Gαq and Gβγ Regulate PAR-1 Signaling of Thrombin-Induced NF-κB Activation and ICAM-1 Transcription in Endothelial Cells
As thrombin binding to the G protein–coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-κB activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, Gαq, and the Gβγ dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-κB activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits Gαq, or α-transducin (Gαt), a scavenger of the Gβγ, markedly decreased NF-κB activity induced by PAR-1 activation. We determined the downstream signaling targets activated by Gαq and Gβγ that mediate NF-κB activation. Expression of the kinase-defective protein kinase C (PKC)-δ mutant inhibited NF-κB activation induced by the constitutively active Gαq mutant, but had no effect on NF-κB activity induced by Gβ1γ2. In related experiments, NF-κB as well as ICAM-1 promoter activation induced by Gβ1γ2 were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated Gαq. Cotransfection of the catalytically inactive Akt mutant inhibited the NF-κB activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of Gαq and PKC-δ. These results support a model in which ligation of PAR-1 induces NF-κB activation and ICAM-1 transcription by the engagement of parallel Gαq/PKC-δ and Gβγ/PI3-kinase pathways that converge at Akt.
The procoagulant thrombin, a serine protease activated during thrombosis, induces the expression of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells.1 ICAM-1, the ligand for the leukocyte β2 integrins (CD11/CD18), promotes the firm adhesion of leukocytes, and facilitates their migration across the endothelial barrier.2 ICAM-1 is constitutively present on the endothelial cell surface, but its expression can be induced by the activation of the transcription factor nuclear factor (NF)-κB.1,3⇓ NF-κB is typically a heterodimer of 50-kDa (p50) and 65-kDa (RelA) subunits sequestered in the cytoplasm in association with IκB proteins that mask the NF-κB nuclear localization signal.4 Stimulation with proinflammatory mediators such as thrombin results in serine phosphorylation (Ser32 and Ser36) of IκBα by IκBβ kinase (IKKβ).5 Phosphorylation targets IκBα for ubiquitination and proteasome-mediated degradation.6 The released NF-κB translocates to the nucleus where it binds to cis-regulatory element present in the ICAM-1 promoter.1
Thrombin activates protease activated receptor-1 (PAR-1) by the cleavage of its NH2-terminal domain at Arg-41 and Ser-42, unmasking the tethered ligand (SFLLRN),7 which interacts with the extracellular loop 2 of the receptor (amino acids 248 to 268).8 PAR-1 activation induces endothelial adhesivity toward human neutrophils (PMNs) and monocytes.1,9⇓ The coupling of PAR-1 with Gq, Gi, and G12/13 in endothelial cells is required for the activation of multiple cellular responses.10 In the present study, we addressed the pathways by which PAR-1 signals NF-κB activation and ICAM-1 transcription in endothelial cells. The results demonstrate that ligation of PAR-1 mediates NF-κB activation and ICAM-1 transcription through the heterotrimeric G protein, Gαq, and the released Gβγ dimer. The downstream signaling involves activation of parallel protein kinase C (PKC)-δ and PI 3-kinase pathways, which converge at Akt to induce NF-κB–dependent ICAM-1 transcription.
Materials and Methods
Human thrombin (activity of 3170 NIH U/mg) was purchased from Enzyme Research Laboratories (South Bend, Ind). PAR-1 agonist peptide (TFLLRNPNDK) and inactive control peptide (FTLLRNPNDK) were synthesized.11 Superfect and plasmid maxi kit were from QIAGEN Inc. DEAE-dextran was purchased from Sigma. The construct pTKRLUC containing Renilla luciferase gene driven by the constitutively active thymidine kinase promoter and Dual Luciferase Assay System were purchased from Promega (Madison, Wis). Plasmids encoding Gβ1 and Gγ2,12 α subunit of bovine retinal transducin (Gαt),13 activated forms of Gαq, pGαqR138C (GαqRC),14 and Gα13, pGα13Q226L (Gα13QL)15 were prepared as described. ICAM-1-LUC construct containing 1393 bp of 5′-regulatory region of ICAM-1 promoter linked to firefly luciferase reporter gene was provided by Dr C. Stratowa (Ernst Boehringer Institut, Vienna, Austria).16 Expression vectors of the constitutively active catalytic subunit (p110*) and dominant-negative regulatory subunit (*p85) of PI 3-kinase were prepared as described.17,18⇓ Constructs of wild-type, constitutively active, and kinase-defective mutants of Akt/PKB and expression vector encoding RGS3T are described.19–21⇓⇓ PKC-α, -ε, and -δ mutants lacking the functional catalytic domain due to substitution of lysine 368, 437, or 376 for arginine, respectively, were gifts from Dr J. Soh (Columbia University, New York, NY).22 Construct encoding kinase-defective mutant of IKKβ is described.19 Construct pNF-κB-LUC containing 5 copies of consensus NF-κB sequences linked to a minimal E1B promoter-firefly luciferase gene was purchased from Stratagene.
Endothelial Cell Culture
Human umbilical vein endothelial cells (HUVECs; Clonetics) were cultured23 in gelatin-coated flasks using endothelial basal medium 2 (EBM2) with bullet kit additives. Confluent cells were incubated for 2 to 12 hours in heat inactivated 0.5% to 1% FBS containing EBM2 before thrombin or PAR-1 agonist peptide challenge. All experiments were made in cells under the 8th passage, except where otherwise indicated.
Total RNA was isolated and Northern blotting was performed using the labeled human ICAM-1 cDNA probe (0.54 kb SalI–PstI fragment).23
Cell Lysis and Immunoblotting
After treatment, cells were lysed in SDS-sample buffer (10 mmol/L Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.4% dithiothreitol, 1 mmol/L orthovanadate with bromphenol blue). Cell lysates were analyzed for ICAM-1 expression by immunoblotting.23
Transfection and Luciferase Assay
Transfections were performed using DEAE-dextran method.23 Briefly, 5 μg DNA was mixed with 50 μg/mL DEAE-dextran in serum-free EBM, and the mixture was added onto 70% to 80% confluent cells. We used 0.125 μg pTKRLUC to normalize the transfection variation from plate to plate. After 1 hour, cells were incubated for 4 minutes with 10% dimethyl sulfoxide (DMSO) in serum-free EBM2. Cells were then washed 2× with EBM2 containing 10% FBS and grown to confluence. This protocol resulted in a transient transfection efficiency of 11±2% (mean±SD; n=3). In some experiments, we used Superfect (Qiagen) to transfect the cells.23 Briefly, reporter DNA (1 μg) was mixed with 5 μL of Superfect in 100 μL serum-free EBM2. We used 0.1 μg pTKRLUC to normalize the transfection variation from plate to plate. After 5 to 10 minutes incubation at room temperature, 0.6 mL EBM2 containing 10% FBS was added, and the mixture was applied onto cells prewashed once with PBS. The medium was changed 3 hours later to EBM2 containing 10% FBS and cells were grown to confluence. Using this protocol, we achieved a transient transfection efficiency of 21±2% (mean±SD; n=3). Cell extracts were prepared and assayed for reporter gene activity using the Promega Biotech Dual Luciferase Assay System. Firefly (Photinus pyralis) luciferase activity was normalized to sea pansy (Renilla reniformis) luciferase activity and expressed as relative light units (RLU)/μg cell protein or fold increase. Protein content was determined using the Bio-Rad protein determination kit.
PMN Adhesion Assay
PMN adhesion assay was performed as described.24 HUVECs grown in 12-well plates were stimulated with PAR-1 agonist peptide or thrombin for 6 to 8 hours followed by labeling with 3 μmol/L fluorescent (red) Cell Tracker dye for 30 minutes. Freshly isolated PMNs were stained with 5 μmol/L fluorescent (green) Cell Tracker dye, coincubated with endothelial cells for 20 minutes, washed with PBS, and visualized using a fluorescent microscope. The number of adherent PMN/0.8 mm2 of endothelial cell was counted and expressed as fold increase relative to untreated control.
Data are presented as mean±SEM. Comparisons between groups were made by ANOVA (Tukey’s post-test). Differences were considered significant at P<0.05.
PAR-1 Induces ICAM-1 Expression and Endothelial Adhesivity PAR-1 agonist peptide (TFLLRNPNDK) was used to address the role of PAR-1 in activation of ICAM-1 expression and endothelial adhesivity toward PMNs. PAR-1 agonist peptide mimicked the effects of thrombin in inducing these responses; however, the magnitude of these responses was 30% to 40% less even at the maximal concentration of TFLLRNPNDK (Figures 1A through 1C). In the control experiment, the inactive peptide (FTLLRNPNDK) failed to induce ICAM-1 transcription (Figure 1A). To address the role of PAR-1 in signaling NF-κB activation, HUVECs were transfected with pNF-κB-LUC, in which the firefly luciferase reporter gene is driven by multiple NF-κB sequences. Results showed that TFLLRNPNDK, like thrombin, induced NF-κB–dependent firefly luciferase activity but to a lesser extent (Figure 1D). We also determined whether NF-κB activation leads to increased ICAM-1 promoter activity. Exposure of cells transfected with ICAM-1LUC to thrombin induced ICAM-1 promoter activity (Figure 1D) consistent with its effect on ICAM-1 expression and endothelial adhesivity toward PMNs (Figure 1A through 1C).
Gαq and Gβγ Transduce PAR-1–Induced NF-κB Activation
We used the NF-κB–dependent firefly luciferase activity as the readout to address the PAR-1–activated signaling mechanisms mediating ICAM-1 transcription. Because PAR-1 couples to multiple heterotrimeric G proteins,25,26⇓ we initially addressed the contributions of Gαq, Gαi, and Gα13 in mediating NF-κB activation. RGS3T, a regulator of G-protein signaling that inhibits Gαq,27 was used to address the function of Gαq. RGS3T significantly reduced NF-κB activity after TFLLRNPNDK or thrombin challenge (Figure 2A). As further evidence of the importance of Gαq, the expression of constitutively active Gαq mutant (GαqRC) induced NF-κB activity in the absence of PAR-1 activation (Figure 2B). In contrast, expression of activated Gαi2 (Gαi2QL) or Gα13 (Gα13QL) had no effect on NF-κB activity (Figure 2B). In control experiments, Gα13QL induced SRE-LUC (serum response element-luciferase) (Figure 2B) and Gαi2QL activated PAR-1-Luc (PAR-1 promoter-luciferase)26 (data not shown).
As the released Gβγ dimer of heterotrimeric G proteins can activate signaling pathways that may lead to NF-κB activation, we studied the effects of α-transducin (Gαt), which sequesters Gβγ and antagonizes Gβγ signaling.13 Cotransfection of Gαt inhibited the NF-κB activity induced by thrombin or TFLLRNPNDK (Figure 2C). In another experiment, cotransfection of equimolar concentrations of constructs encoding Gβ1 and Gγ2 with pNF-κB-LUC induced NF-κB activity in the absence of PAR-1 activation (Figure 2D); this response was inhibited by expression of Gαt (Figure 2D). Coexpression of Gβ1γ2 also induced ICAM-1 promoter activity in a Gαt-sensitive manner (Figure 2E). These data indicate that both Gαq and Gβγ can independently transduce NF-κB activation after PAR-1 activation.
Gαq Induces NF-κB Activation Through PKC-δ
Because PKC-δ is required for thrombin-induced NF-κB activity,23 we addressed the possibility that PKC-δ functions downstream of Gαq in signaling NF-κB activation. Cotransfection of kinase-defective mutant of PKC-δ (PKC-δmut) abrogated the NF-κB activity induced by GαqRC, indicating the requirement of PKC-δ in the response (Figure 3A). Cotransfection of kinase-defective mutants of PKC-α (PKC-αmut) and PKC-ε (PKC-εmut) also reduced the GαqRC-induced NF-κB activity, although to a significantly lesser extent (P<0.05) than the PKC-δmut (Figure 3A).
We also addressed the possibility that PKC-δ contributes to NF-κB activity induced by Gβγ. Cotransfection of PKC-δmut failed to inhibit the Gβ1γ2-induced NF-κB activity (Figure 3B), suggesting that PKC-δ did not contribute to the Gβ1γ2-dependent component of the response.
Gβγ Induces NF-κB Activation Through PI 3-Kinase
Dominant-negative mutant of the regulatory 85-kDa subunit of PI 3-kinase (δp85) was used to address the role of PI 3-kinase in the mechanism of NF-κB activation. Coexpression of δp85 inhibited NF-κB activity (Figure 4A) induced by thrombin or TFLLRNPNDK. Inhibition of PI 3-kinase activity by δp85 also inhibited ICAM-1 promoter activity (data not shown). We also determined the effects of constitutively active mutant of PI 3-kinase (p110*), created by joining the catalytic (p110) and regulatory (p85) subunits through a hinge peptide.17 Expression of p110* induced NF-κB and ICAM-1 promoter activities in the absence of PAR-1 activation (Figures 4B and 4C). These data show that PI 3-kinase is required for PAR-1–induced NF-κB activity and ICAM-1 promoter activation.
To address whether PI 3-kinase functions downstream of Gβγ, we determined the effects of δp85 on the Gβγ-induced NF-κB activity. Cotransfection of δp85 prevented the Gβ1γ2–induced activation of NF-κB (Figure 5A). We also addressed the possibility that PI 3-kinase is a downstream effector of Gαq in mediating NF-κB activation. However, this study showed that coexpression of δp85 failed to prevent NF-κB activity induced by GαqRC (Figure 5B), suggesting that PI 3-kinase does not participate in Gαq-activated signaling of NF-κB activation. These data demonstrate that PI 3-kinase lies downstream of Gβγ in signaling NF-κB activity.
Gαq/PKC-δ and Gβγ/PI 3-Kinase Pathways Converge at Akt
We determined whether the serine/threonine kinase Akt lies downstream of both PKC-δ and PI 3-kinase in mediating PAR-1–induced NF-κB activation. Inhibition of Akt by the kinase-defective Akt mutant (Aktmut) inhibited the NF-κB activity in response to thrombin or TFLLRNPNDK challenge, indicating the involvement of Akt in the response (Figure 6A). Cotransfection of Aktmut also prevented thrombin-induced ICAM-1 promoter activity (data not shown). In another experiment, we determined the effects of inhibition of Akt on NF-κB activity induced by constitutively active mutant of PI 3-kinase (p110*). We found that cotransfection of the Aktmut prevented the NF-κB activity induced by p110* (Figure 6B). We also addressed the other possibility that Akt functions downstream of Gαq/PKC-δ in signaling NF-κB activation. Expression of wild-type Akt (Aktwt) augmented GαqRC-induced NF-κB activity, whereas expression of Aktmut inhibited the response (Figure 7A). These findings show a critical role of Akt in signaling the Gαq-transduced response. As PKC-δ functions downstream of Gαq (Figure 3), we next determined the effects of inhibition of Akt on NF-κB activity induced by the constitutively active PKC-δ (PKC-δCAT). Results of this experiment showed that cotransfection of Aktmut inhibited the PKC-δCAT–induced NF-κB activity (Figure 7B). In contrast, expression of PKC-δmut had no effect on NF-κB activity induced by the constitutively active Akt (AktCAT) (Figure 7C). Taken together, these data show that Gαq/PKC-δ and Gβγ/PI 3-kinase pathways converge at Akt to activate NF-κB.
Akt Signals PAR-1–Induced NF-κB Activation Through IKKβ
As NF-κB activation involves the degradation of IκBα requiring its phosphorylation by IKKβ, we determined the involvement of Akt in mediating the PAR-1–induced NF-κB activity through the activation of IKKβ. HUVECs were cotransfected with pNF-κBLUC in combination with the kinase-defective IKKβ mutant (IKKβmut). Results showed that expression of IKKβmut prevented the PAR-1–induced NF-κB activity (Figure 8A), demonstrating the requirement of IKKβ in the response. We next addressed the function of IKKβ in the Akt-mediated NF-κB activation. Cotransfection of AktCAT activated NF-κB in the absence of PAR-1 activation (Figure 8B). Moreover, cotransfection of IKKβmut prevented AktCAT-induced NF-κB activity (Figure 8B), indicating the critical role of Akt in mediating the PAR-1–induced NF-κB activity through the activation of IKKβ.
Thrombin induces endothelial cell adhesivity and firm adhesion of leukocytes by the expression of ICAM-1.1,9⇓ ICAM-1 expression involves the activation of the transcription factor NF-κB1; however, the signaling mechanisms responsible for NF-κB–dependent ICAM-1 transcription remain unclear. In the present study, we addressed the role of the heterotrimeric G proteins coupled to PAR-1 in transducing NF-κB activation in endothelial cells. The data showed that PAR-1 induces NF-κB activation and resultant ICAM-1 expression through Gαq and the Gβγ dimer. We observed that Gαq and Gβγ independently signal through PKC-δ and PI 3-kinase, with the signals converging at Akt to induce NF-κB activation (Figure 8C).
We used the selective PAR-1 agonist (TFLLRNPNDK)11 to address the role of PAR-1 in mediating NF-κB activation. The present study focused on PAR-1 because it is the predominant thrombin receptor in endothelial cells.26,28⇓ PAR-1 agonist peptide induced NF-κB activation and ICAM-1 expression in endothelial cells, much like thrombin itself. PAR family consists of 4 described receptors of which 3 are activated by thrombin (PAR-1, PAR-3, and PAR-4); PAR-2 is activated by trypsin or mast cell tryptase.29 In addition to PAR-1, endothelial cells express PAR-2 and PAR-3.30,31⇓ As TFLLRNPNDK is not a ligand for PAR-2 and PAR-3,28 NF-κB activation and ICAM-1 expression in the present study can only be ascribed to PAR-1. However, because the magnitude of NF-κB activation and ICAM-1 expression induced by TFLLRNPNDK was less than thrombin, we cannot exclude the involvement of other PARs in contributing to the thrombin response.
PAR-1 may trigger signaling in endothelial cells via its coupled heterotrimeric G proteins, Gi, Gq, and G12/13.10,25,26⇓⇓ We addressed the role of Gi2 and Gα13 by expression of constitutively active Gi2 (Gi2QL) or Gα13 (Gα13QL) forms. Both Gi2QL and Gα13QL failed to activate NF-κB, indicating that Gi2 and Gα13 are not required for this response in endothelial cells. The inability of Gi2QL and Gα13QL to activate NF-κB cannot be the result of a lack of activity of these constructs because in positive control experiments the constructs activated PAR-1-Luc (the PAR-1 promoter-luciferase construct)26 and SRE-LUC (serum response element-luciferase), respectively. Because Gα13QL activates NF-κB in HeLa cells, 32 its lack of effect in endothelial cells suggests that Gα13QL may function in a cell-specific manner. To address the role of Gαq, we used RGS3T, a regulator of G-protein signaling that inhibits Gαq.21,27⇓ These findings showed that inhibition of Gαq signaling prevented NF-κB activation after PAR-1 stimulation. Moreover, the expression of constitutively active Gαq mutant (GαqRC) or Gβ1γ2 dimer was sufficient in itself to activate NF-κB. Because the released Gβγ complex can induce G protein–coupled receptor signaling, 12,33⇓ we investigated the possible role of Gβγ in the mechanism of PAR-1–induced NF-κB activity. The inhibition of Gβγ signaling by α-transducin (Gαt), which sequesters the Gβγ subunits,13 prevented the Gβγ- as well as PAR-1–induced NF-κB activation responses. Thus, these studies show that both Gαq and Gβγ are important in signaling the PAR-1–induced NF-κB activation.
Because the novel PKC isoform PKC-δ activated by thrombin is required for NF-κB activation and ICAM-1 transcription in endothelial cells,23 we addressed its contribution in signaling NF-κB activation downstream of Gαq. The results showed that PKC-δ is in fact critical in signaling NF-κB activation in this sequence. The results also show that PKC-α and PKC-ε contribute to the Gαq-activated response but to a lesser extent. Thus, the data are consistent with an important role of Gαq (which activates both classical and novel PKC isoforms PKC-α, PKC-δ, and PKC-ε34) in signaling the PAR-1–mediated NF-κB activation. These results also support the notion of possible synergism between PKC isoforms to activate NF-κB.
In contrast to Gαq, the stimulatory effect of Gβγ on NF-κB activity was the result of PI 3- kinase, which catalyzes the addition of phosphate to the 3-OH position of the inositol ring of phosphatidylinositol lipids.35 Two of the ubiquitously expressed PI 3-kinase catalytic subunits, p110α and p110β, form a heterodimer complex with the regulatory subunits of p85 family.35 Another PI 3-kinase catalytic subunit, p110γ, lacks the binding site for p85, but instead associates with the regulatory subunit p101.36 Studies have shown that these PI 3-kinase isoforms are activated by Gβγ released from ligand-activated G protein–coupled receptors.19,36,37⇓⇓ The present data show that the expression of δp85 (ie, dominant-negative mutant of p85 subunit) inhibited both thrombin- and TFLLRNPNDK- as well as Gβγ-induced NF-κB activation. Similar results were obtained with the PI 3-kinase inhibitors, LY294002 and wortmannin (data not shown). These findings are consistent with Trumel et al38 showing the signaling function PI 3-kinase in PAR-1–induced platelet aggregation. Thus, the present studies point to an important role of the p110/p85 heterodimer in mediating PAR-1–induced NF-κB activation.
We observed that Akt functions as the downstream effector of both PKC-δ and PI 3-kinase in mediating the PAR-1–induced NF-κB activation. Inhibition of Akt prevented the NF-κB activation, indicating that Akt is required for the induction of this response. In addition, inhibition of Akt prevented NF-κB activation induced by either GαqRC or PKC-δCAT. Although there is controversy concerning the role of Gαq in regulating Akt activation in COS-7 cells,33,39⇓ our evidence indicates that Akt signals downstream of GαqRC/PKC-δCAT in mediating NF-κB activation in endothelial cells. As additional evidence of the importance of Akt, we showed that inhibition of Akt prevented NF-κB activation induced by the constitutively active PI 3-kinase mutant (p110*). These data demonstrate that the Gαq/PKC-δ and Gβγ/PI 3-kinase pathways converge at Akt to activate NF-κB. The data are consistent with other studies showing that both PI 3-kinase and PKC can independently activate Akt.40 We showed that each pathway contributes equally to the PAR-1–induced activation of Akt and the downstream activation of NF-κB.
We addressed possible mechanisms by which Akt induces NF-κB activation. Expression of the kinase-defective mutant of IKKβ (IKKβmut) prevented NF-κB activity induced by thrombin, TFLLRNPNDK, and AktCAT. These data are in accord with the transient association of Akt with IKK in vivo that induces IKK activation leading to phosphorylation and degradation of IκBα, and the subsequent NF-κB DNA-binding activity.41 Another mechanism by which Akt can regulate NF-κB activity may involve phosphorylation of RelA/p65 subunit of NF-κB.42 Although the precise basis of Akt-induced NF-κB activation is not clear, the present studies indicate that Akt serves as a node for PAR-1–activated signaling in endothelial cells. Thus, the targeting of Akt may be useful in preventing the thrombin-activated inflammatory responses.
This work was supported by NHLBI grants HL67424, HL46350, HL64573, and HL45638.
↵*Both authors contributed equally to this study.
Original received February 8, 2002; resubmission received July 9, 2002; accepted July 31, 2002.
- ↵Rahman A, Anwar KN, True AL, Malik AB. Thrombin-induced p65 homodimer binding to downstream NF-κB site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion. J Immunol. 1999; 162: 5466–5476.
- ↵Baldi L, Brown K, Franzoso G, Siebenlist U. Critical role for lysines21 and 22 in signal-induced, ubiquitin-mediated proteolysis of IκB-α. J Biol Chem. 1996; 271: 376–379.
- ↵Kaplanski G, Martin V, Fabrigoule M, Boulay V, Benoliel AM, Bongrand P, Kaplanski S, Farnarier C. Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106). Blood. 1998; 92: 1259–1267.
- ↵Grand RJA, Turnell AS, Grabham P. Cellular consequences of thrombin-receptor activation. Biochem J. 1996; 313: 353–368.
- ↵Damiano BP, Cheung WM, Santulli RJ, Fung-Leung WP, Ngo K, Ye RD, Darrow AL, Derian C K, de Garavilla L, Andrade-Gordon P. Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1. J Pharmacol Exp Ther. 1999; 288: 671–678.
- ↵Faure M, Voyno-Yasenetskaya TA, Bourne HR. cAMP and βγ subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in Cos-7 cells. J Biol Chem. 1994; 269: 7851–7854.
- ↵Conklin BR, Chabre O, Wong YH, Federman AD, Bourne HR. Recombinant Gqα: mutational activation and coupling to receptors and phospholipase C. J Biol Chem. 1992; 267: 31–34.
- ↵Voraberger G, Schafer R, Stratowa C. Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5′-regulatory region. Induction by cytokines and phorbol ester. J Immunol. 1991; 147: 2777–2786.
- ↵Hu Q, Klippel A, Muslin AJ, Fantl WJ, Williams LT. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995; 268: 100–102.
- ↵Hara K, Yonezawa K, Sakaue H, Ando A, Kotani K, Kitamura T, Kitamura Y, Ueda H, Stephens L, Jackson TR, Hawkins PT, Dhand R, Clark AE, Holman GD, Waterfield MD, Ksuga M. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A. 1994; 91: 7415–7419.
- ↵Xie P, Browning DD, Hay N, Mackman N, Ye RD. Activation of NF-κB by bradykinin through a Gαq- and Gβγ-dependent pathway that involves phosphoinositide 3-kinase and Akt. J Biol Chem. 2000; 275: 24907–24914.
- ↵Gingras AC, Kennedy SG, O’Leary MA, Sonenberg N, Hay N. 4E-BP1, a repressor of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signaling pathway. Genes Dev. 1998; 12: 502–513.
- ↵Dulin NO, Sorokin A, Reed E, Elliott S, Kehrl JH, Dunn MJ. RGS3 inhibits G protein-mediated signaling via translocation to the membrane and binding to Gα11. Mol Cell Biol. 1999; 19: 714–723.
- ↵Soh JW, Lee EH, Prywes R Weinstein IB. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol. 1999; 19: 1313.25.
- ↵Rahman A, Anwar KN, Uddin S, Xu N, Ye RD, Platanias LC, Malik AB. Protein kinase C-δ regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase. Mol Cell Biol. 2001; 21: 5554–5565.
- ↵Bhunia AK, Arai T, Bulkley G, Chatterjee S. Lactosylceramide mediates tumor necrosis factor-α–induced intercellular adhesion molecule-1 expression and the adhesion of neutrophils in human umbilical vein endothelial cells. J Biol Chem. 1998; 273: 34349–34357.
- ↵Gilchrist A, Vanhauwe JF, Li A, Thomas TO, Voyno-Yasenetskaya T, Hamm HE. Gα minigenes expressing c-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation. J Biol Chem. 2001; 276: 25672–25679.
- ↵Ellis CA, Malik AB, Gilchrist A, Hamm H, Sandoval R, Voyno-Yasenetskaya T, Tiruppathi C. Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/Mitogen-activated protein kinase pathway. J Biol Chem. 1999; 274: 13718–13727.
- ↵Chatterjee TK, Eapen AK, Fisher RA. A truncated form of RGS3 negatively regulates G protein–coupled receptor stimulation of adenylyl cyclase and phosphoinositide phospholipase C. J Biol Chem. 1997; 272: 15481–15487.
- ↵Vogel SM, Gao X, Mehta D, Ye RD, John TA, Andrade-Gordon P, Tiruppathi C, Malik AB. Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice. Physiol Genomics. 2000; 4: 137–145.
- ↵Bohm SK, McConalogue K, Kong W, Bunnett NW. Proteinase-activated receptors: new functions for old enzymes. News Physiol Sci. 1998; 13: 231–240.
- ↵Schmidt VA, Nierman WC, Maglott DR, Cupit LD, Moskowitz KA, Wainer JA, Bahou WF. The human proteinase-activated receptor-3 (PAR-3) gene: identification within a Par gene cluster and characterization in vascular endothelial cells and platelets. J Biol Chem. 1998; 273: 15061–15068.
- ↵Shepard LW, Yang M, Xie P, Browning DD, Voyno-Yasenetskaya T, Kozasa T, Ye RD. Constitutive activation of NF-κB and secretion of interleukin-8 induced by the G protein–coupled receptor of Kaposi’s sarcoma-associated herpesvirus involve Gα13 and RhoA. J Biol Chem. 2001; 276: 45979–45987.
- ↵Murga C, Laguinge L, Wetzker R, Cuadrado A, Gutkind JS. Activation of Akt/protein kinase B by G protein–coupled receptors: a role for α and βγ subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinaseγ. J Biol Chem. 1998; 273: 19080–19085.
- ↵Leopoldt D, Hanck T, Exner T, Maier U, Wetzker R, Nurnberg B. Gβγ stimulates phosphoinositide 3-kinase-γ by direct interaction with two domains of the catalytic p110 subunit. J Biol Chem. 1998; 273: 7024–7029.
- ↵Trumel C, Payrastre B, Plantavid M, Hechler B, Viala C, Presek P, Martinson EA, Cazenave JP, Chap H, Gachet C. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR1-activating peptide through the late activation of phosphoinositide 3-kinase. Blood. 1999; 94: 4156–4165.
- ↵Bommakanti RK, Vinayak S, Simonds WF. Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits. J Biol Chem. 2000; 275: 38870–38876.
- ↵Kroner C, Eybrechts K, Akkerman JW. Dual regulation of platelet protein kinase B. J Biol Chem. 2000; 275: 27790–27798.
- ↵Madrid LV, Mayo MW, Reuther JY, Baldwin AS Jr. Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-κB through utilization of the IκB kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem. 2001; 276: 18934–18940.