Ghil S , McCoy KL , Hepler JR .

T32 GM008602 NIGMS NIH HHS , R01NS049195 NINDS NIH HHS , R01NS37112 NINDS NIH HHS , R01 NS037112 NINDS NIH HHS , R01 NS049195 NINDS NIH HHS

	Figure 2. PAR1 forms a complex with RGS2 and Gαq/11.A, Top panel: Cartoon illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells transfected with RGS2-Luc (35 ng) and either Gαq-YFP or Gαs-YFP (0.75 µg) were subjected to the BRET assay in both the absence and presence of 0.5 µg of untagged PAR1-FLAG and 30 µM of TFLLR. Net BRET signals are shown between RGS2-Luc and either Gαq-YFP or Gαs-YFP. B, Top panel, Cartoon illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells transfected with RGS2-Luc (35 ng) and either PAR1-Ven or PAR1R205A-Ven (1.5 µg) were subjected to the BRET assay in both the absence and presence of 0.5 µg of untagged Gα11 and TFLLR. Net BRET signals are shown between RGS-Luc and either PAR1-Ven or PAR1R205A-Ven.
	Figure 3. RGS4 interacts with a PAR1/Gαo complex in live cells.A, Top panel: Cartoon illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells were transfected with increasing amounts of PAR1-Ven (0, 0.1, 0.25, 0.5, 1.0, 1.5 µg), together with fixed amount of RGS4-Luc (45 ng) (Red symbols) or RGS14-Luc (5 ng) (Black symbols), and the cells were subjected to BRET analysis in both the absence and presence of 30 µM of TFLLR. Net BRET signals are shown between PAR1-Ven and either RGS4-Luc or RGS14-Luc. B, Top panel: Cartoon illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells were transfected with an increasing amount of PAR1-Ven together with fixed amount of RGS4-Luc (Red symbols) or RGS14-Luc (Black symbols) as in A, and were subjected to BRET analysis in both the absence or presence of untagged Gαo (Blue symbols) and TFLLR. Net BRET signals are shown between PAR1-Ven and either RGS4-Luc or RGS14-Luc. (NOTE: The black and red plots in (B) were identical to those in (A)). C, Top panel: Cartoon illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells were transfected with both fixed amount of PAR1-Ven (1.5 µg) and either RGS4-Luc (45 ng) or RGS14-Luc (5 ng), and the cells were subjected to the BRET assay in both the absence and presence of 0.5 µg of untagged Gα and TFLLR. Net BRET signals are shown between PAR1-Ven and either RGS4-Luc or RGS14-Luc. Abbreviations used are R14-L  =  RGS14-Luc; R4-L  =  RGS4-Luc.
	Figure 4. RGS2 and RGS4 bind directly and selectively to the i3 loop of PAR1.Purified RGS1-His, RGS2-His, RGS4-His or RGS16-His were incubated with equal amounts of GST alone, GST-PAR1-i2, or with GST-PAR1-i3 bound to glutathione-Sepharose beads. After centrifugation, bound RGS proteins were eluted in 2X sample buffer and subjected to SDS-PAGE. Immunoblots were performed using an anti-His antibody.
	Figure 5. RGS2 and RGS4 selectively inhibit PAR1/Gα-mediated signaling in live cells.A, RGS2 and RGS4, but not RGS1, reduce PAR1-evoked calcium activated chloride currents in oocytes. PAR1 cRNA alone or mixed with individual RGS protein cRNA was injected into X. laevis oocytes, which were sustained in 1x Barth’s solution. 4–5 days after injection, Ica (Cl) measurements were obtained from the oocytes in response to activation with 30 mM TFLLR. A two electrode voltage clamp was used to obtain the current changes, as described in Materials and Methods. Data were entered into a Microsoft Excell spreadsheet which was used to calculate the mean change in Ica (Cl) + S.E.M. (n≥11 oocytes). B, RGS2 and RGS4 differentially block PAR1-stimulated ERK1/2 phosphorylation. Vector alone or PAR1 alone, or pairs of PAR1 and the indicated RGS protein were separately transfected into COS-7 cells. Cells were either stimulated with 20% serum or 30 µM TFLLR for 5 min. Immunoblots were performed with either phospho-ERK1/2, total ERK1/2, or an anti-HA antibody, followed by a goat-anti rabbit secondary antibody and detected by ECL. C, PAR1-mediated RhoA activation is not regulated by RGS proteins. As described in the materials and methods section, RhoA activation was measured using a RhoA G-LISA Assay kit. Vector alone, PAR1 alone, or PAR/RGS pairs were separately transfected into COS-7 cells for 5 h before an overnight period of serum starvation. The next day, cells were stimulated with 30 µM TFLLR for 2 min prior to cell lysis. The manufacturer’s protocol was followed throughout the experiment, and the absorbance of each well was read with a spectrophotometer wavelength of 490 nm. Data were entered into a Microsoft Excel spreadsheet which was used to calculate the mean fold change in absorbance (bars) over basal levels plus the S.E.M (error bars), n = 3 for each condition.
	Figure 1. RGS2 interacts with PAR1 in live cells. A, Top panel: Cartoon illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells transfected with an increased amount of PAR1-Ven (0, 0.25, 0.5, 1.0, 1.5, 2.0 µg), together with a fixed amount of RGS2-Luc (35 ng) or RGS14-Luc (5 ng), were subjected to the BRET assay in both the absence and presence of 30 µM of TFLLR. Net BRET signals are shown between PAR1-Ven and either RGS2-Luc or RGS14-Luc. B and C, Top panel: Cartoons illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells transfected with an increased amount of PAR1-Ven, together with fixed amount of RGS2-Luc or RGS14-Luc, were subjected to the BRET assay in both the absence and presence of untagged Gαq (B), Gα11 (C) and TFLLR. Bottom panel, net BRET signals are shown between PAR1-Ven and either RGS2-Luc or RGS14-Luc. The black and red plots in (B) and (C) were identical to those in (A). D, Top panel: Cartoon illustrating proteins and conditions used in the experiment. Bottom panel: COS7 cells were transfected with both fixed amount of PAR1-Ven (2.0 µg) and either RGS2-Luc (35 ng) or RGS14-Luc (5 ng), and the cells were subjected to the BRET assay in both the absence and presence of 0.5 µg of untagged Gα and TFLLR. Bottom panel, net BRET signals are shown between PAR1-Ven and either RGS2-Luc or RGS14-Luc. Right panel, shows a representative immunoblot of the different untagged Gα subunits used in the BRET experiment. All BRET graphs are representative of at least three independent experiments. Abbreviations: R14-L, RGS14-Luc; R2-L, RGS2-Luc.

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