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• W2155430832 abstract "G protein-coupled receptors mediate cell responses to extracellular stimuli and likely function in the context of a larger signal transduction complex. Utilizing the third intracellular loop of a G protein-coupled receptor in glutathione S-transferase pulldown assays from rat brain lysates coupled with high sensitivity detection methods and subsequent functional studies, we report the identification of SET as a regulator of muscarinic receptor signaling. SET is a putative oncogene reported to inhibit protein phosphatase 2A and regulate gene transcription. SET binds the carboxyl region of the M3-muscarinic receptor i3 loop, and endogenous SET co-immunoprecipitates with intact M3 muscarinic receptor expressed in cells. Small interfering RNA knockdown of endogenous SET in Chinese hamster ovary cells stably expressing the M3 muscarinic receptor augmented receptor-mediated mobilization of intracellular calcium by ∼35% with no change in agonist EC50, indicating that interaction of SET with the M3 muscarinic receptor reduces its signaling capacity. SET knockdown had no effect on the mobilization of intracellular calcium by the P2-purinergic receptor, ionomycin, or a direct activator of phospholipase C, indicating a specific regulation of M3 muscarinic receptor signaling. These data provide expanded functionality for SET and a previously unrecognized mechanism for regulation of GPCR signaling capacity. G protein-coupled receptors mediate cell responses to extracellular stimuli and likely function in the context of a larger signal transduction complex. Utilizing the third intracellular loop of a G protein-coupled receptor in glutathione S-transferase pulldown assays from rat brain lysates coupled with high sensitivity detection methods and subsequent functional studies, we report the identification of SET as a regulator of muscarinic receptor signaling. SET is a putative oncogene reported to inhibit protein phosphatase 2A and regulate gene transcription. SET binds the carboxyl region of the M3-muscarinic receptor i3 loop, and endogenous SET co-immunoprecipitates with intact M3 muscarinic receptor expressed in cells. Small interfering RNA knockdown of endogenous SET in Chinese hamster ovary cells stably expressing the M3 muscarinic receptor augmented receptor-mediated mobilization of intracellular calcium by ∼35% with no change in agonist EC50, indicating that interaction of SET with the M3 muscarinic receptor reduces its signaling capacity. SET knockdown had no effect on the mobilization of intracellular calcium by the P2-purinergic receptor, ionomycin, or a direct activator of phospholipase C, indicating a specific regulation of M3 muscarinic receptor signaling. These data provide expanded functionality for SET and a previously unrecognized mechanism for regulation of GPCR signaling capacity. G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; i3 loop, third intracellular loop; MALDI-TOF, matrix-assisted laser-desorption ionization time-of-flight; MR, muscarinic receptor; M3-i3, i3 loop of M3-MR; PP2A, protein phosphatase 2A; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; FBS, fetal bovine serum; NMS, N-methyl scopolamine. 2The abbreviations used are: GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; i3 loop, third intracellular loop; MALDI-TOF, matrix-assisted laser-desorption ionization time-of-flight; MR, muscarinic receptor; M3-i3, i3 loop of M3-MR; PP2A, protein phosphatase 2A; siRNA, small interfering RNA; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase; FBS, fetal bovine serum; NMS, N-methyl scopolamine. represent the largest family of membranous receptors and process signals from a great diversity of endogenous and exogenous stimuli including biogenic amines, peptides, glycoproteins, lipids, nucleotides, ions, proteases, photons, odors, and taste. GPCRs possess a characteristic seven transmembrane domains linked by three extracellular and three intracellular loops. Interaction of agonist with the receptor initiates conformational changes within the receptor that are propagated to intracellular domains of the receptor, resulting in G protein activation and initiation of intracellular events. A long-term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation from GPCRs to G protein and perhaps function in the context of a larger signal transduction complex. Such proteins may influence receptor targeting to specific subcellular compartments, assembly of the receptor into functional complexes (i.e. receptosomes), and receptor trafficking to and from the plasma membrane as well as effector activation (1Sato M. Blumer J.B. Simon V. Lanier S.M. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 151-187Crossref PubMed Scopus (131) Google Scholar, 2Bockaert J. Fagni L. Dumuis A. Marin P. Pharmacol. Ther. 2004; 103: 203-221Crossref PubMed Scopus (219) Google Scholar).The third intracellular loop (i3 loop) and the carboxyl terminus tail of the receptor are key sites for signal initiation and termination for most members of GPCRs. These regions can be of considerable size and represent potential sites for protein interaction.Carboxyl-terminal domains of GPCRs contain discrete motifs that efficiently interact with cellular proteins such as the PDZ (PSD95-disc large-Zonula occludens) recognition motif (3Bockaert J. Marin P. Dumuis A. Fagni L. FEBS Lett. 2003; 546: 65-72Crossref PubMed Scopus (185) Google Scholar). Other proteins interact with the carboxyl termini of some GPCRs via Src homology 2 (SH2) and SH3, pleckstrin homology, or Ena/VASP homology (EVH) domains (3Bockaert J. Marin P. Dumuis A. Fagni L. FEBS Lett. 2003; 546: 65-72Crossref PubMed Scopus (185) Google Scholar). Although the i3 loop is one of the most important domains of GPCRs for their interaction with G proteins, only a few proteins have been identified as binding to this region (2Bockaert J. Fagni L. Dumuis A. Marin P. Pharmacol. Ther. 2004; 103: 203-221Crossref PubMed Scopus (219) Google Scholar, 4Wu G. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Wu G. Bogatkevich G.S. Mukhin Y.V. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 2000; 275: 9026-9034Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 7DeGraff J.L. Gurevich V.V. Benovic J.L. J. Biol. Chem. 2002; 277: 43247-43252Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Wang Q. Limbird L.E. J. Biol. Chem. 2002; 277: 50589-50596Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 9Bernstein L.S. Ramineni S. Hague C. Cladman W. Chidiac P. Levey A.I. Hepler J.R. J. Biol. Chem. 2004; 279: 21248-21256Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 10Lucas J.L. Wang D. Sadee W. Pharm. Res. (N. Y.). 2006; 23: 647-653Crossref PubMed Scopus (23) Google Scholar, 11Budd D.C. McDonald J.E. Tobin A.B. J. Biol. Chem. 2000; 275: 19667-19675Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).We previously identified interaction sites for Gβγ subunits and β arrestins within the i3 loops of M2 and M3 muscarinic receptors (M2-/M3-i3) (4Wu G. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Wu G. Bogatkevich G.S. Mukhin Y.V. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 2000; 275: 9026-9034Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The binding of Gβγ to the i3 loop results in formation of a ternary complex consisting of the i3 loop, Gβγ, and G protein-coupled receptor kinase 2, postulated to position the enzyme on its substrate. In addition to its role in receptor regulation, the interaction of Gβγ or arrestin with the i3 loop may also facilitate the interface of GPCRs to diverse signaling pathways via complex formation with additional Gβγ- and arrestin-binding proteins. Other proteins including calmodulin, RGS2, and casein kinase 1α also interact with the M2-i3 and/or M3-i3 loops (9Bernstein L.S. Ramineni S. Hague C. Cladman W. Chidiac P. Levey A.I. Hepler J.R. J. Biol. Chem. 2004; 279: 21248-21256Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 10Lucas J.L. Wang D. Sadee W. Pharm. Res. (N. Y.). 2006; 23: 647-653Crossref PubMed Scopus (23) Google Scholar, 11Budd D.C. McDonald J.E. Tobin A.B. J. Biol. Chem. 2000; 275: 19667-19675Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Casein kinase 1α phosphorylates the M3-MR controlling the magnitude of M3 muscarinic signaling.The i3 loop does not generally contain obvious predicted binding motifs or structural properties other than the two α helixes located at the juxtamembrane regions of the i3 loop. The small number of proteins interacting with the i3 loop relative to the carboxyl-terminal tail of GPCRs may be due to the weakness of the interaction itself and/or limited sensitivity of techniques used to isolate binding proteins.As part of a broader effort to define protein complexes with the i3 loop, we initially used the i3 loop of the M2-MR, which is characterized by a larger i3 loop (180 amino acids), as a GST fusion protein to isolate binding partners from brain lysates. We took advantage of recent technologies that enhance sensitivity for detecting specific interactions utilizing fluorescent protein labeling and two-dimensional gel electrophoresis. Using this approach we report the identification of the protein SET as a surprising binding partner with the i3 loop of M2-MR. SET, also called template-activating factor I, was first identified as a partner of the SET-CAN fusion gene, a putative oncogene associated with an acute undifferentiated leukemia (12von Lindern M. van Baal S. Wiegant J. Raap A. Hagemeijer A. Grosveld G. Mol. Cell. Biol. 1992; 12: 3346-3355Crossref PubMed Scopus (354) Google Scholar). SET is reported to regulate transcription and inhibit protein phosphatase 2A (PP2A) (13Li M. Makkinje A. Damuni Z. J. Biol. Chem. 1996; 271: 11059-11062Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 14Seo S.B. McNamara P. Heo S. Turner A. Lane W.S. Chakravarti D. Cell. 2001; 104: 119-130Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), which may be of particular interest for receptor regulation and signaling. In this study we report that SET directly interacts with the i3 loop of the M2- and M3-MRs, co-immunoprecipitates with intact receptor expressed in cells and specifically inhibits M3-MR-dependent signaling. These data suggest that SET/i3 loop interaction within the M3-MR leads to reduced signaling capacity for the receptor, providing an unexpected mode of regulation for GPCR coupling to downstream signaling pathways.EXPERIMENTAL PROCEDURESRecombinant Protein Preparations—The cDNAs encoding the i3 loop of the human M2- and rat M3-MRs (His208-Arg387 and Arg252-Gln490, respectively) were subcloned into pGEX-2T or pGEX-4T-1 vectors, respectively (4Wu G. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The M3-MR i3 loop subdomains (Arg252-Gln389, Gly308-Gln389, Val390-Gln490, Lys-369-Thr424, Lys425-Gln490, Thr450-Gln490, Lys425-Gln474) were generated using the full-length M3-MR i3 loop as template and cloned into the BamHI and EcoRI restriction sites of the PGEX-4T-1 vector as described previously (5Wu G. Bogatkevich G.S. Mukhin Y.V. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 2000; 275: 9026-9034Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). To generate the M3-MR Ile474-Gln490 construct, complementary oligonucleotides from this region were synthesized and annealed before ligation into the BamHI and EcoRI restriction sites of the PGEX-4T-1 vector. GST fusion proteins were expressed in BL21 cells and purified on glutathione-Sepharose 4B (GE Healthcare) as described previously (5Wu G. Bogatkevich G.S. Mukhin Y.V. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 2000; 275: 9026-9034Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Immobilized fusion proteins were stored at 4 °C, and each batch of fusion proteins used for experiments was first analyzed by SDS-PAGE and Coomassie Blue staining. The full-length encoding sequence of human SET cloned into the pQE30 vector was kindly provided by Dr. R. Z. Qi (Hong Kong University of Science and Technology). The His-tagged SET protein was expressed in M15 bacteria and purified on Ni2+-nitrilotriacetic acid beads (Qiagen Inc.) according to the manufacturer's instructions.Protein Interaction Assays—Rat brains were homogenized on ice with a Dounce homogenizer in 3 ml of hypotonic lysis buffer (5 mm Tris-HCl, pH 7.5, 5 mm EDTA, 5 mm EGTA)/g of tissue. All buffers contained a protease inhibitor mixture (1 tablet/10 ml, Complete Mini, EDTA-free, Roche Diagnostics). The tissue homogenate was centrifuged at 100,000 × g for 30 min at 4 °C to generate a cytosolic fraction. To reduce nonspecific interactions with GST alone or glutathione resin, brain cytosol was pre-cleared by successive incubations with GST bound to the glutathione-Sepharose 4B matrix followed by an incubation with the glutathione-Sepharose 4B matrix alone (Fig. 1A). Pre-cleared brain cytosol (20 mg) was diluted 5 times (4 mg/ml) in buffer A (50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 5 mm EGTA, 5 mm EDTA) and incubated overnight at 4 °C with GST or GST-M2-i3 (∼400 nm) pre-complexed with the glutathione-Sepharose 4B matrix (40 μl). Resins were then washed 2 times with 5 ml of buffer A containing 150 mm NaCl followed by 2 washes in the same buffer without NaCl. Bound proteins were eluted with 35 μl of 30 mm Tris-HCl, pH 8.5, 7 m urea, 2 m thiourea, and 4% CHAPS by rotation for 2 h at 4°C.Two-dimensional Fluorescence Difference Gel Electrophoresis—Brain cytosol proteins eluted from GST or GST-M2-i3 resins were labeled with 400 pmol of the CyDye fluorochromes Cy2 or Cy5, respectively (GE Healthcare), by coupling of the dye to ϵNH2 group of lysine residues in a volume of 35 μl. The stoichiometry of CyDye labeling is specifically intended to achieve a minimal labeling of lysines (1-2% of all proteins). As an additional control, proteins eluted from GST-M2-i3 resin alone were labeled with Cy3. Labeling was performed on ice in the dark at pH 8.5 for 30 min incubation, after which the reaction was quenched by the addition of 1 μl of 10 mm lysine for 10 min. An equal volume of rehydration buffer (30 mm Tris, pH 8.5, 7 m urea, 2 m thiourea, 4% CHAPS, 130 mm dithiothreitol) containing 2% Bio-Lyte 3/10 ampholytes (Bio-Rad) was added, and samples were incubated at 24 °C for 10 min. The three samples labeled with Cy2, Cy3, or Cy5 were combined in the presence of an equal volume of DeStreak (GE Healthcare), and proteins were separated on first and second dimensions.Two-dimensional Gel Electrophoresis—Proteins were first separated according to their isoelectric point along a linear immobilized pH-gradient strip (pH 3-10, 24 cm long) using the PROTEAN IEF cell (Bio-Rad). Sample loading was performed by active in-gel re-swelling (50 V), and protein separated by applying the following parameters: 200 V for 3 h and 1000 V for 4 h gradient; 8000 V for 8 h gradient; 8000 V for 4 h; 200 V for 48 h. After the first dimension the immobilized pH-gradient strips were equilibrated for 15 min in a buffer containing 6 m urea, 0.375 mm Tris, pH 8.8, 2% SDS, 20% glycerol, 2% dithiothreitol, and then for 15 min in the same buffer containing 2.5% iodoacetamide in place of dithiothreitol. For the second dimension, the strips were loaded onto manual cast 12.5% SDS-polyacrylamide DALT gels (GE Healthcare). Proteins were separated using the following parameters: 30 min at 5 watts/gel and 4 h at 17 watts/gel. After electrophoresis gels were fixed in a solution of 7% acetic acid and 10% methanol for 30 min.Image Acquisition and Two-dimensional Gel Spot Pattern Analysis—Fluorophore-labeled protein gels were scanned using a Typhoon 9400 Variable Mode Imager at 100 μm resolution, with the photomultiplier tube set in the 425-500 V range (GE Healthcare). CyDyes are optimally detected using the following wavelength settings: Cy2, excitation 488 nm, emission 520 nm; Cy3, excitation 532 nm, emission 580 nm; Cy5, excitation 633 nm, emission 670 nm. Spot detection and quantification were performed using the DeCyder differential analysis software DIA, Version 5.0 (GE Healthcare). Gels were then post-stained with Sypro Ruby, and images were captured again using a Typhoon 9400 Variable Mode Imager. Protein spots of interest were excised using the Ettan Spot Handling Work station with a 2-mm diameter spot-picking head (GE Healthcare).Protein Identification by Matrix-assisted Laser-desorption Ionization Time-of-flight (MALDI-TOF) Mass Spectrometry—Gel plugs were destained by incubation in 20 mm ammonium bicarbonate and 50% acetonitrile (3 times, 30 min each) followed by dehydration with 100% acetonitrile (20 min). Dehydrated gel plugs were automatically digested via the Spot Handling Work station (GE Healthcare) in-gel with 20 mm ammonium bicarbonate solution containing 20 ng/ml porcine modified trypsin protease (Promega). The tryptic peptide mixtures were extracted from the gel plugs in 2 cycles of 50% acetonitrile, 0.1% trifluoroacetic acid and dried by evaporation. Peptides were reconstituted in 50% acetonitrile, 0.1% trifluoroacetic acid and mixed with an equal volume of 10 mg/ml α-cyano-4-hydroxycinnamic acid for spotting in duplicate onto a MALDI plate.MALDI-TOF-mass spectrometry and tandem TOF/TOF mass spectrometry were performed on a Voyager 4700 (Applied BioSystems). Ions specific for each sample (discrete from background and trypsin-derived ions) were then used to interrogate sequences entered in the SWISS-PROT and NCBI non-redundant databases using MASCOT. Tandem mass spectrometry was used to generate limited amino acid sequence information on selected ions if additional confirmation was required. Searches were performed without constraining protein molecular weight or isoelectric point and allowed for carbamidomethylation of cysteine, partial oxidation of methionine residues, and one missed trypsin cleavage. Highest confidence identifications have a statistically significant search score(s) and are consistent with the gel region from which the protein was excised (Mr and pI).GST Pulldown Assay with Purified SET—Human recombinant His-tagged SET protein (30 nm) was gently mixed for 1 h at 4 °C with GST-M2-i3, GST-M3-i3 (full-length or truncated fragments), or GST fusion proteins (∼300 nm) bound to the glutathione-Sepharose 4B (12.5 μl) in 750 μl of buffer B (25 mm Tris-HCl, pH 7.5, 1 mm EDTA, 100 mm NaCl, 1 mm dithiothreitol, 0.1% Nonidet P-40, and protease inhibitors). Resins were washed 3 times with 500 μl of buffer B. The retained proteins were eluted from the resin with 25 μl of 5× loading buffer, placed in a boiling water bath for 5 min, and applied to 10% SDS-polyacrylamide gels. Separated proteins were then transferred to a polyvinylidene difluoride membrane and process for immunoblotting with a polyclonal anti-SET antibody (1:1000) kindly provided by Dr. T. D. Copeland (15Adachi Y. Pavlakis G.N. Copeland T.D. J. Biol. Chem. 1994; 269: 2258-2262Abstract Full Text PDF PubMed Google Scholar) (NCI-Frederick, National Institutes of Health). Membranes were systematically re-probed with an anti-GST antibody (1:5000, GE Healthcare) to confirm equal amounts of GST fusion proteins and to control for protein loading.Immunoprecipitation—100-mm dishes of COS-7 cells were transfected with 12 μg of empty vector (pcDNA3) or 12 μg of pcDNA3::M3MR with Lipofectamine 2000 according to manufacturer's instructions (Invitrogen). After 72 h cells were lysed in Nonidet P-40 lysis buffer and incubated on ice for 1 h. The lysate was centrifuged at 12,000 × g for 30 min at 4 °C and precleared with Gamma-Bind Sepharose (GE Healthcare). The precleared lysates (5 mg of protein in 1 ml of lysis buffer) were incubated with 25 μl of monoclonal M3-MR antibody overnight at 4 °C. Gamma-Bind G-Sepharose (12.5 μl) was added, and incubation was continued for 1 h. The resin was pelleted, washed 3 times with Nonidet P-40 lysis buffer, resuspended in 2× protein sample buffer, and incubated at room temperature for 30 min followed by SDS-PAGE and immunoblotting with polyclonal anti-M3MR antibody (provided by Dr. Jurgen Wess, National Institutes of Health, Bethesda) (16Zeng F.Y. Wess J. J. Biol. Chem. 1999; 274: 19487-19497Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) and SET antibody. Confluent 100-mm plates of CHO cells stably expressing the M3-MR (CHO-M3, ∼400 fmol/mg protein) and control CHO cells were lysed and processed for M3-MR immunoprecipitation as described above.The mouse monoclonal M3-MR antibody was generated against the mouse M3-MR third intracellular loop (Arg252-Thr491) purified as a GST fusion protein (17Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar) and prepared by Harlan Sera Labs (United Kingdom). The antibody recognizes the rat, mouse, and human M3-MR. siRNA-mediated Gene Silencing of SET—siRNA duplexes 5′-CCAUCCACAAGUGUCUGCACUGCUU (bp 327-351) and 5′-CCGAAAUCAAAUGGAAAUCUGGAAA (bp 530-554) targeted to the human SET mRNA sequence (GenBank™ accession number D45198) were selected based upon optimal sequences as predicted by computational programs provided by Invitrogen. Control CHO cells and CHO-M3 cells at 30-40% confluency were transfected with a combination of SET siRNA duplexes (bp 327-351 and 530-554) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). In control experiments cells were transfected with the corresponding predicted oligonucleotide control for the siRNA duplex bp 327-351 (5′-CCAACACGUGAGUCUUCACGCUCUU) together with a random low GC oligonucleotide control provided by Invitrogen. The transfection mixture was removed 5 h later and replaced by F-12 medium containing 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin-streptomycin (Invitrogen).To assess SET siRNA knockdown, cells were homogenized in lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40) and incubated for 1 h on ice. Cell homogenates were centrifuged at 10,000 × g at 4 °C, and 12.5 μg of the supernatant were separated on a 10% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. Expression of SET was assessed by immunoblotting with a polyclonal anti-SET antibody. The specificity of the knockdown for SET was validated by reprobing blots with antisera to α-actin (Chemicon international) and Gq.The Gq antibody was generated against the carboxyl-terminal 10 amino acids of Gq (QLNLKEYNLV) cross-linked to KLH via a cysteine added to the amino terminus when the peptide was synthesized. Rabbits were immunized with the conjugate, and the antibody was purified on a protein A-Sepharose resin (GE Healthcare) (18Arthur J.M. Collinsworth G.P. Gettys T.W. Quarles L.D. Raymond J.R. Am. J. Physiol. 1997; 273: F129-F135PubMed Google Scholar).Measurement of Cell Calcium Content—To evaluate receptor-effector coupling, we determined the ability of agonist to increase intracellular calcium in CHO control and CHO-M3 cells using a fluorometric imaging plate reader system (Molecular Devices Corp.). Seventy-two hours after siRNA transfection, CHO-M3 cells were seeded in F-12 medium containing 2% FBS and 1% penicillin-streptomycin (35,000 cell/100 μl/well) in 96-well clear-bottomed black microplates (Corning Costar Corp.) precoated with 100 μg/ml poly-d-lysine (Sigma-Aldrich). Four hours later cells were dye-loaded (FLIPR calcium 3 assay kit, Molecular Devices) with 100 μl of the dyeloading buffer containing 2.5 mm probenecid for 1 h at 37°C in a 5% CO2 incubator. During a data run cells in different wells were exposed to different concentrations of drugs, and the system recorded fluorescent signals for all 96 wells simultaneously every 5 s for 5 min. Increases in intracellular calcium were observed as sharp peaks above the basal fluorescent levels typically 10 s after drug addition. The increases in intracellular calcium levels were determined by subtracting the base-line to the peak values (heights). Data were plotted, and t½ to peak values and for signal decay were determined using Graphpad Prism (version 4.0). Data are representative of 4-7 independent experiments; the data point for any drug concentration is an average from 3 to 4 wells.Quantification of Cell Surface M3-muscarinic Receptor—Cell surface receptor number in CHO-M3 transfectants was determined by radioligand binding using [3H]NMS as described previously (5Wu G. Bogatkevich G.S. Mukhin Y.V. Benovic J.L. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 2000; 275: 9026-9034Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Seventy-two hours after siRNA transfection, ∼500,000 cells from one 100-mm plate were plated in a 12-well plate. Parallel immunoblots indicated effective knockdown of SET as described above. Four hours later cells were placed on ice and washed 3 times with 1 ml of ice-cold cell washing solution (137 mm NaCl, 2.6 mm KCl, 1.8 mm KH2PO4, 10 mm Na2HPO4) and incubated with the muscarinic plasma membrane-impermeable antagonist [3H]NMS (2 nm) in the presence or absence of atropine (10 μm) at 4 °C for 4 h. Cells were then washed 3 times with 1 ml of ice-cold cell washing solution, and 0.5 ml of cell washing solution containing 1% Triton X-100 was added to each well. Cells were scraped and transferred to vials containing 3.5 ml of Ecoscint A (National Diagnostics) for analysis by scintillation spectrometry. Under these experimental conditions, total [3H]NMS binding in intact cells ranged from 20,000 to 30,000 disintegrations/min, and nonspecific [3H]NMS binding was <3% of the total binding.RESULTSIsolation of Proteins Interacting with the i3 Loop of M2-muscarinic Receptor—As part of a broader effort to identify components of a GPCR signaling complex, we focused on the subgroup of GPCRs possessing a larger i3 loop such as found in the muscarinic receptors. We constructed a GST fusion protein containing the i3 loop of the M2-MR (180 amino acids, GST-M2-i3) and used it as an affinity matrix to pull down proteins from rat brain cytosol (Fig. 1A). GST-M2-i3 or GST fusion proteins were expressed in BL21 bacteria and purified by binding to glutathione-Sepharose 4B beads (Fig. 1A). We took advantage of recent technologies that enhance the sensitivity for detecting specific interactions and which involve fluorescent protein labeling and two-dimensional gel electrophoresis (Fig. 1B). We labeled proteins bound to the GST control or to the GST-M2-i3 affinity resins with different CyDye fluorochromes. We labeled proteins bound to GST-M2-i3 with Cy5 and proteins bound to the GST control with Cy2 (Fig. 1B). As an additional control, we used Cy3 to label the GST-M2-i3 fusion protein itself without preincubation with brain cytosol. The three sets of labeled proteins were then mixed and separated on a single two-dimensional gel to eliminate intergel variability often encountered by processing individual samples on different gels. This strategy allowed us to clearly identify proteins that interact specifically with the M2-i3 loop with a high degree of sensitivity by virtue of the CyDye fluorescent tags.Fig. 1C presents the results of a representative CyDye labeling experiment with the two-dimensional gel pattern obtained for each set of labeled proteins. Each spot corresponds to a protein separated according to its apparent molecular weight and its isoelectric point (pI). The green and red signals with Mr ∼ 56,000 correspond to the GST-M2-i3 fusion protein (Cy3 and Cy5 pictures). The green spots below the Mr ∼ 56,000 likely correspond to fragments of the Mr ∼ 56,000 species, GST-M2-i3 fusion protein (Cy3 picture). The blue signal with an Mr ∼ 29,000 in the Cy2 picture corresponds to the GST fusion protein. All these three fluorescent pictures derive from the same single two-dimensional gel.On the overlay picture containing the three fluorescent signals (Fig. 2, left panel), the merged colors yellow and purple (i.e. spots a and b) correspond to proteins present not only in the sample labeled with Cy5 but also in the sample labeled with Cy3 or Cy2. This is illustrated on the right panel of Fig. 2 where enlarged areas of the two-dimensional gel containing the Cy5 nonspecific spots a and b are shown in the three channels, Cy5, Cy3, and Cy2. The corresponding quantification of each spot in all three channels is represented by the histograms. Spot a is present in the Cy5 and Cy3 channels and relates to the GST-M2-i3 fusion protein itself, whereas spot b present in the Cy5 and Cy2 channels relates to a brain protein that interacts with the GST fusion protein (control). We identified two red spots, spots c and d, present only in the Cy5 channel (Fig. 2, right panel, Cy5-specific spots). These spots correspond to two proteins that bind specifically the M2-i3 loop. To identify these tw" @default.
• W2155430832 created "2016-06-24" @default.
• W2155430832 creator A5016119036 @default.
• W2155430832 creator A5017271161 @default.
• W2155430832 creator A5020328262 @default.
• W2155430832 creator A5035925879 @default.
• W2155430832 creator A5082070342 @default.
• W2155430832 date "2006-12-01" @default.
• W2155430832 modified "2023-10-18" @default.
• W2155430832 title "The Proto-oncogene SET Interacts with Muscarinic Receptors and Attenuates Receptor Signaling" @default.
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