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β-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, G-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT angiotensin receptor (ATR). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated ATR in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusβ-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusβ-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Rod tetrameric arrestin 1 (tet-ARR1), stored in the outer nuclear layer/inner segments in the dark, modulates photoreceptor synaptic activity; light exposure stimulates a reduction via translocation to the outer segments for terminating G-protein coupled phototransduction signaling. Here, we test the hypothesis that intraretinal spin-lattice relaxation rate in the rotating frame (1/T1ρ), an endogenous MRI contrast mechanism, has high potential for evaluating rod tet-ARR1 and its reduction via translocation. Dark- and light-exposed mice (null for the ARR1 gene, overexpressing ARR1, diabetic, or wild type with or without treatment with Mn2+, a calcium channel probe) were studied using 1/T1ρ MRI. Immunohistochemistry and single-cell recordings of the retinas were also performed. In wild-type mice with or without treatment with Mn2+, 1/T1ρ of avascular outer retina (64% to 72% depth) was significantly (P < 0.05) greater in the dark than in the light; a significant (P < 0.05) but opposite pattern was noted in the inner retina (<50% depth). Light-evoked outer retina Δ1/T1ρ was absent in ARR1-null mice and supernormal in overexpressing mice. In diabetic mice, the outer retinal Δ1/T1ρ pattern suggested normal dark-to-light tet-ARR1 translocation and chromophore content, conclusions confirmed ex vivo. Light-stimulated Δ1/T1ρ in inner retina was linked to changes in blood volume. Our data support 1/T1ρ MRI for noninvasively assessing rod tet-ARR1 and its reduction via protein translocation, which can be combined with other metrics of retinal function in vivo.
T-cell activation requires signaling by T-cell receptors (TCRs) that bind antigen on the antigen-presenting cells (APCs) at the immunological synapse (IS). Sustained signaling requires continuous supply of new TCRs to the IS. In this issue of The EMBO Journal, Fernández-Arenas et al (2014) describe a novel role of β-arrestin-1 at the IS periphery: endocytosis of TCRs and chemokine CXCR4 receptors. Internalized TCRs are then delivered to the IS, where they engage antigen and support prolonged signaling, whereas CXCR4 internalization stops T-cell migration.
Programmed cell death (apoptosis) is a coordinated set of events eventually leading to the massive activation of specialized proteases (caspases) that cleave numerous substrates, orchestrating fairly uniform biochemical changes than culminate in cellular suicide. Apoptosis can be triggered by a variety of stimuli, from external signals or growth factor withdrawal to intracellular conditions, such as DNA damage or ER stress. Arrestins regulate many signaling cascades involved in life-or-death decisions in the cell, so it is hardly surprising that numerous reports document the effects of ubiquitous nonvisual arrestins on apoptosis under various conditions. Although these findings hardly constitute a coherent picture, with the same arrestin subtypes, sometimes via the same signaling pathways, reported to promote or inhibit cell death, this might reflect real differences in pro- and antiapoptotic signaling in different cells under a variety of conditions. Recent finding suggests that one of the nonvisual subtypes, arrestin-2, is specifically cleaved by caspases. Generated fragment actively participates in the core mechanism of apoptosis: it assists another product of caspase activity, tBID, in releasing cytochrome C from mitochondria. This is the point of no return in committing vertebrate cells to death, and the aspartate where caspases cleave arrestin-2 is evolutionary conserved in vertebrate, but not in invertebrate arrestins. In contrast to wild-type arrestin-2, its caspase-resistant mutant does not facilitate cell death.
Arrestins bind ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) and terminate the activation of G proteins. Additionally, nonvisual arrestin/GPCR complex can initiate G protein-independent intracellular signals through their ability to act as scaffolds that bring other signaling molecules to the internalized GPCR. Like nonvisual arrestins, vertebrate visual arrestin (ARR1) terminates G protein signaling from light-activated, phosphorylated GPCR, rhodopsin. Unlike nonvisual arrestins, its role as a transducer of signaling from internalized rhodopsin has not been reported in the vertebrate retina. Formation of signaling complexes with arrestins often requires recruitment of the endocytic adaptor protein, AP-2. We have previously shown that Lys296 → Glu (K296E), which is a naturally occurring rhodopsin mutation in certain humans diagnosed with autosomal dominant retinitis pigmentosa, causes toxicity through forming a stable complex with ARR1. Here we investigated whether recruitment of AP-2 by the K296E/ARR1 complex plays a role in generating the cell death signal in a transgenic mouse model of retinal degeneration. We measured the binding affinity of ARR1 for AP-2 and found that, although the affinity is much lower than that of the other arrestins, the unusually high concentration of ARR1 in rods would favor this interaction. We further demonstrate that p44, a splice variant of ARR1 that binds light-activated, phosphorylated rhodopsin but lacks the AP-2 binding motif, prevents retinal degeneration and rescues visual function in K296E mice. These results reveal a unique role of ARR1 in a G protein-independent signaling cascade in the vertebrate retina.
Arrestin-1 selectively binds active phosphorylated rhodopsin (P-Rh*), demonstrating much lower affinity for inactive phosphorylated (P-Rh) and unphosphorylated active (Rh*) forms. Receptor interaction induces significant conformational changes in arrestin-1, which include large movement of the previously neglected 139-loop in the center of the receptor binding surface, away from the incoming receptor. To elucidate the functional role of this loop, in mouse arrestin-1 we introduced deletions of variable lengths and made several substitutions of Lys-142 in it and Asp-72 in the adjacent loop. Several mutants with perturbations in the 139-loop demonstrate increased binding to P-Rh*, dark P-Rh, Rh*, and phospho-opsin. Enhanced binding of arrestin-1 mutants to non-preferred forms of rhodopsin correlates with decreased thermal stability. The 139-loop perturbations increase P-Rh* binding of arrestin-1 at low temperatures and further change its binding profile on the background of 3A mutant, where the C-tail is detached from the body of the molecule by triple alanine substitution. Thus, the 139-loop stabilizes basal conformation of arrestin-1 and acts as a brake, preventing its binding to non-preferred forms of rhodopsin. Conservation of this loop in other subtypes suggests that it has the same function in all members of the arrestin family.
Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations.
Arrestins are multifunctional signaling adaptors originally discovered as proteins that "arrest" G protein activation by G protein-coupled receptors (GPCRs). Recently GPCR complexes with arrestins have been proposed to activate G protein-independent signaling pathways. In particular, arrestin-dependent activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has been demonstrated. Here we have performed in vitro binding assays with pure proteins to demonstrate for the first time that ERK2 directly binds free arrestin-2 and -3, as well as receptor-associated arrestins-1, -2, and -3. In addition, we showed that in COS-7 cells arrestin-2 and -3 association with β(2)-adrenergic receptor (β2AR) significantly enhanced ERK2 binding, but showed little effect on arrestin interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule-associated. Using conformationally biased arrestin mutants we found that ERK2 preferentially binds two of these: the "constitutively inactive" arrestin-Δ7 mimicking microtubule-bound state and arrestin-3A, a mimic of the receptor-bound conformation. Both rescue arrestin-mediated ERK1/2/activation in arrestin-2/3 double knockout fibroblasts. We also found that arrestin-2-c-Raf1 interaction is enhanced by receptor binding, whereas arrestin-3-c-Raf1 interaction is not.
We have previously reported on the differential regulation of the human δ-opioid receptor (hDOR) by alkaloid (etorphine) and peptidic (DPDPE and deltorphin I) ligands, in terms of both receptor desensitization and post-endocytic sorting. Since ßarrestins are well known to regulate G protein-coupled receptors (GPCRs) signaling and trafficking, we therefore investigated the role of ßarrestin1 (the only isoform expressed in our cellular model) in the context of the hDOR. We established clonal cell lines of SK-N-BE cells over-expressing ßarrestin1, its dominant negative mutant (ßarrestin1(319-418)), and shRNA directed against endogenous ßarrestin1. Interestingly, both binding and confocal microscopy approaches demonstrated that ßarrestin1 is required for hDOR endocytosis only when activated by etorphine. Conversely, functional experiments revealed that ßarrestin1 is exclusively involved in hDOR desensitization promoted by the peptides. Taken together, these results provide substantial evidence for a ßarrestin1-biased agonism at hDOR, where ßarrestin1 is differentially involved during receptor desensitization and endocytosis depending on the ligand.
Copyright © 2011 Elsevier Inc. All rights reserved.