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.

The regulators of G protein signaling RGS16 and RGS18 inhibit protease‐activated receptor 2/Gi/o signaling through distinct interactions with Gα in live cells

“…RGS8 directly binds to the N terminus and the third intracellular loop of the M1-muscarinic acetylcholine receptor to regulate receptor-mediated Ga q signaling [33]. RGS2 and RGS4 additionally inhibit PAR1-mediated Ga signaling through interactions with distinct Ga proteins [14]. ERK activity was determined by immunoblotting using p-ERK and total ERK antibodies.…”

“…Gpr161, an orphan GPCR, contains a motif for A-kinase anchoring protein in its C-terminal tail and binds the regulatory subunits of PKA, leading to the modulation of cAMP signaling [13]. Using a bioluminescence resonance energy transfer (BRET) technique, we previously reported that RGS2 and RGS4 interact with PAR1 in a Ga-dependent manner to regulate PAR1/Ga-mediated signaling [14]. Using a bioluminescence resonance energy transfer (BRET) technique, we previously reported that RGS2 and RGS4 interact with PAR1 in a Ga-dependent manner to regulate PAR1/Ga-mediated signaling [14].…”

“…GPCRs interact directly or indirectly with RGS proteins as well as Ga. Adrenergic, muscarinic acetylcholine and opioid receptors have been identified as binding factors of RGS proteins [5]. Using a bioluminescence resonance energy transfer (BRET) technique, we previously reported that RGS2 and RGS4 interact with PAR1 in a Ga-dependent manner to regulate PAR1/Ga-mediated signaling [14]. We additionally suggested that RGS8 inhibits PAR1/Ga i/o -mediated signaling by forming a distinct Ga complex [15].…”

Direct visualization of interaction between calmodulin and connexin45

“…The yellow fluorescent fusion protein expression vector Venus-C1 and the Renilla luciferase (Rluc) fusion protein expression vectors were used [52]. Construction of luciferase fusion protein vector mCx45-Rluc was achieved by insertion of the PCR product of the mouse Cx45 into vector Rluc-N1 at XhoI and HindIII enzyme sites upstream of luciferase.…”

A highly conserved δ‐opioid receptor region determines RGS4 interaction

“…Other observations have shown that RGS4 and RGS2 are recruited to the plasma membrane by G proteins and/or the expressed receptors [8,10]. Recent evidence indicates that RGS2 and RGS4 interact directly with PAR1 in a Ga-dependent manner to modulate PAR1/Ga-mediated signalling [23]. All these observations indicate that selectivity of RGS proteins for a given GPCR or G protein is influenced by different parameters ranging from the nature and abundance of G and RGS proteins present in a certain cellular milieu to the activation state of each receptor.…”