title: Regulator of G protein signaling 2 (RGS2) and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1) in live cells.
abstract: Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.
authors.affiliation: Department of Life Science, Kyonggi University, Suwon, Republic of Korea.
authors.forename: Sungho
authors.initials: S
authors.lastname: Ghil
authors.affiliation: Department of Pharmacology, Rollins Research center, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: McCoy
authors.affiliation: Department of Pharmacology, Rollins Research center, Emory University School of Medicine, Atlanta, Georgia, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Hepler
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D011485:Protein Binding; D020710:RGS Proteins / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D015398:Signal Transduction; D014982:Xenopus laevis
chemical_list: D020710:RGS Proteins; D044463:Receptor, PAR-1
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