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Sequences and three-dimensional structures of the four vertebrate arrestins are very similar, yet in sharp contrast to other subtypes, arrestin-1 demonstrates exquisite selectivity for the active phosphorylated form of its cognate receptor, rhodopsin. The N-terminus participates in receptor binding and serves as the anchor of the C-terminus, the release of which facilitates arrestin transition into a receptor-binding state. We tested the effects of substitutions of fourteen residues in the N-terminus of arrestin-1 on the binding to phosphorylated and unphosphorylated light-activated rhodopsin of wild-type protein and its enhanced mutant with C-terminal deletion that demonstrates higher binding to both functional forms of rhodopsin. Profound effects of mutations identified lysine-15 as the main phosphate sensor and phenylalanine-13 as the key anchor of the C-terminus. These residues are conserved in all arrestin subtypes. Substitutions of five other residues reduced arrestin-1 selectivity for phosphorylated rhodopsin, indicating that wild-type residues participate in fine-tuning of arrestin-1 binding. Differential effects of numerous substitutions in wild-type and an enhanced mutant arrestin-1 suggest that these two proteins bind rhodopsin differently.

Arrestins were discovered for their role in homologous desensitization of G-protein-coupled receptors (GPCRs). Later non-visual arrestins were shown to regulate several signaling pathways. Some of these pathways require arrestin binding to GPCRs, the regulation of others is receptor independent. Here, we demonstrate that arrestin-3 binds the E3 ubiquitin ligase parkin via multiple sites, preferentially interacting with its RING0 domain. Identification of the parkin domains involved suggests that arrestin-3 likely relieves parkin autoinhibition and/or stabilizes the enzymatically active "open" conformation of parkin. Arrestin-3 binding enhances ubiquitination by parkin of the mitochondrial protein mitofusin-1 and facilitates parkin-mediated mitophagy in HeLa cells. Furthermore, arrestin-3 and its mutant with enhanced parkin binding rescue mitofusin-1 ubiquitination and mitophagy in the presence of the Parkinson's disease-associated R275W parkin mutant, which is defective in both functions. Thus, modulation of parkin activity via arrestin-3 might be a novel strategy of anti-parkinsonian therapy., (© 2024 International Society for Neurochemistry.)

In rodents with unilateral ablation of neurons supplying dopamine to the striatum, chronic treatment with the dopamine precursor L-DOPA induces a progressive increase of behavioral responses, a process known as behavioral sensitization. This sensitization is blunted in arrestin-3 knockout mice. Using virus-mediated gene delivery to the dopamine-depleted striatum of these mice, we find that the restoration of arrestin-3 fully rescues behavioral sensitization, whereas its mutant defective in c-Jun N-terminal kinase (JNK) activation does not. A 25-residue arrestin-3-derived peptide that facilitates JNK3 activation in cells, expressed ubiquitously or selectively in direct pathway striatal neurons, also fully rescues sensitization, whereas an inactive homologous arrestin-2-derived peptide does not. Behavioral rescue is accompanied by the restoration of JNK3 activity, as reflected by JNK-dependent phosphorylation of the transcription factor c-Jun in the dopamine-depleted striatum. Thus, arrestin-3-assisted JNK3 activation in direct pathway neurons is a critical element of the molecular mechanism underlying sensitization upon dopamine depletion and chronic L-DOPA treatment., Competing Interests: Declaration of interests E.V.G. and V.V.G. have a patent related to this work: “Peptide Regulators Of JNK Family Kinases”. Patent no.: US 10,369,187 B2, date of patent: Aug. 6, 2019., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Biological activity of free arrestins is often overlooked. Based on available data, we compare arrestin-mediated signaling that requires and does not require binding to G-protein-coupled receptors (GPCRs). Receptor-bound arrestins activate ERK1/2, Src, and focal adhesion kinase (FAK). Yet, arrestin-3 regulation of Src family member Fgr does not appear to involve receptors. Free arrestin-3 facilitates the activation of JNK family kinases, preferentially binds E3 ubiquitin ligases Mdm2 and parkin, and facilitates parkin-dependent mitophagy. The binding of arrestins to microtubules and calmodulin and their function in focal adhesion disassembly and apoptosis also do not involve receptors. Biased GPCR ligands and the phosphorylation barcode can only affect receptor-dependent arrestin signaling. Thus, elucidation of receptor dependence or independence of arrestin functions has important scientific and therapeutic implications., Competing Interests: Declaration of interests The authors declare no conflicts of interest, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In rodents with unilateral ablation of the substantia nigra neurons supplying dopamine to the striatum, chronic treatment with the dopamine precursor L-DOPA or dopamine agonists induces a progressive increase of behavioral responses, a process known as behavioral sensitization. The sensitization is blunted in arrestin-3 knockout mice. Using virus-mediated gene delivery to the dopamine-depleted striatum of arrestin-3 knockout mice, we found that the restoration of arrestin-3 fully rescued behavioral sensitization, whereas its mutant defective in JNK activation did not. A 25-residue arrestin-3-derived peptide that facilitates JNK3 activation in cells, expressed ubiquitously or selectively in the direct pathway striatal neurons, fully rescued sensitization, whereas an inactive homologous arrestin-2-derived peptide did not. Behavioral rescue was accompanied by the restoration of JNK3 activity and of JNK-dependent phosphorylation of the transcription factor c-Jun in the dopamine-depleted striatum. Thus, arrestin-3-dependent JNK3 activation in direct pathway neurons is a critical element of the molecular mechanism underlying sensitization., Competing Interests: Disclosures. Eugenia V. Gurevich and Vsevolod V. Gurevich have a patent related to this work: “PEPTIDE REGULATORS OF JNK FAMILY KINASES” Patent No.: US 10,369,187 B2, Date of Patent: Aug. 6, 2019. The authors declare no other competing interests.

The two β -arrestins, β -arrestin-1 and -2 (systematic names: arrestin-2 and -3, respectively), are multifunctional intracellular proteins that regulate the activity of a very large number of cellular signaling pathways and physiologic functions. The two proteins were discovered for their ability to disrupt signaling via G protein-coupled receptors (GPCRs) via binding to the activated receptors. However, it is now well recognized that both β -arrestins can also act as direct modulators of numerous cellular processes via either GPCR-dependent or -independent mechanisms. Recent structural, biophysical, and biochemical studies have provided novel insights into how β -arrestins bind to activated GPCRs and downstream effector proteins. Studies with β -arrestin mutant mice have identified numerous physiologic and pathophysiological processes regulated by β -arrestin-1 and/or -2. Following a short summary of recent structural studies, this review primarily focuses on β -arrestin-regulated physiologic functions, with particular focus on the central nervous system and the roles of β -arrestins in carcinogenesis and key metabolic processes including the maintenance of glucose and energy homeostasis. This review also highlights potential therapeutic implications of these studies and discusses strategies that could prove useful for targeting specific β -arrestin-regulated signaling pathways for therapeutic purposes. SIGNIFICANCE STATEMENT: The two β-arrestins, structurally closely related intracellular proteins that are evolutionarily highly conserved, have emerged as multifunctional proteins able to regulate a vast array of cellular and physiological functions. The outcome of studies with β-arrestin mutant mice and cultured cells, complemented by novel insights into β-arrestin structure and function, should pave the way for the development of novel classes of therapeutically useful drugs capable of regulating specific β-arrestin functions., (U.S. Government work not protected by U.S. copyright.)

Only 1 out of 4 mammalian arrestin subtypes, arrestin-3, facilitates the activation of c-Jun N-terminal kinase (JNK) family kinases. Here, we describe two different sets of protocols used for elucidating the mechanisms involved. One is based on reconstitution of signaling modules from the following purified proteins: arrestin-3, MKK4, MKK7, JNK1, JNK2, and JNK3. The main advantage of this method is that it unambiguously establishes which effects are direct because only intended purified proteins are present in these assays. The key drawback is that the upstream-most kinases of these cascades, ASK1 or other MAP3Ks, are not available in purified form, limiting reconstitution to incomplete two-kinase modules. The other approach is used for analyzing the effects of arrestin-3 on JNK activation in intact cells. In this case, signaling modules include ASK1 and/or other MAP3Ks. However, as every cell expresses thousands of different proteins, their possible effects on the readout cannot be excluded. Nonetheless, the combination of in vitro reconstitution from purified proteins and cell-based assays makes it possible to elucidate the mechanisms of arrestin-3-dependent activation of JNK family kinases. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Construction of arrestin-3-scaffolded MKK4/7-JNK1/2/3 signaling modules in vitro using purified proteins Alternate Protocol 1: Characterization of arrestin-3-mediated JNK1/2 activation by MKK4/7 by measurement of JNK1/2 phosphorylation using immunoblotting with anti-phospho-JNK antibody Support Protocol 1: Expression, purification, and activation of GST-MKK4 Support Protocol 2: Expression, purification, and activation of GST-MKK7-His 6 Support Protocol 3: Expression, purification, and activation of tagless JNK1Α1 Support Protocol 4: Expression, purification, and activation of tagless JNK2Α2 Basic Protocol 2: Analysis of the role of arrestin-3 in ASK1/MKK4/MKK7-induced JNK activation in intact cells Alternate Protocol 2: Analysis of the role of arrestin-3 in MKK4-induced JNK activation in intact cells Basic Protocol 3: Characterization of the biphasic effect of arrestin-3 on ASK1/MKK7-stimulated JNK phosphorylation in cells., (© 2023 Wiley Periodicals LLC.)

Arrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells. In available structures, Lys-295 in the lariat loop of arrestin-3 and its homologue Lys-294 in arrestin-2 directly interact with the activator-attached phosphates. We compared the roles of arrestin-3 conformational equilibrium and Lys-295 in GPCR binding and JNK3 activation. Several mutants with enhanced ability to bind GPCRs showed much lower activity towards JNK3, whereas a mutant that does not bind GPCRs was more active. The subcellular distribution of mutants did not correlate with GPCR recruitment or JNK3 activation. Charge neutralization and reversal mutations of Lys-295 differentially affected receptor binding on different backgrounds but had virtually no effect on JNK3 activation. Thus, GPCR binding and arrestin-3-assisted JNK3 activation have distinct structural requirements, suggesting that facilitation of JNK3 activation is the function of arrestin-3 that is not bound to a GPCR.

Arrestins were first discovered as proteins that selectively bind active phosphorylated GPCRs and suppress (arrest) their G protein-mediated signaling. Nonvisual arrestins are also recognized as signaling proteins regulating a variety of cellular pathways. Arrestins are highly flexible; they can assume many different conformations. In their receptor-bound conformation, arrestins have higher affinity for a subset of binding partners. This explains how receptor activation regulates certain branches of arrestin-dependent signaling via arrestin recruitment to GPCRs. However, free arrestins are also active molecular entities that regulate other signaling pathways and localize signaling proteins to particular subcellular compartments. Recent findings suggest that the two visuals, arrestin-1 and arrestin-4, which are expressed in photoreceptor cells, not only regulate signaling via binding to photopigments but also interact with several nonreceptor partners, critically affecting the health and survival of photoreceptor cells. Detailed in this overview are GPCR-dependent and independent modes of arrestin-mediated regulation of cellular signaling. © 2023 Wiley Periodicals LLC., (© 2023 Wiley Periodicals LLC.)

When the rat D3 dopamine receptor (D3R) was cloned and the distribution of its mRNA examined in 1990-1991, it attracted attention due to its peculiar distribution in the brain quite different from that of its closest relative, the D2 receptor. In the rat brain, the D3R mRNA is enriched in the limbic striatum as opposed to the D2 receptor, which is highly expressed in the motor striatal areas. Later studies in the primate and human brain confirmed relative enrichment of the D3R in the limbic striatum but also demonstrated higher abundance of the D3R in the primate as compared to the rodent brain. Additionally, in the rodent brain, the D3R in the dorsal striatum appears to be co-expressed with the D1 dopamine receptor-bearing striatal neurons giving rise to the direct output striatal pathway, although the picture is less clear with respect to the nucleus accumbens. In contrast, in the primate striatum, the D3R co-localizes with the D2 receptor throughout the basal ganglia as well as in extrastriatal brain areas. The relative abundance of the D3R in the limbic striatum, its output structures, secondary targets, and some of the other connected limbic territories may underpin its role in reward, drug dependence, and impulse control. Selective expression of D3R in the brain proliferative areas may point to its important role in the neural development as well as in neurodevelopmental abnormalities associated with schizophrenia and other developmental brain disorders., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Three classes of G-protein-coupled receptor (GPCR) partners - G proteins, GPCR kinases, and arrestins - preferentially bind active GPCRs. Our analysis suggests that the structures of GPCRs bound to these interaction partners available today do not reveal a clear conformational basis for signaling bias, which would have enabled the rational design of biased GRCR ligands. In view of this, three possibilities are conceivable: (i) there are no generalizable GPCR conformations conducive to binding a particular type of partner; (ii) subtle differences in the orientation of individual residues and/or their interactions not easily detectable in the receptor-transducer structures determine partner preference; or (iii) the dynamics of GPCR binding to different types of partners rather than the structures of the final complexes might underlie transducer bias., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Three out of four subtypes of arrestin proteins expressed in mammals self-associate, each forming oligomers of a distinct kind. Monomers and oligomers have different subcellular localization and distinct biological functions. Here we summarize existing evidence regarding arrestin oligomerization and discuss specific functions of monomeric and oligomeric forms, although too few of the latter are known. The data on arrestins highlight biological importance of oligomerization of signaling proteins. Distinct modes of oligomerization might be an important contributing factor to the functional differences among highly homologous members of the arrestin protein family.

Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds of G protein-coupled receptors, certain receptors of other types, and numerous non-receptor partners. Arrestins have no enzymatic activity and work by interacting with other proteins, often assembling multi-protein signaling complexes. Arrestin binding to every partner affects cell signaling, including pathways regulating cell survival, proliferation, and death. Thus, targeting individual arrestin interactions has therapeutic potential. This requires precise identification of protein-protein interaction sites of both participants and the choice of the side of each interaction which would be most advantageous to target. The interfaces involved in each interaction can be disrupted by small molecule therapeutics, as well as by carefully selected peptides of the other partner that do not participate in the interactions that should not be targeted., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mammalian arrestins are a family of four highly homologous relatively small ~ 45 kDa proteins with surprisingly diverse functions. The most striking feature is that each of the two non-visual subtypes can bind hundreds of diverse G protein-coupled receptors (GPCRs) and dozens of non-receptor partners. Through these interactions, arrestins regulate the G protein-dependent signaling by the desensitization mechanisms as well as control numerous signaling pathways in the G protein-dependent or independent manner via scaffolding. Some partners prefer receptor-bound arrestins, some bind better to the free arrestins in the cytoplasm, whereas several show no apparent preference for either conformation. Thus, arrestins are a perfect example of a multi-functional signaling regulator. The result of this multi-functionality is that reduction (by knockdown) or elimination (by knockout) of any of these two non-visual arrestins can affect so many pathways that the results are hard to interpret. The other difficulty is that the non-visual subtypes can in many cases compensate for each other, which explains relatively mild phenotypes of single knockouts, whereas double knockout is lethal in vivo, although cultured cells lacking both arrestins are viable. Thus, deciphering the role of arrestins in cell biology requires the identification of specific signaling function(s) of arrestins involved in a particular phenotype. This endeavor should be greatly assisted by identification of structural elements of the arrestin molecule critical for individual functions and by the creation of mutants where only one function is affected. Reintroduction of these biased mutants, or introduction of monofunctional stand-alone arrestin elements, which have been identified in some cases, into double arrestin-2/3 knockout cultured cells, is the most straightforward way to study arrestin functions. This is a laborious and technically challenging task, but the upside is that specific function of arrestins, their timing, subcellular specificity, and relations to one another could be investigated with precision.

G protein-coupled receptors (GPCRs) are the largest family of signaling proteins targeted by more clinically used drugs than any other protein family. GPCR signaling via G proteins is quenched (desensitized) by the phosphorylation of the active receptor by specific GPCR kinases (GRKs) followed by tight binding of arrestins to active phosphorylated receptors. Thus, arrestins engage two types of receptor elements: those that contain GRK-added phosphates and those that change conformation upon activation. GRKs attach phosphates to serines and threonines in the GPCR C-terminus or any one of the cytoplasmic loops. In addition to these phosphates, arrestins engage the cavity that appears between trans-membrane helices upon receptor activation and several other non-phosphorylated elements. The residues that bind GPCRs are localized on the concave side of both arrestin domains. Arrestins undergo a global conformational change upon receptor binding (become activated). Arrestins serve as important hubs of cellular signaling, emanating from activated GPCRs and receptor-independent., (Copyright © 2019. Published by Elsevier B.V.)

Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cell-based functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.

Arrestins are soluble relatively small 44-46 kDa proteins that specifically bind hundreds of active phosphorylated GPCRs and dozens of non-receptor partners. There are binding partners that demonstrate preference for each of the known arrestin conformations: free, receptor-bound, and microtubule-bound. Recent evidence suggests that conformational flexibility in every functional state is the defining characteristic of arrestins. Flexibility, or plasticity, of proteins is often described as structural disorder, in contrast to the fixed conformational order observed in high-resolution crystal structures. However, protein-protein interactions often involve highly flexible elements that can assume many distinct conformations upon binding to different partners. Existing evidence suggests that arrestins are no exception to this rule: their flexibility is necessary for functional versatility. The data on arrestins and many other multi-functional proteins indicate that in many cases, "order" might be artificially imposed by highly non-physiological crystallization conditions and/or crystal packing forces. In contrast, conformational flexibility (and its extreme case, intrinsic disorder) is a more natural state of proteins, representing true biological order that underlies their physiologically relevant functions.

Until the late 1990s, class A G protein-coupled receptors (GPCRs) were believed to function as monomers. Indirect evidence that they might internalize or even signal as dimers has emerged, along with proof that class C GPCRs are obligatory dimers. Crystal structures of GPCRs and their much larger binding partners were consistent with the idea that two receptors might engage a single G protein, GRK, or arrestin. However, recent biophysical, biochemical, and structural evidence invariably suggests that a single GPCR binds G proteins, GRKs, and arrestins. Here we review existing evidence of the stoichiometry of GPCR interactions with signal transducers and discuss potential biological roles of class A GPCR oligomers, including proposed homo- and heterodimers., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Actions of the neurotransmitter dopamine in the brain are mediated by dopamine receptors that belong to the superfamily of G protein-coupled receptors (GPCRs). Mammals have five dopamine receptor subtypes, D1 through D5. D1 and D5 couple to Gs/olf and activate adenylyl cyclase, whereas D2, D3, and D4 couple to Gi/o and inhibit it. Most GPCRs upon activation by an agonist are phosphorylated by GPCR kinases (GRKs). The GRK phosphorylation makes receptors high-affinity binding partners for arrestin proteins. Arrestin binding to active phosphorylated receptors stops further G protein activation and promotes receptor internalization, recycling or degradation, thereby regulating their signaling and trafficking. Four non- visual GRKs are expressed in striatal neurons. Here we describe known effects of individual GRKs on dopamine receptors in cell culture and in the two in vivo models of dopamine-mediated signaling: behavioral response to psychostimulants and L-DOPA- induced dyskinesia. Dyskinesia, associated with dopamine super-sensitivity of striatal neurons, is a debilitating side effect of L-DOPA therapy in Parkinson's disease. In vivo, GRK subtypes show greater receptor specificity than in vitro or in cultured cells. Overexpression, knockdown, and knockout of individual GRKs, particularly GRK2 and GRK6, have differential effects on signaling of dopamine receptor subtypes in the brain. Furthermore, deletion of GRK isoforms in select striatal neuronal types differentially affects psychostimulant-induced behaviors. In addition, anti-dyskinetic effect of GRK3 does not require its kinase activity: it is mediated by the binding of its RGS-like domain to Gαq/11, which suppresses Gq/11 signaling. The data demonstrate that the dopamine signaling in defined neuronal types in vivo is regulated by specific and finely orchestrated actions of GRK isoforms., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa (L-dopa) therapy for Parkinson's disease (PD). L-dopa-induced dyskinesia (LID) are ultimately experienced by the vast majority of patients. In addition, psychiatric conditions often manifested as compulsive behaviours, are emerging as a serious problem in the management of L-dopa therapy. The present review attempts to provide an overview of our current understanding of dyskinesia and other L-dopa-induced dysfunctions, a field that dramatically evolved in the past twenty years. In view of the extensive literature on LID, there appeared a critical need to re-frame the concepts, to highlight the most suitable models, to review the central nervous system (CNS) circuitry that may be involved, and to propose a pathophysiological framework was timely and necessary. An updated review to clarify our understanding of LID and other L-dopa-related side effects was therefore timely and necessary. This review should help in the development of novel therapeutic strategies aimed at preventing the generation of dyskinetic symptoms., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Most proteins have multiple functions. Obviously, conventional methods of manipulating the level of the protein of interest in the cell, such as over-expression, knockout or knockdown, affect all of its functions simultaneously. The key advantage of these methods is that over-expression, knockout or knockdown does not require any knowledge of the molecular mechanisms of the function(s) of the protein of interest. The disadvantage is that these approaches are inadequate to elucidate the role of an individual function of the protein in a particular cellular process. An alternative is the use of re-engineered proteins, in which a single function is eliminated or enhanced. The use of mono-functional elements of a multi-functional protein can also yield cleaner answers. This approach requires detailed knowledge of the structural basis of each function of the protein in question. Thus, a lot of preliminary structure-function work is necessary to make it possible. However, when this information is available, replacing the protein of interest with a mutant in which individual functions are modified can shed light on the biological role of those particular functions. Here, we illustrate this point using the example of protein kinases, most of which have additional non-enzymatic functions, as well as arrestins, known multi-functional signaling regulators in the cell.

Among four vertebrate arrestins, only two are ubiquitously expressed. Arrestins specifically bind active phosphorylated G protein-coupled receptors (GPCRs), thereby precluding further G protein activation. Recent discoveries suggest that the formation of the arrestin-receptor complex initiates the second round of signaling with comparable biological importance. Despite having virtually no recognizable sequence motifs known to mediate protein-protein interactions, arrestins bind a surprising variety of signaling proteins with mind-boggling range of functional consequences. High conformational flexibility allows arrestins to show many distinct 'faces' to the world, which allows these relatively small ∼45kDa proteins to bind various partners under different physiological conditions, organizing multi-protein signaling complexes and localizing them to distinct subcellular compartments., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

The activity of all mitogen-activated protein kinases (MAPKs) is stimulated via phosphorylation by upstream MAPK kinases (MAPKK), which are in their turn activated via phosphorylation by MAPKK kinases (MAPKKKs). The cells ensure the specificity of signaling in these cascades by employing a variety of scaffolding proteins that bind matching MAPKKKs, MAPKKs, and MAPKs. All four vertebrate arrestin subtypes bind JNK3, but only arrestin-3 serves as a scaffold, promoting JNK3 activation in intact cells. Arrestin-3-mediated JNK3 activation does not depend on arrestin-3 interaction with G protein-coupled receptors (GPCRs), as demonstrated by the ability of some arrestin mutants that cannot bind receptors to activate JNK3, whereas certain mutants with enhanced GPCR binding fail to promote JNK3 activation. Recent findings suggest that arrestin-3 directly binds both MAPKKs necessary for JNK activation and facilitates JNK3 phosphorylation at both Thr (by MKK4) and Tyr (by MKK7). JNK3 is expressed in a limited set of cell types, whereas JNK1 and JNK2 isoforms are as ubiquitous as arrestin-3. Recent study showed that arrestin-3 facilitates the activation of JNK1 and JNK2, scaffolding MKK4/7-JNK1/2/3 signaling complexes. In all cases, arrestin-3 acts by bringing the kinases together: JNK phosphorylation shows biphasic dependence on arrestin-3, being enhanced at lower and suppressed at supraoptimal concentrations. Thus, arrestin-3 regulates the activity of multiple JNK isoforms, suggesting that it might play a role in survival and apoptosis of all cell types.

Virtually all currently used therapeutic agents are small molecules, largely because the development and delivery of small molecule drugs is relatively straightforward. Small molecules have serious limitations: drugs of this type can be fairly good enzyme inhibitors, receptor ligands, or allosteric modulators. However, most cellular functions are mediated by protein interactions with other proteins, and targeting protein-protein interactions by small molecules presents challenges that are unlikely to be overcome with these compounds as the only tools. Recent advances in gene delivery techniques and characterization of cell type-specific promoters open the prospect of using reengineered signaling-biased proteins as next-generation therapeutics. The first steps in targeted engineering of proteins with desired functional characteristics look very promising. As quintessential scaffolds that act strictly via interactions with other proteins in the cell, arrestins represent a perfect model for the development of these novel therapeutic agents with enormous potential: custom-designed signaling proteins will allow us to tell the cell what to do and when to do it in a way it cannot disobey.

A variety of heritable and acquired disorders is associated with excessive signaling by mutant or overstimulated GPCRs. Since any conceivable treatment of diseases caused by gain-of-function mutations requires gene transfer, one possible approach is functional compensation. Several structurally distinct forms of enhanced arrestins that bind phosphorylated and even non-phosphorylated active GPCRs with much higher affinity than parental wild-type proteins have the ability to dampen the signaling by hyperactive GPCR, pushing the balance closer to normal. In vivo this approach was so far tested only in rod photoreceptors deficient in rhodopsin phosphorylation, where enhanced arrestin improved the morphology and light sensitivity of rods, prolonged their survival, and accelerated photoresponse recovery. Considering that rods harbor the fastest, as well as the most demanding and sensitive GPCR-driven signaling cascade, even partial success of functional compensation of defect in rhodopsin phosphorylation by enhanced arrestin demonstrates the feasibility of this strategy and its therapeutic potential.

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.

The complexity of living systems exceeds everything else studied by natural sciences. Sophisticated networks of intimately intertwined signaling pathways coordinate cellular functions. Clear understanding how the integration of multiple inputs produces coherent behavior is one of the major challenges of cell biology. Integration via perfectly timed highly regulated protein-protein interactions and precise targeting of the "output" proteins to particular substrates is emerging as a common theme of signaling regulation. This often involves specialized scaffolding proteins, whose key function is to ensure that correct partners come together in an appropriate place at the right time. Defective or faulty signaling underlies many congenital and acquired human disorders. Several pioneering studies showed that ectopic expression of existing proteins or their elements can restore functions destroyed by mutations or normalize the signaling pushed out of balance by disease and/or current small molecule-based therapy. Several recent studies show that proteins with new functional modalities can be generated by mixing and matching existing domains, or via functional recalibration and fine-tuning of existing proteins by precisely targeted mutations. Using arrestins as an example, we describe how manipulation of individual functions yields signaling-biased proteins. Creative protein redesign generates novel tools valuable for unraveling the intricacies of cell biology. Engineered proteins with specific functional changes also have huge therapeutic potential in disorders associated with inherited or acquired signaling errors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

G protein-coupled receptor (GPCR) kinases (GRKs) are best known for their role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors and promote high affinity binding of arrestins, which precludes G protein coupling. GRKs have a multidomain structure, with the kinase domain inserted into a loop of a regulator of G protein signaling homology domain. Unlike many other kinases, GRKs do not need to be phosphorylated in their activation loop to achieve an activated state. Instead, they are directly activated by docking with active GPCRs. In this manner they are able to selectively phosphorylate Ser/Thr residues on only the activated form of the receptor, unlike related kinases such as protein kinase A. GRKs also phosphorylate a variety of non-GPCR substrates and regulate several signaling pathways via direct interactions with other proteins in a phosphorylation-independent manner. Multiple GRK subtypes are present in virtually every animal cell, with the highest expression levels found in neurons, with their extensive and complex signal regulation. Insufficient or excessive GRK activity was implicated in a variety of human disorders, ranging from heart failure to depression to Parkinson's disease. As key regulators of GPCR-dependent and -independent signaling pathways, GRKs are emerging drug targets and promising molecular tools for therapy. Targeted modulation of expression and/or of activity of several GRK isoforms for therapeutic purposes was recently validated in cardiac disorders and Parkinson's disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Animals with the neonatal ventral hippocampal lesion (NVHL) demonstrate altered responsiveness to stress and various drugs reminiscent of that in schizophrenia. Post-pubertal onset of abnormalities suggests the possibility of sex differences in NVHL effects that may model sex differences in schizophrenia. Here we demonstrate that novelty- and MK-801-induced hyperactivity is evident in both male and female NVHL rats, whereas only NVHL males were hyperactive in response to apomorphine. Next, we examined the sex- and NVHL-dependent differences in the activity of the ERK and Akt pathways. The basal activity of both pathways was higher in females than in males. NVHL reduces the level of phosphorylation of ERK1/2, Akt, and GSK-3 in both sexes, although males show more consistent down-regulation. Females had higher levels of G-protein-coupled kinases [G-protein-coupled receptor kinase (GRK)] 3 and 5, whereas the concentrations of other GRKs and arrestins were the same. In the nucleus accumbens, the concentration of GRK5 in females was elevated by NVHL to the male level. The data demonstrate profound sex differences in the expression and activity of signalling molecules that may underlie differential susceptibility to schizophrenia.

Dimerization is fairly common in the G-protein-coupled receptor (GPCR) superfamily. First attempts to rationalize this phenomenon gave rise to an idea that two receptors in a dimer could be necessary to bind a single molecule of G protein or arrestin. Although GPCRs, G proteins and arrestins were crystallized only in their inactive conformations (in which they do not interact), the structures appeared temptingly compatible with this beautiful model. However, it did not survive the rigors of experimental testing: several recent studies unambiguously demonstrated that one receptor molecule is sufficient to activate a G protein and bind arrestin. Thus, to figure out the biological role of receptor self-association we must focus on other functions of GPCRs at different stages of their functional cycle.

Dopamine and other G protein-coupled receptors (GPCRs) represent the major target of antipsychotic drugs. GPCRs undergo desensitization via activation-dependent phosphorylation by G protein-coupled receptor kinases (GRKs) followed by arrestin binding. Arrestins and GRKs are major regulators of GPCR signaling. We elucidated changes in expression of two arrestins and four GRKs following chronic (21 days) treatment with haloperidol (1 mg/kg i.p.) or clozapine (20 mg/kg i.p.) 2 or 24 h after the last injection in 11 brain regions. Haloperidol decreased GRK3 in ventrolateral caudate-putamen and transiently down-regulated GRK5 in globus pallidus and caudal caudate-putamen. Clozapine also caused a short-term suppression of the GRK5 expression in the caudal caudate-putamen and globus pallidus, but, unlike haloperidol, elevated GRK5 in the caudal caudate-putamen after 24 h. Unlike haloperidol, clozapine decreased arrestin2 and GRK3 in hippocampus and GRK3 in globus pallidus but increased arrestin2 in the core of nucleus accumbens and ventrolateral caudate-putamen and GRK2 in prefrontal cortex. Clozapine, but not haloperidol, induced long-term activation of extracellular signal-regulated kinase (ERK) 2 in ventrolateral caudate-putamen and transient in prefrontal cortex. The data demonstrate that haloperidol and clozapine differentially affect the expression of arrestins and GRKs and ERK activity, which may play a role in determining their clinical profile.

Accumulating evidence of G-protein-coupled receptor (GPCR) oligomerization on the one hand and perfect functionality of monomeric receptors on the other creates an impression of controversy. However, the GPCR superfamily is extremely diverse, both structurally and functionally. The life cycle of each receptor includes many stages: synthesis, quality control in the endoplasmic reticulum, maturation in the Golgi, delivery to the plasma membrane (where it can be in the inactive or active state, in complex with cognate G protein, G-protein-coupled receptor kinase or arrestin), endocytosis and subsequent sorting in endosomes. Different GPCR subtypes, and even the same receptor at different stages of its life cycle, most likely exist in different oligomerization states, from monomers to dimers and possibly higher-order oligomers.

Arrestins (Arrs) are ubiquitous regulators of the most numerous family of signaling proteins, G protein-coupled receptors. Two models of the Arr-receptor interaction have been proposed: the binding of one Arr to an individual receptor or to two receptors in a dimer. To determine the binding stoichiometry in vivo, we used rod photoreceptors where rhodopsin (Rh) and Arr are expressed at comparably high levels and where Arr localization in the light is determined by its binding to activated Rh. Genetic manipulation of the expression of both proteins shows that the maximum amount of Arr that moves to the Rh-containing compartment exceeds 80%, but not 100%, of the molar amount of Rh present. In vitro experiments with purified proteins confirm that Arr "saturates" Rh at a 1:1 ratio. Thus, a single Rh molecule is necessary and sufficient to bind Arr. Remarkable structural conservation among receptors and Arrs strongly suggests that all Arr subtypes bind individual molecules of their cognate receptors.

In vertebrates, the arrestins are a family of four proteins that regulate the signaling and trafficking of hundreds of different G-protein-coupled receptors (GPCRs). Arrestin homologs are also found in insects, protochordates and nematodes. Fungi and protists have related proteins but do not have true arrestins. Structural information is available only for free (unbound) vertebrate arrestins, and shows that the conserved overall fold is elongated and composed of two domains, with the core of each domain consisting of a seven-stranded beta-sandwich. Two main intramolecular interactions keep the two domains in the correct relative orientation, but both of these interactions are destabilized in the process of receptor binding, suggesting that the conformation of bound arrestin is quite different. As well as binding to hundreds of GPCR subtypes, arrestins interact with other classes of membrane receptors and more than 20 surprisingly diverse types of soluble signaling protein. Arrestins thus serve as ubiquitous signaling regulators in the cytoplasm and nucleus.

Arrestin proteins play a key role in desensitizing G-protein-coupled receptors and re-directing their signaling to alternative pathways. The precise timing of arrestin binding to the receptor and its subsequent dissociation is ensured by its exquisite selectivity for the activated phosphorylated form of the receptor. The interaction between arrestin and the receptor involves the engagement of arrestin sensor sites that discriminate between active and inactive and phosphorylated and unphosphorylated forms of the receptor. This initial interaction is followed by a global conformational rearrangement of the arrestin molecule in the process of its transition into the high-affinity receptor-binding state that brings additional binding sites into action. In this article, we discuss the molecular mechanisms that underlie the sequential multi-site binding that ensures arrestin selectivity for the active phosphoreceptor and high fidelity of signal regulation by arrestin proteins.