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• W2045681007 abstract "Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10–30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of32P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor U73122. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells. Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10–30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of32P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor U73122. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells. thyrotropin-releasing hormone TRH receptor G protein-coupled receptor GPCR kinase N-glycosidase F hemagglutinin casein kinase II The TRH1 receptor belongs to the superfamily of seven-transmembrane-helix G protein-coupled receptors (GPCRs) and plays a key role in maintaining proper function of the thyroid gland (1Gershengorn M.C. Osman R. Physiol. Rev. 1996; 76: 175-191Crossref PubMed Scopus (142) Google Scholar, 2Hinkle P.M. Ann. N. Y. Acad. Sci. 1989; 553: 176-187Crossref PubMed Google Scholar). Two subtypes of TRH receptors, termed type 1 and 2, have been identified (3Straub R.E. Frech G.C. Joho R.H. Gershengorn M.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9514-9518Crossref PubMed Scopus (211) Google Scholar, 4Cao J. O'Donnell D., Vu, H. Payza K. Pou C. Godbout C. Jakob A. Pelletier M. Lembo P. Ahmad S. Walker P. J. Biol. Chem. 1998; 273: 32281-32287Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 5Itadani H. Nakamura T. Itoh J. Iwaasa H. Kanatani A. Borkowski J. Ihara M. Ohta M. Biochem. Biophys. Res. Commun. 1998; 250: 68-71Crossref PubMed Scopus (87) Google Scholar). Although both receptor types are detected in various tissues at different levels (6O'Dowd B.F. Lee D.K. Huang W. Nguyen T. Cheng R. Liu Y. Wang B. Gershengorn M.C. George S.R. Mol. Endocrinol. 2000; 14: 183-193Crossref PubMed Scopus (95) Google Scholar, 7Calza L. Giardino L. Ceccatelli S. Zanni M. Elde R. Hokfelt T. Neuroscience. 1992; 51: 891-909Crossref PubMed Scopus (65) Google Scholar, 8Heuer H. Schafer M.K. O'Donnell D. Walker P. Bauer K. J. Comp. Neurol. 2000; 428: 319-336Crossref PubMed Scopus (159) Google Scholar), the type 1 TRH receptor is primarily expressed in thyrotrophs and lactotrophs in the anterior pituitary gland, and its activation stimulates the secretion of TSH and prolactin, at least in part, by raising the intracellular calcium concentration (9Yu R. Ashworth R. Hinkle P.M. Thyroid. 1998; 8: 887-894Crossref PubMed Scopus (26) Google Scholar, 10Hinkle P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar). The TRH receptor, once occupied by agonist, activates phospholipase C through Gq/11, leading to the formation of inositol 1,4,5-trisphosphate (InsP3), which causes an elevation of intracellular calcium by mobilizing an InsP3-sensitive Ca2+ store in the endoplasmic reticulum (10Hinkle P.M. Nelson E.J. Ashworth R. Trends Endocrinol. Metab. 1996; 7: 370-374Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 11Aragay A.M. Katz A. Simon M.I. J. Biol. Chem. 1992; 267: 24983-24988Abstract Full Text PDF PubMed Google Scholar). In conventional models, GPCRs have been thought to function as monomers that bind one molecule of ligand and then activate one heterotrimeric G protein to turn on the cognate signaling pathway (12Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (509) Google Scholar, 13Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (578) Google Scholar, 14Salahpour A. Angers S. Bouvier M. Trends Endocrinol. Metab. 2000; 11: 163-168Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Overton M.C. Blumer K.J. Curr. Biol. 2000; 10: 341-344Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 16Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (507) Google Scholar). However, evidence indicating that GPCRs can form homo- or heterodimers, pairing with the same receptor type, different subtypes within the same receptor family, or even distinct classes of receptors, has begun to emerge. Receptor dimerization has been documented for a wide variety of GPCRs including the β2-adrenergic receptor (17Angers S. Salahpour A. Joly E. Hilairet S. Chelsky D. Dennis M. Bouvier M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3684-3689PubMed Google Scholar, 18Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar), Ca2+-sensing receptor (19Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 20Ward D.T. Brown E.M. Harris H.W. J. Biol. Chem. 1998; 273: 14476-14483Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), muscarinic m3 receptor (21Zeng F.Y. Wess J. J. Biol. Chem. 1999; 274: 19487-19497Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), γ-aminobutyric acid GABAB receptor (22White 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 (1000) Google Scholar, 23Kaupmann 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 (1007) Google Scholar, 24Jones 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 (913) Google Scholar, 25Kuner R. Kohr G. Grunewald S. Eisenhardt G. Bach A. Kornau H.C. Science. 1999; 283: 74-77Crossref PubMed Scopus (493) Google Scholar), opioid receptor (25Kuner R. Kohr G. Grunewald S. Eisenhardt G. Bach A. Kornau H.C. Science. 1999; 283: 74-77Crossref PubMed Scopus (493) Google Scholar, 26George S.R. Fan T. Xie Z. Tse R. Tam V. Varghese G. O'Dowd B.F. J. Biol. Chem. 2000; 275: 26128-26135Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 27Cvejic S. Devi L.A. J. Biol. Chem. 1997; 272: 26959-26964Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar), GnRH receptor (28Cornea A. Janovick J.A. Maya-Nunez G. Conn P.M. J. Biol. Chem. 2001; 276: 2153-2158Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 29Horvat R.D. Roess D.A. Nelson S.E. Barisas B.G. Clay C.M. Mol. Endocrinol. 2001; 15: 695-703Crossref PubMed Scopus (55) Google Scholar), and others (26George S.R. Fan T. Xie Z. Tse R. Tam V. Varghese G. O'Dowd B.F. J. Biol. Chem. 2000; 275: 26128-26135Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar,30Ng G.Y. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (243) Google Scholar, 31Rocheville 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 (462) Google Scholar, 32AbdAlla S. Zaki E. Lother H. Quitterer U. J. Biol. Chem. 1999; 274: 26079-26084Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 33Romano C. Miller J.K. Hyrc K. Dikranian S. Mennerick S. Takeuchi Y. Goldberg M.P. O'Malley K.L. Mol. Pharmacol. 2001; 59: 46-53Crossref PubMed Scopus (122) Google Scholar, 34Tsuji Y. Shimada Y. Takeshita T. Kajimura N. Nomura S. Sekiyama N. Otomo J. Usukura J. Nakanishi S. Jingami H. J. Biol. Chem. 2000; 275: 28144-28151Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 35Nimchinsky E.A. Hof P.R. Janssen W.G. Morrison J.H. Schmauss C. J. Biol. Chem. 1997; 272: 29229-29237Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 36Xie Z. Lee S.P. O'Dowd B.F. George S.R. FEBS Lett. 1999; 456: 63-67Crossref PubMed Scopus (108) Google Scholar). The ligand binding and signaling properties of a number of GPCRs are modified as a result of receptor dimerization, suggesting functional relevance to this phenomenon (18Hebert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 271: 16384-16392Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar, 22White 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 (1000) Google Scholar, 26George S.R. Fan T. Xie Z. Tse R. Tam V. Varghese G. O'Dowd B.F. J. Biol. Chem. 2000; 275: 26128-26135Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 31Rocheville 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 (462) Google Scholar, 37Liu F. Wan Q. Pristupa Z.B., Yu, X.M. Wang Y.T. Niznik H.B. Nature. 2000; 403: 274-280Crossref PubMed Scopus (1) Google Scholar, 38Gines S. Hillion J. Torvinen M., Le Crom S. Casado V. Canela E.I. Rondin S. Lew J.Y. Watson S. Zoli M. Agnati L.F. Verniera P. Lluis C. Ferre S. Fuxe K. Franco R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8606-8611Crossref PubMed Scopus (386) Google Scholar, 39Jordan B.A. Devi L.A. Nature. 1999; 399: 697-700Crossref PubMed Scopus (966) Google Scholar, 40Pfeiffer 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 (289) Google Scholar, 41Ciruela F. Escriche M. Burgueno J. Angulo E. Casado V. Soloviev M.M. Canela E.I. Mallol J. Chan W.Y. Lluis C. McIlhinney R.A. Franco R. J. Biol. Chem. 2001; 276: 18345-18351Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 42Bai M. Trivedi S. Kifor O. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2834-2839Crossref PubMed Scopus (163) Google Scholar). Thus, GPCR dimerization appears to underscore a novel mechanism of modulating GPCR-mediated signal transduction or mediating “cross-talk” between different receptor families. Eidne and co-workers (43Kroeger K.M. Hanyaloglu A.C. Seeber R.M. Miles L.E. Eidne K.A. J. Biol. Chem. 2001; 276: 12736-12743Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) recently concluded that the TRH receptor forms dimers. Using transiently transfected COS cells, they found that bioluminescence resonance energy transfer occurs between receptors labeled with luciferase and yellow fluorescent protein and that TRH increases energy transfer (43Kroeger K.M. Hanyaloglu A.C. Seeber R.M. Miles L.E. Eidne K.A. J. Biol. Chem. 2001; 276: 12736-12743Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), implying that ligand binding either alters receptor conformation to bring the two reporter groups in closer proximity or promotes receptor dimerization. TRH receptors have also been reported to run at the molecular weight of dimers on SDS-PAGE (44Willars G.B. Heding A. Vrecl M. Sellar R. Blomenrohr M. Nahorski S.R. Eidne K.A. J. Biol. Chem. 1999; 274: 30146-30153Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). One caveat in the interpretation of these experiments is that the receptors were overexpressed, which would be expected to favor oligomerization. In the present study, we characterize dimerization and phosphorylation of epitope-tagged TRH receptors biochemically using human embryonic kidney HEK293 cells and a pituitary cell model system, both expressing receptors at levels no higher than those typical of endogenous pituitary receptors. We show that TRH receptor monomers and dimers are isolated from non-stimulated cells and that TRH increases receptor phosphorylation and dimerization in a dose- and time-dependent manner independent of signaling. HEK293 cells were obtained from the American Type Culture Collection. Sources of equipment and reagents were: pcDNA3 (Invitrogen), primers (synthesized by Genosys), DeepVent DNA polymerase (New England BioLabs), restriction enzymes and LipofectAMINE (Invitrogen), Geneticin, TRH, GF109203X (bisindolylmaleimide I), and protease inhibitor mixture (Calbiochem), alkaline phosphatase, M2 monoclonal anti-FLAG antibody, apigenin, and protein A-conjugated Sepharose 4B-CL beads (Sigma), peptide N-glycosidase F (PNGaseF) (Roche Molecular Biochemicals), U73122 (Biomol), chlordiazepoxide (ICN Pharmaceuticals, Inc.), HA11 monoclonal anti-HA antibody (Covance), horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences), wheat germ agglutinin (Vector Labs), Renaissance chemiluminescence reagent (PerkinElmer Life Sciences), [3H]MeTRH, [32P]orthophosphate, and [3H]inositol (PerkinElmer Life Sciences), fura2/AM (Molecular Probes), and mini-gel electrophoresis system (Bio-Rad). GHFT cells (45Lew D. Brady H. Klausing K. Yaginuma K. Theill L.E. Stauber C. Karin M. Mellon P.L. Genes Dev. 1993; 7: 683-693Crossref PubMed Scopus (116) Google Scholar) were provided by Dr. Richard N. Day (University of Virginia Medical School, Charlottesville, VA), plasmids encoding GRK2 and dominant negative GRK2-K220R were provided by Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA), and an HA-tagged β2-adrenergic receptor was provided by Dr. Richard Clark (University of Texas Health Science Center, Houston, TX). Type 1 rat TRH receptor was tagged at its amino terminus with either two repeats of hemagglutinin (HA) nonapeptide (YPYDVPDYA) separated by a Gly residue or with two repeats of a FLAG sequence (DYKDDDDK), also separated by a Gly residue, using polymerase chain reaction as described previously (46Kolodziej P.A. Young R.A. Methods Enzymol. 1991; 194: 508-519Crossref PubMed Scopus (423) Google Scholar). The forward primer for HA tagging carried a BamHI recognition site at its 5′ end followed by a Kozak sequence (47Kozak M. J. Mol. Biol. 1987; 196: 947-950Crossref PubMed Scopus (992) Google Scholar), HA-coding sequences, and a sequence derived from the first 18 nucleotides of the TRH receptor cDNA. The reverse primer was complimentary to the last 23 nucleotides (for making the full-length receptor) or to the region between nucleotides 978 and 1002, with the addition of a stop codon (for making a mutant receptor truncated after Leu-334) of the receptor cDNA with a flanking XbaI recognition site at its 5′ end. The fragments were amplified from a plasmid carrying type 1 rat TRH receptor cDNA (48Zhao D. Yang J. Jones K.E. Gerald C. Suzuki Y. Hogan P.G. Chin W.W. Tashjian A.H., Jr. Endocrinology. 1992; 130: 3529-3536Crossref PubMed Scopus (75) Google Scholar), digested withBamHI and XbaI, and subcloned into a mammalian expression vector, pcDNA3, yielding p2HA-TRHR andp2HA-CTTRHR, respectively, which encode full-length and carboxyl domain-truncated 2HA-tagged type 1 rat TRH receptors. Because the receptor containing two FLAG epitopes at the amino terminus did not localize to the cell membrane based on radioligand binding and immunolocalization, we introduced a prolactin signal peptide preceding the FLAG sequence to create pProl2FLAG-TRHR. Immunocytochemical analysis showed that the receptor encoded by this construct was expressed primarily on the cell membrane. All sequences were confirmed by nucleotide sequencing. HEK293 cell monolayers were grown in 6-cm dishes containing Dulbecco's modified Eagle's medium (DMEM) with 7.5% fetal bovine serum as described previously (49Yu R. Hinkle P.M. Mol. Endocrinol. 1998; 12: 737-749Crossref PubMed Google Scholar). GHFT cells were grown in DMEM/F-12 medium supplemented with 10% fetal bovine serum. Medium was changed every 2–5 days and replaced with serum-free medium 4–12 h before the cells were challenged with stimuli. Results were not different when serum starvation was omitted. For transient transfection, cell monolayers at ∼80% confluence were washed twice with serum-free medium and then overlaid with 1.6 ml/dish of transfecting complex prepared from 2 μg of plasmid DNA and 16 μl of LipofectAMINE in serum-free medium. After a 5-h incubation, cells were washed once with serum-containing medium, and culture was subsequently resumed in 2.5 ml of the same medium. Experiments were conducted 2 or 5 days after transfection. To create cell lines stably expressing TRH receptors, cells were transfected as described above and either 400 (GHFT cells) or 500 (HEK293 cells) μg/ml Geneticin was added to the culture medium to start selection 24 h after transfection (50Yu R. Hinkle P.M. Mol. Pharmacol. 1997; 51: 785-793Crossref PubMed Scopus (20) Google Scholar). Geneticin-resistant colonies were amplified and then screened for expression of TRH receptors; pools were then cloned. The established cell lines were maintained in the same media as the parental lines. Cells in 6-cm dishes were detached and harvested in 1 ml/dish of buffer containing 155 mm NaCl, 10 mm HEPES, and 1 mmEDTA, pH 7.4. After centrifugation at 500 × g for 1 min, cell pellets were resuspended in 300 μl of homogenization buffer containing 5 mm Tris-HCl, 2 mm EDTA, pH 7.4, plus 1:200 protease inhibitor mixture and then disrupted by 30 passages through 25-gauge needles. The unbroken cells were removed after centrifugation at 500 × g for 2 min, and the resulting supernatant was centrifuged at 14,000 × g for 10 min. The pellets were resuspended in 50 μl of homogenization buffer, and protein concentrations were quantified. Finally, the sample was mixed with an equal volume of sample buffer containing 100 mmTris-HCl, 200 mm dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol, pH 6.8, for further analysis. Cells in 6-cm dishes were solubilized by incubation for 30 min in 1 ml/dish of ice-cold lysis buffer containing 150 mm NaCl, 50 mm Tris base, 1 mm EDTA, 1% Triton X-100, pH 8.0, plus 1:200 protease inhibitor mixture. In the experiments shown in Figs. Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, phosphatase inhibitors were also included (10 mm sodium fluoride, 10 mm sodium pyrophosphate, 100 nmsodium orthovanadate, and 100 nm okadaic acid). The cell lysates were centrifuged at 14,000 × g, 4 °C, for 10 min. The supernatant fractions were collected and then incubated for 2–18 h at 4 °C with HA11 (1:5,000), a monoclonal antibody against HA, or M2 (1:5000), a monoclonal antibody against FLAG. The incubation was continued for another 2 h in the presence of protein A-conjugated Sepharose CL-4B (protein A beads) (5 mg/sample). The beads were washed 3 times with 1 ml of lysis buffer and, finally, resuspended in sample buffer, usually 75 μl.Figure 5Dimerization and phosphorylation of the carboxyl-terminally truncated mutant TRH receptor. Monolayers of HEK293 cells stably expressing HA-tagged CT-TRH receptors were stimulated with 1 μm TRH for 0, 1, 5, or 30 min. Cells were then lysed, immunoprecipitated and immunoblotted with HA11 anti-HA antibody. Arrowheads denote TRH receptors. HC, heavy chain.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6TRH receptor phosphorylation. A, monolayers of HEK293 cells stably expressing HA-tagged TRH receptors were untreated (left lane) or stimulated with 1 μm TRH for 5 min and then subjected to immunoprecipitation with anti-HA antibody, HA11. The immunopurified receptors were incubated with 0, 4, 20, or 100 units (U)/ml alkaline phosphatase (APP) for 1 h at 37 °C and were then analyzed in immunoblots with HA11. B, cells were preincubated for 2 h with vehicle or 10 μmchlordiazepoxide (CDE) and then incubated with 10 nm TRH for 0, 1, or 5 min. Samples were analyzed as inA. C, cells were preincubated for 1 h with no inhibitor, 10 μmU73122, 100 μmapigenin, or 10 μm GF109203X and then treated with or without 1 μm TRH for 5 min in the continued presence of drug. Receptors were absorbed to wheat germ agglutinin, deglycosylated, and immunoblotted with anti-HA antibody without immunoprecipitation.Arrowheads denote TRH receptors.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Effect of GRKs on TRH receptor phosphorylation. A, monolayers of HEK293 cells were co-transfected with plasmids encoding HA-tagged TRH receptor and either wild type GRK2 (wt GRK), dominant negative GRK2-K220R (dn GRK), or empty vector (Mock). The cells were stimulated with or without 1 μm TRH for 5 min and lysed and immunoblotted with HA11 anti-HA antibody without immunoprecipitation. Arrowheads denote TRH receptors.B and C, HEK293 cells were co-transfected with either HA-tagged β2-adrenergic receptor (B) or HA-tagged TRH receptor together (C) with wild type GRK2, dominant negative GRK2-K220R, or empty vector. B, cells expressing β2-adrenergic receptor were incubated for 30 min with or without 100 μm isoproterenol before the cells were fixed and stained with monoclonal anti-HA antibody. C, cells expressing TRH receptors were incubated for 30 min with or without 1 μm TRH before immunostaining. Slides were scored for the fraction of cells with receptors predominantly on the plasma membrane or cytoplasmic vesicles by observers unaware of the treatment group; 22–84 cells were scored. In each case, the open bars show untreated cells, and the dark bars show cells after agonist stimulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8TRH stimulation of incorporation of32P into receptors. A, HEK293 cells stably transfected with HA-tagged TRH receptors were labeled with [32P]orthophosphate and then treated with or without 1 μm TRH for 5 min. Cells were lysed and immunoprecipitated with HA11 anti-HA antibody. Immunoprecipitates were incubated without or with PNGaseF to deglycosylate receptors and then run on SDS-PAGE for standard immunoblotting with anti-HA antibody (left panels) or phosphorimaging (right panels).B, cells were treated as in A, except that U73122or vehicle was present during the last 30 min of the incubation with [32P]orthophosphate and during TRH stimulation.C, HEK293 cells were co-transfected with HA-tagged TRH receptors and wild type (wt GRK) or dominant negative (dn GRK) GRK2 or empty plasmid (none), as described for Fig. 6. In experiments shown in panels B andC, receptors were deglycosylated before SDS-PAGE; deglycosylation was incomplete, and a glycosylated monomer band can be seen. The dark circles show major 32P-labeled bands corresponding to receptor monomer and dimer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For deglycosylation (51Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), TRH receptors were immunopurified from a 6-cm dish of cells, and then the protein A beads were boiled for 2 min in 10 μl of 1% SDS. After dilution with 90 μl of 20 mmphosphate-buffered saline (pH 7.2), 50 mm EDTA, 0.5% Nonidet P-40, and 10 mm NaN3, the receptors were incubated at 37 °C for 10 h in the presence of 0.5 units of peptide PNGaseF. Alternatively, immunoprecipitates were incubated for 2 h at 37 °C in 50 μl of lysis buffer containing 0.14 m β-mercaptoethanol, 10 mmEDTA, 10 mm sodium azide, 5 mm EDTA, and 500 units of PNGaseF. For dephosphorylation (52Merello S. Parodi A.J. Couso R. J. Biol. Chem. 1995; 270: 7281-7287Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), immunopurified receptors from a 6-cm dish of cells were resuspended in 50 μl of 10 mm Tris-HCl, pH 8.0, and then incubated with up to 100 units/ml alkaline phosphatase at 37 °C for 30 min. Where noted, receptors were absorbed for 2–18 h at 4 °C on wheat germ agglutinin (20-μl suspension/1 ml of cell lysate) and then deglycosylated. In all enzymatic steps, control lanes show preparations that were incubated identically but without enzyme. Membrane preparations or immunopurified receptors were boiled for 2 min and resolved along with prestained molecular mass markers on 10% SDS-PAGE as described previously (36Xie Z. Lee S.P. O'Dowd B.F. George S.R. FEBS Lett. 1999; 456: 63-67Crossref PubMed Scopus (108) Google Scholar). When the boiling step was omitted and samples were run without heating or after heating to 37 °C, the ratio of monomers to dimers was not altered, but much of the receptor did not enter the gel. Proteins were transferred onto a nitrocellulose membrane, which was then subjected to two sequential 1-h incubations with primary (1:10,000 HA11, or M2) and secondary (1:2,000 horseradish peroxidase-conjugated anti-mouse IgG) antibodies, respectively, and immunoreactivity was detected by chemiluminescence. All blots are representative of experiments repeated 2–6 times. In some experiments, the apparent intensity of TRH receptor bands increased after TRH treatment. Control experiments confirmed that equal amounts of protein had been loaded per lane and that all immunoreactive receptors had been solubilized by lysis buffer and entered the gel, suggesting that the immunoreactivity of receptor may be increased after TRH treatment. Cells on 6-cm dishes were washed twice and incubated for 1–2 h in phosphate-free Dulbecco's modified Eagle's medium. Cells were then incubated for 3–4 h in the same buffer containing 0.2–0.5 mCi/ml [32P]orthophosphate, washed 3 times, lysed, and treated as described above in buffer with protease and phosphatase inhibitors. Immunoprecipitates from each dish were suspended in 50–65 μl of sample buffer, in some cases after deglycosylation with PNGaseF or dephosphorylation with potato acid phosphatase; 5–10 μl were subsequently used for Western blots, and 10–30 μl were used for phosphorimaging. Lanes contained equal amounts of trichloroacetic acid-precipitable 32P. Samples were transferred to nitrocellulose paper and either immunoblotted or analyzed for 32P-containing bands on a Molecular Dynamics PhosphorImager. To identify TRH receptors by immunocytochemistry, cells were grown on glass coverslips, fixed with paraformaldehyde, and stained with HA11 (1:1000) followed by rhodamine-labeled anti-mouse IgG (1:200) as previously described (49Yu R. Hinkle P.M. Mol. Endocrinol. 1998; 12: 737-749Crossref PubMed Google Scholar). To measure calcium responses, cells were grown on glass coverslips and loaded with fura2/AM in buffered Hanks' balanced salt solution at room temperature. The cells were washed and incubated in the same buffer at 37 °C. Cells were alternately excited with 340- and 380-nm light, and fluorescence emission was measured at 490 nm; 340/380 fluorescence ratios were determined every 1200 ms (53Nelson E.J. Hinkle P.M. Endocrinology. 1994; 135: 1084-1092Crossref PubMed Scopus (23) Google Scholar). Proteins were determined by the Lowry or Bradford methods using bovine serum albumin as standard. Spe" @default.
• W2045681007 created "2016-06-24" @default.
• W2045681007 creator A5005557653 @default.
• W2045681007 creator A5008490648 @default.
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• W2045681007 date "2002-08-01" @default.
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• W2045681007 cites W1548575930 @default.
• W2045681007 cites W1571586310 @default.
• W2045681007 cites W1595449036 @default.
• W2045681007 cites W1600425874 @default.
• W2045681007 cites W1606732154 @default.
• W2045681007 cites W1654156243 @default.
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• W2045681007 cites W1821817384 @default.
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• W2045681007 cites W1976637929 @default.
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• W2045681007 cites W2035103640 @default.
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• W2045681007 cites W2062742229 @default.
• W2045681007 cites W2063562477 @default.
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