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

McLaughlin, Joseph N.; Shen, Lixin; Holinstat, Michael; Brooks, Joshua D.; DiBenedetto, Emmanuele; Hamm, Heidi E.

doi:10.1074/jbc.m414090200

Cited by 179 publications

(200 citation statements)

References 108 publications

(58 reference statements)

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“…Calcium mobilization was determined as before (36). Briefly, after siRNA treatment, HPAEC were grown to confluence and serum deprived in EBM-2 containing 0.1% serum 3 h. Cells were then loaded with the FLIPR Calcium Plus kit dye and fluorescence measured on the FlexStation (Molecular Devices, Sunnyvale, CA).…”

Section: Methodsmentioning

confidence: 99%

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

McLaughlin

Patterson

Malik

2007

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

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161

164

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Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased G␣13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.T hrombin generation during thrombosis contributes to the pathophysiology of multiple thrombosis-related diseases such as acute lung injury resulting increased endothelial permeability (1), vascular inflammation (1, 2), and edema formation (for recent reviews, see refs. 3 and 4). These complications are the result of thrombin activation of protease-activated receptor-1 (PAR1) (5). Cleavage of the extracellular N terminus of PAR1 initiates signaling (6) by the concomitant activation of heterotrimeric GTP-binding proteins G␣ q and G␣ 12/13 (7-9). The mechanisms by which PAR1 selectively activates G protein signaling remain unknown. Because the nonsignaling receptor PAR3 is also expressed in endothelial cells (10), we surmised that PAR1 and PAR3 are capable of interacting in such manner as to regulate PAR1 signaling. We found that PAR3 directly dimerizes with PAR1 to induce a specific PAR1/G␣ 13 -binding conformation that favors G␣ 13 activation. We propose a model of PAR1 activation involving the interaction with PAR3, which alters the selectivity of PAR1 for G␣ 13 coupling and promotes endothelial barrier dysfunction. PAR3 functions as an essential allosteric modulator of PAR1 signaling through PAR1/3 dimerization and favors a distinct G␣ 13 -activated downstream pathway. Results PAR3 Knockdown in Endothelial Cells Shifts the Potency of ThrombinActivation of PAR1. Endothelial cells express PAR1, -2, and -3, but not PAR4 (10), and of these only PAR1 and PAR3 are thrombinsensitive (11). To address whether PAR3 alters thrombin signaling in endothelial cells, siRNA knockdown was used to reduce thrombin receptor expression in human pulmonary artery endothelial cells (HPAECs). PAR1 and PAR3 suppression was determined by semiquantitative RT-PCR. siRNA against PAR3 did not affect PAR1 expression, whereas it reduced PAR3 expression 8-fold. Conversely, siRNA against PAR1 only modestly affected PAR3 expression, whereas it suppres...

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Section: Methodsmentioning

confidence: 99%

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

McLaughlin

Patterson

Malik

2007

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

Self Cite

161

164

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“…PAR1, which is widely studied in this subfamily of GPCRs, couples to multiple G proteins including Gi, Gq, and G12/13. The selectivity for activation of these G proteins depends on the agonists that bind to the receptor [61] . In endothelial cells, thrombin binding to PAR1 leads to activation of p115 RhoGEF, which provides a functional link between G13 and RhoA [62] .…”

Section: Gpcrs and Regulation Of Vascular Endothelial Permeabilitymentioning

confidence: 99%

Role of G protein-coupled receptors in inflammation

2012

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“…Theoretical simulation of the reactions was performed by using MatLab (The Math Works, Natick, MA) on a Linux-based operating system (Ogdensburg, NY;McLaughlin et al, 2005).…”

Section: Mathematical Modelingmentioning

confidence: 99%

RACK1 Regulates Directional Cell Migration by Acting on Gβγ at the Interface with Its Effectors PLCβ and PI3Kγ

Chen

Lin

Shin

et al. 2008

MBoC

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Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein ␤␥ (G␤␥) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with G␤␥ and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains G␤␥-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKC␤ and Src. Rather, RACK1 selectively inhibits G␤␥-stimulated phosphatidylinositol 3-kinase ␥ (PI3K␥) and phospholipase C (PLC) ␤ activity, due to the competitive binding of RACK1, PI3K␥, and PLC␤ to G␤␥. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with G␤␥-dependent activation of key effectors critical for chemotaxis.

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

Cited by 179 publications

References 108 publications

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

Role of G protein-coupled receptors in inflammation

RACK1 Regulates Directional Cell Migration by Acting on Gβγ at the Interface with Its Effectors PLCβ and PI3Kγ

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