Thrombin Receptors Activate Go Proteins in Endothelial Cells to Regulate Intracellular Calcium and Cell Shape Changes

Abstract

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. 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 has been shown by Gilchrist A. B. A. J. W.R. Hamm H.E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google or acid the of to block receptor-mediated activation of signaling To specific functions Gi and in the signaling of thrombin we have these minigenes into indicate that 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 The involvement of Go proteins, but not Gi proteins, was further in pathways that are to be downstream of calcium such as stress fiber formation and endothelial barrier permeability. demonstrate the importance of Go proteins in the signaling of thrombin receptors in endothelial of the cell were from The was from was from vectors and were from of the were from The and were from and the were from were from Manning of and were from endothelial monolayer were from the and were from S. PAR1 were from C. of was from Sf9 cells were at and at an in in a and as H. Y. E. R. G. Yang R. Hamm H.E. 1998; PubMed Scopus Google Scholar). Sf9 cells were with in and with of a at The was at at The was in and in at binding were as M. de M. 2000; PubMed Scopus Google Scholar). of Sf9 cell protein was in and and with at in a of in a of in was to the and the were further at The were by which the were with of and was in a binding was in the of and of binding was in the of we a human microvascular endothelial cell that was from E. The cells were in with and in an of at The cells were at and with of the cell minigene were designed as 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 has been shown to be a and specific Gβγ J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The of the minigenes were as The the of human Gα subunits and or the C terminus in were with and The a by the sequence a and a to the and the peptide was at the the The was in was in at and then to to The were at into a in with the were to to the The was into of cells and the cells were into of at was on and at To that was several were in and their was The was at and on a with in and with the was to the the vectors were the cell with an from the a we the into the minigenes were by the cells with minigenes and to from the the was by of and the by the cells was by through and in at The were with or a transfection To the of the cells were with a green fluorescent protein to stress fiber or a fluorescent protein [Ca2+]i the was and was the cells with the fluorescent proteins were and a fluorescent to determine the of and monolayer permeability were cells with minigenes. were with minigene The of the minigene peptide was with and the were of minigenes led to transfection This was of the which of in cell not cell were by on a the proteins were to The were blocked with in at The were with in at the were at with and and were at The cells were with minigene and the the cells were into a the cells were with a and further in of the of with was to To the thrombin or was with was in the of and The were by the of of The were and and of was to the formation of the at the were at The of in was with a In cell the was to the were with or minigene and with or with the cells were to at a in a and to at The was and was at in of and The cells were with and in The were in the that was on the of an The was at The cells were identified a green by The were of thrombin were and stimulation with The were the a cells, the the green fluorescent protein was with minigene as were on with and with The were in in to and the by in The cells were with and in the cells were at with to The were in and with to the The were in and on a of The cells were with a and lines to and with the The were and cell in in response to thrombin was as C. Malik A.B. I. S. A. PubMed Scopus Google Scholar). were with minigene and on and to The and were to a of was by a signal to between the and The between the and the was by a and by a The same the of the and the to in the of the to the the were with and in The were were a The were at Thrombin is to couple to G proteins including Gi, Gq, and Manning D.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of the thrombin receptor in an of signal transduction pathways such as of of stress fiber and activation of 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, S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2110) Google Scholar). signaling pathways regulated by thrombin have been shown to be sensitive to intracellular calcium activation of acid of PAR1 von and endothelial (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). the interaction between receptors and members of the Gi through ADP-ribosylation of the in the C terminus of the Gα The effect of has been to Gi proteins, Go proteins are generally believed to play a role in the of the protein J. Biol. Chem. Full Text PDF PubMed Google Scholar). To determine whether PAR1, the thrombin can couple to Go proteins, we this receptor in Sf9 cells with and and binding to from these a negative we co-expressed PAR1, and not in Sf9 that binding to the in a in Sf9 PAR1 and proteins. In Sf9 only PAR1 and binding to a In addition, we that could stimulate binding to from Sf9 cells PAR1 and or The of stimulation in the latter was in the we not determine the of PAR1 of the of we could not conclude whether the stimulation by a coupling or a of receptor or G we coupling of PAR1 to Go1 proteins when co-expressed in Sf9 cells, we investigated the of Go proteins in were to and membranes. The of Go proteins was with an that Go1 and proteins, but not Gi proteins G. Yang L. Raj S. Manning D.R. Williamson J.R. J. Biol. Chem. 1994; 269: 8483-8487Abstract Full Text PDF PubMed Google Scholar). that Go proteins. This led to further the coupling of Go proteins to thrombin receptors in To determine whether Gi or Go proteins are in thrombin-mediated of were with or minigenes. We could not demonstrate thrombin-mediated of or stimulation of This be to a of PubMed Scopus Google Scholar). We several including with thrombin the cells with and out thrombin in the or receptor of protein kinase of or of the or of these we demonstrate of or formation by thrombin not thrombin-mediated of has only been in specific endothelial cell lines M.M. J. Biochem. PubMed Scopus Google Scholar, J. Biochem. Res. PubMed Scopus Google Scholar, Res. PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar). To whether were to of we in a cell that which couple to proteins. show that stimulation of receptors in cells these cells were with minigene and the of the 11-amino acid C-terminal peptide of Gi1/2 or Go1 proteins was the this of receptors on formation was blocked of a that a peptide based on the C-terminal sequence of not have an effect on the of formation through receptors. This blockade of was in cells with or minigene that proteins can couple to receptors to Previously, we have shown that thrombin-mediated stimulation of [Ca2+]i was blocked in with but not minigene which a role Gi proteins in this signaling of thrombin receptors (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 with or minigene vectors with and the [Ca2+]i in that with minigene a in their calcium response to but with or minigene vectors were This that Go proteins, but not Gi proteins, play a role in the thrombin-stimulated [Ca2+]i To further the Go proteins in thrombin-mediated calcium signaling we a of the β-adrenergic receptor kinase C-terminal J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google which to the role of the Gβγ subunits of We with minigene and [Ca2+]i in minigene cells led to the same of of [Ca2+]i as in Go minigene cells, suggesting that Go is in activation of PLCβ. the of the with the Gq minigene had no further effect on which is through the Gα of but not Gi, activation by PAR1 in cells to G activation of PLCβ. Gi can couple to PAR1 when in Sf9 cells, these of a cell coupling of PAR1 to PLCβ. The that Go proteins are in the calcium response to thrombin in led to further the importance of Go proteins in downstream signaling has been shown that thrombin-mediated stress fiber formation can in be regulated by [Ca2+]i J. 2001; PubMed Scopus Google Scholar). were with and or minigene and stress fiber formation was The cells were to thrombin and with and their were with cells were from cells by stress fiber formation is blocked in Go1 minigene In the of the Gi1/2 minigene not the of thrombin-stimulated stress fiber formation as with cells with the minigene Go is stress fiber then pertussis which as Gi stress fiber of with pertussis toxin in a blockade of stress fiber formation these show the involvement of Go proteins, but not in stress fiber stress fiber formation may to and of the endothelial This to an in permeability of the endothelial monolayer and may have physiological in and wound To the involvement of Go proteins in endothelial we on were and with thrombin cell through of the a transient in can be the in on with cells with or the Gi sequence in of the a in permeability was when were with thrombin treatment in with minigene or with in a blockade of the with or Gi1/2 minigene Go proteins are the proteins in the endothelial barrier The role of Go proteins has been in the of the protein J. Biol. Chem. Full Text PDF PubMed Google Scholar). Although the role of proteins had been several reports indicate distinct and effectors these Gα which acid with Gi proteins. effectors or such as or have been that Go specifically or differentially with Gi 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, R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. Thomas B. I. Biochem. J. 2000; PubMed Scopus Google Scholar). ADP-ribosylation of Gi or their interaction with G protein-coupled and several thrombin-mediated effects have been shown to be sensitive to (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, 17Rydholm H.E. Falk P. Eriksson E. Risberg B. Scand. J. Clin. Lab. Invest. 1998; 58: 347-352Crossref PubMed Scopus (3) Google such as stimulation of PAR1 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 release C.M. Vanhoutte P.M. J. Vasc. Res. 1997; 34: 175-185Crossref PubMed Scopus (50) Google endothelial N.A. Vanhoutte P.M. Blood Vessels. 1990; 27: 218-229PubMed Google and release of and 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). To determine whether serve a specific role in the signaling pathways of thrombin receptors in HMECs, we investigated whether PAR1 has the potential to activate Go proteins in stimulation of PAR1 a in binding to Sf9 PAR1 and the This stimulation of binding was in PAR1 with the We in that to Sf9 PAR1 and or not no are to determine the of we could not distinguish whether PAR1 to a specific G protein we conclude that PAR1 has the to couple to and activate Go proteins, as as Gi proteins. Although PAR1 is to couple to Gi in Sf9 cells or in HMECs, was no of by thrombin a variety of In a cell that a of formation could be and this effect was blocked by of or minigene these that thrombin receptors not use Gi or Go proteins to in PAR1 does couple to Gi to regulate in cells 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 endothelial Tiruppathi C. Gilchrist A. Hamm H.E. Malik A.B. J. Biol. 2000; PubMed Scopus Google or endothelial F. T. R. C. A. Li, A. A. B. and H. E. in we that Go proteins are in This further that Go proteins may play a role in the signaling of thrombin receptors in calcium signaling has been shown to be sensitive to in several cell we to whether transfection of minigene vectors the C-terminal sequence of Gi1/2 or could block thrombin-stimulated intracellular calcium Previously, we have shown that the but not the Gi1/2 minigene vectors can block thrombin-stimulated hydrolysis and intracellular calcium (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, we that a minigene the Gq C-terminal peptide in which the were could not block these that Gα based minigenes can specifically block G protein We in that Go minigenes caused a in [Ca2+]i response to thrombin with Gi1/2 or minigenes. indicate that but not Gi, serves to couple thrombin receptors to calcium This transient is mediated by Gβγ activation of a was by transfection with a of Gβγ the peptide, and their effects were not is a of coupling of to PLCβ. To further demonstrate the importance of in signal transduction pathways downstream from calcium we thrombin-stimulated stress fiber formation in with minigene vectors or with fiber formation has been shown to on a of signaling including intracellular calcium release activation of protein kinase C J. 2001; PubMed Scopus Google Scholar, H. Malik A.B. J. 1997; PubMed Scopus Google Scholar). stress fiber formation was blocked in cells as as in cells with but not in cells with minigene the involvement of Go proteins, but not Gi proteins, in this signaling of stress to cell in the of endothelial cells, the endothelial we endothelial monolayer a physiological response downstream of stress fiber Thrombin a in and this effect was blocked in cells with The cells with the minigene a thrombin The cells with the had a response to the cells with a we that Go proteins play an role in the signaling of thrombin receptors distinct from Gi proteins, and we its involvement in of the signaling from calcium to stress fiber formation and endothelial barrier In we have a role in the signal transduction of thrombin receptors in in which regulate calcium signaling and cytoskeletal rearrangements. In addition, we have shown that the minigene can be to dissect out the effects of pertussis toxin and determine whether are mediated by Gi or Go proteins.