, 16).

We have previously shown that the sst2A receptor is rapidly phosphorylated, internalized, and desensitized after SS binding (17). However, as with most GPCRs, sst2A receptor phosphorylation has been observed only in cells transfected to express high levels of receptor (17). In this study, we developed a sensitive, nonradioactive assay for sst2A receptor phosphorylation and used this assay to determine the phosphorylation state of native sst2A receptors both in cultured cells and in human tumors that had been subjected to different degrees of autocrine receptor stimulation in situ. In addition, we determined the relationship between the extent of sst2A receptor phosphorylation and its subcellular distribution.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents, cells, and patient samples

All reagents were of the best grade available and purchased from common suppliers. The generation and specificity of the sst2A-receptor antiserum (R2–88) has been described (18). The clonal CHO-R2 cell line was produced by stable transfection of CHO-K1 cells with the rat sst2A receptor, followed by clonal selection, and grown as previously described (19). Prestained protein standards were obtained from either Bio-Rad (high-range) or Amersham Biosciences (Piscataway, NJ) (low-range rainbow molecular weight markers).

Three human tumor samples were investigated: a liver metastasis derived from a somatostatinoma of unknown origin, a primary carcinoid tumor from the ileum, and a pheochromocytoma situated around the celiac truncus. These tumors were selected by the following criteria: 1) a sufficient amount of frozen tumor tissue was available for analysis; 2) the tissue was typical of its tumor type; and 3) the tissue was found to express sst2 either by radioligand autoradiography or in situ hybridization, or both. SS receptor scintigraphy (Octreoscan, Mallinckrodt Inc., St. Louis, MO) was performed with all three tumor patients before surgery. Receptor scintigraphy was negative in the case of the somatostatinoma, was positive for the carcinoid, and was positive for most, but not all, parts of the pheochromocytoma. Informed consent was obtained for all tissue samples. The study conformed to the ethical guidelines of the Institute of Pathology, University of Berne.

Immunolabeling

For immunofluorescence, CHO-R2 cells were grown on glass coverslips coated with poly-D-lysine (50 µg/ml). After preincubation for 20 min in serum-free F12 medium containing 5 mg/ml lactalbumin hydrolysate and 20 mmol/liter HEPES, pH 7.4, the cells were incubated with or without 1 µmol/liter SS at 37 C for various times. After washing twice with ice-cold Tris-buffered saline (TBS), cells were immediately fixed with 4% paraformaldehyde in PBS pH 7.2 (PBS) for 20 min at room temperature. Fixed cells were washed twice with TBS (twice for 5 min each) and incubated at room temperature with 3% normal goat serum in TBS for 30 min. Receptors were detected by incubation at 4 C with R2–88 antibody (1:1000 in TBS containing 0.2% Tween-20, 10% nonfat dry milk, and 10% glycerol). Cells were subsequently rinsed, twice for 5 min each, with TBS and incubated at room temperature for 30–60 min with fluorescein-isothiocyanate-conjugated goat antirabbit antibody (1:100). After two more rinses with TBS (5 min each), the cover slips were mounted on glass slides with Mowiol/DABCO. The cells were imaged by epifluorescence microscopy using a Zeiss (Thornwood, NY) Axophot equipped with a Hamamatsu (Bridgewater, NJ) Model C5810 CCD camera. No immunostaining was observed in untransfected CHO-K1 cells, which did not express sst2A, or in CHO-R2 cells in the absence of primary antibody.

Immunohistochemical staining for sst2A, using R2–88 antibodies, was performed on tumor sections as reported previously (10).

Purification and deglycosylation of 32PO4-labeled sst2A receptor

Metabolic labeling of cells and subsequent immunoprecipitation of the sst2A receptor were carried out as described before (17). Briefly, cells (one 100-mm dish/treatment) were incubated for 3 h in phosphate-free DMEM containing 1 mCi of [32P]orthophosphate. SS was then added directly to the labeling medium, and the cells were incubated at 37 C under 5% CO2 for an additional 15 min. Cells were then scraped into cold HEPES-buffer saline with protease and phosphatase inhibitors (HBS-I: 150 mmol/liter NaCl, 20 mmol/liter HEPES, pH 7.4, 1 mmol/liter phenylmethylsulfonylfluoride (PMSF), 10 mg/ml soybean trypsin inhibitor, 10 mg/ml leupeptin, 50 mg/ml bacitracin, 5 mmol/liter EDTA, 3 mmol/liter EGTA, 10 mmol/liter sodium pyrophosphate, 10 mmol/liter sodium fluoride, 0.1 mmol/liter orthovanadate, 100 nmol/liter okadaic acid). After centrifugation, the cell pellet was solubilized in lysis buffer [HBS-I containing 4 mg/ml dodecyl ß-D-maltoside (DßM)] for 60 min at 4 C. The detergent lysates were centrifuged at 100,000 x g for 30 min, and the protein content of the supernatant was assessed by the method of Bradford (20).

Radiolabeled sst2A receptors were usually subjected to a two-step purification consisting of: 1) adsorption to wheat germ agglutinin (WGA)-agarose; and 2) immunoprecipitation with receptor antibody (17). Between these two steps, some aliquots of the receptor were treated with peptide-N-glycosidase F (PNGase F, E.C.3.5.1.52, Roche Molecular Biochemicals, Indianapolis, IN) (21). In brief, equal amounts of lysate protein were incubated at 4 C for at least 90 min with 100 µl (packed volume) of washed WGA-agarose (Vector Laboratories, Inc., Burlingame, CA). After centrifugation, the WGA-agarose was washed with lysis buffer and divided into replicate aliquots. In some samples, adsorbed glycoproteins were eluted at 37 C for 30 min with 250 µl of lysis buffer containing 3 mmol/liter N,N',N''-triacetyl-chitotriose (TACT, Sigma, St. Louis, MO) and 0.5% sodium dodecyl sulfate (SDS). In replicate samples, the washed WGA-agarose was incubated overnight in 250 µl lysis buffer containing 0.1% SDS and 10 U/ml PNGase F at 37 C (21). In both cases, the supernatants were subsequently incubated with the anti-sst2A receptor antibody R2–88 (1:400 dilution) at 4 C for at least 90 min. The samples were then incubated at 4 C for 60 min with 25 µl (packed volume) of protein A-Sepharose and centrifuged. After extensive washing, the immunoprecipitated proteins were solubilized in sample buffer [62.5 mmol/liter Tris-HCl, 2% SDS, 10% 2-mercaptoethanol (vol/vol), 6 mol/liter urea, 20% glycerol, pH 6.8] at 37 C for 60 min, heated for 15 min at 60 C, and then resolved on 10% SDS-polyacrylamide gels.

Membrane preparation

CHO-R2 cells were pretreated in a CO2 incubator with 100 nmol/liter SS or carrier in serum-free F12 medium containing 5 mg/ml lactalbumin hydrolysate and 20 mmol/liter HEPES, pH 7.4. Cells were then washed with and scraped into cold PBS (10 mmol/liter Na2HPO4, 150 mmol/liter NaCl, pH 7.4) containing protease and phosphatase inhibitors (1 mmol/liter PMSF, 10 mmol/liter sodium pyrophosphate, 10 mmol/liter sodium fluoride, 0.1 mmol/liter sodium orthovanadate, and 0.1 mmol/liter okadaic acid). After centrifugation, the cell pellet was resuspended in homogenization buffer (10 mmol/liter Tris-HCl, 5 mmol/liter EDTA, 3 mmol/liter EGTA, pH 7.6) with protease and phosphatase inhibitors and lysed with a stirred type RZR3 polytron homogenizer (Caframo Ltd., Warton, Ontario, Canada). After a brief low-speed centrifugation, cell membranes were pelleted by centrifugation at 10,000 x g for 30 min, resuspended in cold gly-gly buffer (20 mmol/liter glycylglycine, 1 mmol/liter MgCl2, 250 mmol/liter sucrose, pH 7.2) and stored at -80 C until use.

Frozen tumor samples were partially thawed in ice-cold homogenization buffer containing 250 mmol/liter sucrose, 0.1 mmol/liter okadaic acid, and protease inhibitors (1 mmol/liter PMSF, 10 mg/ml soybean trypsin inhibitor, 10 mg/ml leupeptin, and 50 mg/ml bacitracin). The samples were cut into small pieces on ice, transferred to a cold Dounce homogenizer, and lysed with a stirrer type RZR3 polytron homogenizer. After a brief low-speed centrifugation, cell membranes were pelleted at 10,000 x g for 45 min, resuspended in cold gly-gly buffer, and stored at -80 C until use.

Purification and deglycosylation of unlabeled sst2A receptor from membranes and detection by immunoblotting

Membranes from both CHO-R2 cells and from tumors were solubilized with DßM for 60 min at 4 C. Lysates were clarified by centrifugation at 6000 x g for 10 min and then subjected to partial purification by WGA and deglycosylation by PNGase F as described above. After deglycosylation, proteins in the supernatant were precipitated for 90 min at 4 C by 12.5% trichloroacetic acid (TCA) using 20 µg ribonuclease A as a carrier. For phosphatase treatments, the TCA pellets were washed once with phosphatase buffer (20 mmol/liter HEPES, pH 8.0, 25 mmol/liter KCl, 15 mmol/liter MgCl2) and then incubated overnight at 37 C with 5 U bacterial alkaline phosphatase (BAP) in 100 µl phosphatase buffer containing 4 mg/ml DßM and 0.1 SDS%. Dephosphorylated proteins were then incubated in SDS-PAGE sample buffer containing 10% 2-mercaptoethanol, for 60 min at 37 C, followed by heating at 60 C for 15 min and then resolved on 10% SDS-polyacrylamide gels.

Resolved proteins were transferred to polyvinylidene difluoride membrane as described previously (17). The membrane was blocked for 2 h with Blotto (10 mmol/liter NaH2PO4, 10% nonfat dry milk, 10% glycerol, 0.2% Tween 20) and incubated overnight at 4 C with anti-sst2A antibody R2–88 (1:10,000) in Blotto. Immunoreactive proteins were detected with a goat-antirabbit antibody conjugated with horseradish peroxidase (1:5,000) and the ECL chemiluminescent antibody detection system (Amersham Biosciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Agonist-stimulated phosphorylation of the sst2A receptor

We previously showed that the sst2A receptor is rapidly phosphorylated, internalized, and desensitized after SS treatment of GH-R2 pituitary cells (17). However, GH-R2 cells express sst1 receptors as well as sst2A receptors, and we could not exclude the possibility of heterologous receptor interactions in this cell line (22). Therefore, we investigated CHO-K1 cells, which do not express SS receptors endogenously, as a model for examining homologous regulation of the sst2A receptor. Previous studies had shown that the sst2A receptor stably expressed in CHO-R2 cells is functionally coupled to adenylyl cyclase via G{alpha}i proteins (18, 19). Further, using both photoaffinity labeling and immunoblotting with a receptor-subtype specific antibody, we identified the sst2A receptor in CHO-R2 cells as a broad band migrating between 60 and 90 kDa on SDS polyacrylamide gels (18).

To determine the effect of SS on sst2A receptor phosphorylation, CHO-R2 cells were prelabeled with 32P-orthophosphate and then incubated for 15 min in the absence or presence of 100 nmol/liter SS. After detergent solubilization, the sst2A receptor was purified by lectin chromatography, immunoprecipitated with receptor antiserum, and then analyzed by SDS-PAGE and autoradiography. Although the sst2A receptor was slightly phosphorylated under basal conditions, SS treatment markedly stimulated receptor phosphorylation (Fig. 1Go, top left panel). In three independent experiments, SS increased 32P incorporation into the receptor band 2.9 ± 0.1-fold (mean ± SEM).



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  		FIG. 1. The effect of SS on sst2A receptor phosphorylation. Top panel, 32PO4-labeled CHO-R2 cells were incubated in the absence or presence of 100 nmol/liter SS for 15 min. After detergent solubilization, and adsorption to WGA-agarose, the adsorbed glycosylated proteins were eluted by incubation with either TACT (top left panel) or PNGase F (top right panel). The eluted receptors were then immunoprecipitated with R2–88 antiserum and analyzed by SDS-PAGE and autoradiography. Bottom panel, Nonradiolabeled CHO-R2 cells were incubated in the absence or presence of SS. After detergent solubilization, partial purification by lectin chromatography, and elution with either TACT or PNGase F, proteins were precipitated with TCA. Replicate samples were then incubated overnight without or with BAP and subsequently subjected to SDS-PAGE. The sst2A receptor was visualized by immunoblotting with R2–88 receptor antiserum.

 
In previous studies with the sst1 receptor subtype, we had observed a phosphorylation-induced shift in receptor mobility on SDS gels when the receptor was deglycosylated before analysis (21). To develop a biochemical assay for phosphorylated sst2A, we next examined the mobility of the sst2A receptor from both control cells and cells pretreated with 100 nmol/liter SS for 15 min after incubation with PNGase F. As expected, treatment with PNGase F, which catalyzes the cleavage of N-glycosidically linked carbohydrate chains from asparagine, caused a marked sharpening of the receptor bands as well as a large decrease in their apparent molecular weights (Fig. 1Go, top right panel). Further, the amount of radiolabel in the deglycosylated receptor bands paralleled that seen with the glycosylated receptors. In three independent experiments, SS pretreatment increased the 32P incorporated into the deglycosylated sst2A receptor 3.1 ± 0.6-fold (mean ± SEM). Most interestingly, the receptor prepared from SS-pretreated cells migrated somewhat more slowly than the receptor from untreated cells, suggesting that agonist-stimulated phosphorylation was able to change sst2A receptor mobility (Fig. 1Go, top right panel).

We next determined whether the ligand-induced shift in the sst2A receptor could be detected by immunoblot analysis. Interestingly, whereas the migration of the glycosylated receptor was unaffected by SS pretreatment (Fig. 1Go, bottom left panel), the mobility of the deglycosylated receptor was significantly reduced (Fig. 1Go, bottom right panel). The deglycosylated receptor from control cells had an apparent molecular mass of 37 kDa, whereas the receptor from cells pretreated with 100 nmol/liter SS for 15 min migrated with an apparent mass of 40 kDa (Fig. 1Go, bottom left panel). To confirm that the change in migration of the sst2A receptor after SS treatment was attributable to phosphorylation and not to some other covalent modification, both control and SS-treated samples were incubated with BAP. After phosphatase treatment, the sst2A receptor migrated as a sharp 37-kDa band independent of prior exposure to SS (Fig. 1Go, bottom right panel), showing that phosphorylation was responsible for the altered mobility of the deglycosylated sst2A receptor on SDS-PAGE. Further, because all receptors from the SS-treated cells showed reduced mobility before phosphatase treatment, the phosphorylation induced by SS in CHO-R2 cells must be complete.

SS-induced receptor internalization

Agonist binding can cause rapid internalization of both the sst2A receptor (23) and its bound ligand (17, 24, 25, 26), but the extent of internalization varies substantially among different cell types. We therefore determined the extent to which quantitative phosphorylation of the sst2A receptor in CHO cells was accompanied by receptor internalization. Cells were incubated without or with SS at 37 C, for various times from 5–30 min, then fixed, permeabilized, and labeled with ant-sst2A antibody (Fig. 2Go). In untreated cells, the labeling was diffusely distributed over the cell surface. After 5 min of SS stimulation, the labeling was largely distributed in small punctate vesicles under the plasma membrane and around the nucleus. The perinuclear localization was retained after 10 and 30 min of ligand treatment. These results indicate that SS induced rapid internalization of the majority of the sst2A receptors. Parallel experiments in which we measured radioligand binding at 4 C showed that cell surface sst2A receptors were lost, with a half-time of 5 min, upon SS treatment at 37 C, with a 70% decrease in surface receptor at 30 min (Prejusa, Liu, and Schonbrunn, unpublished observations). These results, taken together with Fig. 1Go, show that the receptors accumulated in intracellular vesicular compartments must be phosphorylated.



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  		FIG. 2. Immunocytochemical localization of the sst2A receptor before and after hormone treatment. CHO-R2 cells were treated with a saturating concentration of SS, at 37 C, for the times shown. The cells were then fixed, permeabilized, and labeled for sst2A with R2–88 antiserum and fluorescein-isothiocyanate-conjugated goat antirabbit second antibody. Under identical conditions and exposures, no labeling was detectable with the parental CHO-K1 cell line, which does not express sst receptors.

 
Phosphorylation of the sst2A receptor in human tumors

Immunocytochemical analysis of a variety of human tumors previously showed that, in most cases, the sst2A receptor is localized at the cell surface (10, 27, 28). However, in a somatostatinoma, which synthesized and secreted SS, the sst2A receptor was found to be intracellular (28). We therefore immunolabeled cryostat sections from a number of human tumors to identify tissues with different patterns of sst2A receptor distribution. In an ileal carcinoid, the sst2A receptor was localized primarily at the cell surface (Fig. 3Go top, panel A), whereas it was mostly intracellular in a somatostatinoma (Fig. 3Go, top, panel B). Only the latter contained SS mRNA by in situ hybridization (Reubi and Waser, data not shown).



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  		FIG. 3. Immunocytochemical localization and phosphorylation state of the sst2A receptor in human tumors. A, Cryostat sections of the SS mRNA-negative ileal carcinoid (A) and the SS mRNA-positive somatostatinoma (B) were immunolabeled with R2–88 antibody without HE counterstaining. Bar, 0.01 mm. B, Membrane proteins were prepared either from CHO-R2 cells incubated with SS (5 µg) or from human tumor samples (300 µg each) as described under Materials and Methods. After detergent solubilization, partial purification by WGA-agarose adsorption, and deglycosylation by PNGase F, the eluted proteins were precipitated with TCA. Replicate samples were then incubated without or with BAP and subjected to SDS-PAGE. The sst2A receptor was visualized by immunoblotting with R2–88 receptor antiserum. Samples shown are from a somatostatinoma (SSoma), an ileal carcinoid (CA), and a pheochromocytoma (Pheo).

 
We next analyzed the phosphorylation state of the endogenous sst2A receptors in these two tumors, as well as in a pheochromocytoma (Fig. 3BGo). Although pheochromocytomas often produce SS, we could detect no SS mRNA in the tumor sample analyzed, and most of the receptor in this sample was localized to the plasma membrane (data not shown). Interestingly, most (but not all) parts of this particular tumor were visualized by Octreoscan, suggesting that it was heterogeneous in its ability to bind radioligand (see Materials and Methods).

Membranes prepared from frozen tumors were subjected to the same purification and enzymatic treatments as CHO-R2 membranes. Immunoblotting of samples, resolved by SDS-PAGE, showed only a single immunoreactive band for each tumor (shown Fig. 3Go, bottom), but this deglycosylated sst2A receptor band exhibited different mobilities, depending on the tumor of origin. The intracellular receptors from the somatostatinoma exhibited an apparent mass of 40 kDa, whereas the cell surface receptors from the ileal carcinoid and pheochromocytoma migrated at 37 kDa (Fig. 3Go). Treatment with phosphatase altered the mobility of the receptor from the somatostatinoma, reducing its apparent molecular mass to 37 kDa. Thus, the same mobility change was produced by phosphorylation of the sst2A receptor in the somatostatinoma as in SS-treated CHO-R2 cells. Phosphatase treatment did not affect the mobility of the receptor from the carcinoid, but seemed to sharpen the band from the pheochromocytoma slightly. The data show that the sst2A receptors in the somatostatinoma were stoichimetrically phosphorylated in situ, in striking contrast to the receptors from the carcinoid tumor. Although sst2A receptors in the pheochromocytoma were mostly unphosphorylated, the sharpening of the receptor band by phosphatase treatment is consistent with phosphorylation of a small fraction of the receptors in this sample and the heterogeneity observed in this tumor by Octreoscan.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By characterizing the phosphorylation state of native sst2A receptors in a variety of neuroendocrine tumors, we here demonstrate, for the first time, that agonist stimulation leads to the phosphorylation of a GPCR in vivo. This provides new evidence for the physiological importance of agonist-stimulated GPCR phosphorylation. Using a nonradioactive, band-shift assay, we show that the extent of receptor phosphorylation observed in different neuroendocrine tumors is correlated with the production of ligand mRNA by the tumors, consistent with phosphorylation resulting from autocrine stimulation. Further, both in somatostatinoma tumor tissue and in SS-treated cultured cells, most of the phosphorylated receptor is intracellular. In CHO-R2 cells, the intracellular accumulation of phosphorylated receptor results from ligand-induced internalization, suggesting that SS-stimulated receptor phosphorylation triggers sst2A receptor endocytosis in vivo, thereby producing the observed intracellular localization of phosphorylated receptor.

Several pieces of evidence support the conclusion that the altered mobility of the sst2A receptor upon agonist stimulation results from phosphorylation. First, SS treatment of CHO-R2 cells leads to the appearance of a new 40-kDa receptor band and the disappearance of the 37-kDa receptor band in immunoblots. The 40-kDa receptor band is heavily labeled with 32PO4, compared with the 37-kDa band. In addition, treatment of samples with alkaline phosphatase increases the mobility of the 40-kDa receptor, so that it migrates at 37 kDa. Although phosphorylation-induced mobility shift assays are extensively used in the signal transduction field to examine the functional role of phosphorylation in the regulation of protein function, they have not been widely used for studies with GPCRs. Yet, this assay provides a simple, sensitive alternative to mass spectrometric analysis, which is the only method successfully used to date to examine GPCR phosphorylation in vivo (6, 29). Like mass spectrometric analysis, and in contrast to radiolabeling studies, the gel shift assay permits quantitation of receptor phosphorylation by comparison of the receptor immunoreactivity in the phosphorylated and unphosphorylated bands. Though the general applicability of phosphorylation-induced mobility shift assays to GPCRs is unknown, such shifts have been shown for several receptors, including the CAR1 cAMP receptor (30), the complement 5a (C5a) anaphylatoxin receptor (31), the 5-HT2C serotonin receptor (32), and the sst1 SS receptor (21). Thus, such assays may provide a more general method than recognized to date for assessing receptor phosphorylation in small tissue samples.

As previously shown in other cell types by receptor immunolabeling (23, 33, 34), we observed that SS stimulation led to rapid internalization of the sst2A receptor in CHO-R2 cells. After 30 min of agonist stimulation, immunofluorescent labeling demonstrated that the receptor was mostly intracellular. Radioligand binding studies, carried out at 4 C, showed that 70% of receptor binding activity had been lost from the cell surface by this time (Prejusa, Liu, and Schonbrunn, unpublished observations). Thirty minutes of SS stimulation also caused essentially stoichiometric receptor phosphorylation. Together, these observations suggest that receptor internalization occurs more rapidly than phosphorylation, thereby preventing accumulation of phosphorylated receptors at the cell surface and leading to the intracellular localization of most of the phosphorylated sst2A.

The high degree of sst2A receptor phosphorylation was surprising. In previous studies, we found that the sst1 SS receptor was only partially phosphorylated upon stimulation with saturating concentrations of agonist (21). Similarly, only partial receptor phosphorylation was observed with the 5HT2C receptor (32) and the C5a anaphylatoxin receptor (31). The factors that regulate the rates of GPCR phosphorylation and dephosphorylation are not well understood at present. However, in the case of sst2A receptors, the observation that essentially all the receptors are phosphorylated after SS stimulation demonstrates that dephosphorylation is rate limiting.

Using the phosphorylation-induced gel-shift assay, we also show here that essentially all the sst2A receptors were phosphorylated in a SS-producing tumor in situ and were intracellular, as observed in SS-stimulated CHO-R2 cells. In contrast, most of the sst2A receptors were unphosphorylated in a carcinoid tumor and a pheochromocytoma which contained no detectable SS mRNA. These results show that the peptide produced by the somatostatinoma functions as an autocrine factor. Further, they suggest that the extent of receptor phosphorylation may be used as a sensitive indicator of tissue stimulation in situ by endogenous ligands, as well as to assess tissue responsiveness to exogenous agents.

In the case of rhodopsin, the only other GPCR whose phosphorylation has been examined in vivo, somewhat variable results have been reported regarding the extent of receptor phosphorylation. Ohguro et al. (29) found that less than 30% of rhodopsin was phosphorylated in the mouse retina after either acute or chronic illumination. In contrast, Kennedy et al. (6) observed that intense illumination, which bleached greater than 99% of retinal rhodopsin in anesthetized mice, resulted in the phosphorylation of approximately 70% of rhodopsin. The molecular mechanisms producing these variations in the extent of receptor phosphorylation remain unknown. However, our results indicate that receptor dephosphorylation must occur much slowly than phosphorylation in the somatostatinoma, as in CHO-R2 cells, to maintain the sst2A receptor in a predominantly phosphorylated form at steady-state.

SS receptors are found in the majority of human tumors originating from SS target tissues, such as pituitary adenomas, hormone-producing gastrointestinal tumors, and tumors of the nervous system, as well as in a variety of other tumors, including breast and prostate cancers (35). Many SS receptor-expressing tumors, such as certain gastroenteropancreatic tumors (somatostatinomas), pheochromocytomas, and medullary thyroid carcinomas (36), can also produce the ligand peptide. Interestingly, in a series of 37 pheochromocytomas, Reubi et al. (37) noted an inverse correlation between SS immunoreactivity and receptor status as determined by ligand autoradiography, although the subcellular localization of the receptor was not determined in those studies because no receptor antibodies were available at that time. Interestingly, they found that they were unable to detect SS receptors in two somatostatinomas that produced high levels of SS mRNA, in contrast to a variety of other neuroendocrine tumors that produced no or low levels of SS (36). More recently, we found that the inability to detect binding in a somatostatinoma was not attributable to the absence of receptors: intracellular sst2A receptors were readily measured by immunolabeling (28). The data presented here, showing that intracellular receptors are phosphorylated, further support the conclusion that agonist-stimulated receptor internalization is responsible for the inability to detect SS receptors by radioligand autoradiography in tumors that produce high levels of SS, and the data help to explain the inability to visualize this particular tumor by Octreoscan. In addition, the observation that sst2A receptors are still present in somatostatinomas suggests that these receptors could be available for re-insertion into the plasma membrane if the autocrine stimulation was blocked. Thus, these results substantially increase our understanding of GPCR dynamics in vivo and provide substantial insight into the utility and limitations of in vivo SS receptor scintigraphy. Moreover, in accordance with recent data indicating a signaling function for internalized GPCRs (38), including SS receptors (33, 39), intracellular phosphorylated sst2A receptors may be responsible for triggering regulatory events which modify tumor behavior.

Autocrine signaling leading to the regulation of tumor cell growth is well recognized in human cancers (40). Neuropeptides that are known to play important roles in such autocrine regulation include gastrin-releasing peptide, neuromedin B, neurotensin, gastrin, and cholecystokin, as well as SS. These peptides all bind to seven transmembrane receptors whose activation and regulation in vivo is still poorly understood. Yet, these GPCRs provide attractive targets for controlling tumor cell growth and secretion and for tumor localization, using radioscintigraphy for imaging (41). Hence, the use of phosphorylation-induced gel shift assays to understand the activation state of GPCRs in human tumors subject to either autocrine or pharmacological regulation is likely to provide important insights for cancer therapy and visualization.


   Footnotes
 
This investigation was supported, in part, by research grants from the National Institute of Arthritis, Diabetes, Digestive, and Kidney Disease (DK32234); by the Welch Foundation (AU-1410); and by the Texas Advanced Technology Program (Grant no. 011618-0032-2001).

Abbreviations: BAP, Bacterial alkaline phosphatase; C5a, complement 5a; DßM, dodecyl-ß-D-maltoside; GPCR, G protein-coupled receptor; PMSF, phenylmethylsulfonylfluoride; PNGase, peptide-N-glycosidase; SDS, sodium dodecyl sulfate; SS, somatostatin; sst, somatostatin receptor subtype; TACT, N,N',N'' triacetyl-chitotriose; TBS, Tris-buffered saline; TCA, trichloroacetic acid; WGA, wheat germ agglutinin.

Received June 9, 2003.

Accepted September 10, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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