Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

Chen, Buxin; Siderovski, David P.; Neubig, Richard R.; Lawson, Mark A.; Trejo, JoAnn

doi:10.1074/jbc.m113.528273

Cited by 15 publications

(9 citation statements)

References 39 publications

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“…RGS2 and RGS4 selectively bind to and inhibit PAR1/G q/11 - and PAR1/G i/o -mediated signals, respectively, and there is a difference in the binding properties of RGS proteins with PAR1 depending on the Gα. Our findings are consistent with a very recent report showing that PAR1 signaling is modulated by R4 family members of RGS proteins that include RGS2 and RGS4 [41] , and provide new insights into molecular mechanisms for how GPCR, RGS and Gα form functional preferred signaling complexes within cells. These studies could be of value in developing small molecule modulators of PAR1 signaling pathways.…”

Section: Discussionsupporting

confidence: 93%

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

2014

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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.

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Section: Discussionsupporting

confidence: 93%

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

2014

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“…Tyrosine Y-485 (Y485F) was mutated to phenylalanine (F) by site-directed mutagenesis. NEDD4–2 siRNA resistant wild-type and Y485F were cloned into a pSLIK lentiviral vector and EA.hy926 stable lines generated as described ( Chen et al, 2014 ).…”

Section: Star⋆methodsmentioning

confidence: 99%

A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling

et al. 2018

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SUMMARYUbiquitination is essential for protein degradation and signaling and pivotal to many physiological processes. Ubiquitination of a subset of G-protein-coupled receptors (GPCRs) by the E3 ligase NEDD4–2 is required for p38 activation, but how GPCRs activate NEDD4–2 to promote ubiquitinmediated signaling is not known. Here, we report that the GPCR protease-activated receptor-1 (PAR1) stimulates c-Src-mediated tyrosine phosphorylation and activation of NEDD4–2 to promote p38 signaling and endothelial barrier disruption. Using mass spectrometry, we identified a unique phosphorylated tyrosine (Y)-485 within the 2,3-linker peptide between WW domain 2 and 3 of NEDD4–2 in agonist-stimulated cells. Mutation of NEDD4–2 Y485 impaired E3 ligase activity and failed to rescue PAR1-stimulated p38 activation and endothelial barrier permeability. The purinergic P2Y1 receptor also required c-Src and NEDD4–2 tyrosine phosphorylation for p38 activation. These studies reveal a novel role for c-Src in GPCR-induced NEDD4–2 activation, which is critical for driving ubiquitin-mediated p38 inflammatory signaling.

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“…The role of GPCRs in cancer has been recently reviewed (60) and highlights the role of many GPCRs in cancer, including protease-activated receptors, G-protein estrogen receptor, lysophosphatidic acid receptor, prostaglandin E2 receptors, sphingosine 1-phosphate receptors, angiotensin II receptor, gonadotropin-releasing hormone receptors, somatostatin receptors, endothelin receptors, among many others (61,62). Some of these receptors have established RGS-mediated signaling pathways; for example, the protease-activated receptor is regulated by multiple R4 family RGS proteins (63)(64)(65)(66). Because of this, many have examined RGS proteins and their contributions to oncogenic processes.…”

Section: Rgs In Cancermentioning

confidence: 99%

Regulator of G-protein signaling (RGS) proteins as drug targets: Progress and future potentials

O’Brien

Wilkinson

Roman

2019

Journal of Biological Chemistry

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Edited by Henrik G. Dohlman G protein-coupled receptors (GPCRs) play critical roles in regulating processes such as cellular homeostasis, responses to stimuli, and cell signaling. Accordingly, GPCRs have long served as extraordinarily successful drug targets. It is therefore not surprising that the discovery in the mid-1990s of a family of proteins that regulate processes downstream of GPCRs generated great excitement in the field. This finding enhanced the understanding of these critical signaling pathways and provided potentially new targets for pharmacological intervention. These regulators of G-protein signaling (RGS) proteins were viewed by many as nodes downstream of GPCRs that could be targeted with small molecules to tune signaling processes. In this review, we provide a brief overview of the discovery of RGS proteins and of the gradual and continuing discovery of their roles in disease states, focusing particularly on cancer and neurological disorders. We also discuss high-throughput screening efforts that have led to the discovery first of peptide-based and then of small-molecule inhibitors targeting a subset of the RGS proteins. We explore the unique mechanisms of RGS inhibition these chemical tools have revealed and highlight the most up-to-date studies using these tools in animal experiments. Finally, we discuss the future opportunities in the field, as there are clearly more avenues left to be explored and potentials to be realized.

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Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

Cited by 15 publications

References 39 publications

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling

Regulator of G-protein signaling (RGS) proteins as drug targets: Progress and future potentials

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