title: Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells.
abstract: Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs-protease-activated receptors (PARs)-is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition.
authors.affiliation: Department of Life Science, Kyonggi University, Suwon 443-760, Republic of Korea.
authors.forename: Jinyong
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Life Science, Kyonggi University, Suwon 443-760, Republic of Korea. Electronic address: shghil@kyonggi.ac.kr.
authors.forename: Sungho
authors.initials: S
authors.lastname: Ghil
mesh_terms: D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020710:RGS Proteins / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D015398:Signal Transduction; D010975:Platelet Aggregation Inhibitors
chemical_list: D020710:RGS Proteins; D044463:Receptor, PAR-1; D048049:Extracellular Signal-Regulated MAP Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D010975:Platelet Aggregation Inhibitors
keywords: Adenylyl cyclase; Bioluminescence resonance energy transfer; Extracellular signal-regulated kinase; Necdin