On in PAR32/2 platelets, which affects the Gq-dependent signaling, but not

On in PAR32/2 platelets, which affects the Gq-dependent signaling, but not G12/13-dependent signaling, we considered the following hypotheses. First, PAR3 can regulate Gq signaling indirectlyFigure 7. Western blot analysis of Akt phosphorylation in mouse platelets. (A) The level of Akt phosphorylation at Ser473 in response to increasing concentrations of thrombin (1?00 nM) was K162 determined by western blotting with phospho-Akt (Ser473) antibody. The membrane was re-probed for total Akt to demonstrate protein loading. The blots shown are from a representative of three independent experiments. (B) 6R-Tetrahydro-L-biopterin dihydrochloride custom synthesis Quantitation of Akt phosphorylation at (Ser 473) in response to thrombin is represented at the mean (6 SD, n = 3) (* p,0.05). doi:10.1371/journal.pone.0055740.gthrough PAR4. The absence of PAR3 may induce a conformational change in PAR4, which increases the activity of Gq. However, PAR4 is also coupled to G12/13 and a conformational change in PAR4 would also affect the signaling downstream of G12/13. A global change in PAR4 activity by PAR3 is not consistent with our results since the G12/13 signaling pathway was not affected in PAR32/2 mice. A second hypothesis is that the expression or distribution of proteins such as RGS (Regulator of G-protein Signaling) is altered in the PAR32/2 mice. A recent study showed that preventing RGS/Spinophilin/and tyrosine phosphatase SHP-1 complex formation in platelet produced a gain in function and increase Gq-mediated signaling [35]. However, as PAR3+/2 platelets produced an intermediate level of Ca2+ mobilization, it is unlikely that RGS expression, function, or both are affected in PAR32/2. A third hypothesis is that PAR3 selectively regulates Gq signaling by direct contact with PAR4 and this is altered in PAR32/2 mice due to a change in the ratio of PAR4 homodimers to PAR3-PAR4 heterodimers. This hypothesis is supported by the study carried out on the serotonin 5hydroxytryptamine2C (5-HT2C) receptors one of the largest subfamilies of GPCRs expressed in platelets [36]. The results of this study show that 5-HT2C homodimer interacts with a single Gq protein and the dimerization plays a functional role in regulating the activity of 5-HT2C receptors expressed in HEK293 cells. Recent studies from our laboratory have shown that human PAR4 forms constitutive homodimers at the plasma membrane [21]. Mutations at the PAR4-PAR4 homodimer interface disrupted dimer formation and reduced Ca2+ mobilization in response to the PAR4 agonist peptide [21]. PAR3-PAR4 heterodimer may have reduced capacity in comparison to thePAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 8. Bioluminescence resonance energy transfer (BRET) analysis of PAR3 homodimers, PAR4 homodimers, and PAR3-PAR4 heterodimers. The HEK293 cells were transfected with: (A) PAR4-Luc (1 mg) and PAR3-GFP (0?.5 mg), (B) PAR3-Luc (1 mg) and PAR3-GFP (0?.5 mg), or (C) PAR4-Luc (1 mg) and PAR4-GFP (0?.5 mg). As a control experiment, the HEK293 cells were transfected with: (D) PAR3-Luc (1 mg) and rho-GFP (0?.12 mg), or (E) PAR4-Luc (1 mg) and rho-GFP (0?.12 mg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3? independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. (G) V5-PAR4-GFP and V5-PAR3-GFP were detected with a.On in PAR32/2 platelets, which affects the Gq-dependent signaling, but not G12/13-dependent signaling, we considered the following hypotheses. First, PAR3 can regulate Gq signaling indirectlyFigure 7. Western blot analysis of Akt phosphorylation in mouse platelets. (A) The level of Akt phosphorylation at Ser473 in response to increasing concentrations of thrombin (1?00 nM) was determined by western blotting with phospho-Akt (Ser473) antibody. The membrane was re-probed for total Akt to demonstrate protein loading. The blots shown are from a representative of three independent experiments. (B) Quantitation of Akt phosphorylation at (Ser 473) in response to thrombin is represented at the mean (6 SD, n = 3) (* p,0.05). doi:10.1371/journal.pone.0055740.gthrough PAR4. The absence of PAR3 may induce a conformational change in PAR4, which increases the activity of Gq. However, PAR4 is also coupled to G12/13 and a conformational change in PAR4 would also affect the signaling downstream of G12/13. A global change in PAR4 activity by PAR3 is not consistent with our results since the G12/13 signaling pathway was not affected in PAR32/2 mice. A second hypothesis is that the expression or distribution of proteins such as RGS (Regulator of G-protein Signaling) is altered in the PAR32/2 mice. A recent study showed that preventing RGS/Spinophilin/and tyrosine phosphatase SHP-1 complex formation in platelet produced a gain in function and increase Gq-mediated signaling [35]. However, as PAR3+/2 platelets produced an intermediate level of Ca2+ mobilization, it is unlikely that RGS expression, function, or both are affected in PAR32/2. A third hypothesis is that PAR3 selectively regulates Gq signaling by direct contact with PAR4 and this is altered in PAR32/2 mice due to a change in the ratio of PAR4 homodimers to PAR3-PAR4 heterodimers. This hypothesis is supported by the study carried out on the serotonin 5hydroxytryptamine2C (5-HT2C) receptors one of the largest subfamilies of GPCRs expressed in platelets [36]. The results of this study show that 5-HT2C homodimer interacts with a single Gq protein and the dimerization plays a functional role in regulating the activity of 5-HT2C receptors expressed in HEK293 cells. Recent studies from our laboratory have shown that human PAR4 forms constitutive homodimers at the plasma membrane [21]. Mutations at the PAR4-PAR4 homodimer interface disrupted dimer formation and reduced Ca2+ mobilization in response to the PAR4 agonist peptide [21]. PAR3-PAR4 heterodimer may have reduced capacity in comparison to thePAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 8. Bioluminescence resonance energy transfer (BRET) analysis of PAR3 homodimers, PAR4 homodimers, and PAR3-PAR4 heterodimers. The HEK293 cells were transfected with: (A) PAR4-Luc (1 mg) and PAR3-GFP (0?.5 mg), (B) PAR3-Luc (1 mg) and PAR3-GFP (0?.5 mg), or (C) PAR4-Luc (1 mg) and PAR4-GFP (0?.5 mg). As a control experiment, the HEK293 cells were transfected with: (D) PAR3-Luc (1 mg) and rho-GFP (0?.12 mg), or (E) PAR4-Luc (1 mg) and rho-GFP (0?.12 mg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3? independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. (G) V5-PAR4-GFP and V5-PAR3-GFP were detected with a.