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J. Biol. Chem., Vol. 276, Issue 28, 25672-25679, July 13, 2001

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G Minigenes Expressing C-terminal Peptides Serve as Specific Inhibitors of Thrombin-mediated Endothelial Activation^*

Annette Gilchrist, Jurgen F. Vanhauwe, Anli Li, Tarita O. Thomas, Tatyana Voyno-Yasenetskaya^§, and Heidi E. Hamm^§¶

From the  Institute for Neuroscience and the  Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, Illinois 60611 and the ^§ Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60610

Received for publication, January 31, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The C termini of G protein  subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G_i peptide to bind the A₁ adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912-14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G_i could specifically inhibit downstream responses to agonist stimulation of the muscarinic M₂ receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610-6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G subunits. Minigene vectors expressing G C-terminal peptides (G_i, G_q, G₁₂, and G₁₃) or the control minigene vector, which expresses the G_i peptide in random order (G_iR), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The endothelium participates actively and dynamically in the control of vascular tone, inflammation, angiogenesis, and hemostasis (see reviews in Refs. 1-3). The local regulation of a hemostatic event is critical for the maintenance of vascular integrity and is best achieved by expression of receptors with opposing pro- or anti-thrombotic activities under the control of environmental stimuli (see reviews in Refs. 4 and 5). Thrombin is a multifunctional enzyme that plays a central role in the regulation of biochemical, transcriptional, and functional responses of endothelial cells (see reviews in Refs. 5-8). Its activities are mediated by a family of G protein-coupled receptors (GPCR)¹ designated protease-activated receptors or PARs. Three of the four PARs cloned thus far can be activated by thrombin (PAR1, PAR2, PAR4), and the resulting intracellular signaling pathways initiated are via heterotrimeric G proteins. Of the four PARs, the signal transduction pathways of PAR1 have been the most extensively studied (9-12).

Thrombin mediates a number of physiological changes in cultured endothelial cells including increased surface expression of platelet-activating factor and P-selectin (13), enhanced secretion of von Willebrand factor (14-16), elevated production of cytokines and growth factors (17, 18), augmented PAR1 gene transcription (19), changes in cell shape (20, 21), increased permeability, and disintegration of the monolayer (22-25). In addition, thrombin is a potent mitogen for endothelial cells (17, 26). Receptor activation occurs following proteolytic cleavage of the N terminus of the thrombin receptor by thrombin (see reviews in Refs. 27 and 28). A new N terminus is exposed on the receptor that serves as a tethered peptide ligand, which results in signaling across the membrane. Because proteolytic cleavage of PAR is irreversible, and the tethered ligand cannot diffuse away from the receptor, classical antagonists have been difficult to identify, and alternative targets for blocking downstream consequences of thrombin-mediated cellular activation must be considered (see reviews in Refs. 29-31). One alternative is at the site of receptor-G protein interaction.

The molecular interactions that occur between the receptor and the G protein are fundamental to the transduction of environmental signals into specific cellular responses. As is observed with many GPCRs, PAR1 is promiscuous, coupling to multiple G proteins in the same cell including G_i, G_q, and G_12/13. A variety of studies have implicated the C terminus of the G subunit in mediating receptor-G protein interaction and selectivity (32-35). We and others (33, 36, 37) have shown that peptides corresponding to the C terminus of the G subunit can be used to block receptor signaling. This interaction appears to be quite specific, as a single amino acid difference can annul the ability of the G_i1/2 peptide to bind the A₁ adenosine receptor (33).

Using a dominant negative strategy we designed a minigene vector that encodes the C-terminal 11 amino acid sequence from G (36). We speculated that when the C-terminal G peptides were produced inside cells, they would serve as competitive inhibitors of downstream responses, presumably by blocking the binding site on the receptor as had been observed when synthetic peptides were employed (19, 36, 38). We demonstrated that the G_i C-terminal minigene vector (pcDNA-G_i1/2) could specifically inhibit G protein-coupled inwardly rectifying K⁺ channel activity mediated through the muscarinic M₂ receptor. To determine the specificity of the receptor-G protein interaction, we have now constructed minigene vectors encoding each of the unique human G C-terminal sequences. As controls we constructed a minigene vector with the G_i1/2 sequence in random order (pcDNA-G_iR) and a minigene vector in which the final two C-terminal amino acids from the G_q sequence are mutated (pcDNA-G_q).

To determine the G proteins that underlie a given thrombin-mediated downstream signaling response, G C-terminal minigene vectors were systematically introduced into human microvascular endothelial cells (HMEC) by transient transfection. Our results indicate that the C-terminal minigenes can appropriately inhibit thrombin-mediated activation events mediated by a particular G protein. Furthermore, the various C-terminal minigenes give us a general idea of which G proteins control subsequent signaling events, as well as highlighting the specificity of the receptor-G protein interface. Together the findings point out the power of the C-terminal minigenes as a tool, and suggest the minigene approach may allow us to define which amino acid residues play a critical role in receptor interaction.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents-- All cell culture reagents were purchased from Life Technologies, Inc. The parent pcDNA 3.1() vector was obtained from Invitrogen (Carlsbad, CA), and the enhanced green fluorescent probe plasmid vector (pEGFP) was from CLONTECH (Palo Alto, CA). Oregon Green Bapta-1 acetoxymethyl ester, Pluronic F127, and rhodamine-phalloidin were purchased from Molecular Probes (Eugene, OR). All restriction enzymes were procured from New England Biolabs (Beverly, MA). The highly purified -thrombin (~2000 units/mg) and quinpirole were obtained from Calbiochem. [ ³H]Myoinositol (specific activity 22.2 Ci/mmol) and [³H]adenine (specific activity 24 Ci/mmol) were purchased from PerkinElmer Life Sciences. Isobutyl-methylxanthine and isoproterenol were from Sigma.

Endothelial Cell Culture-- For our studies we used a human dermal microvascular endothelial cell line that was transformed using SV-40 (HMEC-1; obtained from Dr. Asrar Malik, University of Illinois, Chicago). The cells were maintained in MCDB 131 medium supplemented with 5% fetal bovine serum, penicillin/streptomycin (5000 units/ml, 5000 µg/ml), hydrocortisone (1 µg/ml), epidermal growth factor (0.01 mg/ml), and L-glutamine (2 mM) in an atmosphere of 95% air, 5% CO₂ at 37 °C. Cells were seeded at 1 × 10⁵ cells/ml and subcultured after detachment with trypsin/EDTA (0.05%/0.5 mM). All studies utilized cell passages 18-26.

Plasmid Constructs-- cDNA minigene constructs were designed as described previously (36). Oligonucleotide sequences corresponding to the C terminus of each G were synthesized and ligated into the mammalian expression vector pCDNA 3.1(). Multiple clones for each G C-terminal minigene were selected, grown overnight in LB medium supplemented with ampicillin (100 µg/ml), and the plasmid DNA was purified using miniprep kits from Qiagen (Valencia, CA). The DNA was cut with NcoI and separated on a 1.5% agarose gel to determine whether an insert was present. A shift in the digest band pattern from three bands (3345, 1352, and 735 base pairs) to four bands (3345, 1011, 735, and 380 base pairs) indicates that an insert is present. Clones with the proper digest pattern were verified by dideoxy sequencing. All G minigene constructs used for transfection experiments were purified from 1-liter cultures using endotoxin-free maxi-prep kits (Qiagen) as described (36).

Transfections-- HMEC were transiently transfected with DNA (2 µg/100-mm plate or 0.5 µg/well for 6-well plate) using Effectene transfection reagent (Qiagen). To monitor the efficiency of transfection, cells were cotransfected with pEGFP, a plasmid vector containing enhanced green fluorescent protein. After 6 h, the medium was changed, and fresh medium added. After 48 h, cells cotransfected with pEGFP were replated onto coverslips and analyzed using a fluorescence microscope to determine the efficiency of transfection (39). Typically, 40-60% of the cells were transfected.

cAMP Assay-- HMEC were seeded onto 6-well plates at 1 × 10⁵ cells/well 24 h before transfection. Cells were transiently transfected with pcDNA3.1, pcDNA-G_i, or pcDNA-G_iR (1 µg/well) and were used to assay cAMP accumulation as described (40). After 24 h, cells were labeled with 3 µCi/ml [³H]adenine for 24 h. Thereafter, cells were washed once with serum-free media containing 1 mM isobutyl-methylxanthine, a phosphodiesterase inhibitor. To stimulate cAMP accumulation, cells were treated with 1 µM isoproterenol for 30 min at 37 °C. To determine the inhibitory effect of thrombin, cells were pretreated with 50 nM thrombin or 1 µM quinpirole for 15 min prior to the addition of isoproterenol. The reactions were terminated by the aspiration of media followed by the addition of ice-cold 5% trichloroacetic acid, and the acid-soluble nucleotides were separated on ion exchange columns.

Inositol Phosphate Accumulation-- Experiments to measure inositol phosphate (IP) accumulation were performed as described (41). Briefly, HMEC were seeded onto 6-well plates 18 h before transfection at 2 × 10⁵/well. Cells were transiently transfected with pcDNA3.1, pcDNA-G_i, pcDNA-G_iR, or pcDNA-G_q as described above. After 24 h cells were incubated in 2 ml of culture medium containing 4 µCi/ml [ ³H]myoinositol to obtain steady state labeling of cellular inositol lipids. Transiently transfected cells were assayed for IP accumulation 48 h after transfection. Two hours prior to stimulation cells were washed, and the medium was replaced with medium containing 5 mM LiCl. Cells were stimulated with 10 nM -thrombin for 10 min. Aspiration of the medium and addition of ice-cold methanol (final concentration 5%) stopped IP formation. Acid-lysed cells were centrifuged at 2500 rpm, 4 °C for 5 min. The supernatant containing IP was eluted through a Poly-prep chromatography column (Bio-Rad) containing 1.6-ml anion exchange resin (Dowex AG1-X8, formate form, 200-400 mesh). The perchloric acid-precipitated pellets that contained phosphatidylinositols and lipids were resuspended with 1 ml of chloroform-methanol, 10 M HCl (200:100:1, v/v/v). These suspensions were mixed with 350 µl of HCl and 350 µl of chloroform and centrifuged for 5 min at 2500 rpm to separate the phases. The lower hydrophobic phase was recovered and dried in counting vials to determine the amount of radioactivity in total phosphatidylinositols. The radioactivity contained in the eluate and phospholipid extracts was counted with a Packard Tricarb 1500 counter (Downers Grove, IL) after the addition of scintillation fluid.

MAPK Activity-- HMEC were seeded onto 100-mm plates 18 h before transfection at 1 × 10⁵/ml. Cells were co-transfected with 1 µg of pcDNA3.1, pcDNA-G_i, pcDNA-G_iR, or pcDNA-G_q and 1 µg of hemagglutinin-tagged MAPK (HA-MAPK) as described above. After 30 h, the cells were serum-starved for 18 h before being treated with 10 nM thrombin for 20 min. Cells were lysed in radioimmune precipitation buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 10% glycerol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), and HA-MAPK protein was immunoprecipitated using 12CA5 antibody (Berkeley Antibodies, Berkeley, CA) for 1 h at 4 °C. Protein A-Sepharose beads (Amersham Pharmacia Biotech) were then added and the tubes rotated for 3 h at 4 °C. Immune complexes were washed three times in radioimmune precipitation buffer before being used in the kinase assay (kit obtained from Upstate Biotechnology Inc., Lake Placid, NY). Kinase activity of HA-MAPK (ERK1) was measured using myelin basic protein as the substrate (42).

[Ca²⁺]_i Response-- HMEC cells were transfected with empty vector (pcDNA), pcDNA-G_i, pcDNA-G_q, or pcDNA-G_iR minigene DNA. After 48 h, the cells were transferred to coverslips at a low confluency in a 24-well plate and allowed to adhere for 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 mM CaCl₂, 1 mM MgSO₄, 0.83 mM Na₂HPO₄, 0.17 mM NaH₂PO₄, 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 mM KCl, 0.5 mM CaCl₂, 0.55 mM MgCl₂). The coverslips were placed in the chamber mounted on the stage of an inverted microscope. The experiment was performed at room temperature in Ca²⁺ buffer. Basal conditions were established for 40 s before the addition of thrombin (~ 70 nM). Recordings (1000-ms exposure time) were made every 10 s and continued for 170 s after stimulation with thrombin. Images were quantified using the NIH Image Program.

Immunofluorescent Microscopy-- As a marker for transfected cells, the pEGFP plasmid containing the gene for enhanced green fluorescent protein was co-transfected together with minigene constructs as described above. Cell were grown on gelatin-coated coverslips, serum-starved for 24 h, washed with phosphate-buffered saline, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Thereafter, cells were incubated for 30 min at room temperature with 1 mM rhodamine-phalloidin to visualize polymerized F-actin. Cells were washed extensively and mounted using Vectashield anti-fade mounting medium (Vector Laboratories, Inc., Burlingame, CA). Cells were observed with an inverted microscope (Diaphot 200, Nikon, Inc., Melville, NY) equipped for both differential interference contrast microscopy and epifluorescence observation using a 60× oil immersion objective. Fluorescence and differential interference contrast images were recorded for each cell field with a cooled, integrating Photometric Imagepoint charge-coupled device array camera (CCD; Roper Scientific, Trenton, NJ) connected to the microscope.

Data Analysis-- Statistical comparisons were made using a two-tailed Student's t test. Experimental values were considered significant at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previously, we have shown that a minigene vector expressing the G_i1/2 C-terminal peptide (pcDNA-G_i1/2) can almost completely inhibit G protein inwardly rectifying K⁺ channel activity following agonist stimulation of the M₂ muscarinic receptor (36). This inhibition was specific as transfection of the pcDNA-G_s or pcDNA-G_q C-terminal minigene vector had no effect (36). More recently, we demonstrated that a minigene vector encoding the C-terminal sequence from G_i1/2 could inhibit downstream responses to thrombin stimulation (19, 38) in endothelial cells. To determine whether we could selectively antagonize thrombin-mediated signal transduction, we generated minigene plasmid constructs that encode the C-terminal peptide sequences for several of the different G subunits. Short oligonucleotide sequences corresponding to the C terminus of each G were synthesized and ligated into the mammalian expression vector pCDNA 3.1(). As controls we used minigene vectors containing the G_i1/2 C-terminal peptide in random order (pcDNA-G_iR; Table I) and a G_q minigene vector in which the final two amino acids have been mutated (pcDNA-G_q; Table I).

View this table: [in this window] [in a new window]   Table I Sequences of expressed C-terminal minigene peptides The human C-terminal G peptides are shown with the Met and Gly at their N termini as would be expressed by the minigene vectors. GiR is the Gi1/2 peptide in random order. Gq is the Gq peptide in which the final two amino acids (italicized) have been changed.

In our studies, we used a human dermal microvascular cell line transformed with SV-40 (HMEC-1), as these cells are a readily available, easily transfected cell line that represents an established model system for the study of endothelial functions (43). To determine whether the C-terminal peptides are being expressed, the transfected cells were analyzed using reverse-phase HPLC as described previously (36). We estimate, given the efficiency of transfection for the experiment and the amount of lyophilized peptide obtained from reverse-phase HPLC, that the transfected HMEC have a concentration of ~100 nM peptide after 48 h (data not shown). Following verification of peptide production by the minigenes, we systematically introduced different C-terminal G minigenes into the endothelial cells and examined the downstream consequences on biochemical and functional end points following thrombin stimulation.

The pcDNA-G_i Minigene Vector Decreases Thrombin-mediated Inhibition of cAMP-- Thrombin stimulation is known to affect cAMP levels, and this effect can be inhibited by pertussis toxin indicating that G_i/G_o is involved in the signaling pathway (44). To determine whether the G_i1/2 C-terminal minigene could affect intracellular cAMP levels following thrombin stimulation, we transfected HMEC with pcDNA-G_i or the control vectors pcDNA (vector only) or pcDNA-G_iR (the G_i sequence in random order). As shown in Fig. 1 we found that basal cAMP levels were essentially equivalent for all conditions tested. Endothelial cells stimulated with isoproterenol to activate -adrenergic receptors increase their cAMP levels through the G_s pathway. Cells transfected with pcDNA, pcDNA-G_i, or pcDNA-G_iR showed little difference in isoproterenol-mediated cAMP accumulation, with an 82, 64, and 77-fold increase, respectively. When the endothelial cells are pre-incubated with 50 nM thrombin for 15 min prior to the addition of isoproterenol, a decrease in cAMP levels was observed. Endothelial cells transfected with the control pcDNA vector and pre-incubated with thrombin showed a 39% decrease in cAMP level over cells stimulated with only isoproterenol. Similarly, cells transfected with the pcDNA-G_iR minigene vector and pre-incubated with thrombin had a 43% decrease over cells stimulated with isoproterenol only. However, cells transfected with the pcDNA-G_i minigene vector and pre-incubated with thrombin had virtually no decrease in cAMP levels (0.1%) as compared with cells stimulated with isoproterenol only. Thus, cells expressing the G_i C-terminal peptide appear to be unable to inhibit adenylyl cyclase following activation with thrombin. Our results support our previous work and indicate that we can specifically block thrombin-mediated G_i signaling with transfection of the pcDNA-G_i minigene into endothelial cells (19).

View larger version (34K): [in this window] [in a new window]   Fig. 1.   The Gi minigene vector blocks thrombin-mediated inhibition of isoproterenol-stimulated cAMP accumulation in endothelial cells. HMEC were seeded onto 6-well plates 24 h before transfection. Cells were transiently transfected with pcDNA, pcDNA-GiR, or pcDNA-Gi DNA using Effectene (Qiagen) according to the manufacturer's protocol. After 24 h, cells were labeled with 3 µCi/ml [3H]adenine. After another 24 h, cells were washed with serum-free medium containing 1 mM isobutyl-methylxanthine. To stimulate cAMP accumulation, cells were treated with 1 µM isoproterenol for 30 min at 37 °C. To see the inhibitory effect of thrombin, cells were pretreated with 50 nM thrombin for 15 min prior to the addition of isoproterenol. Three separate experiments were performed in duplicate. **, indicates p < 0.005.

The G_q Minigene Decreases Thrombin-mediated Calcium Response and IP Accumulation-- Thrombin stimulation has been shown previously to initiate a calcium response in cells (45-47). To determine whether any of the C-terminal minigenes could affect [Ca²⁺]_i levels we transfected HMEC with pcDNA-G_q, pcDNA-G_i, or the control vectors (pcDNA or pcDNA-G_iR). As shown in Fig. 2 we found that following cell activation by thrombin there was a transient increase in intracellular [Ca²⁺]_i levels. Thirty seconds after the addition of thrombin, cells transfected with pcDNA-G_q had a calcium response that was decreased 44% as compared with cells transfected with pcDNA (Fig. 2A). In fact, pcDNA-G_q-transfected cells had a 45% decrease compared with those transfected with pcDNA when all time points measured after thrombin stimulation were averaged (Fig. 2B). This decrease appears to be specific, as cells transfected with pcDNA-G_i or pcDNA-G_iR did not have any change in thrombin-stimulated [Ca²⁺]_i levels (Fig. 2, A and B). Thus, cells expressing the G_q C-terminal peptide appear to be inhibited in their ability to stimulate intracellular [Ca²⁺]_i levels following activation with thrombin, indicating that this downstream mediator can be blocked specifically with transfection of the G_q minigene into endothelial cells.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   The Gq minigene vector modulates thrombin-mediated [Ca2+]i flux in endothelial cells. HMEC were transfected with pcDNA, pcDNA-GiR, pcDNA-Gi, or pcDNA-Gq minigene vectors. After 48 h, cells were transferred to coverslips at a low confluency in a 24-well plate and allowed to adhere for 2 h. The medium was aspirated and each coverslip was incubated at 37 °C for 30 min in loading buffer containing 0.1% (v/v) Pluronic F127 and 10 µM Oregon Green Bapta-1 acetoxymethyl ester. Basal conditions were established before the addition of thrombin (~70 nM) in Ca2+-free buffer. Recordings were made every 10 s and were continued for 170 s after stimulation with thrombin. Images were quantitated using NIH Image. Data from at least 70 individually recorded cells were used to calculate the changes in fluorescence. Panel A, fluorescent changes in [Ca2+]i levels 30 s after cell stimulation with thrombin. Basal fluorescence (FB) was recorded for 50 s before the addition of thrombin. Shown is the change in fluorescence (FS) recorded 30 s after thrombin addition to cells. Each bar represents the mean ((FS  FB)/FB) ± S.E. of over 70 individually recorded cells. **, indicates p < 0.005. Panel B, kinetics of [Ca2+]i fluorescence changes after cell stimulation with thrombin. Data presented are the mean ((FS  FB)/FB) ± S.E. at each recording point for cells transfected with pcDNA-GiR or pcDNA-Gq. The arrow indicates the addition of thrombin. Each time point represents more than 70 individually recorded cells.

Thrombin stimulation increases [Ca²⁺]_i by a G_q-mediated activation of PLC, producing IP₃ and diacylglycerol (see review in Ref. 48). We found that HMEC had a nearly 2-fold increase in IP accumulation following activation with thrombin when transfected with pcDNA. As shown in Fig. 3, a similar level of thrombin-mediated IP accumulation was observed in cells transfected with pcDNA-G_iR and pcDNA-G_i (2.1- and 1.9-fold, respectively). However, in cells transfected with pcDNA-G_q, no stimulation of IP accumulation was seen following activation with thrombin (Fig. 3). Thus, endothelial cells expressing the G_q C-terminal peptide are inhibited in their ability to stimulate IP accumulation following activation with thrombin, indicating that transfection of the G_q minigene specifically blocks this downstream signaling event.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Gq minigene inhibits thrombin-mediated inositol phosphate generation. pcDNA, pcDNA-GiR, pcDNA-Gi, pcDNA-Gq, or pcDNA-Gs minigene constructs were transfected into HMEC and used to assay IP accumulation 48 h later. After 24 h cells were reseeded onto 24-well plates and labeled with [3H]myoinositol (2 µCi/ml). After 48 h cells were rinsed and incubated with or without thrombin (10 nM) for 10 min. Total IP accumulation was assayed as described under "Materials and Methods" using Dowex columns to separate [3H]IP. The relative amount of [3H]IP generated was calculated as follows: [3H]IP (cpm)/([3H]IP (cpm) + [3H]inositol (cpm)). Each value was normalized by the basal value (no thrombin stimulation) obtained in pcDNA-transfected cells. The results presented are the normalized mean ± S.E. of at least three independent experiments performed in triplicate. **, indicates p < 0.005.

The G₁₂ and G₁₃ Minigenes Decrease Thrombin-mediated Stress Fiber Formation-- Recent work by Gohla and colleagues (49) elegantly demonstrated that thrombin receptors induce stress fiber accumulation via G₁₂ in an epidermal growth factor receptor-independent manner. The authors suggest the formation of stress fiber formation is Rho-dependent. Both the G₁₂ family (49, 50) as well as the Rho signaling pathway (51-54) have been implicated in stress fiber formation. Thus, the effects of transfecting the G₁₂ or G₁₃ minigenes were compared with control (pcDNA) in confluent monolayers of HMEC for thrombin-induced stress fiber formation by immunostaining for F-actin. After exposure to thrombin, the cells were fixed, permeabilized, and stained for F-actin with rhodamine-phalloidin (Fig. 4). Following serum starvation, cells transfected with pcDNA exhibited a thin cortical F-actin rim at their margins and contained few stress fibers (Fig. 4A). The stress fibers present were inconspicuous and in an apparently random orientation. In HMEC transfected with pcDNA and stimulated with thrombin, the actin is reorganized into prominent stress fibers, typically arranged in a parallel pattern along the longitudinal axis of the cell (Fig. 4B). A very different pattern is observed for cells transfected with pcDNA-G₁₂ (Fig. 4C) or pcDNA-G₁₃ (Fig. 4D) minigenes after exposure to thrombin. In both pcDNA-G₁₂- and pcDNA-G₁₃-transfected cells, thrombin stimulation did not result in the appearance of stress fibers. In cells transfected with pcDNA-G₁₃, the peripheral actin rim appears thicker and more linear, providing a clear outline of cell-cell junctions. Thus, in agreement with earlier reports (21) we found that thrombin induced rapid stress fiber formation in endothelial cells. However, transfection of either pcDNA-G₁₂ or pcDNA-G₁₃ minigenes resulted in cells that no longer showed thrombin-induced stress fiber formation. Given that stress fiber formation is dependent on the small GTPase Rho (21, 55) and that both G₁₂ and G₁₃ are intimately linked to Rho signaling (51-54), our results indicated that transfection of the pcDNA-G₁₂ or pcDNA-G₁₃ minigenes may effect Rho signaling.

View larger version (78K): [in this window] [in a new window]   Fig. 4.   G12 and G13 minigenes inhibit thrombin-mediated stress fiber formation. pcDNA, pcDNA-G12, or pcDNA-G13 minigene constructs (1 µg each/100 mm dish) were transfected into HMEC cells. After 48 h, cells were serum-starved for 18 h before being treated with 10 nM thrombin for 20 min. A, unstimulated cells transfected with pcDNA; B, cells transfected with pcDNA with thrombin stimulation; C, cells transfected with G12 minigene with thrombin stimulation; D, cells transfected with G13 minigene with thrombin stimulation.

Effect of C-terminal Minigenes on Thrombin-mediated MAPK Activation-- The extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein kinases (MAPKs) regulates numerous cell signaling events involved in proliferation and differentiation (see review in Ref. 56). To determine whether the C-terminal minigenes could effect intracellular MAPK activity, we systematically transfected HMEC with minigenes along with HA-MAPK (ERK1). By immunoprecipitating the HA-MAPK, we measured the effects of the minigenes on only those cells that had been transfected. The addition of 10 nM thrombin resulted in a 3.7-fold increase in HA-MAPK activity ((stimulated-basal)/basal) in cells transfected with the pcDNA control vector (Fig. 5). Cells transfected with the pcDNA-G_iR minigene vector had an essentially equivalent increase in thrombin-mediated MAPK activity (4.5-fold increase). However, endothelial cells transfected with the pcDNA-G_i1/2, pcDNA-G_q, pcDNA-G₁₂, or pcDNA-G₁₃ minigene vectors showed a significant decrease in thrombin-mediated HA-MAPK activity (59, 57, 50, and 77%, respectively) as compared with cells transfected with the pcDNA control vector.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   All of the C-terminal minigenes inhibit thrombin-mediated MAPK activity. HA-MAPK (ERK1) and pcDNA, pcDNA-GiR, pcDNA-Gi, pcDNA-Gq, pcDNA-G12, or pcDNA-G13 minigene constructs were transfected into HMEC cells. After 48 h cells were serum-starved for 18 h before being treated with 10 nM thrombin for 20 min. MAPK activity was measured using myelin basic protein as substrate. Proteins were blotted onto nitrocellulose filters in duplicate and counted. MAPK activity (nM/min/mg) was obtained for each, and the relative increase of MAPK activity (thrombin-mediated -fold increase) was calculated as follows: ((stimulated activity  basal activity)/basal activity). The results presented are the mean ± S.E. of at least three independent experiments. *, indicates p < 0.05.

Amino Acid Specificity-- To determine the specificity of our C-terminal G minigenes, a G_q minigene was made in which the final two amino acids (Ala-Val) are mutated (Thr-Lys) (G_q; Table I). Following transient transfection into HMEC, expression of the mutated G_q peptide led to thrombin responses that were much closer to results from cells transfected with the pcDNA-G_iR minigene vector rather than from cells transfected with the pcDNA-G_q minigene vector. In the case of the fluorescent changes in [Ca²⁺]_i we found a 44% reduction observed in cells transfected with pcDNA-G_q, whereas cells transfected with pcDNA-G_q had a 20% reduction when compared with cells transfected with pcDNA (Fig. 6A). When we measured IP accumulation, we found that stimulation with thrombin lead to a nearly 2-fold increase in endothelial cells transfected with pcDNA. This response was completely inhibited in cells transfected with the pcDNA-G_q minigene vector. However, we observed a 1.5-fold increase in thrombin-mediated IP accumulation in cells transfected with the pcDNA-G_q minigene vector, indicating that the presence of the mutated peptide can partially restore the IP response (Fig. 6B). A similar phenomenon was also observed for the MAPK response. In response to thrombin, cells transfected with the control vector (pcDNA) had a 3.7-fold increase in MAPK activity, whereas cells transfected with the pcDNA-G_q minigene vector had a 1.6-fold increase and cells transfected with the pcDNA-G_q minigene vector had a 3.2-fold increase (Fig. 6C). Thus, the 57% decrease observed in thrombin-mediated MAPK activity for cells transfected with pcDNA-G_q is reduced to a 12% decrease in cells transfected with pcDNA-Gq.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Mutation of Gq minigene leads to improved thrombin-mediated [Ca2+]i response, inositol phosphate accumulation, and MAPK activity. pcDNA, pcDNA-GiR, pcDNA-Gq, or pcDNA-Gq minigene constructs were transfected into HMEC as described above. After 48 h cells were assayed for [Ca2+]i flux (A), IP accumulation (B), and MAPK activity (C). **, indicates p < 0.005.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The PARs are a family of GPCR that serve as targets for thrombin cleavage and thus are important therapeutic targets for anti-thrombotic, anti-atherosclerotic, and anti-inflammatory treatments. Three receptors have been shown to be cleaved by thrombin (PAR1, PAR3, PAR4). However, researchers have shown that thrombin receptors other than PAR-1 are either not expressed on endothelial cells or are not able to support a thrombin response on their own (57, 58) (see review in Ref. 59). Thus, for our work we presume that only PAR1 is activated when endothelial cells are stimulated with thrombin.

Although many of the resultant biological consequences of thrombin activation on endothelial cells are known, less is known about which G proteins mediate these events. The G proteins act as molecular switches that couple the thrombin receptors to their relevant effector systems such as enzymes or ion channels. Heterotrimeric G proteins, composed of an  subunit and a dimer, are characterized by the identity of their  subunit. Sixteen unique G subunit genes have been cloned, and on the basis of sequence similarities they are divided into four families: G_i (G_i1, G_i2, G_i3, G_o1, G_o2, G_t, G_gust, G_z), G_s (G_s, G_olf), G_q (G_q, G₁₁, G₁₄, G_15/16), and G₁₂ (G₁₂, G₁₃). The thrombin receptor PAR1, like many other GPCR, can interact with and activate multiple G proteins in the same cell including those of the G_i/o (60, 61), G_q (62), and G_12/13 (63, 64) subtype families.

To date, classical antagonists for PARs have been difficult to identify because of the unique mechanism of thrombin activation via cleavage of the receptor and generation of a tethered ligand. Thus, blockade of thrombin receptors in pathological conditions such as atherosclerosis, thrombosis, stroke, and restenosis has not been possible. Rather than antagonize the ligand binding site, our laboratory has chosen to target an alternative site, that of the receptor-G protein interface. A variety of studies have implicated the G subunit in mediating specific receptor-G protein interactions. The C-terminal region of G has been shown to be critical in determining the specificity of GPCR-G protein interactions (see reviews in Refs. 65 and 66). Conklin et al. (32) demonstrated that substitution of the final three C-terminal amino acids from G_q with the corresponding residues from Gi allowed receptors that signal exclusively through G_i to activate the chimeric  subunits and stimulate the G_q effector, phospholipase C- (PLC-). In addition, synthetic peptides that mimic the C-terminal region of G protein  subunits can be used to block receptor-G protein interactions (33-35, 37). This interaction is quite specific, as changing a single amino acid can annul the ability of the G_i peptide to bind the A₁ adenosine receptor (33).

Recently, our laboratory developed a dominant negative strategy that employs "minigene vectors" to specifically inhibit G protein signaling (36). The minigene vectors are plasmid vectors that express high levels of C-terminal peptides from the G subunit when transfected into mammalian cells. The C-terminal peptides serve as competitive inhibitors for the G protein  subunit binding site on the receptor. Following transfection of the minigene vectors the corresponding G C-terminal peptide is produced inside the cell (36) where it can specifically inhibit agonist-induced GPCR responses (36, 19). To test whether introduction of the minigene vectors into HMEC could inhibit thrombin-mediated responses, we measured biochemical and physiological functions 48 h after transient transfection of the minigene vectors.

Previous studies utilizing pertussis toxin to abolish the inhibitory effect of thrombin on isoproterenol-stimulated cAMP production indicate a role for G_i in thrombin signaling (67). Our results further support the role of G_i as we found that thrombin-stimulated cells transfected with the pcDNA-G_i minigene vector no longer showed inhibition of isoproterenol-mediated cAMP production (Fig. 1) or forskolin-mediated cAMP production (data not shown). This effect appears to be specific, as no difference is observed in cells transfected with control vectors (pcDNA, and pcDNA-G_iR) (Fig. 1).

In endothelial cells, thrombin stimulation has been shown to activate G_q and thereby PLC, producing IP₃ and diacylglycerol and stimulating an increase in intracellular calcium concentration ([Ca²⁺]_i; see review in Ref. 48). Cells transfected with the pcDNA-G_q minigene vector showed an inhibition in their ability to stimulate both IP accumulation and intracellular [Ca²⁺]_i levels following activation with thrombin (Figs. 2 and 3). Thus, it appears we can specifically block this downstream pathway. The effects appear to be specific, as no differences were observed in IP accumulation and intracellular [Ca²⁺]_i levels in cells transfected with the pcDNA-G_i minigene vector or the control vectors (pcDNA, pcDNA-G_iR). That we see a more profound effect on IP accumulation than [Ca²⁺]_i levels following activation with thrombin in cells expressing the G_q peptide suggests that multiple pathways may lead to changes in [Ca²⁺]_i levels, whereas only stimulation of the G_q subunit results in IP accumulation.

Next we examined the signal transduction pathways underlying thrombin-stimulated reorganization of the actin cytoskeleton in HMEC transfected with pcDNA-G₁₂ or pcDNA-G₁₃. Others have shown that receptor activation by thrombin leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding (49, 50). In addition, constitutively active mutants of G₁₂ and G₁₃ are known to induce stress fiber assembly (51). Our data supports the roles of both G₁₂ and G₁₃ in stress fiber formation as transfection of either pcDNA-G₁₂ or pcDNA-G₁₃ minigenes resulted in cells that no longer showed thrombin-induced stress fiber formation (Fig. 4).

Multiple signaling inputs lead to activation of MAPK. Agonist stimulation of many GPCR, including thrombin receptors, leads to MAPK activation via G protein signaling to downstream effector molecules such as Ras and Src. To determine which G protein pathway thrombin receptors use to activate MAPK in endothelial cells, we transfected HMEC with pcDNA-G_i, pcDNA-G_q, pcDNA-G₁₂, or pcDNA-G₁₃ minigene vectors. To our surprise, we found that thrombin activation of MAPK was blocked by minigene vectors encoding C-terminal peptides for all G subunits tested (Fig. 5), whereas the MAPK response for the control vectors (pcDNA, pcDNA-G_iR) was robust. Activation of any particular G complex via agonist stimulation of the thrombin receptor will produce two effectors, namely G·GTP and free G. For instance, MAPK can be activated potentially by either G_q or G (68-70). It is conceivable that non-G_q subunits may serve the negative function of sequestering G and thus prevent activation of the MAPK pathway. Our findings support this idea, as all of the minigenes tested resulted in a decrease in thrombin-mediated MAPK activity as compared with the control vectors (pcDNA or pcDNA-G_iR) (Fig. 5). Thus, there appears to be a redundancy in MAPK signaling. Our results suggest some receptors are multiply coupled to downstream effectors, such that more than one source of input is necessary to drive the signal.

Evidence for the specificity of the minigene action came from a mutant G_q minigene vector constructed with changes in the final two amino acids of the C-terminal G_q peptide (pcDNA-G_q). Our results indicate that transfection of this minigene vector into HMEC resulted in a decreased inhibition of the thrombin-mediated intracellular [Ca²⁺]_i levels, IP accumulation, and MAPK activity as compared with cells transfected with the pcDNA-G_q (Fig. 6). Thus, it appears our C-terminal minigenes may allow us to evaluate the role of specific amino acid residues within the G C terminus that are critical to receptor interactions and antagonism of G protein activation.

Molecular determinants other than the C terminus are involved in the recognition between heterotrimeric G proteins and their cognate receptors (71-73). However, a variety of studies have shown that the G C terminus is critical in both mediating receptor-G protein interaction and in receptor selectivity (see reviews in Refs. 65 and 66). Previous results indicate that introduction of minigene vectors that express C-terminal peptide sequences from G into mammalian cells can selectively block agonist-mediated responses (19, 36, 38). The method appears to be a promising approach for turning off G protein-mediated responses in vitro and in vivo. In this study, systematic transfection of different G C-terminal minigenes allowed us to selectively block signal transduction through a given G protein and thus provided a strategy for determining which G proteins are involved in specific thrombin-mediated physiological responses. In addition, our work begins to address whether G proteins bind to overlapping sites on the receptor.

In conclusion, the studies presented here highlight the complexity of the signaling systems that modulate changes in endothelial cells challenged with thrombin. A fundamental issue is the ability to dissect out the mechanistic and functional differences between G protein subfamilies. Transfection of the different G C-terminal minigenes allowed us to selectively block signal transduction through a given G protein. The experiments presented begin to define the signal transduction pathways that regulate thrombin-mediated endothelial cell activation. This type of detailed analysis is critical for understanding the role of individual G proteins in thrombin-induced vascular injury and for a rational strategy of therapeutic interventions. Inhibition of thrombin activation would have important consequences on thrombogenesis, hemostasis, inflammation, and neointimal vascular response to injury.

	FOOTNOTES

^* This work was supported by Grant HL60678-01A1 (to A. G., T. V-Y., and H. E. H.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, 442 Robinson Research Bldg., 23rd and Pierce Dr., Nashville, TN 37232. Tel.: 615-343-3533; Fax: 615-343-1084; E-mail: heidi.hamm@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M100914200

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; ERK, extracellular receptor kinase; G proteins, guanine nucleotide-binding proteins; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HMEC, human microvascular endothelial cell; HPLC, high pressure liquid chromatography; IP, inositol phosphate; MAPK, mitogen activated protein kinase; PAR, protease-activated receptor; PLC, phospholipase C.

	REFERENCES

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