Communicate with one another to modulate signaling [14,15,21,31]. It has been reported that PAR3 is able to enhance the cleavage of PAR4 with thrombin in cells expressing PAR4 and 
the N-terminal domain of PAR3 linked to CD8 [6]. It is unlikely that the PAR3-CD8 is dimerizing with PAR4. These data indicate that the interaction between PAR3 and PAR4 is not required for enhanced cleavage of PAR4 by thrombin. Our data suggest that PAR3’s ability to enhance PAR4 cleavage is distinct from its influence on PAR4 signaling. We demonstrate in the current study that PAR3 directly interacts with PAR4 and forms constitutive homoZK 36374 site dimers and heterodimers by using bioluminescent resonance energy transfer (BRET) (Figure 8). The balance between PAR3 homodimers, PAR4 homodimers, and PAR3PAR4 heterodimers maybe altered in the absence of PAR3 in mouse platelets, which may influence the Gq signaling pathway. It is widely accepted that PAR3 1326631 does not signal on its own. However, there are two examples of PAR3 regulating signaling from otherPAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 5. Dose response curve of Ca2+ mobilization in the absence of extracellular Ca2+ in mouse platelets. Fura 2-loaded wild type (black line) and PAR32/2 (gray line) platelets were resuspended in Ca2+-free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: (A) thrombin (1?00 nM), (C) AYPGKF (0.15? mM), 
or (E) 3 mM thapsigargin (TG). Quantitation of the change in peak Ca2+ mobilization in platelets stimulated with: (B) thrombin, (D) AYPGKF, or (F) thapsigargin. The results are the mean (6 SD) of three independent experiments (* p,0.05). doi:10.1371/journal.pone.0055740.gPAR family members. McLaughlin et al. showed that the activation of PAR1-PAR3 heterodimers with thrombin induces distinct signaling from PAR1-PAR1 homodimers [17]. A second example is in podocytes where PAR3 influenced activated protein C (APC)-mediated cytoprotective signaling through PAR1 in mice or PAR2 in humans [18]. The debate between monomers, dimers, and oligomers is ongoing in the GPCR field. However, recent technological advances using sophisticated FRET detection systems have suggested that some GPCRs form parallelogramshaped tetramers [32]. In addition, one report using this technique has demonstrated an excess of dimers in addition to tetramers [33]. The authors hypothesize that there are two interfaces of the complexes with differing KDM5A-IN-1 affinities, a high affinity site (dimers) and a lower affinity site for the tetramers (a dimer of dimers). It is quite possible that we are seeing dimers of PAR3 interact with dimers of PAR4. As discussed above, PAR4 also forms heterodimers with the P2Y12 receptor [23]. The P2Y12 receptors may be oligomerizing with dimers of PAR4 in a similar manner. At this point we arePAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 6. RhoA activity measured by G-LISA Kit in mouse platelets. The level of activated RhoA-GTP is measured by absorbance at 490 nm in response to increasing concentrations of thrombin (1?100 nM). The results are from three independent experiments (* p,0.05, ns: not significant). doi:10.1371/journal.pone.0055740.glimited by the technology to detect these higher order structures in a quantitative manner in native platelet membranes. The understanding of how GPCRs cooperate physically to mediate signaling is crucial to understanding their function and s.Communicate with one another to modulate signaling [14,15,21,31]. It has been reported that PAR3 is able to enhance the cleavage of PAR4 with thrombin in cells expressing PAR4 and the N-terminal domain of PAR3 linked to CD8 [6]. It is unlikely that the PAR3-CD8 is dimerizing with PAR4. These data indicate that the interaction between PAR3 and PAR4 is not required for enhanced cleavage of PAR4 by thrombin. Our data suggest that PAR3’s ability to enhance PAR4 cleavage is distinct from its influence on PAR4 signaling. We demonstrate in the current study that PAR3 directly interacts with PAR4 and forms constitutive homodimers and heterodimers by using bioluminescent resonance energy transfer (BRET) (Figure 8). The balance between PAR3 homodimers, PAR4 homodimers, and PAR3PAR4 heterodimers maybe altered in the absence of PAR3 in mouse platelets, which may influence the Gq signaling pathway. It is widely accepted that PAR3 1326631 does not signal on its own. However, there are two examples of PAR3 regulating signaling from otherPAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 5. Dose response curve of Ca2+ mobilization in the absence of extracellular Ca2+ in mouse platelets. Fura 2-loaded wild type (black line) and PAR32/2 (gray line) platelets were resuspended in Ca2+-free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: (A) thrombin (1?00 nM), (C) AYPGKF (0.15? mM), or (E) 3 mM thapsigargin (TG). Quantitation of the change in peak Ca2+ mobilization in platelets stimulated with: (B) thrombin, (D) AYPGKF, or (F) thapsigargin. The results are the mean (6 SD) of three independent experiments (* p,0.05). doi:10.1371/journal.pone.0055740.gPAR family members. McLaughlin et al. showed that the activation of PAR1-PAR3 heterodimers with thrombin induces distinct signaling from PAR1-PAR1 homodimers [17]. A second example is in podocytes where PAR3 influenced activated protein C (APC)-mediated cytoprotective signaling through PAR1 in mice or PAR2 in humans [18]. The debate between monomers, dimers, and oligomers is ongoing in the GPCR field. However, recent technological advances using sophisticated FRET detection systems have suggested that some GPCRs form parallelogramshaped tetramers [32]. In addition, one report using this technique has demonstrated an excess of dimers in addition to tetramers [33]. The authors hypothesize that there are two interfaces of the complexes with differing affinities, a high affinity site (dimers) and a lower affinity site for the tetramers (a dimer of dimers). It is quite possible that we are seeing dimers of PAR3 interact with dimers of PAR4. As discussed above, PAR4 also forms heterodimers with the P2Y12 receptor [23]. The P2Y12 receptors may be oligomerizing with dimers of PAR4 in a similar manner. At this point we arePAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 6. RhoA activity measured by G-LISA Kit in mouse platelets. The level of activated RhoA-GTP is measured by absorbance at 490 nm in response to increasing concentrations of thrombin (1?100 nM). The results are from three independent experiments (* p,0.05, ns: not significant). doi:10.1371/journal.pone.0055740.glimited by the technology to detect these higher order structures in a quantitative manner in native platelet membranes. The understanding of how GPCRs cooperate physically to mediate signaling is crucial to understanding their function and s.
