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

Cell Signal. 2016 May;28(5):391-400. doi: 10.1016/j.cellsig.2016.01.015. Epub 2016 Jan 29.

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.

Keywords: Adenylyl cyclase; Bioluminescence resonance energy transfer; Extracellular signal-regulated kinase; Necdin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • GTP-Binding Protein alpha Subunits, Gi-Go / antagonists & inhibitors
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • HEK293 Cells
  • Humans
  • RGS Proteins / metabolism*
  • Receptor, PAR-1 / antagonists & inhibitors
  • Receptor, PAR-1 / metabolism*
  • Signal Transduction*

Substances

  • RGS Proteins
  • Receptor, PAR-1
  • Extracellular Signal-Regulated MAP Kinases
  • GTP-Binding Protein alpha Subunits, Gi-Go