Heterodimerization of Somatostatin and Opioid Receptors Cross-modulates Phosphorylation, Internalization, and Desensitization
2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês
10.1074/jbc.m110373200
ISSN1083-351X
AutoresManuela Pfeiffer, Thomas Koch, Helmut Schröder, Magdalena Laugsch, Volker Höllt, Stefan Schulz,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoHeterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst2A and sst3 somatostatin receptors results in inactivation of sst3 receptor function (Pfeiffer, M., Koch, T., Schröder, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., Höllt, V., and Schulz, S. (2001) J. Biol. Chem.276, 14027–14036). Here we examine dimerization of the sst2A somatostatin receptor and the μ-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst2A and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst2A and sst3, sst2A-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst2A-MOR1 heterodimer to the sst2A-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst2A as well as MOR1. Similarly, exposure of the sst2A-MOR1 heterodimer to the μ-selective ligand [d-Ala2,Me-Phe4,Gly5-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst2A but not internalization of sst2A. Cross-phosphorylation and cross-desensitization of the sst2A-MOR1 heterodimer were selective; they were neither observed with the sst2A-sst3 heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors. Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst2A and sst3 somatostatin receptors results in inactivation of sst3 receptor function (Pfeiffer, M., Koch, T., Schröder, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., Höllt, V., and Schulz, S. (2001) J. Biol. Chem.276, 14027–14036). Here we examine dimerization of the sst2A somatostatin receptor and the μ-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst2A and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst2A and sst3, sst2A-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst2A-MOR1 heterodimer to the sst2A-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst2A as well as MOR1. Similarly, exposure of the sst2A-MOR1 heterodimer to the μ-selective ligand [d-Ala2,Me-Phe4,Gly5-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst2A but not internalization of sst2A. Cross-phosphorylation and cross-desensitization of the sst2A-MOR1 heterodimer were selective; they were neither observed with the sst2A-sst3 heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors. Recent biochemical, biophysical, and functional studies suggest that G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCRG protein-coupled receptorERKextracellular signal-regulated kinasesHEKhuman embryonic kidneyMOR1μ-opioid receptorPAGEpolyacrylamide gel electrophoresisPMAphorbol 12-myristate 13-acetatePKCprotein kinase CSS-14somatostatinsstsomatostatin receptorDAMGO[d-Ala2,Me-Phe4,Gly5-ol]enkephalinHAhemagglutinin can assemble as homo- or heterodimeric complexes (1Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (581) Google Scholar, 2Devi L.A. Trends Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Heterodimerization has been shown to alter both ligand binding affinity and signaling efficacy of GPCRs (1Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (581) Google Scholar, 2Devi L.A. Trends Pharmacol. Sci. 2001; 22: 532-537Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). δ- and κ-opioid receptors form stable heterodimers with ligand binding and signaling properties resembling that of the κ2 receptor (3Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (978) Google Scholar). Formation of heterodimers between the sst1 and sst5 somatostatin receptors has been found to modulate the pharmacology and signaling of both receptors (4Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). The γ-aminobutyric acid receptor B is unique in that heterodimerization of the nonfunctional γ-aminobutyric acid receptors B1 and B2 is required for native affinity for ligands and complete functional activity (5White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1015) Google Scholar, 6Ng G.Y. Clark J. Coulombe N. Ethier N. Hebert T.E. Sullivan R. Kargman S. Chateauneuf A. Tsukamoto N. McDonald T. Whiting P. Mezey E. Johnson M.P. Liu Q. Kolakowski L.F., Jr. Evans J.F. Bonner T.I. O'Neill G.P. J. Biol. Chem. 1999; 274: 7607-7610Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 7Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1017) Google Scholar, 8Kuner R. Kohr G. Grunewald S. Eisenhardt G. Bach A. Kornau H.C. Science. 1999; 283: 74-77Crossref PubMed Scopus (502) Google Scholar, 9Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.J. Johnson M. Gunwaldsen C. Huang L.Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (925) Google Scholar). Heteromeric assembly of fully functional AT1 angiotensin II and B2 bradykinin receptors results in increased efficacy of angiotensin II and decreased efficacy of bradykinin (10AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (436) Google Scholar). G protein-coupled receptor extracellular signal-regulated kinases human embryonic kidney μ-opioid receptor polyacrylamide gel electrophoresis phorbol 12-myristate 13-acetate protein kinase C somatostatin somatostatin receptor [d-Ala2,Me-Phe4,Gly5-ol]enkephalin hemagglutinin Heterodimerization has also been shown to alter endocytotic trafficking of GPCRs (3Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (978) Google Scholar, 4Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem. 2000; 275: 7862-7869Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 10AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (436) Google Scholar, 11Jordan B.A. Trapaidze N. Gomes I. Nivarthi R. Devi L.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 343-348PubMed Google Scholar). The κ-δ heterodimer exhibited a decrease in agonist-mediated receptor endocytosis (3Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (978) Google Scholar). Oligomerization of δ- and κ-opioid receptors with the distantly related β2-adrenergic receptor results in increased and decreased receptor endocytosis, respectively (11Jordan B.A. Trapaidze N. Gomes I. Nivarthi R. Devi L.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 343-348PubMed Google Scholar). AT1-B2heterodimerization induced a switch to a clathrin- and dynamin-dependent endocytotic pathway for both receptors (10AbdAlla S. Lother H. Quitterer U. Nature. 2000; 407: 94-98Crossref PubMed Scopus (436) Google Scholar). Signaling of GPCRs is often terminated by phosphorylation of intracellular serine and threonine residues. After phosphorylation of the receptor, arrestins are frequently recruited to the plasma membrane, at which they facilitate endocytosis by serving as scaffolding proteins that bind to clathrin. Although changes in trafficking have been clearly documented, agonist-induced phosphorylation and desensitization of these GPCR heterodimers has not been examined. We have recently shown that the sst2A and sst3somatostatin receptors exist as constitutive homodimers when expressed alone and as constitutive heterodimers when coexpressed in human embryonic kidney (HEK) 293 cells (12Pfeiffer M. Koch T. Schroder H. Klutzny M. Kirscht S. Kreienkamp H.J. Hollt V. Schulz S. J. Biol. Chem. 2001; 276: 14027-14036Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Whereas the sst2A-sst3 heterodimer behaved like the sst2A homodimer, it did not reproduce the pharmacological characteristics of the sst3 homodimer, suggesting that physical interaction of sst3 with sst2A induced functional inactivation of the sst3 subtype (12Pfeiffer M. Koch T. Schroder H. Klutzny M. Kirscht S. Kreienkamp H.J. Hollt V. Schulz S. J. Biol. Chem. 2001; 276: 14027-14036Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Here we report that the sst2A receptor also forms stable heterodimers with the μ-opioid receptor (MOR1), a member of a closely related GPCR family. Unlike that observed for the sst2A-sst3 heterodimer, sst2A-MOR1 heterodimerization did not significantly affect the ligand binding or coupling properties but promoted cross-modulation of phosphorylation, internalization, and desensitization of these receptors. The sst2-selective ligand L-779,976 and the sst3-selective ligand L-796,778 were kindly provided by Dr. Susan Rohrer (13Rohrer S.P. Birzin E.T. Mosley R.T. Berk S.C. Hutchins S.M. Shen D.M. Xiong Y. Hayes E.C. Parmar R.M. Foor F. Mitra S.W. Degrado S.J. Shu M. Klopp J.M. Cai S.J. Blake A. Chan W.W. Pasternak A. Yang L. Patchett A.A. Smith R.G. Chapman K.T. Schaeffer J.M. Science. 1998; 282: 737-740Crossref PubMed Scopus (443) Google Scholar) (Merck). The radioligand [125I-Tyr11]SS-14 (74 TBq/mmol) was fromAmersham Biosciences, and [3H]DAMGO was from (PerkinElmer Life Sciences). Mouse monoclonal anti-T7 tag antibody was obtained from Novagen (Madison, WI), rat monoclonal anti-HA tag antibody was from Roche Molecular Biochemicals, and polyclonal rabbit anti-T7 and anti-HA antibodies were from Gramsch Laboratories (Schwabhausen, Germany). In addition, rabbit anti-sst2A antibody (6291), guinea-pig anti-sst2A antibody (GP3), rabbit anti-MOR1 antibody (9998), and rabbit anti-sst3 antibody (7986) were used and have been characterized extensively (12Pfeiffer M. Koch T. Schroder H. Klutzny M. Kirscht S. Kreienkamp H.J. Hollt V. Schulz S. J. Biol. Chem. 2001; 276: 14027-14036Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 14Schulz S. Schreff M. Koch T. Zimprich A. Gramsch C. Elde R. Hollt V. Neuroscience. 1998; 82: 613-622Crossref PubMed Scopus (72) Google Scholar, 15Schulz S. Schreff M. Schmidt H. Handel M. Przewlocki R. Hollt V. Eur. J. Neurosci. 1998; 10: 3700-3708Crossref PubMed Scopus (98) Google Scholar, 16Koch T. Schulz S. Schroder H. Wolf R. Raulf E. Hollt V. J. Biol. Chem. 1998; 273: 13652-13657Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). All polyclonal rabbit antisera were affinity-purified against their immunizing peptides using the Sulfo-Link coupling gel according to the instructions of the manufacturer (Pierce). The wild-type rat sst2A receptor was tagged at its amino terminus with the T7 epitope tag sequence MASMTGGQQMG using polymerase chain reaction and subcloned into a pcDNA3.1 expression vector (Invitrogen) containing a neomycin resistance as described previously. The wild-type rat μ-opioid receptor MOR1 was tagged at its amino terminus with the HA epitope tag sequence YPYDVPDYA using polymerase chain reaction and subcloned into a pEAK10 expression vector (Edge Bio Systems, Gaithersburg, MD) containing a puromycin resistance as described previously (17Koch T. Schulz S. Pfeiffer M. Klutzny M. Schroder H. Kahl E. Hollt V. J. Biol. Chem. 2001; 276: 31408-31414Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). HEK 293 cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 10% CO2. The cells were first transfected with plasmids containing the neomycin resistance using the calcium phosphate precipitation method. Stable transfectants were selected in the presence of 500 μg/ml G418 (Invitrogen). To generate lines coexpressing two differentially epitope-tagged receptors, the cells were subjected to a second round of transfection using FuGENE 6 (Roche Diagnostics) and selected in the presence of 500 μg/ml G418 and 1 μg/ml puromycin (Sigma). Three clones expressing T7sst2A alone, six clones expressing HAMOR1 alone, and four clones coexpressing T7sst2Aand HAMOR1 were generated. Receptor expression was monitored using saturation ligand binding assays as described below. In addition, quantitative Western blot analysis was carried out to ensure that clones coexpressing ∼ 1:1 ratio of sst2A and MOR1 were selected. Finally, double immunofluorescent staining was performed to validate that sst2A and MOR1 were coexpressed within the same cells. The Bmax and KDvalues of the cells that were used throughout this study are given in Table I.Table ILigand binding properties of sst2A, MOR1, and sst2A-MOR1 receptors[125I]SS-14 bindingKDBmaxT7sst2A-HAMOR1T7sst2AT7sst2A-HAMOR1T7sst2Anmfmol/mg protein0.48 ± 0.080.17 ± 0.022458 ± 418820 ± 19[3H]DAMGO bindingKDBmaxT7sst2A-HAMOR1HAMOR1T7sst2A-HAMOR1HAMOR11.20 ± 0.071.22 ± 0.142986 ± 135574 ± 20Saturation binding assays were performed on membranes prepared from stably transfected cells. The dissociation constant (KD) and number of [3H]DAMGO-binding sites (Bmax) were calculated by Scatchard analysis as described under "Experimental Procedures." The data are presented as the means ± S.E. of three or four independent experiments performed in triplicate. Open table in a new tab Saturation binding assays were performed on membranes prepared from stably transfected cells. The dissociation constant (KD) and number of [3H]DAMGO-binding sites (Bmax) were calculated by Scatchard analysis as described under "Experimental Procedures." The data are presented as the means ± S.E. of three or four independent experiments performed in triplicate. Stably transfected HEK 293 cells were plated onto poly-l-lysine-coated 150-mm dishes and grown to 80% confluence. The cells were exposed to the cross-linking agents bis(sulfosuccinimidyl)suberate or dithiobis-(succinimidylpropionate) (both from Pierce) and subsequently lysed as described (12Pfeiffer M. Koch T. Schroder H. Klutzny M. Kirscht S. Kreienkamp H.J. Hollt V. Schulz S. J. Biol. Chem. 2001; 276: 14027-14036Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 17Koch T. Schulz S. Pfeiffer M. Klutzny M. Schroder H. Kahl E. Hollt V. J. Biol. Chem. 2001; 276: 31408-31414Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The cell membranes were prepared and solubilized in detergent buffer (20 mm HEPES, pH 7.4, 150 mm NaCl, 5 mmEDTA, 3 mm EGTA, 4 mg/ml β-dodecylmaltoside, 10 mm iodoacetamide, 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 10 μg/ml bacitracin) for 1 h on ice. Alternatively, the cells were lysed in radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and proteinase inhibitors) as described below. The receptor proteins were then immunoprecipitated with 100 μl of protein A-agarose beads preloaded with 10 μg of anti-HA, anti-T7, or anti-sst2A (6291) antibodies. Immunocomplexes were eluted from the beads using SDS sample buffer for 20 min at 60 °C and resolved by SDS-PAGE. After electroblotting, membranes were incubated with either mouse monoclonal anti-T7, rat monoclonal anti-HA, affinity-purified rabbit anti-sst2A (6291), or anti-MOR1 (9998) antibodies at a concentration of 1 μg/ml for 12 h at 4 °C, followed by detection using an enhanced chemiluminescence detection system. When indicated, the membranes were placed in stripping buffer (100 mm 2-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.7) for 30 min at 55 °C and subsequently reprobed. The cells expressing T7sst2A, T7sst3, or HAMOR1 alone as well as cells coexpressing T7sst2A and Mycsst3 or T7sst2A and HAMOR1 were plated onto 100-mm dishes and grown to 80% confluence. The cells were washed with serum- and phosphate-free medium and then labeled with 200 μCi/ml carrier-free [32P]orthophosphate (285 Ci/mg Pi; ICN, Eschwege, Germany) for 60 min at 37 °C. The labeled cells were exposed to either 100 nm L-779,976, 1000 nmL-796,778, 100 nm SS-14, or 1000 nm DAMGO for 20 min. After incubation, the cells were placed on ice and washed with ice-cold phosphate-buffered saline and then scraped into 1 ml of radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 10 mm NaF, 10 mm disodium pyrophosphate, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 10 μg/ml bacitracin). The cells were solubilized for 1 h at 4 °C on a rotating platform. The supernatants were obtained by centrifugation at 13,000 ×g for 60 min at 4 °C, after which aliquots were taken to determine the total protein content in the supernatant of each sample to be immunoprecipitated. Immunoprecipitations were carried out by adding 10 μg of affinity-purified polyclonal rabbit anti-HA tag, anti-T7 tag, or anti-sst3 (7986) antibodies as described above. Immunocomplexes were eluted from the beads using SDS sample buffer for 20 min at 60 °C. The amount of receptor in each sample was calculated as the function of receptor expression times the total protein content of the solubilized fraction of each sample subjected to immunoprecipitation. The receptor content of each sample was normalized to the sample with the least receptor content by dilution with sample buffer. The samples were then subjected to 8% SDS-polyacrylamide gel electrophoresis followed by autoradiography. The extent of phosphorylation of receptor monomers was quantitated using a Fuji PhosphorImaging system and BAS 1000 software. The cells were grown on poly-l-lysine-treated coverslips overnight and then exposed to agonists. The cells were fixed and permeabilized as described (12Pfeiffer M. Koch T. Schroder H. Klutzny M. Kirscht S. Kreienkamp H.J. Hollt V. Schulz S. J. Biol. Chem. 2001; 276: 14027-14036Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). For single immunofluorescence, the cells were then incubated with either mouse monoclonal anti-T7, rat monoclonal anti-HA, affinity-purified rabbit anti-HA, affinity-purified rabbit anti-sst2A (6291), or affinity-purified rabbit anti-MOR1 (9998) antibody at a concentration of 1 μg/ml in Tris/phosphate-buffered saline and 1% normal goat serum overnight. Bound primary antibody was detected with biotinylated secondary antibodies (1:100; Vector, Burlingame, CA) followed by cyanine 3.18-conjugated streptavidin (Amersham Biosciences). For double immunofluorescence, the cells were incubated either with a mixture of rat monoclonal anti-HA and affinity-purified rabbit anti-sst2A (6291) or affinity-purified rabbit anti-HA and guinea pig anti-sst2A (GP3) antibodies. Bound primary antibodies were detected with biotinylated anti-rabbit antibodies, followed by a mixture of cyanine 2.18-conjugated streptavidin and cyanine 5.18-conjugated anti-rat or anti-guinea pig antibodies (1:200, Jackson ImmunoResearch, West Grove, PA). The cells were then dehydrated, cleared in xylol, and permanently mounted in DPX (Fluka, Neu-Ulm, Germany). Male Wistar rats (n = 3, 200–250 g; Tierzucht, Schönwalde, Germany) were deeply anesthetized with chloral hydrate and transcardially perfused with Tyrode's solution followed by Zamboni's fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 m phosphate buffer, pH 7.4. The brains were rapidly dissected and post-fixed in the same fixative for 2 h at room temperature. For all animal procedures ethical approval was sought prior to the experiments according to the requirements of the German National Act on the Use of Experimental Animals. The tissue was cryoprotected by immersion in 30% sucrose before sectioning using a freezing microtome. Free-floating sections (30–40 μm) were incubated with a mixture of guinea pig anti-sst2A (GP3) and rabbit anti-MOR1 (9998) antibodies for 48–72 h at room temperature. Bound primary antibodies were detected as above, and sections were permanently mounted in DPX. The specimens were examined using a Leica TCS-NT laser scanning confocal microscope (Heidelberg, Germany) equipped with a krypton/argon laser. Cyanine 2.18 was imaged with 488-nm excitation and 500–560-nm band pass emission filters, cyanine 3.18 was imaged with 568-nm excitation and 570–630-nm band pass emission filters, and cyanine 5.18 was imaged with 647-nm excitation and 665-nm long pass emission filters. The cells were seeded at a density of 2 × 105/well onto poly-l-lysine-treated 24-well plates. The next day, the cells were preincubated with 1 μg of affinity-purified rabbit anti-T7 or anti-HA antibody for 2 h in OPTIMEM 1 (Invitrogen) at 4 °C. The cells were then treated with 100 nm L-779,976, 1000 nm DAMGO, or 100 nm PMA in OPTIMEM for 60 min. Subsequently, the cells were fixed and incubated with peroxidase-conjugated anti-rabbit antibody (1:1000; AmershamBiosciences) for 2 h at room temperature. After washing, the plates were developed with 250 μl of ABTS solution (Roche). After 10–30 min, 200 μl of the substrate solution from each well was transferred to a 96-well plate and analyzed at 405 nm using a microplate reader (Bio-Rad). Saturation binding assays were performed on membrane preparations from stably transfected cells as described previously. The dissociation constant (KD) and number of [3H]DAMGO binding sites (Bmax) was calculated by Scatchard analysis using at least six concentrations of [3H]DAMGO in a range from 0.3 to 9 nm. Nonspecific binding was determined as radioactivity bound in the presence of 1 μm unlabeled DAMGO. KD and Bmax values of somatostatin binding sites were calculated by Scatchard analysis with increasing concentrations of [125I-Tyr11]SS-14 ranging from 0.025 to 0.3 nm. Nonspecific binding was defined as that not displaced by 1 μm SS-14. For competition binding assays, aliquots of membrane preparation containing 25 μg of protein were incubated with either 0.05 nm[125I-Tyr11]SS-14 or 0.05 nm[3H]DAMGO in the presence or absence of L-779,976 or DAMGO in concentrations ranging from 10−12 to 10−5m. Transfected cells were seeded at a density of 1.5 × 105/well onto poly-l-lysine-treated 22-mm 12-well dishes. On the next day, the cells were incubated in the presence or absence of 100 nm L-779,976 or 100 nm DAMGO for 0, 0.5, 1, 2, 4, or 6 h in OPTIMEM 1. The medium was then removed and replaced with 0.5 ml of serum-free RPMI medium containing 25 μmforskolin or 25 μm forskolin plus either L-779,976 or DAMGO in concentrations ranging from 10−14 to 10−6 M. The cells were incubated at 37 °C for 15 min. The reaction was terminated by removal of the culture medium and subsequent addition of 1 ml of ice-cold HCl/ethanol (1 volume of 1n HCl with 100 volumes of ethanol). After centrifugation, the supernatant was evaporated, the residue was dissolved in TE buffer (50 mm Tris-EDTA, pH 7.5), and the cAMP content was determined using a commercially available radioimmunoassay kit (Amersham Biosciences). The cells were seeded at a density of 1.5 × 105/well onto poly-l-lysine-treated 22-mm 12-well dishes, grown in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum overnight, and then pretreated with OPTIMEM 1 for 2 h. The cells were then incubated in the presence or absence of 100 nm L-779,976 or 100 nm DAMGO for 1, 2, 4, or 6 h in OPTIMEM 1 and then exposed to either L-779,976, DAMGO, or lysophosphatidic acid in concentrations ranging from 10−14 to 10−6 M for 5 min in RPMI medium at 37 °C. Incubation was terminated by removal of the culture medium and subsequent addition of 250 μl of boiling SDS sample buffer. Equal amounts of protein of each sample were separated on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes. The protein content was determined using the BCA method. After blocking, the membranes were incubated with mouse monoclonal phospho-specific anti-ERK1/2 antibody clone E10 (New England Biolabs, Beverly, MA) or phosphorylation-independent rabbit polyclonal anti-ERK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Densitometric analysis of total ERK2 and phospho-ERK1/2 levels on Western blots exposed in the linear range of the x-ray film was performed using National Institutes of Health Image 1.57 software. Phospho-ERK1/2 levels were normalized to total ERK1/2 per lane and expressed as the fold ERK1/2 phosphorylation over the basal value of untreated cells. Data from ligand binding, cAMP, and ERK assays were analyzed by nonlinear regression curve fitting using GraphPad Prism 3.0 software. Statistical analysis was carried out using the two-tailed paired t test or two-way analysis of variance followed by the Bonferroni test. p values < 0.05 were considered to be statistically significant. To directly examine the association between the sst2A and the MOR1 receptor, we stably coexpressed T7-tagged sst2A receptors and HA-tagged MOR1 receptors in HEK 293 cells. Saturation binding experiments revealed that these cells coexpressed ∼1:1 ratio of somatostatin binding sites (Bmax 2,458 ± 418 fmol/mg membrane protein) and DAMGO binding sites (Bmax 2,986 ± 135 fmol/mg membrane protein) (Table I). When membrane extracts from these cells were prepared with detergent buffer and immunoprecipitated using the rat anti-HA antibody, the carboxyl-terminal anti-sst2A antibody (6291) detected a single band migrating at 160 kDa, suggesting that this band represents a T7sst2A-HAMOR1 heterodimer (Fig. 1A). Immunoprecipitation of sst2A-MOR1 heterodimers was facilitated when cells were preincubated with the cross-linking agent bis(sulfosuccinimidyl)suberate (Fig. 1A). In contrast, no bands were detectable in immunoprecipitates prepared under identical conditions from cells expressing only T7sst2A or HAMOR1 or from a mixture of T7sst2A- and HAMOR1-expressing cells. These data strongly suggest that sst2A-MOR1 heterodimers pre-existed in cells prior to cell lysis and were not artificially formed during sample preparation. When the blot shown in Fig. 1A was stripped and reprobed with rabbit anti-HA antibody, MOR1 monomers and dimers were revealed in immunoprecipitates from HAMOR1 cells, from T7sst2A-HAMOR1 cells, and from a mixture of T7sst2A and HAMOR1 cells (Fig. 1A′). To test the stability of sst2A-MOR1 heterodimers under conditions used for whole cells phosphorylation assays, the cells were lysed in radioimmune precipitation buffer, immunoprecipitated using anti-HA antibody, and detected with anti-sst2A antibody. As shown in Fig. 1B, sst2A-MOR1 heterodimers were not detectable under these conditions. When the blot shown in Fig. 1B was stripped and reprobed with rabbit anti-HA antibody, it was apparent that cell lysis in SDS-containing radioimmune precipitation buffer in the absence of cross-linking agents leads to nearly complete dissociation of sst2A-MOR1 heterodimers (Fig. 1B′). We compared the ligand binding properties of sst2A-MOR1 heterodimers with those of sst2A and MOR1 homodimers by examining the ability of the selective agonists to compete with [125I-Tyr11]SS-14 or [3H]DAMGO binding in membranes prepared from cells expressing either T7sst2A or HAMOR1 or coexpressing both T7sst2A and HAMOR1. The results in Table II show that T7sst2A-HAMOR1 cells had a 2-fold lower affinity for the sst2-selective agonist L-779,976 than T7sst2A cells. In contrast, HAMOR1 and T7sst2A-HAMOR1 cells had similar high affinities for the μ-selective agonist DAMGO. Moreover, L-779,976 did not compete with [3H]DAMGO binding, and DAMGO did not compete with [125I-Tyr11]SS-14 binding in membranes prepared from T7sst2A-HAMOR1 cells. Ligand binding assays also revealed that T7sst2A cells had no affinity for DAMGO and that HAMOR1 cells had no affinity for L-779,976. The activation of somatostatin and opioid receptors by agonists results in decreased levels of intracellular cAMP as well as in a rapid and transient stimulation of ERK1/2 phosphorylation. As shown in Table II, the sst2-selective agonist L-779,976 produced 2–4-fold more robust responses in cells coexpressing T7sst2A and HAMOR1 compared with cells expressing T7sst2A alone. Conversely, the MOR1-selective agonist DAMGO produced slightly more robust responses in cells coexpressing T7sst2A and HAMOR1 as compared with cells expressing HAMOR1 alone. In contrast, L-779,976 neither inhibited forskolin-stimulated cAMP accumula
Referência(s)