Modulation of G-protein-coupled receptor 55-mediated signaling by regulator of G-protein signaling 2

https://doi.org/10.1016/j.bbrc.2020.09.123Get rights and content

Highlights

  • GPR55 interacts with RGS2 alone and also forms a ternary complex with RGS2 and either Gαq or Gα12.

  • RGS2 inhibits GPR55-mediated cellular signaling.

  • RGS2 prohibits GPR55-induced cancer cell proliferation and migration.

  • Our findings suggest a novel physiological function of RGS2 as a regulator of GPR55 signaling.

Abstract

Activation of seven-transmembrane G-protein coupled receptor (GPCR) mediates extracellular signals into intracellular responses. G-protein coupled receptor 55 (GPR55) is one of GPCRs and activated by endogenous cannabinoids. A family of regulators of G-protein signaling (RGS) stimulates GTP hydrolysis of alpha subunit of G-protein (Gα) and inhibits GPCR/Gα-mediated signaling. RGS2 is member of R4 RGS family and mainly attenuates GPCR/Gαq signaling. Although RGS2 is known to modulate some GPCR signaling, the specific effects of RGS2 on GPR55-mediated signaling are not fully understood at present. Previously, we reported some RGS proteins interact with protease-activated receptors, one of GPCRs, and modulate their functions. Here, we investigated whether GPR55 interacts with RGS2, employing bioluminescence resonance energy transfer and co-immunoprecipitation analyses. Interestingly, GPR55 interacted with RGS2 alone and also formed a ternary complex with RGS2 and either Gαq or Gα12. In the presence of GPR55 alone and together with Gαq or Gα12, RGS2 translocated from the cytoplasm to plasma membrane while RGS1 remained in the cytoplasm. GPR55 activation significantly induced ERK phosphorylation and intracellular calcium mobilization, which were markedly inhibited by RGS2 in HCT116 colon cancer cell line. Furthermore, GPR55-mediated cell proliferation and migration of HCT116 cells, was significantly attenuated by RGS2. Our collective findings highlight a novel physiological function of RGS2, supporting its utility as a therapeutic target to control GPR55-induced pathophysiology.

Introduction

G-protein coupled receptor (GPCR) activates heterotrimeric GTP-binding protein (G-protein) by responding to a variety of extracellular signals. G-proteins form a heterotrimeric complex (Gαβγ) in the inactivated state. GPCR activation by binding to specific ligand triggers dissociation of the G-protein complex into Gα and Gβγ dimers. The dissociated subunits regulate activation of downstream effectors, including adenlylyl cyclases, phospholipases and ion channels. Characteristics of G-protein is specified by types of Gα subunit which is classified into several families [1]. G-protein coupled receptor 55 (GPR55) is one of orphan GPCRs and was initially reported as a putative receptor for endocannabinoids [2]. GPR55 also acts as a receptor for lysophosphatidylinositol (LPI), a bioactive lipid. GPR55 couples with Gαq and Gα12/13, in turn, activating RhoA and MEK1/2, and reported to be involved in various pathophysiologies, including cancer, obesity, pain, and inflammation [3]. The receptor is highly expressed in several cancer cell lines and significantly involved in cell proliferation, migration and invasion [4]. Knockdown of GPR55 is associated with a decrease in cell proliferation and migration while its overexpression or activation has the opposite effect [5,6].

An alpha subunit of G-protein (Gα) possess intrinsic GTPase activity which is accelerated by GTPase-activating proteins (GAP). The most prominent GAP types are regulators of G protein signaling (RGS). Several RGS subfamilies have been identified based on amino acid sequences and protein structures. All RGS families contain an RGS domain that serves as the interacting site for GAP activity [7]. RGS2 was initially characterized as a member of the R4 RGS family, with reports of variable expression patterns in multiple tissues. RGS2 strongly inhibits GPCR-mediated signaling through binding to Gαq, manifested by changes in activation of phospholipase C and intracellular Ca2+ mobilization [8].

Previously, we showed that some RGS proteins interact with protease-activated receptor (PAR), a GPCR, together with Gα, to modulate PAR-mediated downstream signaling pathways. Notably, specific types of RGS protein form ternary complex with PARs and distinct Gα subunits [[9], [10], [11], [12]]. It has been also known that RGS proteins modulate GPCR-mediated signaling with binding to the receptors both directly and indirectly [13,14]. While RGS proteins are clearly involved in regulation of GPCR activity, their specific effects on GPR55-mediated signaling are not fully understood at present. In the current study, we investigated the RGS isoforms specifically interacting with GPR55 in live cells using bioluminescence resonance energy transfer (BRET) and examined their inhibitory effects on GPR55-mediated signaling.

Section snippets

Cell culture and transfection

293T and HCT116 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 mg/mL streptomycin and 50 mg/mL gentamycin, and were incubated at 37 °C in a humidified incubator under 5% CO2. Cell transfection was performed using polyethyleneimine [11].

BRET assay

293T cells were plated at a density of 3.5×105 cells/well onto six-well tissue culture plates. Cells were transfected with luciferase-tagged BRET donor and Venus-tagged acceptor.

GPR55 interacts with RGS2 in live cells

Employing BRET analysis, we determine interaction between GPR55 and RGS2 in live cells. Plasmids encoding GPR55-luciferase (GPR55-Luc) and RGS-Venus (RGS-Ven) as the BRET donor and acceptor, respectively, was used. 293T cells were transfected with GPR55-Luc (0.05 μg) and either RGS1-Ven or RGS2-Ven (0–3.0 μg) (Fig. 1a). Analysis of BRET signals revealed binding of GPR55 to RGS2 (Fig. 1a, red line) but not RGS1 (Fig. 1a, black line). To investigate the potential effects of interactions with RGS2

Discussion

In the current study, we determined whether RGS2 interacts with GPR55 in live cells and it affects GPR55/Gα-mediated signaling. Our findings indicate that GPR55 formed a ternary complex with RGS2 together with Gαq or Gα12/13, as evident from BRET and co-immunoprecipitation analyses. Subcellular localization of RGS2 was altered by expression of GPR55 from the cytoplasm to plasma membrane. Additional expression of Gαq or Gα12 did not alter membrane localization of RGS2. In contrast to RGS2, RGS1

Declaration of competing interest

The authors have no conflicts of interest to declare.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2018R1D1A1B07045284 to SG).

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      Bioluminescence resonance energy transfer (BRET) is a powerful experimental tool for determining interactions between receptors, and between GPCR and Gα. Earlier BRET experiments by our group demonstrated that a number of GPCRs, including protease-activated receptors (PAR) and GPR55, form distinct G protein-dependent complexes with RGS protein [10–14]. More recently, we showed physical and functional crosstalk between GPR55 and lysophosphatidic acid receptor 2 (LPA2) [15].

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