Agonist-induced Desensitization, Internalization, and Phosphorylation of the sst2A Somatostatin Receptor*

(Received for publication, October 25, 1996, and in revised form, January 30, 1997)

R. William Hipkin Dagger §, Jacqueline Friedman Dagger , Richard B. Clark Dagger , C. Mark Eppler and Agnes Schonbrunn Dagger par

From the Dagger  Department of Pharmacology, University of Texas Medical School, Houston, Texas 77225 and the  Agricultural Research Center, American Cyanamid Co., Princeton, New Jersey 08543

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Cellular responsiveness to the inhibitory peptide somatostatin (SRIF) or its clinically used analogs can desensitize with agonist exposure. While desensitization of other seven-transmembrane domain receptors is mediated by receptor phosphorylation and/or internalization, the mechanisms mediating SRIF receptor (sst) desensitization are unknown. Therefore, we investigated the susceptibility of the sst2A receptor isotype to ligand-induced desensitization, internalization, and phosphorylation in GH-R2 cells, a clone of pituitary tumor cells overexpressing this receptor. A 30-min exposure of cells to either SRIF or the analog SMS 201-995 (SMS) reduced both the potency and efficacy of agonist inhibition of adenylyl cyclase. Internalization of receptor-bound ligand was rapid (t1/2 = 4 min) and temperature-dependent. SRIF and SMS increased the phosphorylation of the 71-kDa sst2A protein 25-fold within 15 min. Receptor phosphorylation was dependent on both the concentration and time of agonist exposure and was not affected by pertussis toxin pretreatment, indicating that receptor occupancy rather than second messenger formation was required. Receptor phosphorylation was also stimulated by phorbol 12-myristate 13-acetate activation of protein kinase C. Both ligand-stimulated and phorbol 12-myristate 13-acetate-stimulated receptor phosphorylation occurred primarily on serine. These studies are the first demonstration of agonist-dependent desensitization, internalization, and phosphorylation of the sst2A receptor and suggest that phosphorylation may mediate the homologous and heterologous regulation of this receptor.


INTRODUCTION

The somatostatin peptides (SRIF-14 and SRIF-28)1 influence endocrine, exocrine, and neuronal function through binding to a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, and sst5) (1, 2). Within the SRIF receptor family, sst2A receptor mRNA has been detected in many tissues including the brain, pituitary, pancreas, spleen, small intestine, and stomach (1, 2), and the receptor protein has recently been shown to be widely distributed in the mammalian brain (3). Thus, this receptor isotype mediates many of the central and peripheral actions of SRIF.

Early studies on the signal transduction mechanisms activated by SRIF showed that sst receptors elicited their actions predominantly via pertussis toxin-sensitive G proteins (1, 2, 4). Thus, SRIF inhibition of adenylyl cyclase and Ca2+ channels, as well as SRIF stimulation of K+ channels, phospholipase C, serine/threonine and tyrosine phosphatases, arachidonic acid release, and mitogen-activated protein kinases are inhibited by pertussis toxin treatment (5-12). However, some actions of SRIF, such as stimulation of other tyrosine phosphatases as well as inhibition of Na/H exchange, are pertussis toxin-insensitive (13, 14). The network of signaling pathways activated by individual sst receptor isotypes is largely unknown. Signaling mechanisms have been especially difficult to elucidate in the native environment of the receptors because most SRIF target cells express multiple sst receptor isotypes that cannot be individually activated with the analogs currently available.

For most G protein-coupled receptors, hormone treatment decreases receptor responsiveness (desensitization), receptor levels (down-regulation), or both. However, relatively little is known about sst receptor regulation, especially following acute hormonal challenge. Exposure to SRIF or to selective SRIF agonists such as SMS 201-995 (SMS) has been reported to lead to desensitization in pituitary cells over the course of hours, days, or weeks (15-18). Desensitization occurring over both hours (19, 20) and minutes (21) has been reported in the AtT20 corticotropic pituitary cell line, depending on the signaling pathway being examined. However, SRIF receptor desensitization was not detected in the mammotropic GH4C1 pituitary cell line (22) and does not occur during long term treatment of many human pituitary tumors with SMS (23). SRIF receptors can be either down-regulated (24) or up-regulated (22) by hormone pretreatment depending on the cell type examined. The SRIF receptor isotypes involved in these varying effects are unknown although sst2 receptors are expressed, along with other sst receptor subtypes, in the normal pituitary, in pituitary tumors, and in the AtT20 and GH4C1 cell lines (2, 25).2 Interestingly, for sst2A receptors exogenously expressed in Chinese hamster ovary cells, receptor binding has been reported to be either decreased (27, 28) or increased (29) by SRIF pretreatment. However, no desensitization studies have been reported with this receptor isotype.

Ligand-dependent and -independent desensitization of other G protein-coupled receptors is mediated by receptor phosphorylation (30). Examination of sst2A receptor phosphorylation requires a cell line expressing high levels of functional receptor protein. However, identification of appropriate cell lines for studies of exogenously expressed sst2A receptors has proven problematic. Although it is well established that SRIF inhibits adenylyl cyclase activity in native cells (1, 2, 4), the coupling of the sst2A receptor to adenylyl cyclase in heterologous cell lines varies with the cell model (1) suggesting that components required for faithful mimicry of the normal function of the sst2A receptor are not ubiquitously expressed. The GH4C1 pituitary tumor cell line, which contains both the sst1 and sst2A receptors (25),2 has been extensively used for studies of SRIF receptor signaling and regulation (4). Recently, a subclone of GH4C1 cells was isolated following transfection with sst2A receptor cDNA (31). This transfected cell line (GH4-R2.20 cells) expresses approximately 100 times as many functional sst2A receptors as the parental GH4C1 cells and has both the elevated sst2A receptor expression required for phosphorylation studies and an environment that allows normal receptor coupling. By taking advantage of the 10,000-fold greater affinity of SMS 201-995 for the sst2 over the sst1 receptor (1), we now demonstrate agonist-dependent desensitization, internalization, and phosphorylation of the sst2A receptor in this pituitary model cell line.


EXPERIMENTAL PROCEDURES

Hormones and Supplies

Cell culture medium and G418 were purchased from Life Technologies, Inc. The sst2A-receptor antiserum (R2-88) has been described.2 Leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, bacitracin, cholesterol hemisuccinate, Nonidet P-40, and Protein A were obtained from Sigma. N-Dodecyl beta -D-maltoside and pertussis toxin were purchased from Calbiochem and List Biological Laboratories, Inc. (Campbell, CA), respectively. CNBr-activated Sepharose 4B was from Pharmacia Biotech Inc. (Uppsala, Sweden). Bradford reagent and reagents for electrophoresis and Western blotting were obtained from Bio-Rad. Carrier-free Na125I was purchased from Amersham Corp. Phosphate-free Dulbecco's modified Eagle's medium and [32P]orthophosphate were purchased from ICN Biomedicals (Irvine, CA). All other reagents were of the best grade available and purchased from common suppliers.

Cell Culture

The clonal GH4-R2.20 cell line (hereafter referred to as GH-R2 cells) was generated by transfecting GH4C1 pituitary tumor cells with the rat sst2A receptor and was grown as described previously (31). Experimental cultures were plated in medium without G418 and used 2-7 days later with a medium change 18-24 h prior to use. Experiments were carried out with cells plated in 100-mm dishes except whole cell binding experiments, which used cells in 35-mm wells.

Cell Membrane Preparation

GH-R2 cells were washed with and scraped into cold phosphate-buffered saline (PBS: 10 mM Na2HPO4, 150 mM NaCl, pH 7.4) containing protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate, 100 nM okadaic acid). Following centrifugation, the cell pellet was resuspended in homogenization buffer (10 mM Tris-HCl, 5 mM EDTA, 3 mM EGTA, pH 7.6) containing protease and phosphatase inhibitors and membranes were prepared and stored as described previously (25). Membranes used for photoaffinity cross-linking were prepared in the presence of protease inhibitors alone.

Adenylyl Cyclase Measurements

GH-R2 cells were incubated in the absence or presence of 100 nM SMS (Sandoz Pharmaceuticals, Basel, Switzerland) or SRIF for 30 min in fresh growth medium. Membranes were then prepared as described above and assayed in triplicate (2-5 µg of membrane protein/tube) for adenylyl cyclase activity as described previously (32). Cyclase activity was 30-100 pmol/min/mg under basal conditions and 300-800 pmol/min/mg in the presence of 100 nM VIP. Cyclase activity measured in the presence of both 100 nM VIP and varying concentrations of SMS was expressed as a percent of the VIP-stimulated activity, and fitted values for maximal inhibition and EC50 (the concentration required to produce half-maximal inhibition) were obtained by least squares nonlinear regression analysis of dose-response curves using the program D/R (Biomedical Computing, Inc. Houston, TX).

Radioligand Binding and Internalization

[Leu8,D-Trp22,Tyr25]somatostatin-28 (Bachem California, Torrance, CA) and the sst2 receptor-selective somatostatin analog [Tyr3]SMS (Sandoz Pharmaceuticals) were radioiodinated using chloramine T and subsequently purified by reverse-phase high performance liquid chromatography as described previously (33). GH-R2 cells were incubated at 4 °C in 1 ml of binding buffer (F10 medium containing 20 mM HEPES and 5 mg/ml lactalbumin hydrolysate, pH 7.4) containing approximately 100,000 cpm of [125I-Tyr3]SMS with or without 100 nM unlabeled SRIF (34). After 60 min, the cells were rinsed to remove unbound trace and then incubated in fresh 37 °C buffer to allow internalization of the receptor-bound ligand. Cells were subsequently incubated on ice for 5 min in acidic glycine-buffered saline (100 mM glycine, 50 mM NaCl, pH 3.0) to release surface-bound ligand. After collection of the acidic buffer, the cells were dissolved in 0.1 N NaOH. The radioactivity in both the glycine buffer (representing surface-bound ligand) and in the cell lysates (representing internalized ligand) was measured (34). Specific binding was calculated as the difference between the amount of radioligand bound in each fraction in the absence (total binding) and presence of 100 nM SRIF (nonspecific binding).

Detection of sst2A Receptor by Immunoblotting

GH-R2 cells were washed, scraped into cold PBS, pelleted, and solubilized in PBS containing 4 mg/ml dodecyl beta -maltoside, 200 µg/ml cholesterol hemisuccinate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 50 µg/ml bacitracin for 60 min at 4 °C. The detergent lysates were clarified by centrifugation at 100,000 × g for 30 min, and the protein content of the supernatants was assessed by the method of Bradford (35). Protein (10 µg/lane) was dissolved in sample buffer (62.5 mM Tris-HCl, 2% sodium dodecyl sulfate, 10% 2-mercaptoethanol (v/v), 6 M urea, 20% glycerol, pH 6.8) by incubation at 60 °C for 15 min prior to resolution on a 7.5% sodium dodecyl sulfate-polyacrylamide gel.

Resolved proteins were transferred to PVDF membrane as described previously.2 The membrane was then blocked for 2 h with "Blotto" (10 mM NaH2PO4, 10% nonfat dry milk, 10% glycerol, 0.2% Tween 20) and incubated overnight at 4 °C with 1:20,000 dilution of anti-sst2A antibody R2-88 in Blotto. Following repeated washing, the membrane was incubated with 1:5000 dilution of goat-anti-rabbit antibody conjugated with horseradish peroxidase at room temperature for 1 h. Immunoreactive proteins were detected with the ECL chemiluminescent antibody detection system (Amersham Corp.).

Detection of Immunoprecipitated sst2A Receptor by Immunoblotting

GH-R2 cells were incubated in growth medium in the presence or absence of protein kinase activators for 30 min. Cell membranes were prepared as described above and solubilized by agitation at 4 °C for 60 min in HEPES-buffered saline (150 mM NaCl, 20 mM Hepes, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, 50 µg/ml bacitracin, 5 mM EDTA, 3 mM EGTA, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 0.1 mM sodium orthovanadate) containing 4 mg/ml dodecyl beta -maltoside, 200 µg/ml cholesterol hemisuccinate (lysis buffer). After centrifugation at 100,000 × g for 30 min, the supernatant was incubated overnight at 4 °C with anti-sst2A-receptor antiserum covalently coupled to Protein A-Sepharose (final dilution of 1:200). The immunoprecipitated proteins were dissolved in sample buffer without mercaptoethanol for 15 min at 60 °C. After removal of the Sepharose beads and the addition of 10% mercaptoethanol (v/v), proteins were resolved on 7.5% sodium dodecyl sulfate-polyacrylamide gels, transferred to PVDF membrane, and immunoblotted with anti-sst2A antiserum as described above.

Purification of the Phosphorylated sst2A Receptor

Metabolic labeling of cells and subsequent immunoprecipitation of the sst2A receptor was carried out as described previously (36, 37). Briefly, cells (1 dish/treatment) were incubated for 3 h in 3.5 ml of phosphate-free Dulbecco's modified Eagle's medium containing 1 mCi of [32P]orthophosphate and either 1% newborn calf serum or 5 mg/ml lactalbumin hydrolysate. Hormones and pharmacological agents were then added directly to the labeling medium, and the cells were further incubated at 37 °C under 5% CO2 for the indicated times. The cells were then scraped into cold Hepes-buffered saline, pelleted, and solubilized in lysis buffer containing phosphatase inhibitors for 60 min at 4 °C. The detergent lysates were centrifuged at 100,000 × g for 30 min, and the protein content of the supernatants was assessed by the method of Bradford (35).

The sst2A receptors were subjected to a two-step purification consisting of lectin affinity chromatography followed by immunoprecipitation with receptor antibody (36).2 Briefly, equal amounts of lysate protein (~2 mg/ml) were incubated at 4 °C for 90 min with 100 µl (packed volume) of washed wheat germ agglutinin-agarose (Vector Laboratories, Inc., Burlingame, CA). Following centrifugation, the wheat germ agglutinin-agarose was washed vigorously with 30 volumes of lysis buffer, and adsorbed glycoproteins were eluted at 4 °C for 90 min with 250 µl of lysis buffer containing 3 mM N,N",N'''-triacetyl-chitotriose (Sigma) and 0.5% SDS (v/v). Eluted proteins were diluted 5-fold and incubated with a 1:200 dilution of the anti-sst2A receptor antibody R2-88 at 4 °C for 90 min.2 The samples were then incubated at 4 °C for 90 min with 25 µl (packed volume) of protein A-Sepharose. Following centrifugation, the beads were washed as described previously (36), and the immunoprecipitated proteins were solubilized in sample buffer (60 °C, 15 min) and resolved on 7.5% sodium dodecyl sulfate-polyacrylamide gels.

For phosphoamino acid analysis, SDS-PAGE-resolved proteins were transferred to PVDF membrane, and the piece of membrane containing the 32P-labeled receptor (detected by autoradiography) was excised and incubated in 100 µl of 5.7 N HCl (Pierce) at 110 °C for 0.5 or 2 h (37). Phosphoamino acids were resolved by two-dimensional thin layer electrophoresis on cellulose plates (37). Receptor phosphorylation and phosphoamino acid analyses were quantitated using a PhosphorImager (37).

Other Methods

Protein A (Sigma) was covalently coupled to CNBr-activated Sepharose B according to the manufacturer's instructions (Pharmacia). Antireceptor IgG was covalently coupled to protein A-Sepharose as described previously.2 Photoaffinity labeling of the membrane sst2A receptor with [Leu8,D-Trp22,125I-Tyr25]somatostatin-28 and N-5'-azido-2'-nitrobenzoyl-N-oxysuccinimide (Pierce) and subsequent immunoprecipitation of the receptor with anti-sst2A was accomplished by published procedures (38).2 Unless otherwise indicated results of a representative experiment are shown. All experiments were repeated at least 2 times.


RESULTS

Desensitization and Internalization of sst2A Receptor Expressed in GH-R2 Cells

Whereas GH4C1 cells contain low levels (~0.1 pmol/mg of cell protein) of a mixture of the somatostatin receptor subtypes sst1 and sst2 (25, 39)3 the transfected GH-R2 cell line expresses approximately 10 pmol of the sst2A receptor/mg of protein (data not shown). Incubation of intact GH-R2 cells for 2 h at 4 °C with the sst2 selective ligand [125I-Tyr3]SMS and increasing concentrations of unlabeled peptides showed dose-dependent inhibition of radioligand binding with both SRIF (EC50 = 2.24 ± 0.31 nM) and SMS (EC50 = 38.9 ± 7.5 nM). The relative affinities of the rat sst2A receptor for these two ligands thus agrees well with those of the human sst2A receptor as determined in membrane binding studies (40).

To investigate the susceptibility of the sst2A receptor to desensitization, GH-R2 cells were incubated in the absence or presence of 100 nM SMS for 30 min at 37 °C. Membranes were then prepared, and the effect of pretreatment on hormonal regulation of adenylyl cyclase activity was determined (Fig. 1). SMS pretreatment did not affect either basal or VIP-stimulated adenylyl cyclase activity (data not shown). In membranes from untreated cells SMS inhibited VIP-stimulated adenylyl cyclase activity with an EC50 of 1.2 ± 0.1 nM. Maximal inhibition was 63.7 ± 1.5%. Preincubation of cells with SMS attenuated both the potency (EC50 = 7.0 ± 0.4 nM) and efficacy of SMS inhibition (maximum inhibition = 36.2 ± 2.3%). Treatment of cells with 100 nM SRIF for 30 min had the same effect as SMS; maximal inhibition was reduced from 60.1 ± 2.0 to 41.2 ± 2.6% while the EC50 for SMS was increased from 1.0 ± 0.2 to 6.9 ± 2.5 nM. Therefore, exposure to agonist results in homologous desensitization of the sst2A receptor.


Fig. 1. The effect of agonist pretreatment on the subsequent inhibition of VIP-stimulated adenylyl cyclase activity by SMS 201-995. GH-R2 cells were incubated in the absence (open circles) or presence of 100 nM SMS 201-995 (SMS; closed circles) for 30 min and then used to prepare membranes. Adenylyl cyclase activity was subsequently assayed in the presence of no additions or with 100 nM VIP in the absence and presence of the indicated concentrations of SMS. Data represent the mean ± range from two independent experiments each assayed in triplicate and are expressed as a percentage of VIP-stimulated membrane adenylyl cyclase activity in the absence of SMS. SMS pretreatment had no effect on either basal or VIP-stimulated adenylyl cyclase activity. VIP increased adenylyl cyclase activity 6.96 ± 1.54 and 5.92 ± 0.78-fold over basal in membranes from control and SMS pretreated cells, respectively. Both the potency and efficacy of SMS to inhibit VIP-stimulated adenylyl cyclase was diminished in membranes isolated from pretreated cells (control: EC50 = 1.21 ± 0.15 nM, maximum inhibition = 63.7 ± 1.5%; pretreated: EC50 = 7.02 ± 0.38 nM, maximum inhibition = 36.2 ± 2.3%).
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To ascertain if peptide binding induced rapid internalization of the ligand-receptor complex, cells were incubated for 1 h at 4 °C with [125I-Tyr3]SMS to occupy cell surface receptors, washed to remove unbound peptide, and then warmed to 37 °C for different periods of time (Fig. 2). A rapid, time-dependent internalization of the receptor-bound ligand occurred at 37 °C reaching a steady state by 90 min (Fig. 2, upper panel). The rate of internalization was fit to the sum of two first order reactions, giving a value of 4.0 ± 0.7 min for the half-time of internalization of the receptor-ligand complex (Fig. 2, lower panel). These results show that following binding to cell surface sst2A receptors, the bound ligand is rapidly internalized in a temperature-dependent manner.


Fig. 2. Internalization of receptor-bound [125I-Tyr3]SMS. Top panel, GH-R2 cells were incubated for 60 min at 4 °C with [125I-Tyr3]SMS (100,000 cpm/ml) in the absence and presence of 100 nM SRIF, rapidly washed, and then warmed to 37 °C. At the times shown replicate dishes were chilled, washed to remove unbound [125I-Tyr3]SMS and incubated for 5 min at 4 °C with acidic glycine-buffered saline to release surface bound ligand. Following removal of the glycine buffer, the cells were dissolved in 0.1 N NaOH. The radioactivity in both the acid wash (bullet ), representing surface-bound ligand, and the cell lysates (open circle ), representing internalized ligand, were then measured. Specific binding was calculated as the difference between the amount of radioligand bound in the absence and presence of 100 nM SRIF. The data represent the distribution of the specifically bound [125I-Tyr3]SMS between the two fractions and show the mean ± range of two independent experiments. At zero time (t = 0), the specific binding was 3656 ± 593 cpm/dish. Bottom panel, the amount of surface-bound ligand at each time point (Bt) is expressed relative to surface binding at time zero (B0 = 3063 cpm/dish) in a representative experiment. The internalization data were fitted to the equation Bt/Bo = Ae-k1t + (1 - A)e-k2t using KaleidaGraphTM 3.0 and gave the following fitted values: A = 0.584, k1 = 0.213 min-1, and k2 = 0.00123 min-1, where A is the fraction of receptor dissociating with a rate constant of k1 and (1 - A) is the fraction of receptor dissociating with a rate constant of k2.
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Immunodetection of sst2A Receptor in GH-R2 Cells

To determine whether the sst2A receptor was phosphorylated, we next developed and validated methods for the detection and purification of the sst2A receptor protein. The immunoblot in Fig. 3 (left panel) shows that a receptor antibody that specifically recognizes the sst2A receptor isotype reacted with a broad 71-kDa protein band in GH-R2 cell extracts.2 This band was not detected in immunoblots incubated with preimmune sera or with immune sera in the presence of 1 µM antigen peptide. To determine whether this 71-kDa protein was the sst2A receptor, GH-R2 membranes were photoaffinity-labeled with [Leu8,D-Trp22,125I-Tyr25]SRIF-28 and N-5'-azido-2'-nitrobenzoyl-N-oxysuccinimide in the presence or absence of 100 nM SRIF (38). Membranes were then either directly solubilized with sample buffer (Fig. 3, middle panel) or solubilized with a non-denaturing detergent and then immunoprecipitated with preimmune or immune sst2A receptor antisera (Fig. 3, right panel). Subsequent analysis by SDS-PAGE and autoradiography showed that a 71-kDa protein was photoaffinity-labeled in membranes and that radiolabeling was effectively competed with SRIF, as expected for a high affinity sst receptor (middle panel). The photoaffinity-labeled protein was immunoprecipitated by receptor antiserum but not by preimmune antiserum nor by immune serum in the presence of 1 µM antigen peptide (right panel). Therefore, the receptor antibody effectively precipitated the 71-kDa sst2A receptor protein expressed in GH-R2 cells.


Fig. 3. Immunological and biochemical characterization of the sst2A receptor in GH-R2 cells. Left panel, detergent-solubilized GH-R2 cells were subjected to SDS-PAGE (10 µg of protein/lane). Following electrophoretic transfer of the resolved proteins to a PVDF membrane, immunoblotting was performed with either immune R2-88 serum (I = 1:20,000 dilution), preimmune serum (PI = 1:20,000) or immune serum in the presence of 1 µM antigen peptide (I+Ag). Middle and right panels, autoradiograms of GH-R2 cell membranes (0.1 mg/ml) that were incubated for 120 min at 30 °C with [Leu8, D-Trp22, 125I-Tyr25]SRIF-28 (400,000 cpm/ml) in the presence or absence of 100 nM SRIF as described under "Experimental Procedures." After centrifugation to remove unbound ligand, photoaffinity cross-linking was performed with 30 µM N-5'-azido-2'-nitrobenzoyl-N-oxysuccinimide. To detect affinity-labeled receptors, membranes (100 µg/lane) were solubilized in sample buffer and subjected to SDS-PAGE and autoradiography (middle panel). To determine the reactivity of the sst2A antiserum with the photoaffinity-labeled receptor (right panel), affinity-labeled membranes were solubilized with dodecyl-beta -D-maltoside/cholesterol hemisuccinate (lysis buffer) and then incubated with either sst2A antiserum (I) or preimmune serum (PI) coupled to Protein A-Sepharose (final dilution of 1:200) in the absence or presence of 1 µM antigen peptide (Ag). Immunoprecipitated proteins were solubilized in sample buffer, resolved by SDS-PAGE, and subjected to autoradiography. All three panels show the portion of the gel containing immunoreactive proteins.
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Agonist-induced Phosphorylation of sst2A Receptor in Intact Cells

To determine if agonist binding stimulated sst2A receptor phosphorylation, GH-R2 cells were labeled with [32P] orthophosphate and incubated in the absence or presence of 100 nM SRIF for 15 min. Following detergent solubilization and partial purification by lectin affinity chromatography, the sst2A receptor was immunoprecipitated with receptor antibody and analyzed by SDS-PAGE and autoradiography. A low level of basal receptor phosphorylation was detectable with long film exposure (Figs. 6 and 8) or by analysis with a PhosphorImager, although it is difficult to discern in Fig. 4. Treatment of cells with SRIF increased the phosphorylation of the 71-kDa sst2A receptor protein 22 ± 6-fold over basal (n = 5). The 71-kDa phosphoprotein was not immunoprecipitated with either preimmune serum or with immune serum in the presence of antigen peptide (data not shown). Interestingly, it was necessary to solubilize the immunoprecipitated receptor by heating in sample buffer with 6 M urea to dissociate receptor aggregates. When either phosphorylated (Fig. 4, lane 3) or photoaffinity-labeled (data not shown) receptors were immunoprecipitated and then solubilized in SDS sample buffer under reducing conditions but without urea and heating, a higher molecular weight band was observed in addition to the 71-kDa receptor. This receptor aggregate appeared to be generated during the immunoprecipitation procedure since it was not present in GH-R2 cell extracts as analyzed by immunoblotting (Fig. 3, left panel) or in solubilized photoaffinity-labeled membranes (data not shown).


Fig. 6. Concentration dependence for SRIF stimulation of sst2A receptor phosphorylation. 32PO4-labeled GH-R2 cells were incubated with the indicated concentration of SRIF for 30 min. Following detergent solubilization and partial purification by lectin chromatography, the sst2A receptor was immunoprecipitated with receptor antiserum at a final dilution of 1:200. Immunoprecipitated proteins were analyzed by SDS-PAGE and either autoradiography (gel inset) or phosphorimaging (graph).
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Fig. 8. The effect of heterologous agents on sst2A receptor phosphorylation. Top panel, 32PO4-labeled GH-R2 cells were incubated for 15 min with either no additions (Control), 100 nM SRIF, 10 µM carbachol (CARB) or 10 µM (-)N6-(R-phenyl-isopropyl)-adenosine (PIA). Bottom panel, 32PO4-labeled GH-R2 cells were incubated for 15 min with either no additions (Control), 100 nM SRIF, 200 nM PMA, or 10 µM forskolin (Fsk). In both panels, cell proteins were solubilized with dodecyl-beta -D-maltoside/cholesterol hemisuccinate, and the sst2A receptor was purified by lectin chromatography followed by immunoprecipitation with receptor antiserum. Immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography.
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Fig. 4. The effect of SRIF on sst2A receptor phosphorylation. 32PO4-labeled GH-R2 cells were incubated in the absence or presence of 100 nM SRIF for 15 min. Following detergent solubilization and partial purification by lectin chromatography, the sst2A receptor was immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were solubilized either in sample buffer containing 6 M urea for 15 min at 60 °C or in the same sample buffer without urea for 15 min at room temperature and then analyzed by SDS-PAGE and autoradiography.
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If sst2A receptor phosphorylation plays a role in either agonist-stimulated sst2A receptor desensitization or internalization, phosphorylation should be increased within the time frame of these regulatory events. As shown in Fig. 5, stimulation of sst2A receptor phosphorylation was half-maximal after a 2-min incubation with 100 nM SRIF, was maximal by 5 min, and was then maintained for at least 30 min. SMS also increased receptor phosphorylation by 2 min (data not shown) and was as effective as SRIF (Fig. 5).


Fig. 5. Time course for SRIF stimulation of sst2A receptor phosphorylation. 32PO4-labeled GH-R2 cells were incubated either with 100 nM SRIF for the times shown or with 100 nM SMS 201-995 for 30 min. Following detergent solubilization and partial purification by lectin chromatography, the sst2A receptor was immunoprecipitated with receptor antiserum at a final dilution of 1:200. Immunoprecipitated proteins were analyzed by SDS-PAGE and either autoradiography (gel inset) or phosphorimaging (graph).
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Agonist stimulation of sst2A receptor phosphorylation was also concentration-dependent (Fig. 6). Phosphorylation of the sst2A receptor was significanctly elevated by 3 nM SRIF and was further increased with higher doses of SRIF up to a maximal effect with 100 nM peptide. Incubation of 32P-labeled GH-R2 cells with concentrations of SRIF greater than 100 nM did not produce any additional stimulation (data not shown). Thus, phosphorylation of the sst2A receptor increases in response to ligand stimulation in a time- and concentration-dependent manner.

To assess the importance of sst2A receptor-Gi/o coupling for SRIF-induced receptor phosphorylation, GH-R2 cells were pretreated with 100 ng/ml pertussis toxin for 24 h. This treatment abolished SRIF inhibition of VIP-stimulated cAMP accumulation (data not shown). However, SRIF-induced phosphorylation of the sst2A receptor was unaffected by pertussis toxin pretreatment (Fig. 7). Therefore, functional interaction of the sst2A receptor with pertussis toxin-sensitive G proteins is not necessary for agonist-induced sst2A receptor phosphorylation.


Fig. 7. The effect of pertussis-toxin pretreatment on SRIF stimulation of sst2A receptor phosphorylation. GH-R2 cells were incubated in the absence or presence of 100 ng/ml pertussis toxin (PTX) for 24 h prior to and during cell labeling with [32P]orthophosphate. 32PO4-labeled cells were then incubated in the absence or presence of 100 nM SRIF for 15 min. Following detergent solubilization and lectin chromatography, the sst2A receptor was immunoprecipitated with receptor antiserum at a final dilution of 1:200. Immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography.
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GH4C1 rat pituitary cells express m2 muscarinic and A1-adenosine receptors that couple to the same pertussis toxin-sensitive effector pathways as SRIF (41). However, incubation of 32P-labeled GH-R2 cells with either the muscarinic agonist carbachol or with the adenosine receptor agonist PIA did not cause any increase in sst2A receptor phosphorylation (Fig. 8, top panel). Thus heterologous activation of SRIF-stimulated second messenger cascades is not sufficient to stimulate sst2A receptor phosphorylation.

The Effect of Protein Kinase Activators on sst2A Receptor Phosphorylation

To assess the effect of second messenger-regulated protein kinases on sst2A receptor phosphorylation, 32P-labeled GH-R2 cells were incubated for 15-min with either no additions (Control), 100 nM SRIF, 200 nM phorbol 12-myristate 13-acetate (PMA), or 10 µM forskolin prior to sst2A receptor purification. As shown in Fig. 8 (bottom panel), a 15 min incubation with the protein kinase C activator PMA stimulated a 32-fold increase in sst2A receptor phosphorylation whereas stimulation of cAMP synthesis with forskolin had no effect. Incubation with the Ca2+ ionophore ionomycin (10 nM for 15 min) also did not alter sst2A receptor phosphorylation (data not shown). Therefore, heterologous activation of protein kinase C increased sst2A receptor phosphorylation whereas stimulation of either protein kinase A or Ca2+-dependent protein kinases apparently did not.

The signal intensity observed following immunoprecipitation of the 32PO4-labeled receptor is determined by 1) the stoichiometry of receptor phosphorylation and 2) the receptor concentration. The latter depends, in turn, on receptor immunoprecipitation efficiency. The sst2A antibody used in this study recognizes a region in the sst2A receptor cytoplasmic tail containing potential phosphorylation sites. Hence phosphorylation in this region could decrease the efficiency with which the sst2A receptor is immunoprecipitated and thereby prevent the detection of 32PO4 labeling as was shown to occur with the bombesin receptor (37). To test whether receptor phosphorylation affected the ability of the antibody to recognize the receptor protein, GH-R2 cells were incubated for 15 min with no additions or with either 100 nM SRIF, 200 nM PMA, or 10 µM forskolin. Membranes were then prepared, and the ability of the antibody to recognize the different phosphorylated forms of the receptor was assessed both by immunoblotting (Fig. 9, upper panel) and by immunoprecipitation (Fig. 9, lower panel). Incubation of GH-R2 cells with SRIF, PMA, or forskolin for 15 min did not affect cellular sst2A receptor levels and did not alter the immunoprecipitation efficiency of sst2A receptor (Fig. 9). Therefore, the 32PO4 incorporation into the purified receptor (Figs. 3, 4, 5, 6, 7, 8) accurately reflects its phosphorylation state.


Fig. 9. The effect of protein kinase activators on antibody binding to the sst2A receptor. GH-R2 cells were incubated for 15 min with no additions (Control), 100 nM SRIF, 200 nM PMA, or 10 µM forskolin (Fsk). Membranes were then prepared and split into two groups. Top panel, to determine whether the antibody recognized both unphosphorylated and phosphorylated forms of the receptor on a Western blot, membrane proteins were dissolved in sample buffer and subjected to SDS-PAGE (10 µg of protein/lane). Resolved proteins were electrophoretically transferred to PVDF membrane and immunoblotted with sst2A receptor antiserum (final dilution of 1:20,000). Bottom panel, to determine whether protein kinase activators altered the efficiency with which the receptor was immunoprecipitated, membranes were solubilized with dodecyl-beta -D-maltoside/cholesterol hemisuccinate (25 µg of protein/lane) and immunoprecipitated with receptor antiserum covalently coupled to protein A-Sepharose (final dilution of 1:200) in the absence or presence of 1 µM antigen peptide (Ag). Immunoprecipitated proteins were resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-sst2A receptor antiserum.
[View Larger Version of this Image (43K GIF file)]

Phosphoamino Acid Analysis of Phosphorylated sst2A

To identify the phosphorylated residues in the sst2A receptor, phosphoamino acid analysis was carried out with receptor from control cells and from cells incubated with either SRIF or PMA. Following acid hydrolysis of the receptor for 30 min, phosphoserine and phosphotyrosine residues were detected (Fig. 10, top panel). Hydrolysis for 2 h facilitated detection of phosphoserine and phosphothreonine residues (Fig. 10, bottom panel). Under all three treatment conditions, the most heavily labeled residue was phosphoserine. However, all three phosphoamino acid species were detectable following analysis of sst2A receptor from unstimulated cells, and phosphorylation of all three residues was increased in a concentration-dependent manner in response to agonist stimulation (data not shown). These studies show that basal and SRIF- and PMA-stimulated sst2A receptor phosphorylation occur primarily on serine residues, although an increase in the phosphorylation of threonine and tyrosine also occurs.


Fig. 10. Phosphoamino acid analysis of the phosphorylated sst2A receptor. 32PO4-labeled GH-R2 cells were incubated for 15 min with either 100 nM SRIF or 200 nM PMA and then solubilized with dodecyl-beta -D-maltoside/cholesterol hemisuccinate as described under "Experimental Procedures.". The sst2A receptor was sequentially purified by lectin chromatography and immunoprecipitation with receptor antiserum (final dilution of 1:200) and then subjected to SDS-PAGE. Following electrophoretic transfer of the resolved proteins to a PVDF membrane, the phosphorylated receptor was located by autoradiography. The portions of the PVDF membrane containing the receptor were excised and incubated at 110 °C with 5.7 N HCl for either 30 (top panel) or 120 min (bottom panel). The hydrolysed samples were analyzed by two-dimensional thin layer chromatography in the presence of unlabeled carrier phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) standards as described under "Experimental Procedures." The figure shows an autoradiogram of the TLC plate. The migration of the unlabeled standards was determined by staining with ninhydrin and is shown by the circles.
[View Larger Version of this Image (69K GIF file)]


DISCUSSION

The studies presented here demonstrate for the first time that hormone binding leads to rapid desensitization, internalization, and phosphorylation of the sst2A receptor.

Desensitization to native SRIF peptides as well as to the clinically used analog SMS or octreotide has been observed in some tissues and cancers but not in others, suggesting that susceptibility to desensitization depends on the sst receptor subtypes present. For example, in GH4C1 cells, which express both the sst1 and sst2 receptors (4, 25), prolonged exposure to SRIF does not attenuate subsequent SRIF inhibition of adenylyl cyclase (22). Since SRIF binds with high affinity to all sst receptor isotypes, the lack of effect of SRIF pretreatment on GH4C1 cell responsiveness represents the sum of the functional effects on all of the SRIF receptors present. Thus, the behavior of the individual receptor isotypes cannot be deduced from these experiments. However, the analog SMS binds with high affinity only to the sst2 and sst5 receptor subtypes (1). As reverse transcriptase-polymerase chain reaction analysis showed that sst5 receptor mRNA is not expressed in GH-R2 cells4 the diminished potency and efficacy of SMS to inhibit adenylyl cyclase activity following pretreatment of cells with this analog represents homologous desensitization of sst2A receptor function. Taken together, our present and previous (22) data indicate that in GH4C1 cells sst1 and sst2 receptors act redundantly to mediate SRIF inhibition of adenylyl cyclase and that the sst1-receptor isotype is resistant to desensitization. The potential for sst2A receptor desensitization must thus be recognized not only for the physiological actions of SRIF but also when analogs such as SMS/octreotide are used clinically for the treatment of acromegaly and other sst2 receptor-positive tumors (42).

As in the case of desensitization, most studies of SRIF receptor internalization have utilized cell lines expressing multiple sst receptors whose composite behavior was monitored using SRIF analogs that bound to several sst receptor isotypes. Hormone binding did not lead to receptor-mediated internalization in either GH4C1 pituitary cells or in RINm5F insulinoma cells (34, 43). In contrast, variable amounts of receptor-bound peptide were internalized in AtT-20 pituitary cells, human pituitary tumor cells, islet cells, and pancreatic acinar cells (44-47). In these early studies the internalization of the hormone-receptor complex could not be attributed to a specific sst receptor isotype. We show here that agonist binding to the sst2A receptor triggers rapid receptor-mediated internalization. Very recently the sst2 receptor was also reported to mediate internalization of bound [Leu8,D-Trp22,125I-Tyr25]SRIF-28 when expressed in Chinese hamster ovary cells (29). However, in Chinese hamster ovary cells only about 20% of the receptor-bound ligand was internalized even after 60 min. The explanation for the differences in the extent of receptor-mediated internalization in the two studies is not clear; internalization of the receptor-ligand complex could be influenced by both the cellular environment and the nature of the bound ligand.

The mechanisms mediating sst2A receptor desensitization and internalization are not known but, as has been postulated for other G-protein coupled receptors, may involve receptor phosphorylation (30, 48, 49). Indeed, this hypothesis is consistent with our findings that 1) agonist stimulation of sst2A receptor phosphorylation (t1/2 approx  2 min, Fig. 5) occurs concurrently with receptor internalization in GH-R2 cells (t1/2 approx  4 min, Fig. 2) and that 2) maximal sst2A receptor phosphorylation is evident under conditions used to elicit sst2A receptor desensitization.

Phosphorylation of other G protein-coupled receptors is catalyzed by two types of kinases: second messenger-activated kinases and G protein-coupled receptor kinases (GRKs). Our results suggest that agonist-stimulated phosphorylation of the sst2A receptor preferentially involves GRKs. SRIF inhibition of adenylyl cyclase, as well as regulation of other signaling pathways, is blocked by pertussis toxin, which prevents coupling of sst receptors to Gi/o (5-12). However, pertussis toxin pretreatment did not affect SRIF stimulation of sst2A receptor phosphorylation. These results with the sst2A receptor agree with observations with m2 muscarinic receptor, another inhibitory G protein-coupled receptor known to be phosphorylated by GRKs (50). Hormone stimulation of sst2A receptor phosphorylation by a second messenger-independent mechanism thus suggests the involvement of G protein-coupled receptor kinases, athough our experiments do not rule out the possibility that second messenger cascades activated via pertussis toxin-insensitive G proteins mediate SRIF stimulation of sst2A receptor phosphorylation. However, two other observations argue against this possibility. First, even though PIA, carbachol, and SRIF produce the same inhibitory effect on hormone secretion, adenylyl cyclase activity, and intracellular calcium in GH cells (41, 51) neither PIA nor carbachol increased sst2A receptor phosphorylation. Thus activation of adenosine or muscarinic receptors does not produce the same effect as does occupancy of the sst2A receptor with agonist. Second, stimulation of sst2A receptor phosphorylation occurred at relatively high concentrations of hormone. By analogy to the beta -adrenergic receptor, where much lower agonist concentrations are required to stimulate receptor phosphorylation by cAMP-dependent protein kinase than by GRKs (30), the necessity for high SRIF concentrations for sst2A receptor phosphorylation suggests that the agonist-occupied receptor is being preferentially phosphorylated by GRKs. Indeed, Mayor et al. (52) reported that homologous desensitization of S49 mouse lymphoma cells with SRIF occurred concurrently with the translocation of a G protein-coupled receptor kinase from the cytoplasm to the plasma membrane. While these results are highly suggestive, additional experiments will be neccessary to directly demonstrate a role for G protein-coupled receptor kinases in sst2A receptor phosphorylation.

Interestingly, pharmacological activation of several second messenger-regulated kinases showed that phosphorylation of the sst2A receptor was specifically stimulated following a 15-min incubation with the protein kinase C activator, phorbol 12-myristate 13-acetate (Fig. 8). Although heterologous activation of protein kinase C can regulate sst2A receptor function, it is unlikely that this kinase catalyzed the SRIF-stimulated phosphorylation because incubation with SRIF did not increase phospholipase C activity in GH-R2 cells as assayed by inositol trisphosphate accumulation.4 Moreover, in preliminary studies we found that the amount of 32PO4 incorporated into the sst2A receptor following incubation with maximal concentrations of both SRIF (100 nM) and PMA (200 nM) was close to the sum of the incorporation achieved with either agent alone. This observation further argues against the involvement of protein kinase C in ligand-stimulated receptor phosphorylation. Although it is quite likely that the sst2A receptor is a substrate for protein kinase C because the receptor protein contains consensus sequences for protein kinase C phosphorylation (53), it is also possible that protein kinase C indirectly influences the phosphorylation state of the receptor by activating another kinase and/or by inactivating a protein phosphatase.

The functional consequences of protein kinase C-stimulated receptor phosphorylation are unknown. Previous studies showed that a 2-4-h exposure of pancreatic acinar cells (54) or GH4C1 cells (55) to phorbol ester decreased SRIF binding, with no change in binding affinity observed in the acinar cells (54). Incubation with phorbol ester also attenuated SRIF inhibition of adenylyl cyclase activity in GH4C1 cells (26). However, from these early studies one cannot assess the direct impact, if any, of protein kinase C phosphorylation on the sst2A receptor. Our observation that protein kinase C activation leads to a dramatic increase in sst2A receptor phosphorylation paves the way for the critical analysis of the mechanism by which this phosphorylation occurs as well as its biological consequences.

Although the sst2A receptor also contains putative consensus sequences for protein kinase A phosphorylation (53), stimulation of cAMP synthesis with forskolin did not increase 32PO4 incorporation into the sst2A receptor (Fig. 8). The ineffectiveness of forskolin in stimulating sst2A receptor phosphorylation raised the possibility that phosphorylation was not detected because of a potential experimental problem. As was the case for the bombesin receptor (37), phosphorylation of residues within the region of the sst2A receptor recognized by our antibody could inhibit receptor immunoprecipitation. Therefore, it was prudent to assess the ability of the antibody to recognize the receptor both before and after kinase activation. As shown in Fig. 9, neither SRIF, PMA, nor forskolin altered sst2A receptor immunoprecipitation efficiency. Thus, the signal intensity of phosphorylated sst2A receptor in these studies accurately reflects the incorporation of 32PO4 into the purified receptor.

Agonist- and PMA-stimulated sst2A receptor phosphorylations occur at multiple sites as 32PO4 labeling of phosphoserine, phosphothreonine, and phosphotyrosine residues was increased (Fig. 10). However, phosphoserine was the predominantly labeled residue under all hydrolysis conditions tested, suggesting that the sst2A receptor is phosphorylated primarily on serine(s). Identification of these phosphorylation sites within the intracellular regions of the receptor will allow a critical examination of the causal relationship between sst2A receptor phosphorylation, desensitization, and internalization.


FOOTNOTES

*   This investigation was supported by the National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases Research Grants DK32234 (to A. S.) and GM31208 (R. B. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Partially supported by a postdoctoral fellowship from the Juvenile Diabetes Foundation.
par    To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Medical School, P. O. Box 20708, Houston, TX 77225. Tel.: 713-500-7470; Fax: 713-500-7456; E-mail: aschonb{at}farmr1.med.uth.tmc.edu.
1   The abbreviations used are: SRIF, somatostatin; SMS, SMS 201-995 (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol); VIP, vasoactive intestinal peptide; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; GRK, G protein-coupled receptor kinase; PBS, phosphate-buffered saline.
2   Y.-Z. Gu and A. Schonbrunn, Mol. Endrocrinol., in press.
3   Y.-Z. Gu and A. Schonbrunn, unpublished observations.
4   Y. Wang and A. Schonbrunn, unpublished observations.

ACKNOWLEDGEMENTS

We thank Dr. Yi Zhong Gu for advice regarding use of the sst2A receptor antibody and with the photoaffinity labeling studies, Yining Wang for reverse transcriptase-polymerase chain reaction analysis of the sst receptor subtype mRNAs in GH-R2 cells, Dr. Mari Haddox and Barbara Cochran for help with the phosphoamino acid analysis, and Dr. Roger Barber for critical reading of the manuscript.


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