Homo- and Heterodimerization of Somatostatin Receptor Subtypes
2001; Elsevier BV; Volume: 276; Issue: 17 Linguagem: Inglês
10.1074/jbc.m006084200
ISSN1083-351X
AutoresManuela Pfeiffer, Thomas Koch, Helmut Schröder, Marcus Klutzny, Susanne Kirscht, Hans‐Jürgen Kreienkamp, Volker Höllt, Stefan Schulz,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoSeveral recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst2A and sst3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst2A and sst3. The sst2A-sst3 heterodimer exhibited high affinity binding to somatostatin-14 and the sst2-selective ligand L-779,976 but not to the sst3-selective ligand L-796,778. Like the sst2A homodimer, the sst2A-sst3 heterodimer stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst2-selective ligand L-779,976. However, unlike the sst3 homodimer, the sst2A-sst3 heterodimer did not promote GTPγS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst3-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst2A-sst3 heterodimer desensitized at a slower rate than the sst2A and sst3 homodimers. Both sst2A and sst3 homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst2A-sst3 heterodimer separated at the plasma membrane, and only sst2A but not sst3 underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst2A and sst3results in a new receptor with a pharmacological and functional profile resembling that of the sst2A receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst3 receptor function by heterodimerization with sst2A or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst3-specific binding and signaling in mammalian tissues. Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst2A and sst3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst2A and sst3. The sst2A-sst3 heterodimer exhibited high affinity binding to somatostatin-14 and the sst2-selective ligand L-779,976 but not to the sst3-selective ligand L-796,778. Like the sst2A homodimer, the sst2A-sst3 heterodimer stimulated guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst2-selective ligand L-779,976. However, unlike the sst3 homodimer, the sst2A-sst3 heterodimer did not promote GTPγS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst3-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst2A-sst3 heterodimer desensitized at a slower rate than the sst2A and sst3 homodimers. Both sst2A and sst3 homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst2A-sst3 heterodimer separated at the plasma membrane, and only sst2A but not sst3 underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst2A and sst3results in a new receptor with a pharmacological and functional profile resembling that of the sst2A receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst3 receptor function by heterodimerization with sst2A or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst3-specific binding and signaling in mammalian tissues. Although G protein-coupled receptors (GPCRs)1 generally were believed to act as monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. The existence of homodimers has been shown for several GPCRs including the β2-adrenergic receptor (1Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem... 1996; 271: 16384-16392Google Scholar), δ- and κ-opioid receptors (2Cvejic S. Devi L.A. J. Biol. Chem... 1997; 272: 26959-26964Google Scholar, 3Jordan B.A. Devi L.A. Nature.. 1999; 399: 697-700Google Scholar), the metabotrobic glutamate receptor 5 (4Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem... 1996; 271: 28612-28616Google Scholar), the calcium-sensing receptor (5Bai M. Trivedi S. Brown E.M. J. Biol. Chem... 1998; 273: 23605-23610Google Scholar), the m3 muscarinic receptor (6Zeng F.Y. Wess J. J. Biol. Chem... 1999; 274: 19487-19497Google Scholar), and dopamine receptors (7George S.R. Lee S.P. Varghese G. Zeman P.R. Seeman P. Ng G.Y. O'Dowd B.F. J. Biol. Chem... 1998; 273: 30244-30248Google Scholar). GPCRs seem to dimerize via different mechanisms. 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There is also evidence for different, although not mutually exclusive, pathways of intracellular signaling of somatostatin receptor subtypes, e.g. activation of extracellular signal-regulated kinases (ERK) via sst1, sst3, and sst4; activation of phosphotyrosine phosphatases via sst1, sst2, and sst3; activation of phospholipase A2 via sst4; and modulation of K+ channels via sst2 (21Meyerhof W. Rev. Physiol. Biochem. Pharmacol... 1998; 133: 55-108Google Scholar,22Patel Y.C. Front. Neuroendocrinol... 1999; 20: 157-198Google Scholar). Individual target cells often express more than one somatostatin receptor, raising the possibility that the functional diversity of these receptors could be expanded by heterodimerization among somatostatin receptor subtypes. Recently, Rocheville et al. (23Rocheville M. Lange D.C. Kumar U. Sasi R. Patel R.C. Patel Y.C. J. Biol. Chem... 2000; 275: 7862-7869Google Scholar) provided evidence, based on photobleaching fluorescence resonance energy transfer, for homodimerization of the sst5 somatostatin receptor. The sst5 receptor also appears to form heterodimers with sst1 but not sst4. In this study, we have examined dimerization of sst2A and sst3somatostatin receptors. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we show that sst2Aand sst3 associate as dimers, both as homodimers and heterodimers. sst2A-sst3 heterodimerization resulted in a new receptor with enhanced sst2A-like and diminished sst3-like activity. The sst2-selective ligand L-779,976 and the sst3-selective ligand L-796,778 were kindly provided by Dr. Susan Rohrer (24Rohrer 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: 737-740Google Scholar) (Merck). The radioligand 3-[125I]iodotyrosyl-SS-14 (74 TBq/mmol) was from Amersham Pharmacia Biotech, and [35S]GTPγS (46.25 TBq/mmol) was from PerkinElmer Life Sciences. Mouse monoclonal anti-T7 tag antibody was obtained from Novagen (Madison, WI), and polyclonal rabbit anti-T7 and anti-c-Myc antibodies were obtained from Gramsch Laboratories (Schwabhausen, Germany). The rabbit anti-sst2Aantibody (6291) and the guinea pig anti-sst2A antibody (GP3) were generated to the peptide ETQRTLLNGDLQTSI, which corresponds to residues 355–369 of the carboxyl terminus of the rat/mouse/human sst2A and have been characterized extensively (25Schulz S. Schmidt H. Handel M. Schreff M. Hollt V. Neurosci. Lett... 1998; 257: 37-40Google Scholar, 26Schulz S. Schreff M. Schmidt H. Handel M. Przewlocki R. Hollt V. Eur. J. Neurosci... 1998; 10: 3700-3708Google Scholar). The anti-sst3 antibody (7986) was generated to the peptide TAGDKASTLSHL, which corresponds to residues 417–428 of the carboxyl terminus of the rat/mouse sst3 and has also been characterized previously (27Handel M. Schulz S. Stanarius A. Schreff M. Erdtmann-Vourliotis M. Schmidt H. Wolf G. Hollt V. Neuroscience.. 1999; 89: 909-926Google Scholar). All polyclonal rabbit antisera were affinity-purified against their immunizing peptides using the Sulfo-Link coupling gel (Pierce) according to the instructions of the manufacturer. The wild-type rat sst2A and sst3 receptors were tagged at their amino termini either with the c-Myc epitope tag sequence MEEQKLISEEDLLR or the T7 epitope tag sequence MASMTGGQQMG using polymerase chain reaction. Two more amino acids, KL, were added representing aHindIII cloning site encoded by the nucleotide sequence AAGCTT. The resulting fragments were then subcloned into pcDNA3.1 expression vectors containing either a neomycin or a hygromycin resistance (Invitrogen, Groningen, The Netherlands). The integrity of all constructs was verified by dideoxy sequencing. Human embryonic kidney (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. Cells were first transfected with plasmids containing the neomycin resistance using the calcium phosphate precipitation method. Stable tranfectants were selected in the presence of 500 μg/ml G418 (Life Technologies, Inc.). To generate lines coexpressing two differentially epitope-tagged receptors, cells were subjected to a second round of transfection using Effectene (Qiagen, Hilden, Germany) and selected in the presence of 500 μg/ml G418 and 300 μg/ml hygromycin B (Invitrogen). Three clones expressing sst2A alone, four clones expressing sst3 alone, and two clones coexpressing sst2Aand sst3 were generated. Receptor expression was monitored using saturation ligand binding assays as described below. TheBmax values of all selected clones were in the range of 800–1200 fmol/mg of protein, and KD values were in the range of 0.16–0.24 nm. In addition, quantitative Western blot analysis was carried out to ensure that clones coexpressing ∼1:1 ratio of sst2A and sst3 were selected and used throughout this study. Finally, double immunofluorescent staining was performed to validate that sst2A and sst3 were coexpressed within the same cells. Cells were plated onto poly-l-lysine-coated 150-mm dishes and grown to 80% confluence. When indicated, cells were exposed to agonist, treated with reducing agents, or incubated with cross-linking agents. Agonist exposure was performed with either SS-14, L-779,976, or L-796,778 at a concentration of 1 μm for 10, 30, or 60 min at 37 °C. Treatment with reducing agents was performed with 1 mmdl-dithiothreitol (DTT) for 30 min at 37 °C. Incubation with cross-linking agents was performed with either 2 mmbis(sulfosuccinimidyl)suberate (BS3) or 5 mmdithiobis-(succinimidylpropionate) (both from Pierce) for 30 or 60 min in phosphate-buffered saline (PBS) at 4 °C. The reaction was quenched by the addition of 50 mm Tris (pH 7.5) for 15 min. Cells were then washed twice with PBS and harvested into ice-cold lysis buffer (10 mm Tris-HCl, pH 7.6, 5 mm EDTA, 3 mm EGTA, 250 mm sucrose, 10 mmiodoacetamide, and the following proteinase inhibitors: 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, 10 μg/ml bacitracin). Iodoacetamide was included in each buffer used for protein preparation to prevent nonspecific disulfide linkages. Cells were swollen for 15 min on ice and homogenized. The homogenate was spun at 500 ×g for 5 min at 4 °C to remove unbroken cells and nuclei. Membranes were then pelleted at 20,000 × g for 30 min at 4 °C, and pelleted membranes were lysed 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, and proteinase inhibitors as above) for 1 h on ice. The lysate was centrifuged at 20,000 ×g for 30 min at 4 °C. The protein content of the resulting supernatant was determined using the BCA protein assay (Pierce); samples containing equal amounts of protein (300 μg) were then subjected to immunoprecipitation, or glycoproteins were purified using wheat germ lectin. For enrichment of glycoproteins, one ml of the supernatant was incubated with 100 μl wheat germ agglutinin-agarose beads (Amersham Pharmacia Biotech) for 90 min at 4 °C with continuous agitation. Beads were washed five times with detergent buffer, and adsorbed glycoproteins were either subjected to enzymatic deglycosylation or directly eluted into 200 μl of SDS-sample buffer (62.5 mmTris-HCl, pH 6.8, 2% SDS, 20% glycerol, 100 mmdl-dithiothreitol, 0.005% bromphenol blue) at 60 °C for 20 min. Deglycosylation experiments were performed using peptideN-glycosidase F according to the manufacturer's protocol (New England Biolabs, Beverly, MA). For immunoprecipitation, the lysates were precleared with 50 μl of protein A-agarose beads (Calbiochem) for 2 h. After immunoprecipitation with 10 μg of either mouse monoclonal anti-T7, affinity-purified rabbit anti-c-Myc, affinity-purified rabbit anti-sst2A (6291), or affinity-purified rabbit anti-sst3 (7986) antibodies, immunocomplexes were collected using 100 μl of protein A-agarose beads. Beads were washed five times with detergent buffer, and immunoprecipitates were eluted from the beads with 200 μl of SDS-sample buffer at 60 °C for 20 min. Equal amounts of protein of each sample were then loaded onto regular 6% SDS-polyacrylamide gels, which contain 0.1% SDS. When indicated gels containing 2-fold (0.2%) or 4-fold (0.4%) higher SDS concentrations were run to test the sensitivity of dimers to stronger denaturing conditions. After electroblotting, membranes were incubated with either mouse monoclonal anti-T7, affinity-purified rabbit anti-c-Myc, affinity-purified rabbit anti-sst2A (6291) or affinity-purified rabbit anti-sst3 (7986) antibody at a concentration of 1 μg/ml for 12 h at 4 °C, followed by detection using an enhanced chemiluminescence detection system. Densitometric analysis of Western blots exposed in the linear range of the x-ray film was performed as described (31Schulz S. Pauli S.U. Handel M. Dietzmann K. Firsching R. Hollt V. Clin. Cancer Res... 2000; 6: 1865-1874Google Scholar). The amount of immunoreactive material in each lane was quantified using NIH Image 1.57 software. Cells were grown on poly-l-lysine-treated coverslips overnight and then exposed to agonists. Cells were fixed with 4% paraformaldehyde and 0.2% picric acid in phosphate buffer, pH 6.9, for 40 min at room temperature and washed several times in 10 mm Tris, 10 mmphosphate buffer, 137 mm NaCl, and 0.05% thimerosal, pH 7.4 (TPBS). Specimens were then incubated for 3 min in 50% methanol and for 3 min in 100% methanol and subsequently washed several times in TPBS and preincubated with TPBS and 3% normal goat serum for 1 h at room temperature. For single immunofluorescence, cells were then incubated with either mouse monoclonal anti-T7, affinity-purified rabbit anti-c-Myc, affinity-purified rabbit anti-sst2A(6291), or affinity-purified rabbit anti-sst3 (7986) antibody at a concentration of 1 μg/ml in TPBS 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 Pharmacia Biotech). For double immunofluorescence, cells were incubated either with a mixture of mouse monoclonal anti-T7 and affinity-purified rabbit anti-c-Myc or guinea pig anti-sst2A (GP3) and affinity-purified rabbit anti-sst3 (7986) 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-mouse or anti-guinea pig antibodies (1:200; Jackson ImmunoResearch, West Grove, PA). Cells were then dehydrated, cleared in xylol, and permanently mounted in DPX (Fluka, Neu-Ulm, Germany). 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 with 568-nm excitation and 570–630-nm band pass emission filters, and cyanine 5.18 with 647-nm excitation and 665-nm long pass emission filters. Confocal micrographs were taken by a person blinded to the treatments who was instructed to randomly select one colony of 4–12 cells per coverslip. Cells were harvested into PBS and stored at −80 °C. After thawing, cells were centrifuged at 20,000 × g for 5 min at 4 °C and then homogenized in lysis buffer (50 mm Tris-HCl, 3 mm EGTA, 5 mm EDTA, pH 7.4). Cell membranes were pelleted by centrifugation at 50,000 × g for 15 min at 4 °C, washed with lysis buffer, and resuspended in binding buffer (10 mm HEPES, 5 mm MgCl2, 5 μg/ml bacitracin, pH 7.5). For saturation binding assays, aliquots of the membrane preparations containing 25 μg of protein were incubated with increasing concentrations of [125I-Tyr11]SS-14 ranging from 0.025 to 0.3 nm. Receptor densities (Bmax) and ligand affinities (KD) were calculated as mean ± S.E. from four or five determinations performed in duplicate. Nonspecific binding was defined as that not displaced by 1 μm SS-14. For competition binding assays, aliquots of the membrane preparation containing 25 μg of protein were incubated with 0.05 nm [125I-Tyr11]SS-14 in the presence or absence of either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10−12 to 10−5m. The experiment for each concentration was performed in triplicate. Assays were performed in 96-well polypropylene plates in a final volume of 200 μl for 45 min at room temperature. The incubation was terminated by vacuum filtration through glass fiber filters presoaked in 0.1% polyethyleneimine using an Inotech cell harvester (Dittikon, Switzerland). Filters were rinsed twice with washing buffer (50 mm Tris-HCl, pH 7.4) and air-dried. Bound radioactivity was determined using a γ-counter. Protein content was determined by the Lowry method. Cells were harvested and lysed as described above except that a lysis buffer containing 50 mmTris and 10 mm EDTA (pH 7.4) was used. The resulting pellet was resuspended in assay buffer (20 mm HEPES, 100 mm NaCl, 10 mm MgCl2, pH 7.4), and aliquots containing 25 μg of protein were incubated with 3 μm GDP and 0.05 nm [35S]GTPγS in the presence or absence of either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10−12 to 10−6m. Assays were carried out in a final volume of 1 ml for 30 min at 30 °C under continuous agitation. Nonspecific binding was determined in the presence of 10 μm unlabeled GTPγS. The incubation was terminated by vacuum filtration through glass fiber filters as described above. Filters were rinsed twice with washing buffer (20 mm HEPES, pH 7.4). Scintillation mixture was added, and radioactivity was determined using a β-counter. The maximal effector response (Emax) was determined by stimulation with sst agonists at a concentration of 1 μm. 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, cells were either not preincubated or preincubated with 1 μm SS-14 for 1, 2, 4, or 6 h in Opti-MEM 1 (Life Technologies). The medium was then removed and replaced with 0.5 ml of serum-free RPMI medium containing 25 μm forskolin or 25 μm forskolin plus either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10−12 to 10−6m. The cells were incubated at 37 °C for 15 min. The reaction was terminated by removal of the culture medium and the subsequent addition of 1 ml of ice-cold HCl/ethanol (1 volume of 1 n HCl, 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 Pharmacia Biotech). Cells were seeded at a density of 1.5 × 105/well onto poly-l-lysine-treated 22-mm 12-well dishes and grown for 2 days in Dulbecco's modified Eagle's medium containing 0.5% fetal calf serum. Cells were then exposed to either SS-14, L-779,976, or L-796,778 in concentrations ranging from 10−12 to 10−6m for 5 min in RPMI medium without fetal calf serum at 37 °C. Incubation was terminated by removal of the culture medium and the subsequent addition of 300 μl of boiling SDS-sample buffer. Samples were heated to 95 °C for an additional 5 min period. 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 with 5% low fat dried milk dissolved in PBS containing 0.1% Tween 20, membranes were incubated with mouse monoclonal phosphospecific anti-ERK1/2 antibody clone E10 (1:1000; New England Biolabs). Blots were developed using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. Densitometric analysis of phospho-ERK1/2 levels on Western blots exposed in the linear range of the x-ray film was performed using NIH Image 1.57 software. Data from ligand binding, GTPγS, cAMP, and ERK assays were analyzed by nonlinear regression curve fitting using GraphPad Prism 3.0 software. To examine dimerization of the sst2A receptor, we used HEK 293 cells stably expressing either the T7-tagged sst2A alone (Bmax = 820 ± 19 fmol/mg of membrane protein) or stably coexpressing T7-tagged sst2A and Myc-tagged sst2A receptors were used. Western blot analysis of membrane extracts from these cells with the anti-sst2Aantibody (6291) revealed a predominant receptor band migrating at 80 kDa (Fig. 1 A). We also observed an additional band with a higher molecular mass migrating at 160 kDa (Fig. 1 A). These bands were not only detected with antibodies directed against the carboxyl terminus (6291, GP3) but also with antibodies directed against the amino-terminal added T7 or Myc tag (not shown). A similar ratio of these two immunoreactive bands was observed when the cells had been subjected to cross-linking using the cell-impermeable cross-linker BS3 prior to cell lysis (Fig.1 A). However, enzymatic deglycosylation reduced the size of the 80-kDa protein to 55 kDa and the size of the 160-kDa protein to 110 kDa, suggesting that the band with the higher molecular weight may consist of two sst2A receptor proteins (Fig.1 B). Exposure to SS-14 did not grossly modulate the dimer/monomer ratio (Fig. 1 C). The sst2A dimer was stable under reducing conditions. Similar levels of sst2A dimers were detected after incubation of the cells with 1 mm DTT for 30 min prior to cell lysis (Fig. 1 D). The addition of DTT to the SDS-sample buffer also did not reduce the level of sst2A dimers detected on Western blots. sst2A dimers were also stable in the presence of 2% SDS (e.g. heating to 60 °C for 20 min in SDS-sample buffer and loading onto regular SDS-polyacrylamide gels). However, when these samples were run on SDS-polyacrylamide gels containing 2- or 4-fold higher SDS concentrations, the sst2A dimer was destabilized in a concentration-dependent manner, and only the monomeric form of the receptor was detectable under these conditions (Fig.1 D). This suggests that the sst2A dimer is formed by noncovalent interactions of two receptor proteins. To directly examine the presence of sst2A receptor dimers, we used coimmunoprecipitation and Western blotting of differentially epitope-tagged sst2A receptors. HEK 293 cells coexpressing Mycsst2A and T7sst2A were lysed, and the receptors from these cells were immunoprecipitated using anti-c-Myc antibody. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-T7 antibody. As depicted in Fig. 1 E, anti-T7 antibody detected a single band migrating at 160 kDa, which represents the Mycsst2A-T7sst2A dimer. Receptor monomer was not detectable, suggesting that the coprecipitated sst2A dimers w
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