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Originally published In Press as doi:10.1074/jbc.M402084200 on June 25, 2004

J. Biol. Chem., Vol. 279, Issue 35, 36593-36600, August 27, 2004
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{alpha}1B-Adrenoceptor Signaling and Cell Motility

GTPase FUNCTION OF Gh/TRANSGLUTAMINASE 2 INHIBITS CELL MIGRATION THROUGH INTERACTION WITH CYTOPLASMIC TAIL OF INTEGRIN {alpha} SUBUNITS*

Sung Koo Kang{ddagger}, Kye Sook Yi§, Nyoun Soo Kwon¶, Kwang-Hyun Park§, Uh-Hyun Kim§, Kwang Jin Baek¶, and Mie-Jae Im§||

From the {ddagger}Oriental Herbal Research Institute, Dongkuk University, Department of Biochemistry, College of Medicine, Chung-Ang University, Seoul 156-756, and §Department of Biochemistry, Medical School, Chonbuk National University, Keumam-dong, Jeonju 561-182, Republic of Korea

Received for publication, February 25, 2004 , and in revised form, May 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A multifunctional enzyme, Gh, is a GTP-binding protein that couples to the {alpha}1B-adrenoreceptor and stimulates phospholipase C-{delta}1 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 {alpha}1-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 {alpha}1B-adrenoreceptor with the {alpha}1 agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. Gh/TG2 is coimmunoprecipitated by an integrin {alpha}5 antibody and binds to the cytoplasmic tail peptide of integrins {alpha}5, {alpha}v, and {alpha}IIb subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP{gamma}S). Mutation of Lys-Arg residues in the GFFKR motif, present in the {alpha}5-tail, significantly reduces the binding of GTP{gamma}S-Gh/TG2. Moreover, the motif-containing integrin {alpha}5-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 {alpha} subunits.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A bifunctional GTP-binding protein, Gh is tissue transglutaminase 2 (TG2),1 a member of transglutaminase (TGase) family (1, 2). TGases are Ca2+ and thiol-dependent enzymes that catalyze formation of isopeptide bonds between the {gamma}-carboxamide group of protein-bound glutamines and the {epsilon}-amino group of protein-bound lysines or polyamines (2–6). 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) (6, 7). Recent studies demonstrate that cell damage or inflammation causes Gh/TG2 secretion, which leads to formation of autoantibody against Gh/TG2 (8, 9). The Gh/TG2 antibody inhibits transendothelial migration of CD8+ T cell (9). In correlation to the involvement of Gh/TG2 in inflammation, activation of cytosolic phospholipase A2 via posttranslational modification by TGase activity (10, 11) and reactive oxygen species-mediated activation of TGase of Gh/TG2 are also observed (12). In addition, cell surface Gh/TG2 has been implicated to play a role in cell adhesion and migration in association with integrin {beta}1 and {beta}3 subunits by a mechanism that is independent of Gh/TG2 activity (13, 14).

GTPase activity of Gh/TG2 inhibits TGase activity upon binding of GTP or GDP and mediates signals from cell surface receptors to effectors (2, 4). Gh/TG2-coupled receptors include {alpha}1B-adrenoreceptor (AR) (1, 15–17), {alpha}1DAR (16), thromboxane A2 receptor {alpha} subtype (18, 19), and oxytocin receptor (20). Activation of these receptors stimulates GDP/GTP exchange of Gh/TG2 and activates phospholipase C-{delta}1, resulting in an increase of [Ca2+]i (17, 18, 21–24). GTP{gamma}S-bound Gh/TG2 also regulates Maxi K+ channel (Ca2+-activated K+ channel) (25). A study reports that activation of extracellular signal-regulated kinase 1/2 by {alpha}1AR-Gh/TG2 coupling is inhibited by overexpression of calreticulin (CRT) in neonatal cardiomyocytes (26). This calcium-binding protein, CRT, is shown to inhibit both GTP binding and TGase activities of Gh/TG2 (27). 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 (19). 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 (13, 14, 19, 28, 29). 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 {alpha}1BAR 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 {alpha} subunits.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (30), and a TGase active site mutant C277S tTG (C-STG) was obtained by substituting cysteine to serine (31). An {alpha}1BAR interaction site mutant Gh/TG2 (m3TG) was prepared as previously described (15). A deletion mutant ({Delta}N20TG) of Gh/TG2 in which 20 amino acids were deleted from the N terminus was obtained by PCR using human heart Gh{alpha} 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 {beta}AR blocker propranolol (1 µM) and {alpha}2AR 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 x 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 (14). Briefly, cell suspension (1 x 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 {Delta}N20TG 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 x g for 1 h were precleared with protein A-Sepharose (Amersham Biosciences) and incubated with and without 5 µM GTP{gamma}S or 200 µM GDP or GTP{gamma}S 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 {alpha}5 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 {alpha}5 was determined by immunoblotting using a monoclonal anti-Gh/TG2 antibody (CUB7402 from Neomarkers, Fremont, CA) or the polyclonal integrin {alpha}5 antibody as described previously (15). 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 {alpha} and {beta}1 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 x 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 (27).



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  		FIG. 4.
Coimmunoprecipitation of Gh/TG2 by an integrin {alpha}5 antibody and binding of Gh/TG2 to the cytoplasmic tails of integrin {alpha}5 or {beta}1 subunit. HEK 293 cells expressing wtTG were lysed and extracted with 1% Triton X-100 in lysis buffer. Samples were subjected to coimmunoprecipitation under various conditions using a polyclonal anti-integrin {alpha}5 antibody against the extracellular domain. Concentrations of ligands were 30 µM 120-kDa fragment, 1 mM RGD-containing peptide, 1 mM RGE peptide, 20 µM GTP{gamma}S, 200 µM GDP, and 5 mM MgCl2. A, coimmunoprecipitation of Gh/TG2 by the integrin {alpha}5 antibody. The presence of Gh/TG2 in the immunocomplexes was determined by immunoblotting with TG2 antibody. The integrin {alpha}5 subunit in the immunocomplexes was determined with the {alpha}5 antibody using the same blot. B, guanine nucleotide-dependent coimmunoprecipitation of Gh/TG2. Gh/TG2 was immunoprecipitated more in the presence of GTP{gamma}S than GDP or buffer. C, amino acid sequence of cytoplasmic tails of the integrin {alpha}5 and {beta}1 subunits. The cytoplasmic portion is underlined. Bold type in the {beta}1 peptides indicates the overlapping regions. D, direct binding of purified Gh/TG2 to {alpha}5-tail but not to the {beta}1-tail. Purified Gh/TG2 (1.5 µg) was incubated in the presence of 5 µM GTP{gamma}S with the peptide affinity gels (40 µl of 1:1 suspension) at 4 °C for 2 h and washed 3 times. Gh/TG2 was visualized by immunoblotting with TG2 antibody. Representative data from three independent experiments are shown.

 



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  		FIG. 6.
Gh/TG2 interacts with integrin {alpha} subunits via GFFKR motif. A, amino acid alignments of cytoplasmic tails of various integrin {alpha} subunits including {alpha}5. B, CRT blocks Gh/TG2 binding to {alpha}5-, {alpha}v -, and {alpha}IIb-tail peptides. Peptide affinity gels of {alpha}5, {alpha}v, and {alpha}IIb 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 GTP{gamma}S 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 {alpha}5A was mutated to I (K-I{alpha}5A) or T (R-T{alpha}5A), or IT (KR-IT{alpha}5A), 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 {alpha}5 was taken as 100%. The data presented are the means ± S.E. from three independent experiments. *, {alpha}5 versus {alpha}5A or various mutated {alpha}5A, p < 0.001.

 
Assays—Density of {alpha}1ARs 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 (32). Determination of GTP binding to Gh/TG2 was evaluated using 10 µCi of [{alpha}-32P]GTP by photoaffinity labeling (33). The {alpha}1 agonist-mediated GTP binding to Gh/TG2 was determined using a method described previously (34). 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 [{alpha}-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 [{alpha}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of {alpha}1ARs with Epinephrine Inhibits Migratory Activity of HASMC in Collaboration with Integrins—We first examined whether stimulation of the {alpha}1AR 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 {alpha}1 agonist-mediated inhibition was blocked by treatment of the {alpha}1-antagonists prazosin and chloroethylclonidine (CEC). Cell migration to poly-L-lysine was small, and the {alpha}1 agonist-dependent inhibition was not observed, indicating that the {alpha}1 agonist-mediated inhibition of cell migration is integrin-dependent. Moreover, the inhibition of cell migration was significantly enhanced as a function of {alpha}1 agonist concentrations (Fig. 1B). In contrast, spreading of HASMC and formation of stress fibers were greatly stimulated in the presence of {alpha}1 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 {alpha}1 agonist were found (data not shown). These data indicate that stimulation of CEC-sensitive {alpha}1AR in HASMC modulates cell migratory activity and facilitates cytoskeletal reorganization but not cell adhesion and that integrin ligation is indispensable for these {alpha}1AR-mediated cellular responses.



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  		FIG. 1.
Stimulation of {alpha}1-adrenoceptors in human aortic smooth muscle cell inhibits cell migratory activity. A, epinephrine-mediated inhibition of cell migration to various extracellular matrices. HASMC (5 x 105 in 150 µl) were added to the top chamber and incubated in the presence of epinephrine (Epi, 5 µM) or epinephrine + prazosin (Pra, 10 nM) or epinephrine + CEC (10 µM) for 8 h. All experimental sets of cells were pretreated with and without CEC for 10 min and washed with serum-free media. Prazosin and CEC were dissolved in dimethyl sulfoxide (Me2SO) and diluted with water, and the final concentration of Me2SO was 0.01%. The control (–Epi) contains only 0.01% Me2SO. Cell migration assay was performed as described under "Experimental Procedures." The data presented are the means ± S.E. from four independent experiments. *, –epinephrine (control) versus +epinephrine, p < 0.001; #, +epinephrine versus epinephrine + Pra or CEC, p < 0.001. B, migratory activity of HASMC is inhibited as a function of epinephrine concentrations. Data are presented as the means ± S.E. from three independent experiments. C, HASMC spreading and formation of stress fibers in response to epinephrine treatment. HASMC was seeded on Fn-coated glass slides (10 µg/ml Fn) and incubated with 5 µM epinephrine. At the indicated time point, cells were washed, fixed, permeabilized, and stained with tetramethyl rhodamine isothiocyanate-labeled phalloidin. D, epinephrine-mediated HASMC spreading was blocked by treatment with CEC. Cells were incubated in the presence of epinephrine, epinephrine + CEC, or control (–Epi) for 12 h, and cell spreading was visualized by staining stress fibers. Formation of stress fibers is seen in all experimental sets. Representative data from three independent experiments are shown.

 
{alpha}1BAR-Gh/TG2 Coupling and Up-regulation of Gh/TG2 Expression Modulate Cell Motility—The above results indicate that the CEC-sensitive {alpha}1AR is involved in modulation of cell migration. This {alpha}1AR blocker covalently binds to the {alpha}1BAR with high affinity and the {alpha}1BAR couples with Gh/TG2 and Gq (1). To evaluate whether the CEC-sensitive {alpha}1AR-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 (5, 35, 36). 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 [{alpha}-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 {alpha}1 agonist. To ascertain whether the {alpha}1BAR-Gh/TG2 coupling occurs, binding of GTP to Gh/TG2 and changes in [Ca2+]i in response to the {alpha}1AR activation were determined. As shown Fig. 2B, GTP binding of Gh/TG2 by incubation with the {alpha}1 agonist was significantly increased as compared with that in the absence of the {alpha}1 agonist, and the {alpha}1 agonist-mediated increase was blocked by an {alpha}1-antagonist, phentolamine. Moreover, the {alpha}1AR-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).



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  		FIG. 2.
Induction of Gh/TG2 expression in HASMC by retinoic acid inhibits cell migration. Cells were incubated with various concentrations of RA for 24 h. A, inhibition of migratory activity of HASMC to Fn as a function of RA concentrations. Data are presented as the means ± S.E. from three independent experiments. B, {alpha}1 agonist-mediated [{alpha}-32P]GTP binding to Gh/TG2. Experiment was performed as detailed under "Experimental Procedures." Epi, epinephrine; Phen, phentolamine. CEC-treated membrane was prepared from HASMC pretreated with RA for 24 h after incubation with 10 µM CEC for 10 min. An arrow indicates Gh/TG2. C, increase in [Ca2+]i in the RA-treated cells by treatment of epinephrine. [Ca2+]i was determined as described previously (24). A representative [Ca2+]i signal of single cell is shown in each experimental set from three independent experiments. An arrow on the trace indicates the time point of epinephrine addition. Inset, Gh/TG2 expression induced by RA treatment was determined using cell lysates (100 µg of protein) by immunoblotting with a monoclonal anti-TG2 antibody (CUB7402).

 
The observation that coupling of Gh/TG2 to the {alpha}1BAR and/or up-regulation of the Gh/TG2 expression inhibit cell migration was again evaluated using DDT1-MF2, which expresses the {alpha}1BAR subtype among {alpha}1ARs (37), by overexpressing wtTG, C-STG, and m3TG. The mutant C-STG is inert in TGase activity (31), and the mutant m3TG is unable to couple to the {alpha}1BAR effectively as compared with wtTG (15). 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 {alpha}1BAR 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 {alpha}1BAR did not change the migration of m3TG-overexpressed cells. In addition, we also determined whether the inhibition of cell migration by the coupling of {alpha}1BAR 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 shown), indicating that the coupling and overexpression of Gh/TG2 attenuate chemokinesis. Again, no significant changes in cell adhesion among these cell lines were observed, and treatment with the {alpha}1 agonist did not change the numbers of the attached cells.



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  		FIG. 3.
Overexpression of various forms of Gh/TG2 results in inhibition of migratory activity of DDT1-MF2 and HEK 293 cells. A, expression level of wtTG, C-STG, and m3TG in DDT1-MF2. Expression of Gh/TG2 proteins was determined in 100 µg of protein of cell lysates by immunoblotting with TG2 antibody. B, incubation of cells with epinephrine augments the Gh/TG2-mediated inhibition of cell migratory activity. Migratory activity of cells was determined using Fn-coated membranes. The number of the vector control cells migrated in the absence of the {alpha}1 agonist was taken as 100%. The data presented are from three independent experiments. *, –epinephrine versus +epinephrine, p < 0.001; #, DDT1-MF2 (DDT1) versus vector or various forms of Gh/TG2 expressed cells in the absence of epinephrine, p < 0.001. C, expression level of various forms of Gh/TG2 in HEK 293 cells. Expression of wtTG and its mutants was determined using cell lysates (100 µg protein) by immunoblotting with TG2 antibody. D, overexpression of Gh/TG2 proteins reduces migratory activity of HEK 293 cells. Migratory activity of cells was determined using Fn-coated membranes. The number of vector control cells migrated to Fn-coated membranes was taken as 100%. The data are the means ± S.E. from four independent experiments. Migration of HEK 293 cell-expressed Gh/TG2 proteins to bovine serum albumin (BSA) was small and similar with each other. #, vector versus various forms of Gh/TG2 expressed mutants, p < 0.001. E, characterization of expressed wtTG and its mutants. [{alpha}-32P]GTP binding was determined using cell extract (50 µg) as detailed under "Experimental Procedures." Binding of Gh/TG2s to Fn was determined with Fn-Sepharose using the cytosol fraction (500 µg). Coimmunoprecipitation of Gh/TG2s was evaluated in the presence of 20 µM GTP{gamma}S and 5 mM MgCl2using cell extracts (300 µg) and an integrin {alpha}5 antibody against extracellular domain.

 
To further determine whether Gh/TG2 without {alpha}1BAR is involved in the inhibition of cell migration, we utilized HEK 293 cells, which does not express both {alpha}1ARs and Gh/TG2 judged by measurement of [3H]prazosin binding, TGase activity (data not shown), and immunoblotting of Gh/TG2 (see Fig. 3C). It has been shown that cell surface Gh/TG2 inhibits cell migration (13, 14) and that amino acid residues (15 residues) located in N terminus of Gh/TG2 play a critical role in externalization on cell surface (38). To examine whether Gh/TG2-mediated inhibition of cell migration is due to the externalized Gh/TG2, we also expressed a deletion mutant, {Delta}N20TG, by deleting 20 amino acids from N terminus of Gh/TG2 (see "Experimental Procedures"). As shown in Fig. 3D, the migration of wtTG, C-STG, and {Delta}N20TG cells was significantly inhibited as compared with that of the vector cells. The expressed Gh/TG2 proteins exhibited GTP binding activity, and wtTG and C-STG but not {Delta}N20TG were able to bind to Fn (Fig. 3E). Coimmunoprecipitation of these Gh/TG2s by an integrin {alpha}5 subunit antibody against extracellular domain was also observed. Interestingly, {Delta}N20TG, including C-STG, did not exhibit TGase activity. Although the reason for the absence of TGase activity of {Delta}N20TG is not clearly understood, a loss of TGase activity by deletion of N-terminal domain ({beta}-sandwich) has been observed (39). In addition, there were no significant differences in the numbers of the attached cells on Fn among the cell lines stably expressed wtTG, C-STG, {Delta}N20TG, and vector. Together, these results suggest that intracellular Gh/TG2 and Gh function may play roles in the inhibition of cell migratory activity.

Interaction of Integrin {alpha}5 Cytoplasmic Tail with GTP{gamma}S-bound Gh/TG2 Modulates Cell Migratory Activity—To identify the signaling molecule(s) targeted by Gh/TG2 in inhibition of cell migration, we examined whether Gh/TG2 interacts with a prototype Fn receptor, integrin {alpha}5{beta}1. Because the above results suggested that intracellular Gh/TG2 is involved in modulation of cell migration and integrin ligation is critical in this cellular response, coimmunoprecipitation studies were performed using integrin {alpha}5 subunit antibody against extracellular domain under various conditions; cell extract prepared from HEK 293 cell-overexpressed wtTG was incubated in the presence and absence of GTP{gamma}S or a soluble ligand of Fn, a 120-kDa fragment that contains an RGD motif but not the Gh/TG2 binding site or a known blocking ligand (RGD peptide) or non-blocking ligand (RGE peptide) of integrins. The results showed that Gh/TG2 was coimmunoprecipitated more in the presence of both GTP{gamma}S and the 120-kDa fragment than in the absence of GTP{gamma}S (Fig. 4A). RGD peptide but not RGE blocked the coimmunoprecipitation of Gh/TG2, indicating that RGD effect is specific. Coimmunoprecipitation of Gh/TG2 was also blocked by incubation with GDP (Fig. 4B). A possibility that Gh/TG2 may interact with cytoplasmic domain of integrin was directly assessed utilizing the cytoplasmic tail peptides of the integrin {alpha}5 and {beta}1 subunits and purified Gh/TG2 (Figs. 4, C and D). The results revealed that Gh/TG2 interacts with the {alpha}5-tail more tightly than with {beta}1-tail peptides (Fig. 4D). However, a residual binding of Gh/TG2 to {beta}1 tail may be due to the incomplete wash since the {beta}1-tail peptides did not inhibit the Gh/TG2 binding to the {alpha}5-tail (data not shown).

To further ascertain the above observations, the interacting region of Gh/TG2 in the {alpha}5-tail was determined. Three peptides ({alpha}5A, {alpha}5B, and {alpha}5C) of the cytoplasmic tail of the integrin {alpha}5 were synthesized and used as peptide affinity gels (Fig. 5A). The {alpha}5A and full-length {alpha}5-tail but not the {alpha}5B or {alpha}5C were able to bind Gh/TG2 in the presence of GTP{gamma}S (Fig. 5B). Moreover, the Gh/TG2 binding to the {alpha}5-tail was inhibited by the {alpha}5A peptide in a concentration-dependent manner (Fig. 5C) but not by the scrambled {alpha}5A (Fig. 5D). Coimmunoprecipitation of Gh/TG2 by the integrin {alpha}5 antibody was also inhibited by {alpha}5A but not {alpha}5B or {alpha}5C (Fig. 5E). The inhibition of cell migration resulting in the expression of wtTG was significantly blocked by the {alpha}5-tail and {alpha}5A peptides but not by {alpha}5B, {alpha}5C, and scrambled {alpha}5A peptides (Fig. 5F). The inhibition of cell migratory activity of C-STG and {Delta}N20TG cells was also blocked by the {alpha}5-tail and {alpha}5A peptides but not by the scrambled {alpha}5A peptides. These results clearly indicate that the direct interaction of GTP{gamma}S-bound Gh/TG2 with a cytoplasmic tail of integrin {alpha}5 modulates cell migratory activity.



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  		FIG. 5.
Identification of Gh/TG2 interaction site in the {alpha}5 tail. A, amino acid sequence of cytoplasmic tail peptides of the integrin {alpha}5, subunit and a scrambled peptide of. Lysine was inserted at the {alpha}5A end of N terminus to use for cross-linking with CNBr-Sepharose at pH 8.2. Bold type in the peptides indicates the GFFKR motif. B, Gh/TG2 binding to {alpha}5, {alpha}5A, {alpha}5B, or {alpha}5C peptide affinity gels. C, binding of Gh/TG2 to {alpha}5-tail was inhibited by {alpha}5A in a dose-dependent manner. D, binding of Gh/TG2 to {alpha}5-tail was not inhibited by the scrambled {alpha}5A peptide. E, blocking of Gh/TG2 coimmunoprecipitation by {alpha}5A. Extract (300 µg of protein) of wtTG-expressed HEK 293 cells was incubated with or without 100 µM of a5A, {alpha}5B, or {alpha}5C peptide in the presence of 20 µM GTP{gamma}S and 5 mM MgCl2 at 4 °C for 2 h. Coimmunoprecipitation of Gh/TG2 was performed using the integrin {alpha}5 antibody. The {alpha}5A but not {alpha}5B and {alpha}5C inhibits the coimmunoprecipitation of Gh/TG2. E, the {alpha}5 and {alpha}5A peptides reversed Gh/TG2-mediated inhibition of cell migratory activity of HEK 293 cell-overexpressed Gh/TG2. F, cells expressing wtTG, C-STG, {Delta}N20TG, and vector were permeabilized using streptolysine-O (10 µM) for 10 min and washed with DMEM-F-12 (23). Migration assays were performed in the presence of various {alpha}5 peptides (1 mM) at 37 °C for 6 h. Migration of vector-expressed cells was taken as 100%. The data are the means ± S.E. from three independent experiments, and scr.{alpha}5A indicates the scrambled {alpha}5A peptide. #, wtTG or C-STG or {Delta}N20TG versus {alpha}5 or various {alpha}5 peptides; p < 0.01.

 
The GFFKR Motif in the Cytoplasmic Domain of Integrin {alpha} Subunits is the Gh/TG2 Binding Site—To identify the amino acid residues that interacted with Gh/TG2 in the {alpha}5A peptide, we performed several experiments. CRT modulates Gh/TG2 activities, acting as a negative regulator (27), and regulates integrin-mediated Ca2+-influx via binding to the GFFKR motif present in cytoplasmic tail of integrin {alpha} subunits (40, 41). Incubation of HASMC with the {alpha}1 agonist resulted in inhibition of cell migration to both Fn and Vn (see Fig. 1A). These observations suggest that Gh/TG2 may interact with this motif. To examine the possibility, cytoplasmic tail peptides of integrin {alpha}v and {alpha}IIb subunits were also utilized (Fig. 6A). Indeed, Gh/TG2 interacted with cytoplasmic tail peptides of integrin {alpha}v and {alpha}IIb, including {alpha}5, and CRT reduced the binding of Gh/TG2 to these peptides (Fig. 6B). On the basis of these results mutant peptides of the {alpha}5A peptide were synthesized. We substituted Lys by Ile (K-I{alpha}5A) or Arg by Thr (R-T{alpha}5A) or Lys-Arg by Ile-Thr (KR-IT{alpha}5A) considering the same space filling to prevent distortion of the conformation (30). As shown in Fig. 6, C and D, Gh/TG2 binding to the K-I{alpha}5A peptide was ~38% less than those of the intact {alpha}5-tail and {alpha}5A. The Gh/TG2 binding to the R-T{alpha}5A was ~59% less than those to {alpha}5-tail and {alpha}5A. Substitution of both Lys and Arg residues by Ile and Thr in {alpha}5A resulted in an 89% reduction of Gh/TG2 binding. These results show that Gh/TG2 interacts with the cytoplasmic tail of integrin {alpha} subunits via the GFFKR motif and that both Lys and Arg residues in this motif are critical for the interaction of integrin with Gh/TG2.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Involvement of Gh/TG2 in transmembrane signaling of the cell surface receptors and TGase-mediated cross-linking of a variety of proteins after massive elevation of [Ca2+]i are well established cellular functions. However, reduction of cell motility by overexpression of Gh/TG2 and the priming molecular basis involving GTPase or TGase function of Gh/TG2 in this cellular response remain elusive. The present study demonstrates for the first time that {alpha}1BAR-Gh/TG2 coupling and/or increased Gh/TG2 expression modulates cell migratory activity through the interaction of GTP (GTP{gamma}S)-Gh/TG2 with integrins via the GFFKR motif located in the cytoplasmic tail of integrin {alpha} subunits.

Studies in vivo and in vitro indicate that Gh/TG2 plays a role in cell adhesion, migration, hypertrophy, and contractility of heart (13, 14, 19, 28, 29). It has been observed that Gh/TG2 expression is increased in human failing heart (ischemic and dilated cardiomyopathy) and hypertrophic heart (32, 42). The Gh/TG2-coupled receptors have also shown to be involved in smooth muscle contractility and hypertrophy (43, 44). These observations suggest that Gh/TG2 is involved in regulation of cell motility. The present study has indeed revealed that the {alpha}1BAR-Gh/TG2 coupling and/or up-regulation of Gh/TG2 expression modulate integrin-mediated cell migratory activity. Stimulation of {alpha}1AR with the {alpha}1 agonist significantly reduces migration of HASMC to Fn and Vn, and the inhibition is blocked by treatment of the {alpha}1 antagonists. Moreover, induction of Gh/TG2 expression by RA inhibits HASMC migration, and the inhibition is further augmented by the treatment of the {alpha}1 agonist. Similarly, overexpression of Gh/TG2 and its mutants (C-STG and {Delta}N20TG) in DDT1-MF2 or HEK 293 cells also reduces migration of these cells. Furthermore, the observation that the coupling of CEC-sensitive {alpha}1BAR with Gh/TG2 may modulate the cell migratory activity is substantiated by the study with DDT1-MF2, which is known to express the {alpha}1BAR among {alpha}1ARs. These results also indicate that Gh function but not TGase is the leading primer for modulation of cell migratory activity. In the case of Gh/TG2 overexpression, it has been demonstrated that the increase of Gh/TG2 expression in cells does not lead to activation of TGase activity that only occurs by the elevation of [Ca2+]i using a Ca2+-mobilizing agent or by depleting GTP (45). We have also shown that Gh/TG2 free from CRT ({beta}-subunit of Gh/TG2) has high affinity for GTP (GTP{gamma}S) (46). Moreover, it is known that the levels of GTP in cells is higher than GDP (2, 4). Expression of TGase-lacking mutants C-STG and {Delta}N20TG in HEK 293 cells results in inhibition of the cell migratory activity. These findings suggest that inhibition of the cell migratory activity resulting in overexpression of Gh/TG2 is also primed by Gh function. In addition, treatment of the {alpha}1 agonist increases formation of stress fibers and spreading of HASMC, resulting in increases in cell size. Molecular mechanisms of these {alpha}1AR-mediated cellular responses remain to be clarified.

Our studies on molecular basis of the inhibition of cell migratory activity by the {alpha}1BAR-Gh/TG2 coupling or the increased Gh/TG2 protein levels have suggested that direct interaction of GTP (GTP{gamma}S)-bound Gh/TG2 with the cytoplasmic tail of integrin {alpha} subunits modulates cell migratory activity. Evidence for this conclusion is that (a) coimmunoprecipitation of Gh/TG2 by integrin {alpha}5 antibody against the extracellular domain is blocked by {alpha}5A peptide, and Gh/TG2 coimmunoprecipitation by the antibody increases in the presence of GTP{gamma}S, (b) Gh/TG2 binds to the cytoplasmic domain of integrin {alpha}5, {alpha}v, and {alpha}IIb, and the interaction site of Gh/TG2 is the GFFKR motif present in these integrin {alpha} cytoplasmic tails, and (c) the motif-containing {alpha}5-tail and {alpha}5A peptides block the inhibition of HEK 293 cell migration caused by overexpression of wtTG, C-STG, and {Delta}N20TG. Moreover, mutation of Lys-Arg residues in the motif results in significant reduction of Gh/TG2 binding. In the same line as our observations, several studies have indicated that integrin {alpha} subunits are involved in the regulation of cell migration (47–49).

This study uncovers the Gh/TG2-mediated modulation of cell migratory activity involving integrins and the plausible molecular mechanism by which binding of GTP-bound Gh/TG2 to integrin {alpha} subunits via the GFFKR motif reduces cell motility. These findings provide new information on the cellular role of the {alpha}1BAR-Gh/TG2 signaling as well as Gh/TG2 signaling.


   FOOTNOTES
 
* This work was supported by Korea Research Foundation Grant KRF-2003-015-E00065 (to K. J. B.) and Chonbuk National Univerisity Research Fund, 1999 (to U.-H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Biochemistry, Medical School, Chonbuk National University, Keumam-dong, Jeonju 561-182, Republic of Korea. Fax: 82-63-274-9833; E-mail: imm{at}chonbuk.ac.kr.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We deeply appreciate Drs. J.-F. Feng and I. Assad for initiation this work and P. J. Davies for the kind gift of C277S tTG mutant. We thank The Cleveland Clinic Foundation for early support of this project. We also thank Drs. T. Das and G. Sa for the preliminary work of immunofluorescence staining and A. Shukula for excellent technical support.



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T. Shi, Z.-H. Duan, R. Papay, E. Pluskota, R. J. Gaivin, C. A. de la Motte, E. F. Plow, and D. M. Perez
Novel {alpha}1-Adrenergic Receptor Signaling Pathways: Secreted Factors and Interactions with the Extracellular Matrix
Mol. Pharmacol., July 1, 2006; 70(1): 129 - 142.
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K. A. Johnson and R. A. Terkeltaub
External GTP-bound Transglutaminase 2 Is a Molecular Switch for Chondrocyte Hypertrophic Differentiation and Calcification
J. Biol. Chem., April 15, 2005; 280(15): 15004 - 15012.
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K. Y. Chae, J.-H. Kim, W.-J. Park, Y.-G. Kim, H.-Y. Yun, N. S. Kwon, M.-J. Im, and K. J. Baek
Distinct pH Modulation for Dual Function of G{alpha}h (Transglutaminase II)
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