FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2055078479> ?p ?o ?g. }

• W2055078479 endingPage "36600" @default.
• W2055078479 startingPage "36593" @default.
• W2055078479 abstract "A multifunctional enzyme, Gh, is a GTP-binding protein that couples to the α1B-adrenoreceptor and stimulates phospholipase C-δ1 but also displays transglutaminase 2 (TG2) activity. Gh/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of Gh/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the α1-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of Gh/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the α1B-adrenoreceptor with the α1 agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. Gh/TG2 is coimmunoprecipitated by an integrin α5 antibody and binds to the cytoplasmic tail peptide of integrins α5, αv, and αIIb subunits in the presence of guanosine 5′-3-O-(thio)triphosphate (GTPγS). Mutation of Lys-Arg residues in the GFFKR motif, present in the α5-tail, significantly reduces the binding of GTPγS-Gh/TG2. Moreover, the motif-containing integrin α5-tail peptides block Gh/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression Gh/TG2. These results provide evidence that Gh function initiates the modulation of cell motility via association of GTP-bound Gh/TG2 with the GFFKR motif located in integrin α subunits. A multifunctional enzyme, Gh, is a GTP-binding protein that couples to the α1B-adrenoreceptor and stimulates phospholipase C-δ1 but also displays transglutaminase 2 (TG2) activity. Gh/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of Gh/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the α1-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of Gh/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the α1B-adrenoreceptor with the α1 agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. Gh/TG2 is coimmunoprecipitated by an integrin α5 antibody and binds to the cytoplasmic tail peptide of integrins α5, αv, and αIIb subunits in the presence of guanosine 5′-3-O-(thio)triphosphate (GTPγS). Mutation of Lys-Arg residues in the GFFKR motif, present in the α5-tail, significantly reduces the binding of GTPγS-Gh/TG2. Moreover, the motif-containing integrin α5-tail peptides block Gh/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression Gh/TG2. These results provide evidence that Gh function initiates the modulation of cell motility via association of GTP-bound Gh/TG2 with the GFFKR motif located in integrin α subunits. A bifunctional GTP-binding protein, Gh is tissue transglutaminase 2 (TG2), 1The abbreviations used are: TG2, transglutaminase 2; TGase, transglutaminase; HEK cells, human embryonic kidney cells; DMEM, Dulbecco's modified Eagle's medium; wt, wild type; PBS, phosphate-buffered saline; AR, adrenoceptor; CEC, chloroethylclonidine; CRT, calreticulin; [Ca2+]i intracellular calcium concentrations; Fn, fibronectin; Gh/TG2, this terminology is used to represent both G-protein and transglutaminase functions of transglutaminase 2; HASMC, human aortic smooth muscle cell; RA, all-trans retinoic acid; Vn, vitronectin.1The abbreviations used are: TG2, transglutaminase 2; TGase, transglutaminase; HEK cells, human embryonic kidney cells; DMEM, Dulbecco's modified Eagle's medium; wt, wild type; PBS, phosphate-buffered saline; AR, adrenoceptor; CEC, chloroethylclonidine; CRT, calreticulin; [Ca2+]i intracellular calcium concentrations; Fn, fibronectin; Gh/TG2, this terminology is used to represent both G-protein and transglutaminase functions of transglutaminase 2; HASMC, human aortic smooth muscle cell; RA, all-trans retinoic acid; Vn, vitronectin. a member of transglutaminase (TGase) family (1Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar, 2Im M.-J. Russell M.A. Feng J.-F. Cell. Signal. 1997; 9: 477-482Crossref PubMed Scopus (96) Google Scholar). TGases are Ca2+ and thiol-dependent enzymes that catalyze formation of isopeptide bonds between the γ-carboxamide group of protein-bound glutamines and the ϵ-amino group of protein-bound lysines or polyamines (2Im M.-J. Russell M.A. Feng J.-F. Cell. Signal. 1997; 9: 477-482Crossref PubMed Scopus (96) Google Scholar, 3Greenberg C.S. Birckbichler P.J. Rice R.H. FASEB J. 1991; 5: 3071-3077Crossref PubMed Scopus (930) Google Scholar, 4Lorand L. Graham R.M. Nat. Rev. Mol. Cell Biol. 2003; 4: 140-157Crossref PubMed Scopus (1198) Google Scholar, 5Chen J.S. Mehta K. Int. J. Biochem. Cell Biol. 1999; 31: 817-836Crossref PubMed Scopus (176) Google Scholar, 6Griffin M. Casadio R. Bergamini C.M. Biochem. J. 2003; 368: 377-396Crossref Google Scholar). TGase activity of Gh/TG2 covalently cross-links a variety of proteins such as cytoskeletal proteins, signaling proteins, and enzymes after an increase in intracellular Ca2+ concentrations ([Ca2+]i) (6Griffin M. Casadio R. Bergamini C.M. Biochem. J. 2003; 368: 377-396Crossref Google Scholar, 7Orrù S. Caputo I. D'Amato A. Ruoppolo M. Esposito C. J. Biol. Chem. 2003; 278: 31766-31773Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Recent studies demonstrate that cell damage or inflammation causes Gh/TG2 secretion, which leads to formation of autoantibody against Gh/TG2 (8Dieterich W. Ehnis T. Bauer M. Donner P. Volta V. Riecken E.D. Schuppan D. Nat. Med. 1997; 3: 797-801Crossref PubMed Scopus (1774) Google Scholar, 9Mohan K. Pinto D. Issekutz T.B. J. Immunol. 2003; 71: 3179-3186Crossref Scopus (52) Google Scholar). The Gh/TG2 antibody inhibits transendothelial migration of CD8+ T cell (9Mohan K. Pinto D. Issekutz T.B. J. Immunol. 2003; 71: 3179-3186Crossref Scopus (52) Google Scholar). In correlation to the involvement of Gh/TG2 in inflammation, activation of cytosolic phospholipase A2 via posttranslational modification by TGase activity (10Cordella-Miele E. Miele L. Mukherjee A.B. J. Biol. Chem. 1990; 265: 17180-17188Abstract Full Text PDF PubMed Google Scholar, 11Son J. Kim T.-I. Yoon Y.-H. Kim J.-Y. Kim S.-Y. J. Clin. Investig. 2003; 111: 121-128Crossref PubMed Scopus (107) Google Scholar) and reactive oxygen species-mediated activation of TGase of Gh/TG2 are also observed (12Lee Z.-W. Kwon S.-M. Kim S.-W. Yi S.-J. Kim Y.-M. Ha K.-S. Biochem. Biophys. Res. Commun. 2003; 305: 633-640Crossref PubMed Scopus (59) Google Scholar). In addition, cell surface Gh/TG2 has been implicated to play a role in cell adhesion and migration in association with integrin β1 and β3 subunits by a mechanism that is independent of Gh/TG2 activity (13Akimov S.S. Belkin A.M. Blood. 2001; 98: 1567-1576Crossref PubMed Scopus (247) Google Scholar, 14Balklava Z. Verderio E. Collighan R. Gross S. Adams J. Griffin M. J. Biol. Chem. 2002; 277: 16567-16575Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). GTPase activity of Gh/TG2 inhibits TGase activity upon binding of GTP or GDP and mediates signals from cell surface receptors to effectors (2Im M.-J. Russell M.A. Feng J.-F. Cell. Signal. 1997; 9: 477-482Crossref PubMed Scopus (96) Google Scholar, 4Lorand L. Graham R.M. Nat. Rev. Mol. Cell Biol. 2003; 4: 140-157Crossref PubMed Scopus (1198) Google Scholar). Gh/TG2-coupled receptors include α1B-adrenoreceptor (AR) (1Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar, 15Feng J.-F. Gray C.D. Im M.-J. Biochemistry. 1999; 38: 2224-2232Crossref PubMed Scopus (36) Google Scholar, 16Chen S. Lin F. Iismaa S. Lee K.N. Birckbichler P.J. Graham R.M. J. Biol. Chem. 1996; 271: 32385-32391Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 17Wu J. Liu S.-L. Zhu J.-L. Norton P.A. Norjiri S. Hoek J.B. Zern M.A. J. Biol. Chem. 2000; 275: 22213-22219Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), α1DAR (16Chen S. Lin F. Iismaa S. Lee K.N. Birckbichler P.J. Graham R.M. J. Biol. Chem. 1996; 271: 32385-32391Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), thromboxane A2 receptor α subtype (18Vezza R. Habib A. FitzGerald G.A. J. Biol. Chem. 1999; 274: 12774-12779Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Zhang Z. Vezza R. Plappert T. McNamara P. Lawson J.A. Austin S. Pratic D. Sutton M.St.-J. FitzGerald G.A. Cir. Res. 2003; 92: 1153-1161Crossref PubMed Scopus (70) Google Scholar), and oxytocin receptor (20Baek K.J. Kwon N.S. Lee H.S. Kim M.S. Muralidhar P. Im M.-J. Biochem. J. 1996; 315: 739-744Crossref PubMed Scopus (62) Google Scholar). Activation of these receptors stimulates GDP/GTP exchange of Gh/TG2 and activates phospholipase C-δ1, resulting in an increase of [Ca2+]i (17Wu J. Liu S.-L. Zhu J.-L. Norton P.A. Norjiri S. Hoek J.B. Zern M.A. J. Biol. Chem. 2000; 275: 22213-22219Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18Vezza R. Habib A. FitzGerald G.A. J. Biol. Chem. 1999; 274: 12774-12779Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 21Feng J.-F. Rhee S.G. Im M.-J. J. Biol. Chem. 1996; 271: 16451-16454Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 22Park E.-S. Won J.H. Han K.J. Suh P.-G. Ruy S.H. Lee H.S. Yun H.-Y. Kwon N.S. Baek K.J. Biochem. J. 1998; 331: 283-289Crossref PubMed Scopus (71) Google Scholar, 23Baek K.J. Kang S.K. Damron D.S. Im M.-J. J. Biol. Chem. 2001; 276: 5591-5597Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 24Kang S.K. Kim D.K. Damron D.S. Baek K.J. Im M.-J. Biochem. Biophys. Res. Commun. 2002; 293: 383-390Crossref PubMed Scopus (38) Google Scholar). GTPγS-bound Gh/TG2 also regulates Maxi K+ channel (Ca2+-activated K+ channel) (25Lee M.Y. Chung S.K. Bang H.W. Baek K.J. Uhm D.Y. Pfluegers Arch. Eur. J. Physiol. 1997; 433: 671-673Crossref PubMed Scopus (29) Google Scholar). A study reports that activation of extracellular signal-regulated kinase 1/2 by α1AR-Gh/TG2 coupling is inhibited by overexpression of calreticulin (CRT) in neonatal cardiomyocytes (26Lee J.-H. Lee N. Lim S. Jung H. Ko Y.-G. Park H.-Y. Jang Y. Lee H. Hwang K.-C. J. Steroid Biochem. Mol. Biol. 2003; 84: 101-107Crossref PubMed Scopus (31) Google Scholar). This calcium-binding protein, CRT, is shown to inhibit both GTP binding and TGase activities of Gh/TG2 (27Feng J.-F. Readon M. Yadav S.P. Im M.-J. Biochemistry. 1999; 38: 10743-10749Crossref PubMed Scopus (40) Google Scholar). A very recent in vivo study with transgenic mice, which have overexpressed Gh/TG2 in heart, has provided compelling evidence for the Gh/TG2-mediated signaling (19Zhang Z. Vezza R. Plappert T. McNamara P. Lawson J.A. Austin S. Pratic D. Sutton M.St.-J. FitzGerald G.A. Cir. Res. 2003; 92: 1153-1161Crossref PubMed Scopus (70) Google Scholar). This study has demonstrated that Gh/TG2 overexpression results in cardiac hypertrophy, expression of several genes, and apoptosis due to enhanced thromboxane A2 receptor signaling that leads to extracellular signal-regulated kinase 1/2 activation. A major obstacle in Gh/TG2 signaling is defining of GTPase-versus TGase-mediated responses, because the coupling of Gh/TG2 to the receptors increases [Ca2+]i, that may lead to activation of TGase. Overexpression of wild-type Gh/TG2 or TGase activity-ablated Gh/TG2 in vivo and in vitro is shown to affect cell spreading, migration, and contractility of heart (13Akimov S.S. Belkin A.M. Blood. 2001; 98: 1567-1576Crossref PubMed Scopus (247) Google Scholar, 14Balklava Z. Verderio E. Collighan R. Gross S. Adams J. Griffin M. J. Biol. Chem. 2002; 277: 16567-16575Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 19Zhang Z. Vezza R. Plappert T. McNamara P. Lawson J.A. Austin S. Pratic D. Sutton M.St.-J. FitzGerald G.A. Cir. Res. 2003; 92: 1153-1161Crossref PubMed Scopus (70) Google Scholar, 28Jones R.A. Nicholas B. Mian S. Davies P.J. Griffin M. J. Cell Sci. 1997; 110: 2461-2472Crossref PubMed Google Scholar, 29Small K. Feng J.-F. Lorenz J. Donnelly E.T. Yu A. Im M.-J. Dorn II, G.W. Liggett S.B. J. Biol. Chem. 1999; 274: 21291-21296Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). These observations indicate that Gh/TG2 may play a critical role in cell motility. In this study we have attempted to clarify the primary and initiating Gh/TG2 function (G-protein versus TGase function) on cell motility by means of determining cell migratory activity. Our results revealed that the coupling of Gh/TG2 to the α1BAR and/or up-regulation of Gh/TG2 expression inhibit cell migration in an integrin- and Gh-dependent manner. The results also indicate that modulation of the cell migratory activity is mediated by the binding of Gh to the GFFKR motif present in the cytoplasmic tail of various integrin α subunits. Cell Culture—Primary human aortic smooth muscle cells (HASMC) were provided by Dr. P. Dicorletto at The Lerner Research Institute, The Cleveland Clinic Foundation (Cleveland, OH); 6–10 passages were used for the study. Hamster leiomyosarcoma (DDT1-MF2) and human embryonic kidney cells (HEK 293 cells) were obtained from American Type Culture Collection. HASMC and HEK 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)-F-12 supplemented with heat-inactivated 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mg/ml glutamine. DDT1-MF2 cells were grown in DMEM containing 10% fetal bovine serum and the antibiotics indicated above. The cells were maintained in a humidified incubator at 37 °C in the presence of 5% CO2, 95% air. Plasmids and Transfection—Wild-type Gh/TG2 (wtTG) was cloned from a human heart cDNA library (30Hwang K.-C. Gray C.D. Sivasubramaniam N. Im M.-J. J. Biol. Chem. 1995; 270: 27058-27062Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and a TGase active site mutant C277S tTG (C-STG) was obtained by substituting cysteine to serine (31Lee K.N. Arnold S.A. Birchbichler P.J. Patterson Jr, M.K. Fraij B.M. Takekeuchi Y. Carter H.A. Biochim. Biophys. Acta. 1993; 1202: 1-6Crossref PubMed Scopus (77) Google Scholar). An α1BAR interaction site mutant Gh/TG2 (m3TG) was prepared as previously described (15Feng J.-F. Gray C.D. Im M.-J. Biochemistry. 1999; 38: 2224-2232Crossref PubMed Scopus (36) Google Scholar). A deletion mutant (ΔN20TG) of Gh/TG2 in which 20 amino acids were deleted from the N terminus was obtained by PCR using human heart Ghα DNA and two primers: a sense primer, 5′-CCACCATGCACACGGCCGACCTGTGCCG-3′, and an antisense primer, 5′-TGGGACCAGGGGCACATTCCATTTC. A correct PCR product was confirmed by nucleotide sequencing. All Gh/TG2 clones were inserted into pcDNA3.0-neo. Plasmids of wtTG, its mutants, and vector were transfected to DDT1-MF2 and HEK 293 cells using LipofectAMINE as described in the manufacturer's protocol (Invitrogen). Transfected cells were selected using 500 μg/ml G418 (Invitrogen) and maintained in the respective growth media containing 300 μg/ml G418. Determination of Cell Migratory Activity—Cells were detached with 25 mm HEPES (pH 7.4)-buffered saline solution containing 1 mm EDTA and washed three times with DMEM or DMEM-F-12 containing βAR blocker propranolol (1 μm) and α2AR blocker rauwalscine (100 nm). Cell migratory activity was determined in the presence and absence of 5 μm (–)epinephrine using Transwell (Costar, Corning, NY) with 8-μm-poresize membranes. The bottom of the membranes was coated with 10 μg/ml fibronectin (Fn) or vitronectin (Vn) (Roche Applied Science) or poly-l-lysine (Sigma), and cells (5 × 105 in 150 μl) were added to the top chamber. In some cases both sides of membranes were coated with 10 μg/ml Fn. After incubation for 8 h, cells in the top chamber were removed using cotton swaps and frozen at –80 °C for 1 h or more. Cells migrated to the bottom membranes were quantified by determining DNA content using a CyQUANT kit as provided by the manufacturer (Molecular Probes, Eugene, OR). Stress Fiber Staining—Cell spreading and formation of stress fibers in HASMC were performed with and without 5 μm (–)epinephrine in the presence of propranolol (1 μm) and rauwalscine (100 nm). Cells were plated on Fn (10 μg/ml)-coated glass coverslips and incubated in a cell culture incubator. At indicated time points, the cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 5 min and blocked with 4% heat-inactivated fetal bovine serum. After washing with PBS, the cells were incubated with tetramethyl rhodamine isothiocyanate-labeled phalloidin (Sigma) for 1 h and washed with PBS. Formation of stress fibers was examined using a Leica confocal microscope. Cell Adhesion Assay—Cell adhesion assay used was a slight modification of the method described previously (14Balklava Z. Verderio E. Collighan R. Gross S. Adams J. Griffin M. J. Biol. Chem. 2002; 277: 16567-16575Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, cell suspension (1 × 105 cells/well) was seeded in a 48-well plate coated with 5–20 μg/ml Fn, incubated in serum free media, allowed to attach for 30 min or 1 h. The floating cells were gently removed with PBS, and numbers of attached cells were determined by measuring DNA content using Cy-QUANT kit. For the experiments with HASMC and DDT1-MF2, cells were incubated with and without 5 μm (–)epinephrine. Immunoprecipitation and Immunoblotting—For the immunoprecipitation, HEK 293 cells expressed wtTG, C-STG, or ΔN20TG were extracted using 1% Triton X-100 in a lysis buffer (20 mm HEPES (pH 7.4), 1 mm EGTA, 1 mm EDTA, 10% glycerol, 10 μm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) by 3 rounds of freezing in a dry ice bath and thawing at 30 °C. The extracts (500 μgof protein) obtained by centrifugation at 100,000 × g for 1 h were precleared with protein A-Sepharose (Amersham Biosciences) and incubated with and without 5 μm GTPγS or 200 μm GDP or GTPγS plus a 120-kDa soluble Fn fragment (Invitrogen) or poly-RGD peptide or RGE peptide (Sigma) at room temperature for 30 min. A polyclonal antibody against the extracellular domain of integrin α5 subunit (Chemicon, Temecula, CA) and protein A-Sepharose beads (20 μl of 1:1 suspension, Sigma) was added, and the mixtures were incubated with gentle shaking at 4 °C for 4 h. The beads were collected by centrifugation at 3000 rpm and washed 3 times (1 ml/wash) with a washing buffer (25 mm HEPES (pH 7.4), 150 mm NaCl, 5 mm MgCl2, 0.5% Triton X-100, and 0.2% sucrose monolaurate). Immunoprecipitation of Gh/TG2 and/or integrin α5 was determined by immunoblotting using a monoclonal anti-Gh/TG2 antibody (CUB7402 from Neomarkers, Fremont, CA) or the polyclonal integrin α5 antibody as described previously (15Feng J.-F. Gray C.D. Im M.-J. Biochemistry. 1999; 38: 2224-2232Crossref PubMed Scopus (36) Google Scholar). After incubating the blots with the respective secondary antibodies, the immunoreactive proteins were determined using a Super Signal chemiluminescent kit (Pierce) and exposing to BIOMAX films (Eastman Kodak Co.). Determination of Gh/TG2 Binding to Peptide-Sepharose—Equimolar amounts of cytoplasmic tails of the integrin α and β1 subunits or Fn were incubated with CNBr-activated Sepharose 4B using the protocols provided by the manufacturer (Amersham Biosciences). The peptides were synthesized in the Biotechnology Core Facility at The Cleveland Clinic Foundation (Cleveland, OH). The purity of the peptides was assessed by high performance liquid chromatography and mass spectroscopy analyses. The peptide beads (40 μl of a 1:1 suspension) pretreated with 1% bovine serum albumin were incubated with the cytosol fraction prepared from HEK 293 cell-expressed wtTG under the various conditions at 4 °C for 3 h. The beads obtained by centrifugation at 3000 rpm for 5 min were washed 3 times with 1 ml of the washing buffer. Gh/TG2 bound to the peptides was determined by immunoblotting as described above. For the preparation of cytosol fraction, cells were washed with HEPES (pH 7.4)-buffered saline solution and lysed with the lysis buffer. The cytosol fraction was obtained by centrifugation at 100,000 × g for 1 h. In some cases, purified Gh/TG2 and CRT from rat liver were used as specified in the legends to Figs. 4C and 6B. Gh/TG2 and CRT were purified as described previously (27Feng J.-F. Readon M. Yadav S.P. Im M.-J. Biochemistry. 1999; 38: 10743-10749Crossref PubMed Scopus (40) Google Scholar).Fig. 6Gh/TG2 interacts with integrin α subunits via GFFKR motif. A, amino acid alignments of cytoplasmic tails of various integrin α subunits including α5. B, CRT blocks Gh/TG2 binding to α5-, αv -, and αIIb-tail peptides. Peptide affinity gels of α5, αv, and αIIb were incubated CRT (4 μg) at the in presence or absence of 4 °C for 2 h and then further incubated with purified Gh/TG2 (1.5 μg) in the presence of 5 μm GTPγS and 5 mm MgCl2 for 2 h. Gh/TG2 binding was determined by immunoblotting with TG2 antibody. C and D, mutation of Lys-Arg (KR) residues in the GFFKR motif reduces Gh/TG2 binding. Lys (K), Arg (R), or Lys-Arg residues in the GFFKR motif in α5A was mutated to I (K-Iα5A) or T (R-Tα5A), or IT (KR-ITα5A), respectively. Peptide gels were incubated with cytosol prepared from HEK 293 cells expressed wtTG under the same conditions described above. The gels were washed and subjected to immunoblotting with TG2 antibody. Binding of Gh/TG2 to α5 was taken as 100%. The data presented are the means ± S.E. from three independent experiments. *, α5versus α5A or various mutated α5A, p < 0.001.View Large Image Figure ViewerDownload (PPT) Assays—Density of α1ARs was determined using 20 nm [3H]prazosin by a fast filter method, and TGase activity was measured using 1 μCi of [3H]putrescine and 1% N,N′-dimethyl casein as described previously (32Hwang K.-C. Gray C.D. Sweet W.E. Moravec C.S. Im M.-J. Circulation. 1996; 94: 718-726Crossref PubMed Scopus (56) Google Scholar). Determination of GTP binding to Gh/TG2 was evaluated using 10 μCi of [α-32P]GTP by photoaffinity labeling (33Im M.-J. Riek P.R. Graham R.M. J. Biol. Chem. 1990; 265: 18952-18960Abstract Full Text PDF PubMed Google Scholar). The α1 agonist-mediated GTP binding to Gh/TG2 was determined using a method described previously (34Im M.-J. Graham R.M. J. Biol. Chem. 1990; 265: 18944-18951Abstract Full Text PDF PubMed Google Scholar). Briefly, membranes (100 μg) prepared from HASMC treated with 5 μm for 24 h were preincubated in the presence of 5 μm (–)epinephrine or epinephrine plus phentolamine (0.1 mm) at room temperature for 30 min. The samples were transferred to an ice bath, and [α-32P]GTP (20 μCi/tube) was added. After incubation for 5 min, the samples were subjected to UV irradiation at 254 nm for 8 min. The [α-32P]GTP binding was visualized by autoradiography after SDS-PAGE (8% gel). Protein concentrations were determined using a Bio-Rad protein assay kit, and known concentrations of bovine serum albumin were used as the standard. Data Analysis—Data are expressed as means ± S.E. Statistical analysis was performed using one-way analysis of variance. The difference was considered significant at p < 0.05. Stimulation of α1ARs with Epinephrine Inhibits Migratory Activity of HASMC in Collaboration with Integrins—We first examined whether stimulation of the α1AR displays any effects on the migratory activity, spreading, and adhesion of HASMC using integrin ligands Fn and Vn. Poly-l-lysine was used to examine an involvement of integrins in these cellular responses. As presented in Fig. 1, treatment of HASMC with epinephrine inhibited the migratory activity of HASMC to Fn and Vn (Fig. 1A). The α1 agonist-mediated inhibition was blocked by treatment of the α1-antagonists prazosin and chloroethylclonidine (CEC). Cell migration to poly-l-lysine was small, and the α1 agonist-dependent inhibition was not observed, indicating that the α1 agonist-mediated inhibition of cell migration is integrin-dependent. Moreover, the inhibition of cell migration was significantly enhanced as a function of α1 agonist concentrations (Fig. 1B). In contrast, spreading of HASMC and formation of stress fibers were greatly stimulated in the presence of α1 agonist in a time-dependent manner (Fig. 1C) and were visibly blocked by CEC (Fig. 1D). The order of cell size increased was epinephrine ≫ CEC > control (–epinephrine) after a 12-h incubation. Determination of cell adhesion to Fn revealed that although numbers of attached cells were increased as a function of Fn concentration and incubation time, no significant differences in cell adhesion with and without the α1 agonist were found (data not shown). These data indicate that stimulation of CEC-sensitive α1AR in HASMC modulates cell migratory activity and facilitates cytoskeletal reorganization but not cell adhesion and that integrin ligation is indispensable for these α1AR-mediated cellular responses. α1BAR-Gh/TG2 Coupling and Up-regulation of Gh/TG2 Expression Modulate Cell Motility—The above results indicate that the CEC-sensitive α1AR is involved in modulation of cell migration. This α1AR blocker covalently binds to the α1BAR with high affinity and the α1BAR couples with Gh/TG2 and Gq (1Nakaoka H. Perez D.M. Baek K.J. Das T. Husain A. Misono K. Im M.-J. Graham R.M. Science. 1994; 264: 1593-1596Crossref PubMed Scopus (528) Google Scholar). To evaluate whether the CEC-sensitive α1AR-mediated inhibition of cell migration is caused by the coupling of the receptor with Gh/TG2, expression of Gh/TG2 in HASMC was induced by incubation of all-trans retinoic acid (RA), which is one of the well characterized Gh/TG2 inducers (5Chen J.S. Mehta K. Int. J. Biochem. Cell Biol. 1999; 31: 817-836Crossref PubMed Scopus (176) Google Scholar, 35Nagy L. Saydak M. Shipley N. Lu S. Basilion J.P. Yan Z.H. Syka P. Chandraratna R.A.S. Stein J.P. Heyman R.A. Davies P.J.A. J. Biol. Chem. 1996; 271: 4355-4365Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 36Antonyak M.A. Singh V.S. Lee D.A. Boehm J.E. Combs C. Zgola M.M. Page R.L. Cerione R.A. J. Biol. Chem. 2001; 276: 33582-33587Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). An RA concentration-dependent increase of Gh/TG2 was confirmed by immunoblotting (see the inset in Fig. 2C) and measurement of TGase activity, and GTP binding was determined by [α-32P]GTP photoaffinity labeling (data not shown). The cell migratory activity was reduced in a RA concentration-dependent manner (Fig. 2A). Moreover, further augmentation of the Gh/TG2-mediated inhibition was observed by treatment of the α1 agonist. To ascertain whether the α1BAR-Gh/TG2 coupling occurs, binding of GTP to Gh/TG2 and changes in [Ca2+]i in response to the α1AR activation were determined. As shown Fig. 2B, GTP binding of Gh/TG2 by incubation with the α1 agonist was significantly increased as compared with that in the absence of the α1 agonist, and the α1 agonist-mediated increase was blocked by an α1-antagonist, phentolamine. Moreover, the α1AR-stimulated GTP binding of Gh/TG2 was not observed with the membranes prepared from CEC-pretreated HASMC. [Ca2+]i was also increased as Gh/TG2 levels were increased (Fig. 2C). The observation that coupling of Gh/TG2 to the α1BAR and/or up-regulation of the Gh/TG2 expression inhibit cell migration was again evaluated using DDT1-MF2, which expresses the α1BAR subtype among α1ARs (37Cotecchia S. Schwinn D.A. Randall R.R. Lefknowitz R.J. Caron M.G. Kobilika B.K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7159-7163Crossref PubMed Scopus (483) Google Scholar), by overexpressing wtTG, C-STG, and m3TG. The mutant C-STG is inert in TGase activity (31Lee K.N. Arnold S.A. Birchbichler P.J. Patterson Jr, M.K. Fraij B.M. Takekeuchi Y. Carter H.A. Biochim. Biophys. Acta. 1993; 1202: 1-6Crossref PubMed Scopus (77) Google Scholar), and the mutant m3TG is unable to couple to the α1BAR effectively as compared with wtTG (15Feng J.-F. Gray C.D. Im M.-J. Biochemistry. 1999; 38: 2224-2232Crossref PubMed Scopus (36) Google Scholar). Approximately 4-fold overexpression of wtTG and C-STG resulted in significant inhibition of cell migratory activity as compared with the native and vector control cells (Figs. 3, A and B). Stimulation of the α1BAR with epinephrine further augmented the inhibition of the migratory activity of wtTG and C-STG cells, and the migratory activity of native and vector cells was also significantly reduced (Fig. 3B). Moreover, overexpression of m3TG did not reduce the cell migratory activity, probably due to the competition with endogenous Gh/TG2. As expected, activation of the α1BAR did not change the migration of m3TG-overexpressed cells. In addition, we also determined whether the inhibition of cell migration by the coupling of α1BAR with Gh/TG2 or overexpression of wtTG is due to blocking of chemotaxis or reduced chemokinesis. When cell migration was determined using Transwell membranes with both sides coated with Fn or random migration on Fn-coated glass slides, similar inhibitory effects on motility were observed whether the membranes were coated on one or both sides (data not show" @default.
• W2055078479 created "2016-06-24" @default.
• W2055078479 creator A5002575077 @default.
• W2055078479 creator A5006677281 @default.
• W2055078479 creator A5014943644 @default.
• W2055078479 creator A5028584423 @default.
• W2055078479 creator A5032289839 @default.
• W2055078479 creator A5040021434 @default.
• W2055078479 creator A5090556368 @default.
• W2055078479 date "2004-08-01" @default.
• W2055078479 modified "2023-10-17" @default.
• W2055078479 title "α1B-Adrenoceptor Signaling and Cell Motility" @default.
• W2055078479 cites W1509677149 @default.
• W2055078479 cites W1557226477 @default.
• W2055078479 cites W1592181599 @default.
• W2055078479 cites W1595481029 @default.
• W2055078479 cites W1779003769 @default.
• W2055078479 cites W1892184508 @default.
• W2055078479 cites W1964826020 @default.
• W2055078479 cites W1967455040 @default.
• W2055078479 cites W1974222030 @default.
• W2055078479 cites W1981715774 @default.
• W2055078479 cites W1988867634 @default.
• W2055078479 cites W1999453187 @default.
• W2055078479 cites W1999466305 @default.
• W2055078479 cites W2002979980 @default.
• W2055078479 cites W2010824095 @default.
• W2055078479 cites W2011915496 @default.
• W2055078479 cites W2018772944 @default.
• W2055078479 cites W2020006960 @default.
• W2055078479 cites W2023081206 @default.
• W2055078479 cites W2023594113 @default.
• W2055078479 cites W2033653650 @default.
• W2055078479 cites W2039882384 @default.
• W2055078479 cites W2043856196 @default.
• W2055078479 cites W2045255717 @default.
• W2055078479 cites W2050598970 @default.
• W2055078479 cites W2051208708 @default.
• W2055078479 cites W2059154200 @default.
• W2055078479 cites W2061413318 @default.
• W2055078479 cites W2063398165 @default.
• W2055078479 cites W2063708626 @default.
• W2055078479 cites W2065295487 @default.
• W2055078479 cites W2067403820 @default.
• W2055078479 cites W2068131808 @default.
• W2055078479 cites W2070524237 @default.
• W2055078479 cites W2071306432 @default.
• W2055078479 cites W2077187474 @default.
• W2055078479 cites W2077231895 @default.
• W2055078479 cites W2087953477 @default.
• W2055078479 cites W2103627235 @default.
• W2055078479 cites W2114463465 @default.
• W2055078479 cites W2134987889 @default.
• W2055078479 cites W2149646582 @default.
• W2055078479 cites W2158724869 @default.
• W2055078479 cites W2161954973 @default.
• W2055078479 cites W2163222592 @default.
• W2055078479 cites W2414766140 @default.
• W2055078479 cites W4232056108 @default.
• W2055078479 cites W4300803924 @default.
• W2055078479 doi "https://doi.org/10.1074/jbc.m402084200" @default.
• W2055078479 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15220331" @default.
• W2055078479 hasPublicationYear "2004" @default.
• W2055078479 type Work @default.
• W2055078479 sameAs 2055078479 @default.
• W2055078479 citedByCount "44" @default.
• W2055078479 countsByYear W20550784792012 @default.
• W2055078479 countsByYear W20550784792013 @default.
• W2055078479 countsByYear W20550784792014 @default.
• W2055078479 countsByYear W20550784792017 @default.
• W2055078479 countsByYear W20550784792018 @default.
• W2055078479 countsByYear W20550784792020 @default.
• W2055078479 countsByYear W20550784792021 @default.
• W2055078479 crossrefType "journal-article" @default.
• W2055078479 hasAuthorship W2055078479A5002575077 @default.
• W2055078479 hasAuthorship W2055078479A5006677281 @default.
• W2055078479 hasAuthorship W2055078479A5014943644 @default.
• W2055078479 hasAuthorship W2055078479A5028584423 @default.
• W2055078479 hasAuthorship W2055078479A5032289839 @default.
• W2055078479 hasAuthorship W2055078479A5040021434 @default.
• W2055078479 hasAuthorship W2055078479A5090556368 @default.
• W2055078479 hasBestOaLocation W20550784791 @default.
• W2055078479 hasConcept C126322002 @default.
• W2055078479 hasConcept C134018914 @default.
• W2055078479 hasConcept C170493617 @default.
• W2055078479 hasConcept C185592680 @default.
• W2055078479 hasConcept C55493867 @default.
• W2055078479 hasConcept C58207958 @default.
• W2055078479 hasConcept C62478195 @default.
• W2055078479 hasConcept C71924100 @default.
• W2055078479 hasConcept C86803240 @default.
• W2055078479 hasConcept C95444343 @default.
• W2055078479 hasConceptScore W2055078479C126322002 @default.
• W2055078479 hasConceptScore W2055078479C134018914 @default.
• W2055078479 hasConceptScore W2055078479C170493617 @default.
• W2055078479 hasConceptScore W2055078479C185592680 @default.
• W2055078479 hasConceptScore W2055078479C55493867 @default.
• W2055078479 hasConceptScore W2055078479C58207958 @default.

• next