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

Vanhauwe, Jurgen; Thomas, Tarita O.; Minshall, Richard D.; Tiruppathì, Chinnaswamy; Li, Anli; Gilchrist, Annette; Yoon, Eun Ja; Malik, Asrar B.; Hamm, Heidi E.

doi:10.1074/jbc.m204477200

Cited by 53 publications

(48 citation statements)

References 36 publications

(36 reference statements)

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“…2E), suggesting that G␣ i/o has no effect on induced permeability. This result differs from that which has been published previously (19). This difference might be attributed to different lots and passage numbers for the cells used or differences in experimental conditions.…”

Section: Fig 1 Differences Between Thrombin and Agonist Peptide Actcontrasting

confidence: 56%

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Functional Selectivity of G Protein Signaling by Agonist Peptides and Thrombin for the Protease-activated Receptor-1

McLaughlin

Shen

Holinstat

et al. 2005

Journal of Biological Chemistry

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179

182

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Thrombin activates protease-activated receptor-1 (PAR-1) by cleavage of the amino terminus to unmask a tethered ligand. Although peptide analogs can activate PAR-1, we show that the functional responses mediated via PAR-1 differ between the agonists. Thrombin caused endothelial monolayer permeability and mobilized intracellular calcium with EC 50 values of 0.1 and 1.7 nM, respectively. The opposite order of activation was observed for agonist peptide (SFLLRN-CONH 2 or TFLL-RNKPDK) activation. The addition of inactivated thrombin did not affect agonist peptide signaling, suggesting that the differences in activation mechanisms are intramolecular in origin. Although activation of PAR-1 or PAR-2 by agonist peptides induced calcium mobilization, only PAR-1 activation affected barrier function. Induced barrier permeability is likely to be G␣ 12/13 -mediated as chelation of G␣ q -mediated intracellular calcium with BAPTA-AM, pertussis toxin inhibition of G␣ i/o , or GM6001 inhibition of matrix metalloproteinase had no effect, whereas Y-27632 inhibition of the G␣ 12/13 -mediated Rho kinase abrogated the response. Similarly, calcium mobilization is G␣ q -mediated and independent of G␣ i/o and G␣ 12/13 because pertussis toxin and Y-27632 had no effect, whereas U-73122 inhibition of phospholipase C-␤ blocked the response. It is therefore likely that changes in permeability reflect G␣ 12/13 activation, and changes in calcium reflect G␣ q activation, implying that the pharmacological differences between agonists are likely caused by the ability of the receptor to activate G␣ 12/13 or G␣ q . This functional selectivity was characterized quantitatively by a mathematical model describing each step leading to Rho activation and/or calcium mobilization. This model provides an estimate that peptide activation alters receptor/G protein binding to favor G␣ q activation over G␣ 12/13 by ϳ800-fold.

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Section: Fig 1 Differences Between Thrombin and Agonist Peptide Actcontrasting

confidence: 56%

“…Activated by thrombin, PAR-1 exerts its effects on the endothelium by concomitant activation of the G␣ i/o , G␣ q , and G␣ 12/13 families of G proteins (18,19). These signals ultimately integrate to induce profound changes in vascular endothelial cells, including increased endothelial monolayer permeability (20 -22).…”

mentioning

confidence: 99%

Functional Selectivity of G Protein Signaling by Agonist Peptides and Thrombin for the Protease-activated Receptor-1

McLaughlin

Shen

Holinstat

et al. 2005

Journal of Biological Chemistry

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179

182

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“…Alternatively, cysLT 2 -R may couple several G proteins similar to other GPCRs. In recombinant systems, cysLT 2 -R and thrombin receptors appear to couple predominantly through G q -mediated phospholipase C ␤-triggered Ca 2ϩ elevation, but coupling through G i has also been reported for thrombin in ECs (6,48). That G i may participate in cysLT 2 -R signaling in HUVECs was indicated by a partial inhibition of LTD 4 -dependent ERK1͞2 phosphorylation by pertussis toxin (K.L.…”

Section: Discussionmentioning

confidence: 99%

Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells

Uzonyi

Lötzer

Jahn

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway, and the cysLT receptors cysLT 1-R͞cysLT2-R mediate inflammatory tissue reactions. Although endothelial cells (ECs) predominantly express cysLT 2-Rs, their role in vascular biology remains to be fully understood. To delineate cysLT2-R actions, we stimulated human umbilical vein EC with LTD 4 and determined early induced genes. We also compared LTD4 effects with those induced by thrombin that binds to protease-activated receptor (PAR)-1. Stringent filters yielded 37 cysLT 2-R-and 34 PAR-1-up-regulated genes (>2.5-fold stimulation). Most LTD4-regulated genes were also induced by thrombin. Moreover, LTD4 plus thrombin augmented gene expression when compared with each agonist alone. Strongly induced genes were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A transcription factors; E-selectin; CXC ligand 2; IL-8; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS1); Down syndrome critical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygenase 2. Transcripts peaked at Ϸ60 min, were unaffected by a cysLT 1-R antagonist, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD 4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus; LTD4 up-regulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC procoagulant activity. These data show that cysLT 2-R activation results in a proinflammatory EC phenotype. Because LTD 4 and thrombin are likely to be formed concomitantly in vivo, cysLT 2-R and PAR-1 may cooperate to augment vascular injury.cysteinyl leukotriene 2 receptor gene signature ͉ protease-activated receptor 1 gene signature ͉ vascular inflammation L eukotrienes (LTs), i.e., LTB 4 and the cysteinyl LTs (cysLT) LTC 4 , LTD 4 , and LTE 4 constitute a group of lipid mediators derived from the 5-lipoxygenase (5-LO) pathway (1, 2). LTs are either produced by leukocytes at sites of inflammation or formed through transcellular metabolism after uptake and metabolism of leukocyte-derived LTA 4 by downstream enzymes of the 5-LO pathway (LTA 4 hydrolase and LTC 4 synthase) in cells that normally do not express 5-LO, such as endothelial cells (ECs) (3, 4). LTs act through G protein-coupled surface receptors (GPCRs), i.e., the LTB 4 receptors and the cysLT receptors (LT-Rs) (cysLT 1 -R and cysLT 2 -R) (5-10). LT-Rs are expressed on multiple target cells, including leukocytes, smooth muscle cells, and ECs (1). Recent studies implicate the 5-LO pathway in cardiovascular disease (11)(12)(13)(14)(15)(16)(17).Considerable information is available on cysLT 1 -R, whereas little is known about cysLT 2 -R. We have used human umibilical vein (HUV)ECs as a model of vascular cells to study cysLT 2 -R activation by demonstrating that cysLTs exclusively signal through cysLT 2 -R in this cell type (18): In fact, HUVECs are the first primary cell type that s...

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“…2B). Although thrombin is known to couple to a range of other heterotrimeric G proteins (13,14) (including G i , G 12/13 , and G o ), judging from the very strong Ca 2ϩ signal induced by this agonist (see Fig. 2 A), G q may well represent the predominant effector G protein of thrombin in our HEK-293 cells.…”

Section: Trpm7 Is Inhibited By Pertussis Toxin (Ptx)-sensitive G Protmentioning

confidence: 99%

Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain

Takezawa

Schmitz²,

Demeuse

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

166

159

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TRPM7 is a ubiquitously expressed and constitutively active divalent cation-selective ion channel, whose basal activity is regulated by intracellular levels of Mg 2؉ and Mg⅐ATP. We have investigated receptor-mediated mechanisms that may actively regulate TRPM7 activity. We here report that TRPM7 currents are suppressed by intracellular GTP␥S, suggesting the involvement of heterotrimeric G proteins. TRPM7 currents are also inhibited by stimulating endogenous muscarinic receptors, which is mediated by Gi because the inhibitory effect is blunted by pertussis toxin. Conversely, stimulation of endogenous Gs-coupled ␤-adrenergic receptors potentiates TRPM7 currents, whereas Gq-coupled thrombin receptors have little effect. Consistent with the involvement of Gs͞Gi in controlling adenylyl cyclase activity, elevations of intracellular cAMP levels enhance TRPM7 activity and prevent receptor-mediated modulation of TRPM7 activity by muscarinic and adrenergic agonists. This cAMP-dependent effect requires the functional integrity of both protein kinase A (PKA) and the endogenous kinase domain of TRPM7 because cAMP-mediated effects are abolished when treating cells with the PKA inhibitors H89 or KT5720 as well as in cells expressing phosphotransferase-deficient TRPM7 constructs. These mutant channels are also much less susceptible to GTP␥S-mediated inhibition, suggesting that the main regulatory effect occurs through Gi-and Gs-mediated changes in cAMP. Taken together, our results demonstrate that TRPM7 activity is up-and down-regulated through its endogenous kinase in a cAMP-and PKA-dependent manner.B ased on sequence similarities, the mammalian transient receptor potential (TRP) channel family is divided into three subfamilies [TRP classical (formerly short TRP channel)], TRP vanilloid [(formerly osm TRP channel), and TRP melastatin (formerly long TRP channel)] (1-4). TRPM7 (long TRP channel 7, TRP-phospholipase C interacting kinase and channel kinase 1), a Ca 2ϩ -and Mg 2ϩ -permeable divalent cation channel of the TRP melastatin ion channel subfamily, is required for cellular viability and noted for its ubiquitous distribution profile (5, 6). We have previously demonstrated that TRPM7 is regulated by millimolar levels of intracellular Mg 2ϩ and Mg⅐ATP and its activity appears to be linked to cellular energy metabolism (5, 7). In resting cells, physiological levels of these molecules strongly suppress the activity of TRPM7 channels and only a small constitutive activity remains, sufficient to maintain basal divalent cation fluxes. In whole-cell patch-clamp experiments, intracellular solutions that lack added Mg⅐ATP lead to activation of TRPM7-mediated currents (Fig.

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

Cited by 53 publications

References 36 publications

Functional Selectivity of G Protein Signaling by Agonist Peptides and Thrombin for the Protease-activated Receptor-1

Functional Selectivity of G Protein Signaling by Agonist Peptides and Thrombin for the Protease-activated Receptor-1

Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells

Receptor-mediated regulation of the TRPM7 channel through its endogenous protein kinase domain

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