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• W2143517945 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 (t½ = 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. 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 (t½ = 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. The somatostatin peptides (SRIF-14 and SRIF-28) 1The 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. 1The 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. influence endocrine, exocrine, and neuronal function through binding to a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, and sst5) (1Hoyer D. Bell G.I. Berelowitz M. Epelbaum J. Feniuk W. Humphrey P.P.A. O'Carroll A.-M. Patel Y.C. Schonbrunn A. Taylor J.E. Reisine T. Trends Pharmacol. Sci. 1995; 16: 86-88Google Scholar, 2Reisine T. Bell G. Endocr. Rev. 1995; 16: 427-442Google Scholar). Within the SRIF receptor family, sst2A receptor mRNA has been detected in many tissues including the brain, pituitary, pancreas, spleen, small intestine, and stomach (1Hoyer D. Bell G.I. Berelowitz M. Epelbaum J. Feniuk W. Humphrey P.P.A. O'Carroll A.-M. Patel Y.C. Schonbrunn A. Taylor J.E. Reisine T. Trends Pharmacol. Sci. 1995; 16: 86-88Google Scholar, 2Reisine T. Bell G. Endocr. Rev. 1995; 16: 427-442Google Scholar), and the receptor protein has recently been shown to be widely distributed in the mammalian brain (3Dournaud P. Gu Y.-Z. Schonbrunn A. Tannenbaum G. Beaudet A. J. Neurosci. 1996; 16: 4468-4478Google Scholar). 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 (1Hoyer D. Bell G.I. Berelowitz M. Epelbaum J. Feniuk W. Humphrey P.P.A. O'Carroll A.-M. Patel Y.C. Schonbrunn A. Taylor J.E. Reisine T. Trends Pharmacol. Sci. 1995; 16: 86-88Google Scholar, 2Reisine T. Bell G. Endocr. Rev. 1995; 16: 427-442Google Scholar, 4Schonbrunn A. Gu Y.-Z. Brown P.J. Loose-Mitchell D. CIBA Found. Symp. 1995; 190: 204-221Google Scholar). 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 (5Koch B.D. Dorflinger L.J. Schonbrunn A. J. Biol. Chem. 1985; 260: 13138-13145Google Scholar, 6Koch B.D. Blalock J.B. Schonbrunn A. J. Biol. Chem. 1988; 263: 216-225Google Scholar, 7Yatani A. Codina J. Sekura R.D. Birnbaumer L. Brown A.M. Mol. Endocrinol. 1987; 1: 283-289Google Scholar, 8White R.E. Schonbrunn A. Armstrong D.L. Nature. 1991; 351: 570-573Google Scholar, 9Bito H. Mori M. Sakanaka C. Takano T. Honda Z. Gotoh Y. Nishida E. Shimizu T. J. Biol. Chem. 1994; 269: 12722-12730Google Scholar, 10Duerson K. White R.E. Jiang F. Schonbrunn A. Armstrong D.L. Neuropharmacology. 1996; 35: 949-961Google Scholar, 11Florio T. Rim C. Hershberger R.E. Loda M. Stork P.J.S. Mol. Endocrinol. 1994; 8: 1289-1297Google Scholar, 12Tomura H. Okajima F. Akbar M. Abdul Majid M. Sho K. Kondo Y. Biochem. Biophys. Res. Commun. 1994; 200: 986-992Google Scholar). However, some actions of SRIF, such as stimulation of other tyrosine phosphatases as well as inhibition of Na/H exchange, are pertussis toxin-insensitive (13Colas B. Cambillau C. Buscail L. Zeggari M. Esteve J.P. Lautre V. Thomas F. Vaysse N. Susini C. Eur. J. Biochem. 1992; 207: 1017-1024Google Scholar, 14Hou C. Gilbert R.L. Barber D.L. J. Biol. Chem. 1994; 269: 10357-10362Google Scholar). 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 (15Smith M. Yamamoto G. Vale W. Mol. Cell. Endocrinol. 1984; 37: 311-318Google Scholar, 16Welsh J. Szabo M. Endocrinology. 1988; 123: 2230-2234Google Scholar, 17Kehijman M. Frohman L. J. Clin. Endocrinol. Metab. 1990; 71: 157-163Google Scholar, 18Koper J. Hofland L. van Koetsveld P. den Holder F. Lamberts S. Cancer Res. 1990; 50: 6238-6242Google Scholar). Desensitization occurring over both hours (19Reisine T. J. Pharmacol. Exp. Ther. 1984; 229: 14-20Google Scholar, 20Srikant B. Heisler S. Endocrinology. 1985; 117: 78-271Google Scholar) and minutes (21Reisine T. Wang H. Guild S. J. Pharmacol. Exp. Ther. 1988; 245: 225-231Google Scholar) 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 (22Presky D.H. Schonbrunn A. J. Biol. Chem. 1988; 263: 714-721Google Scholar) and does not occur during long term treatment of many human pituitary tumors with SMS (23Ducasse M. Tauber J.P. Tourre T. Bonafe A. Babin T.H. Tauber M.T. Harris A.G. Bayard F. J. Clin. Endocrinol. Metab. 1987; 65: 1042-1046Google Scholar). SRIF receptors can be either down-regulated (24Mahy N. Woolkalis M. Manning D. Reisine T. J. Pharmacol. Exp. Ther. 1988; 247: 390-396Google Scholar) or up-regulated (22Presky D.H. Schonbrunn A. J. Biol. Chem. 1988; 263: 714-721Google Scholar) 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 (2Reisine T. Bell G. Endocr. Rev. 1995; 16: 427-442Google Scholar,25Gu Y.-Z. Brown P.J. Loose-Mitchell D.S. Stork P.J.S. Schonbrunn A. Mol. Pharmacol. 1995; 48: 1004-1014Google Scholar). 2Y.-Z. Gu and A. Schonbrunn, Mol. Endrocrinol., in press. 2Y.-Z. Gu and A. Schonbrunn, Mol. Endrocrinol., in press. Interestingly, for sst2A receptors exogenously expressed in Chinese hamster ovary cells, receptor binding has been reported to be either decreased (27Rens-Domiano S. Law S.F. Yamada Y. Seino S. Bell G.I. Reisine T. Mol. Pharmacol. 1992; 42: 28-34Google Scholar, 28Vanetti M. Vogt G. Hollt V. FEBS Lett. 1993; 331: 260-266Google Scholar) or increased (29Hukovic N. Panetta R. Kumar U. Patel Y. Endocrinology. 1996; 137: 4046-4049Google Scholar) 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 (30Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Google Scholar). 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 (1Hoyer D. Bell G.I. Berelowitz M. Epelbaum J. Feniuk W. Humphrey P.P.A. O'Carroll A.-M. Patel Y.C. Schonbrunn A. Taylor J.E. Reisine T. Trends Pharmacol. Sci. 1995; 16: 86-88Google Scholar, 2Reisine T. Bell G. Endocr. Rev. 1995; 16: 427-442Google Scholar, 4Schonbrunn A. Gu Y.-Z. Brown P.J. Loose-Mitchell D. CIBA Found. Symp. 1995; 190: 204-221Google Scholar), the coupling of the sst2A receptor to adenylyl cyclase in heterologous cell lines varies with the cell model (1Hoyer D. Bell G.I. Berelowitz M. Epelbaum J. Feniuk W. Humphrey P.P.A. O'Carroll A.-M. Patel Y.C. Schonbrunn A. Taylor J.E. Reisine T. Trends Pharmacol. Sci. 1995; 16: 86-88Google Scholar) 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 (25Gu Y.-Z. Brown P.J. Loose-Mitchell D.S. Stork P.J.S. Schonbrunn A. Mol. Pharmacol. 1995; 48: 1004-1014Google Scholar),2 has been extensively used for studies of SRIF receptor signaling and regulation (4Schonbrunn A. Gu Y.-Z. Brown P.J. Loose-Mitchell D. CIBA Found. Symp. 1995; 190: 204-221Google Scholar). Recently, a subclone of GH4C1 cells was isolated following transfection with sst2A receptor cDNA (31Carrick T.A. Bingham B. Eppler C.M. Baumbach W.R. Zysk J.R. Endocrinology. 1995; 136: 4701-4704Google Scholar). 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 (1Hoyer D. Bell G.I. Berelowitz M. Epelbaum J. Feniuk W. Humphrey P.P.A. O'Carroll A.-M. Patel Y.C. Schonbrunn A. Taylor J.E. Reisine T. Trends Pharmacol. Sci. 1995; 16: 86-88Google Scholar), we now demonstrate agonist-dependent desensitization, internalization, and phosphorylation of the sst2A receptor in this pituitary model cell line. 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 β-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. 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 (31Carrick T.A. Bingham B. Eppler C.M. Baumbach W.R. Zysk J.R. Endocrinology. 1995; 136: 4701-4704Google Scholar). 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. GH-R2 cells were washed with and scraped into cold phosphate-buffered saline (PBS: 10 mmNa2HPO4, 150 mm NaCl, pH 7.4) containing protease and phosphatase inhibitors (1 mmphenylmethylsulfonyl 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 (25Gu Y.-Z. Brown P.J. Loose-Mitchell D.S. Stork P.J.S. Schonbrunn A. Mol. Pharmacol. 1995; 48: 1004-1014Google Scholar). Membranes used for photoaffinity cross-linking were prepared in the presence of protease inhibitors alone. 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 (32Yuan N. Friedman J. Whaley B.S. Clark R.B. J. Biol. Chem. 1994; 269: 23032-23038Google Scholar). 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). [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 (33Presky D.H. Schonbrunn A. Mol. Pharmacol. 1988; 34: 651-658Google Scholar). 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 (34Presky D.H. Schonbrunn A. J. Cell Biol. 1986; 102: 878-888Google Scholar). 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 (34Presky D.H. Schonbrunn A. J. Cell Biol. 1986; 102: 878-888Google Scholar). 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). GH-R2 cells were washed, scraped into cold PBS, pelleted, and solubilized in PBS containing 4 mg/ml dodecyl β-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 (35Bradford M.M. Anal. Biochem. 1976; 53: 304-308Google Scholar). 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.). 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 mmNaCl, 20 mm Hepes, pH 7.4, 1 mmphenylmethylsulfonyl 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 β-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. Metabolic labeling of cells and subsequent immunoprecipitation of the sst2A receptor was carried out as described previously (36Hipkin R.W. Sanchez-Yague J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Google Scholar, 37Williams B.Y. Wang Y. Schonbrunn A. Mol. Pharm. 1996; 50: 716-727Google Scholar). 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 (35Bradford M.M. Anal. Biochem. 1976; 53: 304-308Google Scholar). The sst2A receptors were subjected to a two-step purification consisting of lectin affinity chromatography followed by immunoprecipitation with receptor antibody (36Hipkin R.W. Sanchez-Yague J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Google Scholar).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 (36Hipkin R.W. Sanchez-Yague J. Ascoli M. Mol. Endocrinol. 1993; 7: 823-832Google Scholar), 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 the32P-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 (37Williams B.Y. Wang Y. Schonbrunn A. Mol. Pharm. 1996; 50: 716-727Google Scholar). Phosphoamino acids were resolved by two-dimensional thin layer electrophoresis on cellulose plates (37Williams B.Y. Wang Y. Schonbrunn A. Mol. Pharm. 1996; 50: 716-727Google Scholar). Receptor phosphorylation and phosphoamino acid analyses were quantitated using a PhosphorImager (37Williams B.Y. Wang Y. Schonbrunn A. Mol. Pharm. 1996; 50: 716-727Google Scholar). 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 (38Brown P.J. Lee A.B. Norman M.G. Presky D.H. Schonbrunn A. J. Biol. Chem. 1990; 265: 17995-18004Google Scholar).2Unless otherwise indicated results of a representative experiment are shown. All experiments were repeated at least 2 times. Whereas GH4C1 cells contain low levels (∼0.1 pmol/mg of cell protein) of a mixture of the somatostatin receptor subtypes sst1 and sst2 (25Gu Y.-Z. Brown P.J. Loose-Mitchell D.S. Stork P.J.S. Schonbrunn A. Mol. Pharmacol. 1995; 48: 1004-1014Google Scholar,39Schonbrunn A. Tashjian Jr., A.H. J. Biol. Chem. 1978; 253: 6473-6483Google Scholar) 3Y.-Z. Gu and A. Schonbrunn, unpublished observations. 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 (40Kubota A. Yamada Y. Kagimoto S. Shimatsu A. Imamura M. Tsudas K. Imura H. Seino S. Seino Y. J. Clin. Invest. 1994; 93: 1321-1325Google Scholar). 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. 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. 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 (38Brown P.J. Lee A.B. Norman M.G. Presky D.H. Schonbrunn A. J. Biol. Chem. 1990; 265: 17995-18004Google Scholar). 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 μmantigen peptide (right panel). Therefore, the receptor antibody effectively precipitated the 71-kDa sst2A receptor protein expressed in GH-R2 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 6m 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 wei" @default.
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• W2143517945 title "Agonist-induced Desensitization, Internalization, and Phosphorylation of the sst2A Somatostatin Receptor" @default.
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• W2143517945 doi "https://doi.org/10.1074/jbc.272.21.13869" @default.
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