Thrombin Receptors Activate Go Proteins in Endothelial Cells to Regulate Intracellular Calcium and Cell Shape Changes
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m204477200
ISSN1083-351X
AutoresJurgen F.M. Vanhauwe, Tarita O. Thomas, Richard D. Minshall, Chinnaswamy Tiruppathì, Anli Li, Annette Gilchrist, Eun-Ja Yoon, Asrar B. Malik, Heidi E. Hamm,
Tópico(s)Cell Adhesion Molecules Research
ResumoThrombin receptors couple to Gi/o, Gq, and G12/13 proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of Gi, Gq, G12, or G13 proteins in thrombin signaling has been investigated extensively, the role of Go proteins has largely been ignored. To determine whether Go proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of Go1 proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (1Gilchrist A. Vanhauwe J.F., Li, A. Thomas T.O. Voyno-Yasenetskaya T. Hamm H.E. J. Biol. Chem. 2001; 276: 25672-25679Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In this study, we demonstrate that Go proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [35S]guanosine-5′-O-(3-thio)triphosphate binding to Go proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to Go proteins was substantiated by transfection of the Go1 minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca2+]i from intracellular stores. Transfection of the β-adrenergic kinase C terminus blocked the [Ca2+]i response to the same extent as with Go1 minigene peptide, suggesting that this Go-mediated [Ca2+]i transient was caused by Gβγ stimulation of PLCβ. Transfection of a Gi1/2 minigene had no effect on thrombin-stimulated [Ca2+]i signaling in HMEC, suggesting that Gβγ derived from Go but not Gi could activate PLCβ. The involvement of Go proteins on events downstream from calcium signaling was further evidenced by investigating the effect of Go1 minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of Go1minigenes but not Gi1/2 minigenes. We conclude that the Go proteins play a role in thrombin signaling distinct from Gi1/2 proteins, which are mediated through their Gβγ subunits and involve coupling to calcium signaling and cytoskeletal rearrangements. Thrombin receptors couple to Gi/o, Gq, and G12/13 proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of Gi, Gq, G12, or G13 proteins in thrombin signaling has been investigated extensively, the role of Go proteins has largely been ignored. To determine whether Go proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of Go1 proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (1Gilchrist A. Vanhauwe J.F., Li, A. Thomas T.O. Voyno-Yasenetskaya T. Hamm H.E. J. Biol. Chem. 2001; 276: 25672-25679Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In this study, we demonstrate that Go proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [35S]guanosine-5′-O-(3-thio)triphosphate binding to Go proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to Go proteins was substantiated by transfection of the Go1 minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca2+]i from intracellular stores. Transfection of the β-adrenergic kinase C terminus blocked the [Ca2+]i response to the same extent as with Go1 minigene peptide, suggesting that this Go-mediated [Ca2+]i transient was caused by Gβγ stimulation of PLCβ. Transfection of a Gi1/2 minigene had no effect on thrombin-stimulated [Ca2+]i signaling in HMEC, suggesting that Gβγ derived from Go but not Gi could activate PLCβ. The involvement of Go proteins on events downstream from calcium signaling was further evidenced by investigating the effect of Go1 minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of Go1minigenes but not Gi1/2 minigenes. We conclude that the Go proteins play a role in thrombin signaling distinct from Gi1/2 proteins, which are mediated through their Gβγ subunits and involve coupling to calcium signaling and cytoskeletal rearrangements. protease-activated receptor guanosine-5′-O-(3-thio)triphosphate β-adrenergic kinase C terminus Chinese hamster ovary 4′,6-diamidino-2-phenylindole dihydrochloride green fluorescent protein Hank's buffered salt solution human microvascular endothelial cell N-ethyl-5′-carbamoyladenosine pertussis toxin Spodoptera frugiperda 9 Thrombin Receptor Activating Peptide Thrombin is a multifunctional serine protease that catalyzes conversion of fibrinogen to fibrin, a process that is crucial in blood coagulation (1Gilchrist A. Vanhauwe J.F., Li, A. Thomas T.O. Voyno-Yasenetskaya T. Hamm H.E. J. Biol. Chem. 2001; 276: 25672-25679Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In addition, thrombin plays a central role in a variety of biological functions such as platelet aggregation, mitogenesis of fibroblasts, monocytic cell chemotaxis, and endothelial cell monolayer permeability (2Bevilacqua M.P. Gimbrone M.A., Jr. Semin. Thromb. Hemostasis. 1987; 13: 425-433Crossref PubMed Scopus (129) Google Scholar, 3Wu K.K. Thiagarajan P. Annu. Rev. Med. 1996; 47: 315-331Crossref PubMed Scopus (291) Google Scholar, 4Aengevaeren W.R. Atherosclerosis. 1999; 147 (Suppl. 1): S11-S16Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Many of its actions, including the regulation of biochemical, transcriptional, and functional responses in endothelial cells occur through activation of protease-activated receptors (PARs),1 which belong to the superfamily of G protein-coupled receptors. Four PARs have been cloned so far, but only PAR1, PAR3, and PAR4 can be activated by thrombin (5Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2110) Google Scholar). The activation and signal transduction pathways of PAR1, the prototype of the PAR family, have been studied in great detail. Thrombin cleaves the N-terminal extracellular domain of PAR1 at a specific site, which unmasks a new N terminus that then serves as a tethered agonist ligand and activates the receptor by binding intramolecularly to the body of the receptor (6Coughlin S.R. Thromb. Haemostasis. 1993; 70: 184-187Crossref PubMed Scopus (84) Google Scholar). Cleaved,i.e. irreversibly activated, PAR1 can couple to members of the Gi/o, Gq, and G12/13 protein families and regulate a variety of intracellular effectors (1Gilchrist A. Vanhauwe J.F., Li, A. Thomas T.O. Voyno-Yasenetskaya T. Hamm H.E. J. Biol. Chem. 2001; 276: 25672-25679Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Although the role of Go proteins has been generally believed to be confined to the brain and heart, several reports indicate that Go proteins may serve to regulate various intracellular pathways in non-neuronal cell lines (7Kajiyama Y. Murayama T. Kitamura Y. Imai S. Nomura Y. Biochem. J. 1990; 270: 69-75Crossref PubMed Scopus (19) Google Scholar, 8Baffy G. Yang L. Raj S. Manning D.R. Williamson J.R. J. Biol. Chem. 1994; 269: 8483-8487Abstract Full Text PDF PubMed Google Scholar). In addition, several new effectors have been identified that are specifically or differentially regulated by Go proteins (versusGi proteins) (9Luo Y. Denker B.M. J. Biol. Chem. 1999; 274: 10685-10688Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 10Gomez M.P. Nasi E. J. Neurosci. 2000; 20: 5254-5263Crossref PubMed Google Scholar, 11Holtje M. von Jagow B. Pahner I. Lautenschlager M. Hortnagl H. Nurnberg B. Jahn R. Ahnert-Hilger G. J. Neurosci. 2000; 20: 2131-2141Crossref PubMed Google Scholar). Pertussis toxin (PTX)-mediated ADP-ribosylation of the Cys351 residue in the C terminus of Gi and Go proteins disables their interaction with receptors and thus prevents receptor-mediated activation of these G proteins. Many effects of thrombin are mediated through pertussis toxin-sensitive G proteins (12Huang C.L. Cogan M.G. Cragoe E.J., Jr. Ives H.E. J. Biol. Chem. 1987; 262: 14134-14140Abstract Full Text PDF PubMed Google Scholar, 13O'Rourke F. Zavoico G.B. Smith L.H., Jr. Feinstein M.B. FEBS Lett. 1987; 214: 176-180Crossref PubMed Scopus (16) Google Scholar, 14Flavahan N.A. Vanhoutte P.M. Blood Vessels. 1990; 27: 218-229PubMed Google Scholar, 15Boulanger C.M. Vanhoutte P.M. J. Vasc. Res. 1997; 34: 175-185Crossref PubMed Scopus (50) Google Scholar, 16van den Eijnden-Schrauwen Y. Atsma D.E. Lupu F. de Vries R.E. Kooistra T. Emeis J.J. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2177-2187Crossref PubMed Scopus (47) Google Scholar, 17Rydholm H.E. Falk P. Eriksson E. Risberg B. Scand. J. Clin. Lab. Invest. 1998; 58: 347-352Crossref PubMed Scopus (3) Google Scholar, 18Ellis C.A. Malik A.B. Gilchrist A. Hamm H. Sandoval R. Voyno-Yasenetskaya T. Tiruppathi C. J. Biol. Chem. 1999; 274: 13718-13727Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). This method, however, does not distinguish between Gi and Goproteins, and the importance of the latter subtype could be inadequately appreciated (5Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2110) Google Scholar). To dissect out the contribution of Gi1/2 and Go1 proteins in thrombin-regulated signaling pathways in HMECs, we have designed a dominant negative strategy based on minigene vectors that encode the C-terminal 11-amino acid sequence from Gα. Previously, we have shown that these minigenes are quite specific in such a way that Gq-based minigenes blocked only thrombin activation of Gq protein-mediated pathways (phosphatidylinositol bisphosphate hydrolysis and intracellular calcium increase) but not Gi1/2 or G12/13protein-mediated pathways (1Gilchrist A. Vanhauwe J.F., Li, A. Thomas T.O. Voyno-Yasenetskaya T. Hamm H.E. J. Biol. Chem. 2001; 276: 25672-25679Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 19Gilchrist A. Bunemann M., Li, A. Hosey M.M. Hamm H.E. J. Biol. Chem. 1999; 274: 6610-6616Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The specificity of Gα C-terminal peptides has been shown dramatically by Gilchrist et al.(20Gilchrist A. Mazzoni M.R. Dineen B. Dice A. Linden J. Proctor W.R. Lupica C.R. Dunwiddie T.V. Hamm H.E. J. Biol. Chem. 1998; 273: 14912-14919Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), where one or two amino acid substitutions inhibited the ability of peptides to block receptor-mediated activation of signaling pathways. To delineate specific functions for Gi and Goproteins in the signaling of thrombin receptors, we have introduced these minigenes into HMECs. Our findings indicate that HMECs contain Go proteins and that PAR1 has the potential to couple to Gi and Go proteins when co-expressed in Sf9 cell membranes. In addition, we show that Go1minigenes block thrombin-stimulated release of [Ca2+]i, whereas Gi1/2minigenes do not. The involvement of Go proteins, but not Gi proteins, was further established in pathways that are known to be downstream of calcium signaling, such as stress fiber formation and endothelial barrier permeability. Together our data demonstrate the importance of Go proteins in the signaling of thrombin receptors in endothelial cells. All of the cell culture reagents were purchased from Invitrogen. The parent pcDNA 3.1(−) vector was obtained from Invitrogen; pEGFP was from CLONTECH, retroviral Tet-inducible vectors pRevTRE2 and pREvTRE2-dEGFP were from CLONTECH (Palo Alto, CA). All of the restriction enzymes were procured from New England Biolabs (Beverly, MA). The highly purified α-thrombin (∼2000 units/mg) and PTX were obtained from Calbiochem. Alexa Fluor 568 phalloidin, DAPI, Oregon Green Bapta-1 acetoxymethylester, Pluronic F127, and the Prolong Antifade kit were purchased from Molecular Probes (Eugene, OR). Anti-Gαo1/2antibodies were from Dr. D. Manning (University of Pennsylvania, Philadelphia, PA). 3-Isobutyl-1-methylxanthine, forskolin, and isoproterenol were from Sigma. Electrodes for endothelial monolayer resistance measurements were obtained from Applied Biosciences (Troy, NY). Virions producing the rat Gαi1, Gαi2, Gαi3, and Gαo1 were obtained from Dr. S. Graber (West Virginia University, Morgantown, WV), whereas those for PAR1 were obtained from Dr. C. Chinni (University of Cambridge, Cambridge, UK). [35S]GTPγS was from PerkinElmer Life Sciences. Sf9 cells were grown at 27 °C and at an ambient atmosphere in suspension in a shaking incubator and transfected as described before (21Ford C.E. Skiba N.P. Bae H. Daaka Y. Reuveny E. Shekter L.R. Rosal R. Weng G. Yang C.S. Iyengar R. Miller R.J. Jan L.Y. Lefkowitz R.J. Hamm H.E. Science. 1998; 280: 1271-1274Crossref PubMed Scopus (371) Google Scholar). Harvested Sf9 cells were washed with ice-cold 50 mm Tris-HCl buffer, pH 7.4, resuspended in hypotonic 10 mm Tris-HCl buffer, pH 7.4, and homogenized with 10 strokes of a Bio-Homogenizer (BioSpec Products, Inc.) at high speed. The homogenate was centrifuged at 30,000 × g for 20 min at 4 °C. The membrane pellet was resuspended in 50 mmTris-HCl buffer, pH 7.4, containing 10% glycerol and stored in aliquots at −80 °C. [35S]GTPγS binding experiments were performed as described previously (22Vanhauwe J.F. Ercken M. van de Wiel D. Jurzak M. Leysen J.E. Psychopharmacology. 2000; 150: 383-390Crossref PubMed Scopus (20) Google Scholar). Briefly, 10 μg of Sf9 cell membrane protein was diluted in 50 mm Tris-HCl buffer, pH 7.4, containing 5 mm MgCl2, 1 mmEGTA, 100 mm NaCl, 0.1 mm dithiothreitol, 10 μg/ml saponin, and 1 μm GDP and preincubated with TRAP for 15 min at room temperature in a volume of 125 μl in a 96-well plate. Then 25 μl of [35S]GTPγS diluted 1000-fold in assay buffer was added to the wells, and the assay mixtures were further incubated for 30 min at room temperature. The reactions were terminated by rapid filtration, after which the filters were washed four times with 200 μl of 50 mm Tris-HCl buffer, pH 7.4, containing 100 mm NaCl, 5 mmMgCl2, and 1 mm EGTA. Filter-bound radioactivity was counted in a liquid scintillation spectrometer. Nonspecific [35S]GTPγS binding was measured in the presence of 100 μm GTPγS and never exceeded 10% of basal binding. Basal [35S]GTPγS binding was estimated in the absence of TRAP. For our studies we used a human dermal microvascular endothelial cell line that was transformed using SV-40 (HMEC-1; obtained from Dr. E. Ades (Centers for Disease Control, Atlanta, GA). The cells were maintained in MCDB 131 medium supplemented with 5% fetal bovine serum, penicillin/streptomycin (5000 units/ml; 5000 μg/ml), hydrocortisone (500 μg/ml), epidermal growth factor (0.01 μg/ml), and l-glutamine (2 mm) in an atmosphere of 95% air, 5% CO2 at 37 °C. The cells were seeded at 1 × 105 cells/ml and subcultured after detachment with 0.05% trypsin,/0.5 mm EDTA. All of the studies utilized cell passages 18–26. cDNA minigene constructs were designed as described previously (19Gilchrist A. Bunemann M., Li, A. Hosey M.M. Hamm H.E. J. Biol. Chem. 1999; 274: 6610-6616Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The C terminus of the G protein-coupled receptor kinase 2 (βARK-ct) has been shown to be a potent and specific Gβγ inhibitor (23Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar). The βARK-ct construct codes for residues 548–671 of the rat homolog βARK. Retroviral minigenes were constructed as follows. The cDNA encoding the last 11 amino acids of human Gα subunits (Gαi1/2 and Gαo) or the Gαi1/2 C terminus in random order (GαiR) were synthesized (Great American Gene Company) with newly engineered 5′- and 3′-ends. The 5′-end contained a BamHI site followed by the ribosome-binding consensus sequence (5′-GCCGCCACC-3′), a methionine (ATG) for translation initiation, and a glycine (GGA) to protect the ribosome-binding site during translation and the nascent peptide against proteolytic degradation. A HindIII site was synthesized at the 3′-end immediately following the translational stop codon (TGA). The DNA was brought up in sterile double distilled H2O (stock concentration, 100 μm). Complimentary DNA was annealed in 1× NE Buffer 3 (50 mmTris-HCl, 10 mm MgCl2, 100 mm NaCl, 1 mm dithiothreitol; New England Biolabs) at 85 °C for 10 min and then allowed to cool slowly to room temperature. The annealed cDNA were ligated for 1 h at room temperature into a Tet-inducible retroviral vector pRevTRE2 in murine Maloney tumor virus (CLONTECH) previously cut with BamHI andHindIII. Following ligation, the samples were heated to 65 °C for 5 min to deactivate the T4 DNA ligase. The ligation reaction (1 μl) was electroporated into 50 μl of competent ARI814 cells (Bio-Rad Escherichia coli Pulsar), and the cells were immediately placed into 1 ml of superoptimal catabolite medium (Invitrogen). After 1 h at 37 °C, 100 μl was spread on LB/ampicillin plates and incubated at 37 °C for 12–16 h. To verify that insert was present, several colonies were grown overnight in LB/ampicillin, and their plasmid DNA was purified (Qiagen SpinKit). The plasmid DNA was digested withNcoI (New England Biolabs, Inc.) for 1 h at 37 °C and run on a 1.5% (3:1) agarose gel. Vector alone produced one band (6.5 kb), whereas vector with insert resulted in two bands (5.1 and 1.4 kb). DNA with the correct pattern was sequenced (Northwestern University Biotechnology Center) to confirm the appropriate sequence. For optimal results, the retroviral vectors were packaged using the pantropic GP-293 cell line (CLONTECH) with vesicular stomatitis virus glycoprotein, an envelope glycoprotein from the vesicular stomatitis virus. As a control, we used the enhanced GFP inserted into the parental vector (pRevTRE2-dEGFP; CLONTECH). Retroviral minigenes were generated by infecting the packaging cells GP-293 with pRevTRE2 minigenes and vesicular stomatitis virus glycoprotein using Effectene reagent according to instructions from the manufacturer (CLONTECH). 12–16 h later, the medium was replaced by 5 ml of fresh medium/10-cm dish, and the virus produced by the cells was collected 2–3 days post-transfection by filtration through 0.45-μm cellulose acetate filters and stored in aliquots at −80 °C. The virus titer reached 4 × 106plaque-forming units/ml. For pcDNA-based minigenes, HMECs were transiently transfected with DNA (2 μg/100-mm plate or 500 ng/well for a 6-well plate) using Effectene transfection reagent (Qiagen). To monitor the efficiency of transfection, the cells were co-transfected with pEGFP, a plasmid vector containing enhanced green fluorescent protein to monitor stress fiber formation, or DsRED (CLONTECH), a plasmid vector containing red fluorescent protein for [Ca2+]i imaging. After 3 h, the medium was changed, and fresh medium was added. After 48 h, the cells co-transfected with the fluorescent proteins were replated onto coverslips and analyzed using a fluorescent microscope to determine the efficiency of transfection. Adenylyl cyclase and HMEC monolayer permeability experiments were performed using cells infected with retroviral minigenes. HMECs were infected with retroviral minigene virus (2 × 106 plaque-forming units/well for 6-well plate). The expression of the minigene peptide was induced 24 h after viral infection with 2 μg/ml doxycyclin, and the experiments were performed 24 h after induction. Use of retroviral minigenes led to ∼100% transfection efficiency. This was confirmed using infection of the pRevTRE2-dEGFP vector, which exhibited expression of GFP in virtually every cell (data not shown). Endothelial cell lysates were resolved by SDS-PAGE on a 10–20% separating gel under reducing conditions. For immunoblotting analysis, the proteins were transferred to polyvinylidene difluoride membranes using standard semi-dry transfer method. The membranes were blocked with 5% dry milk in phosphate-buffered saline, 0.05% Tween 20 for 1 h at room temperature. The membranes were incubated with indicated primary antibody (diluted in blocking buffer) at 4 °C overnight. Following washes, the membranes were incubated at room temperature with peroxidase-labeled secondary antibodies and detected using luminol-based chemiluminescent detection system (LumiGLO, Kirkegaard and Perry Laboratories, Gaithersburg, MD). HMECs were seeded onto 6-well plates at 1 × 105 cells/well 24 h before transfection. The cells were transfected with retroviral minigene constructs, and 24 h before the assay, the cells were seeded into a 24-well plate. Thereafter, the cells were washed once with serum-free medium containing 1 mm 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and further incubated for 20 min in 500 μl of serum-free medium containing 1 mm3-isobutyl-1-methylxanthine. After the preincubation, 50 μl of preincubation medium supplemented with forskolin (final concentration, 10 μm) was added to each well. To detect the inhibitory effect, 100 nm thrombin or 10 μm NECA was added along with forskolin. Basal cAMP accumulation was measured in the absence of forskolin and compounds. The reactions were terminated by the addition of 100 μl of 1 n HCLO4. The samples were frozen and thawed, and 200 μl of KOH/K3PO4 (0.5 m, pH 13.5) was added to neutralize the samples (final pH, 7.4). After formation of the KClO4 precipitate (30 min at 4 °C), the plates were centrifuged (10 min at 650 × g, 4 °C). The amount of cAMP in each well was determined with a commercial125I-labeled cAMP radioimmunoassay kit (Biomedical Technologies Inc., Stoughton, MA). In single cell fluorescence measurements, the DsRED (CLONTECH) fluorescence reporter gene was used to confirm the transfected cells. HMECs were transfected with pcDNA-Gi1/2, pcDNA-Go1, or pcDNA-GiR minigene DNA and with or without βARK-ct DNA along with DsRED. After 48 h, the cells were transferred to coverslips at a low confluency in a 24-well plate and allowed to adhere for at least 2 h. The medium was aspirated, and each coverslip was incubated at 37 °C for 30 min in 0.5 ml of loading buffer (20 mm Hepes, pH 7.4, 130 mm NaCl, 5 mm KCl, 2 mmCaCl2, 1 mm MgSO4, 0.83 mm Na2HPO4, 0.17 mmNaH2PO4, 1 mg/ml bovine serum albumin, 25 mm mannose) containing 0.1% (v/v) Pluronic F127 and 10 μm Oregon Green Bapta-1 acetoxymethyl ester. The cells were washed twice with and incubated in Ca2+ buffer (10 mm Hepes, pH 7.4, 140 mm NaCl, 5 mmKCl, 0.5 mm CaCl2, 0.55 mmMgCl2). The coverslips were placed in the chamber that was mounted on the stage of an upright microscope. The experiment was performed at room temperature. The transfected cells were identified using a green filter by observing DsRED fluorescence. The basal conditions were established for 40 s before addition of thrombin (∼70 nm). Recordings (exposure time) were made every 10 s and continued for 170 s after stimulation with thrombin. The images were quantified using the NIH Image Program. As a marker for transfected cells, the pEGFP plasmid containing the gene for enhanced green fluorescent protein was transiently co-transfected together with minigene constructs as described above. HMECs were grown on gelatin-coated coverslips, serum-starved for 24 h, washed with HBSS, and fixed with 4% paraformaldehyde. The coverslips were washed three times for 5 min in 100 mm glycine in HBSS to quench and remove the fixative followed by three washes for 10 min in HBSS. The cells were permeabilized with 0.1% Triton X-100 and washed three times for 10 min in HBSS. Thereafter, the cells were incubated for 90 min at room temperature with 200 nm Alexa Fluor 568-phallodin to visualize polymerized F-actin. The coverslips were washed three times for 10 min in HBSS and labeled with 1 μg/ml DAPI for 30 min to visualize the nucleus. The coverslips were finally washed three times for 10 min in HBSS and mounted on a drop of ProLong Antifade mounting medium (Molecular Probes). The cells were observed with a Zeiss 510 laser scanning confocal microscope (New York, NY) using 364-, 488-, and 568-nm excitation laser lines to detect DAPI (BP 385–470 nm emission), fluorescein isothiocyanate/Alexa 488 (BP505–550 emission), and rhodamine/Alexa 568 fluorescence (LP585 emission) with the ×63 1.4 NA water immersion objective. The acquired images were later assembled using Adobe Photoshop, MS PowerPoint, and Macromedia Freehand image processing software. Endothelial cell retraction measured in real time in response to thrombin was measured as described before (24Tiruppathi C. Malik A.B. Del Vecchio P.J. Keese C.R. Giaever I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7919-7923Crossref PubMed Scopus (374) Google Scholar). HMECs were infected with retroviral minigene constructs and seeded on gelatin-coated gold electrodes (4.9 × 104 cm2) and grown to confluence. The small and larger counter electrodes were connected to a phase-sensitive lock-in amplifier. A constant current of 1 μA was applied by a 1 V, 4000 Hz AC signal connected serially to 1mΩ resistor between the small and large counter electrodes. The voltage between the small electrode and the large counter electrode was monitored by a lock-in amplifier, stored, and processed by a personal computer. The same computer controlled the output of the amplifier and switched the measurement to different electrodes in the course of the experiment. Prior to the experiments, the monolayers were washed two times with serum-free medium and incubated for 2 h in 1% serum-supplemented medium. The data were analyzed using GraphPad Prism 2.01 (GraphPad Software, San Diego, CA). Statistical comparisons were made using a two-tailed Student’s t test. The experimental values were considered significant at p < 0.05. Thrombin is known to couple to multiple G proteins including Gi, Gq, and G12/13proteins (25Barr A.J. Brass L.F. Manning D.R. J. Biol. Chem. 1997; 272: 2223-2229Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Activation of the thrombin receptor results in initiating an array of signal transduction pathways such as phosphatidylinositol hydrolysis, mobilization of Ca2+ stores, induction of stress fiber formation, and activation of mitogen-activated protein kinase (1Gilchrist A. Vanhauwe J.F., Li, A. Thomas T.O. Voyno-Yasenetskaya T. Hamm H.E. J. Biol. Chem. 2001; 276: 25672-25679Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 5Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2110) Google Scholar). Some signaling pathways regulated by thrombin have been shown to be sensitive to PTX,e.g. intracellular calcium release, activation of Na+/H+ exchanger, arachidonic acid release, induction of PAR1 gene expression, von Willebrand Factor release, and endothelial relaxation (12Huang C.L. Cogan M.G. Cragoe E.J., Jr. Ives H.E. J. Biol. Chem. 1987; 262: 14134-14140Abstract Full Text PDF PubMed Google Scholar, 13O'Rourke F. Zavoico G.B. Smith L.H., Jr. Feinstein M.B. FEBS Lett. 1987; 214: 176-180Crossref PubMed Scopus (16) Google Scholar, 14Flavahan N.A. Vanhoutte P.M. Blood Vessels. 1990; 27: 218-229PubMed Google Scholar, 15Boulanger C.M. Vanhoutte P.M. J. Vasc. Res. 1997; 34: 175-185Crossref PubMed Scopus (50) Google Scholar, 16van den Eijnden-Schrauwen Y. Atsma D.E. Lupu F. de Vries R.E. Kooistra T. Emeis J.J. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2177-2187Crossref PubMed Scopus (47) Google Scholar, 17Rydholm H.E. Falk P. Eriksson E. Risberg B. Scand. J. Clin. Lab. Invest. 1998; 58: 347-352Crossref PubMed Scopus (3) Google Scholar, 18Ellis C.A. Malik A.B. Gilchrist A. Hamm H. Sandoval R. Voyno-Yasenetskaya T. Tiruppathi C. J. Biol. Chem. 1999; 274: 13718-13727Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). PTX abolishes the interaction between receptors and all members of the Gi subfamily (except Gz) through ADP-ribosylation of the Cys351residue in the C terminus of the Gα subunit. The effect of PTX has often been attributed to Gi proteins, because Go proteins are generally believed to play a role in the brain, where it constitutes about 1% of the total protein content (26Sternweis P.C. Robishaw J.D. J. Biol. Chem. 1984; 259: 13806-13813Abstract Full Text PDF PubMed Google Scholar). To determine whether PAR1, the most characterized thrombin receptor, can potentially couple to Go proteins, we expressed this receptor in Sf9 cells along with Gαo1, Gβ1 and Gγ2, and measured TRAP-stimulated [35S]GTPγS binding to membranes prepared from these cells. As a negative control, we co-expressed PAR1, Gβ1, and Gγ2 (but not Gαo) in Sf9 cells. Fig 1 shows that TRAP stimulated [35S]GTPγS binding up to 700 ± 50% above the basal level in a concentration-dependent manner in Sf9 membranes co-expressing PAR1 and Gαo1β1γ2 proteins. In Sf9 membranes co-expressing only PAR1 and Gβ1γ2, TRAP stimulated [35S]GTPγS binding to a significantly lower level (230 ± 10% above basal level) (p < 0.05). In addition, we found that TRAP could stimulate [35S]GTPγS binding to membranes prepared from Sf9 cells co-expressing PAR1 and Gαi1β1γ2, Gαi2β1γ2, or Gαi3β1γ2 heterotrimers. The level of stimulation in the latter membranes was apparently lower than in membranes co-expressing the Gαo1β1γ2 heterotrimer. Because we did not determine the level of PAR1 expression (because of the lack of commercially available radioligands), we could not conclude whether the lower stimulation level by Gαiβ1γ2 reflected a lower coupling efficiency or a lower expr
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