Heterodimerization of Somatostatin and Opioid Receptors Cross-modulates Phosphorylation, Internalization, and Desensitization (2002)

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. 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. 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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; for 2 h at room temperature. After the were with μl of solution After 200 μl of the solution from each well was to a and at nm using a Saturation binding assays were performed on 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 of [3H]DAMGO in a from to binding was as in the presence of 1 and Bmax of somatostatin binding sites were calculated by Scatchard analysis with of [125I-Tyr11]SS-14 from to binding was as that not by 1 For binding aliquots of containing μg of protein were incubated with either or in the presence or of L-779,976 or DAMGO in from to cells were seeded at a density of × 105/well onto poly-l-lysine-treated the next day, the cells were incubated in the presence or of 100 nm L-779,976 or 100 nm DAMGO for or 6 h in OPTIMEM The medium was then and with ml of medium containing or either L-779,976 or DAMGO in from to M. The cells were incubated at 37 °C for min. The reaction was terminated by of the medium and of 1 ml of ice-cold of with 100 of After the supernatant was the was in buffer (50 mm pH and the content was using a (Amersham Biosciences). The cells were seeded at a density of × 105/well onto poly-l-lysine-treated grown in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum and then with OPTIMEM 1 for 2 The cells were then incubated in the presence or of 100 nm L-779,976 or 100 nm DAMGO for or 6 h in OPTIMEM 1 and then exposed to either L-779,976, DAMGO, or lysophosphatidic acid in from to for 5 min in medium at 37 °C. was terminated by of the medium and of μl of SDS sample buffer. of protein of each sample were on 10% SDS-polyacrylamide and onto The protein content was using the method. After the membranes were incubated with mouse monoclonal antibody or rabbit polyclonal antibody were using peroxidase-conjugated secondary antibodies and enhanced analysis of total and on Western exposed in the of the was performed using National of software. were normalized to total and expressed as the phosphorylation the of cells. from ligand binding, and assays were by using software. analysis was carried out using the or analysis of followed by the were to be To examine the between the sst2A and the MOR1 receptor, we stably coexpressed sst2A receptors and MOR1 receptors in HEK 293 cells. Saturation binding experiments that these cells coexpressed ratio of somatostatin binding sites ± and DAMGO binding sites ± When from these cells were prepared with detergent buffer and immunoprecipitated using the rat anti-HA the anti-sst2A antibody (6291) detected a single band at suggesting that this band a heterodimer of sst2A-MOR1 heterodimers was when cells were preincubated with the cross-linking bis(sulfosuccinimidyl)suberate In were in prepared under from cells expressing T7sst2A or HAMOR1 or from a mixture of and cells. data suggest that sst2A-MOR1 heterodimers in cells prior to cell and were not sample When the blot shown in was and with rabbit anti-HA MOR1 monomers and were in from HAMOR1 from and from a mixture of T7sst2A and HAMOR1 cells To the of sst2A-MOR1 heterodimers under used for cells phosphorylation the cells were lysed in radioimmune precipitation buffer, immunoprecipitated using anti-HA and detected with anti-sst2A shown in sst2A-MOR1 heterodimers were not under these When the blot shown in was and with rabbit anti-HA it was that cell in radioimmune precipitation buffer in the of cross-linking agents to complete dissociation of sst2A-MOR1 heterodimers We the ligand binding properties of sst2A-MOR1 heterodimers with of sst2A and MOR1 homodimers by the of the to with [125I-Tyr11]SS-14 or [3H]DAMGO binding in membranes prepared from cells expressing either T7sst2A or HAMOR1 or coexpressing both T7sst2A and The results in Table II that cells a affinity for the sst2-selective L-779,976 T7sst2A cells. In HAMOR1 and cells for the μ-selective L-779,976 did not with [3H]DAMGO binding, and DAMGO did not with [125I-Tyr11]SS-14 binding in membranes prepared from cells. binding assays also that T7sst2A cells affinity for DAMGO and that HAMOR1 cells affinity for The of somatostatin and receptors by results in decreased of intracellular as well as in a and of shown in Table the sst2-selective L-779,976 in cells coexpressing T7sst2A and HAMOR1 with cells expressing T7sst2A the DAMGO in cells coexpressing T7sst2A and HAMOR1 as with cells expressing HAMOR1 In L-779,976 neither