Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

Brant M Wagener,Eric R Prossnitz,Nicole A Marjon

doi:10.1371/journal.pone.0147442

Brant M Wagener, Eric R Prossnitz + Show 1 more

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https://doi.org/10.1371/journal.pone.0147442

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Journal: PloS one	Publication Date: Jan 20, 2016
Citations: 12	License type: CC BY 4.0

Affiliation: New Mexico Cancer Center, University of New Mexico

Abstract

Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

Highlights

Arrestins were originally described as cytosolic proteins recruited to ligand-activated and phosphorylated G protein-coupled receptors (GPCRs) [1,2,3]
To better understand how arrestins regulate signaling within the context of formyl peptide receptor (FPR) activation and apoptotic signaling, we employed a mutant of arrestin-2 that is deficient in Src kinase binding [20]
In the absence of ligand activation, less than 5% of cells expressing any of the GFP vectors (EGFP vector only, arr2-WT or arr2-P91G/P121E) underwent apoptosis as determined by propidium iodide (PI) staining (Fig 1A), consistent with our previous work demonstrating that apoptosis in these cells is dependent upon FPR activation [33,34]

Summary

Introduction

Arrestins were originally described as cytosolic proteins recruited to ligand-activated and phosphorylated G protein-coupled receptors (GPCRs) [1,2,3]. Signaling proteins, including Src kinase [19,20], ERK1/ 2 [19,21] and JNK3 [22,23,24], bind arrestins and are activated by ligand- and arrestin-bound GPCRs. It has been proposed that arrestins selectively recruit signaling mediators to activated and internalized GPCR signaling scaffolds to prevent crosstalk between unintended proteins through the spatial and temporal localization of signaling complexes [25]. When ERK1/2 is phosphorylated in the context of a GPCR-arrestin scaffold, activated ERK1/2 remains localized to endosome-localized receptors and activates cytosolic effector molecules [19,26,27]

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