Mechanisms of Arrestin-Mediated Signaling.

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.

Conformational flexibility of ß-arrestins - How these scaffolding proteins guide and transform the functionality of GPCRs.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and ß-arrestins. To influence specific GPCR signaling behaviors, ß-arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within ß-arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess ß-arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins.

ß-Arrestins: Structure, Function, Physiology, and Pharmacological Perspectives.

The two ß-arrestins, ß-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 ß-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 ß-arrestins bind to activated GPCRs and downstream effector proteins. Studies with ß-arrestin mutant mice have identified numerous physiologic and pathophysiological processes regulated by ß-arrestin-1 and/or -2. Following a short summary of recent structural studies, this review primarily focuses on ß-arrestin-regulated physiologic functions, with particular focus on the central nervous system and the roles of ß-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 ß-arrestin-regulated signaling pathways for therapeutic purposes. SIGNIFICANCE STATEMENT: The two ß-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 ß-arrestin mutant mice and cultured cells, complemented by novel insights into ß-arrestin structure and function, should pave the way for the development of novel classes of therapeutically useful drugs capable of regulating specific ß-arrestin functions.

A streamlined protocol for expression and purification of wild-type ß-arrestins.

The two isoforms of ß-arrestins namely ß-arrestin 1 and 2 interact with, and regulate a broad repertoire of G protein-coupled receptors (GPCRs). While several protocols have been described in the literature for purification of ß-arrestins for biochemical and biophysical studies, some of these protocols involve multiple complicated steps that prolong the process and yield relatively smaller amounts of purified proteins. Here, we describe a simplified and streamlined protocol for expression and purification of ß-arrestins using E. coli as an expression host. This protocol is based on N-terminal fusion of GST tag and involves a two-step protocol involving GST-based affinity chromatography and size exclusion chromatography. The protocol described here yields sufficient amounts of high-quality purified ß-arrestins suitable for biochemical and structural studies.

Sequestration of Gßγ by deubiquitinated arrestins into the nucleus as a novel desensitization mechanism of G protein-coupled receptors.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCRs) refers to a rapid attenuation of responsiveness that occurs with repeated or continuous exposure to agonists. GRK-mediated phosphorylation and subsequent binding with arrestins in the activated receptor cytoplasmic cavity in competition with G proteins has been suggested as the conventional mechanism of desensitization. Along with widely accepted conventional mechanism of desensitization, studies of various GPCRs including dopamine D2-like receptors (D2R, D3R, D4R) have suggested the existence of another desensitization mechanism. In this study, loss-of-function approaches and D2-like receptor mutants that display different desensitization properties were used to elucidate the molecular mechanisms responsible for desensitization. RESULTS: Desensitization development entailed the signaling cascade composed of Src, PDK1, and Akt, the latter of which in turn interacted with USP33, an arrestin deubiquitinase, to promote arrestin deubiquitination. The deubiquitinated arrestin subsequently formed a complex with Gßγ and translocated to the nucleus via an importin complex, wherein it sequestered Gßγ from the receptor and Gα, thereby attenuating receptor signaling. As in D2-like receptors, both USP33 and importin ß1 were involved in the desensitization of the ß2 adrenoceptor. CONCLUSIONS: In addition to the conventional mechanism of desensitization, which occurs on the plasma membrane and in the cytosol, this study provides a new insight that another desensitization pathway in which nuclear trafficking plays a critical role is operating. It is plausible that multiple, complementary desensitization measures are in place to properly induce desensitization depending on receptor characteristics or the surrounding environment. Video Abstract.

Structures of the TMC-1 complex illuminate mechanosensory transduction.

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

Structural basis of GPCR coupling to distinct signal transducers: implications for biased signaling.

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.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

GPCRsignal: webserver for analysis of the interface between G-protein-coupled receptors and their effector proteins by dynamics and mutations.

GPCRsignal (https://gpcrsignal.biomodellab.eu/) is a webserver devoted to signaling complexes of G-protein-coupled receptors (GPCRs). The recent improvement in cryo-electron microscopy resulted in the determination of a large number of high-resolution structures of GPCRs bound to their effector proteins: G proteins or arrestins. Analyzing the interfaces between receptor and an effector protein is of high importance since a selection of proper G protein or specific conformation of arrestin leads to changes of signaling that can significantly affect action of drugs. GPCRsignal provides a possibility of running molecular dynamics simulations of all currently available GPCR-effector protein complexes for curated structures: wild-type, with crystal/cryo-EM mutations, or with mutations introduced by the user. The simulations are performed in an implicit water-membrane environment, so they are rather fast. User can run several simulations to obtain statistically valid results. The simulations can be analyzed separately using dynamic FlarePlots for particular types of interactions. One can also compare groups of simulations in Interaction frequency analysis as HeatMaps and also in interaction frequency difference analysis as sticks, linking the interacting residues, of different color and size proportional to differences in contact frequencies.

An Eight Amino Acid Segment Controls Oligomerization and Preferred Conformation of the two Non-visual Arrestins.

G protein coupled receptors signal through G proteins or arrestins. A long-standing mystery in the field is why vertebrates have two non-visual arrestins, arrestin-2 and arrestin-3. These isoforms are ~75% identical and 85% similar; each binds numerous receptors, and appear to have many redundant functions, as demonstrated by studies of knockout mice. We previously showed that arrestin-3 can be activated by inositol-hexakisphosphate (IP6). IP6 interacts with the receptor-binding surface of arrestin-3, induces arrestin-3 oligomerization, and this oligomer stabilizes the active conformation of arrestin-3. Here, we compared the impact of IP6 on oligomerization and conformational equilibrium of the highly homologous arrestin-2 and arrestin-3 and found that these two isoforms are regulated differently. In the presence of IP6, arrestin-2 forms "infinite" chains, where each promoter remains in the basal conformation. In contrast, full length and truncated arrestin-3 form trimers and higher-order oligomers in the presence of IP6; we showed previously that trimeric state induces arrestin-3 activation (Chen et al., 2017). Thus, in response to IP6, the two non-visual arrestins oligomerize in different ways in distinct conformations. We identified an insertion of eight residues that is conserved across arrestin-2 homologs, but absent in arrestin-3 that likely accounts for the differences in the IP6 effect. Because IP6 is ubiquitously present in cells, this suggests physiological consequences, including differences in arrestin-2/3 trafficking and JNK3 activation. The functional differences between two non-visual arrestins are in part determined by distinct modes of their oligomerization. The mode of oligomerization might regulate the function of other signaling proteins.

Lysine in the lariat loop of arrestins does not serve as phosphate sensor.

Arrestins demonstrate strong preference for phosphorylated over unphosphorylated receptors, but how arrestins "sense" receptor phosphorylation is unclear. A conserved lysine in the lariat loop of arrestins directly binds the phosphate in crystal structures of activated arrestin-1, -2, and -3. The lariat loop supplies two negative charges to the central polar core, which must be disrupted for arrestin activation and high-affinity receptor binding. Therefore, we hypothesized that receptor-attached phosphates pull the lariat loop via this lysine, thus removing the negative charges and destabilizing the polar core. We tested the role of this lysine by introducing charge elimination (Lys->Ala) and reversal (Lys->Glu) mutations in arrestin-1, -2, and -3. These mutations in arrestin-1 only moderately reduced phospho-rhodopsin binding and had no detectable effect on arrestin-2 and -3 binding to cognate non-visual receptors in cells. The mutations of Lys300 in bovine and homologous Lys301 in mouse arrestin-1 on the background of pre-activated mutants had variable effects on the binding to light-activated phosphorylated rhodopsin, while affecting the binding to unphosphorylated rhodopsin to a greater extent. Thus, conserved lysine in the lariat loop participates in receptor binding, but does not play a critical role in phosphate-induced arrestin activation.

The α-arrestin family of ubiquitin ligase adaptors links metabolism with selective endocytosis.

The regulation of nutrient uptake into cells is important, as it allows to either increase biomass for cell growth or to preserve homoeostasis. A key strategy to adjust cellular nutrient uptake is the reconfiguration of the nutrient transporter repertoire at the plasma membrane by the addition of nutrient transporters through the secretory pathway and by their endocytic removal. In this review, we focus on the mechanisms that regulate selective nutrient transporter endocytosis, which is mediated by the α-arrestin protein family. In the budding yeast Saccharomyces cerevisiae, 14 different α-arrestins (also named arrestin-related trafficking adaptors, ARTs) function as adaptors for the ubiquitin ligase Rsp5. They instruct Rsp5 to ubiquitinate subsets of nutrient transporters to orchestrate their endocytosis. The ART proteins are under multilevel control of the major nutrient sensing systems, including amino acid sensing by the general amino acid control and target of rapamycin pathways, and energy sensing by 5'-adenosine-monophosphate-dependent kinase. The function of the six human α-arrestins is comparably under-characterised. Here, we summarise the current knowledge about the function, regulation and substrates of yeast ARTs and human α-arrestins, and highlight emerging communalities and general principles.

Molecular mechanism of biased signaling in a prototypical G protein-coupled receptor.

Biased signaling, in which different ligands that bind to the same G protein-coupled receptor preferentially trigger distinct signaling pathways, holds great promise for the design of safer and more effective drugs. Its structural mechanism remains unclear, however, hampering efforts to design drugs with desired signaling profiles. Here, we use extensive atomic-level molecular dynamics simulations to determine how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor. The receptor adopts two major signaling conformations, one of which couples almost exclusively to arrestin, whereas the other also couples effectively to a G protein. A long-range allosteric network allows ligands in the extracellular binding pocket to favor either of the two intracellular conformations. Guided by this computationally determined mechanism, we designed ligands with desired signaling profiles.

Continuing challenges in targeting oligomeric GPCR-based drugs.

This chapter will focus on the therapeutic potential of oligomeric GPCR structures. We will discuss the history of these complexes and their connection with treatment. We will highlight several examples from the literature of complexes of therapeutic or theranostic opportunity. As part of this we will discuss different mechanisms that oligomers use to modulate their partner that could be taken advantage of from a drug targeting perspective. We will also discuss the challenges and hurdles to targeting such complexes.

Structure-function analysis of ß-arrestin Kurtz reveals a critical role of receptor interactions in downregulation of GPCR signaling in vivo.

Arrestins control signaling via the G protein coupled receptors (GPCRs), serving as both signal terminators and transducers. Previous studies identified several structural elements in arrestins that contribute to their functions as GPCR regulators. However, the importance of these elements in vivo is unclear, and the developmental roles of arrestins are not well understood. We carried out an in vivo structure-function analysis of Kurtz (Krz), the single ortholog of mammalian ß-arrestins in the Drosophila genome. A combination of Krz mutations affecting the GPCR-phosphosensing and receptor core-binding ("finger loop") functions (Krz-KKVL/A) resulted in a complete loss of Krz activity during development. Endosome recruitment and bioluminescence resonance energy transfer (BRET) assays revealed that the KKVL/A mutations abolished the GPCR-binding ability of Krz. We found that the isolated "finger loop" mutation (Krz-VL/A), while having a negligible effect on GPCR internalization, severely affected Krz function, suggesting that tight receptor interactions are necessary for proper termination of signaling in vivo. Genetic analysis as well as live imaging demonstrated that mutations in Krz led to hyperactivity of the GPCR Mist (also known as Mthl1), which is activated by its ligand Folded gastrulation (Fog) and is responsible for cellular contractility and epithelial morphogenesis. Krz mutations affected two developmental events that are under the control of Fog-Mist signaling: gastrulation and morphogenesis of the wing. Overall, our data reveal the functional importance in vivo of direct ß-arrestin/GPCR binding, which is mediated by the recognition of the phosphorylated receptor tail and receptor core interaction. These Krz-GPCR interactions are critical for setting the correct level of Fog-Mist signaling during epithelial morphogenesis.

Arrestin-3 interaction with maternal embryonic leucine-zipper kinase.

Maternal embryonic leucine-zipper kinase (MELK) overexpression impacts survival and proliferation of multiple cancer types, most notably glioblastomas and breast cancer. This makes MELK an attractive molecular target for cancer therapy. Yet the molecular mechanisms underlying the involvement of MELK in tumorigenic processes are unknown. MELK participates in numerous protein-protein interactions that affect cell cycle, proliferation, apoptosis, and embryonic development. Here we used both in vitro and in-cell assays to identify a direct interaction between MELK and arrestin-3. A part of this interaction involves the MELK kinase domain, and we further show that the interaction between the MELK kinase domain and arrestin-3 decreases the number of cells in S-phase, as compared to cells expressing the MELK kinase domain alone. Thus, we describe a new mechanism of regulation of MELK function, which may contribute to the control of cell fate.

Structural Mechanism of the Arrestin-3/JNK3 Interaction.

Arrestins, in addition to desensitizing GPCR-induced G protein activation, also mediate G protein-independent signaling by interacting with various signaling proteins. Among these, arrestins regulate MAPK signal transduction by scaffolding mitogen-activated protein kinase (MAPK) signaling components such as MAPKKK, MAPKK, and MAPK. In this study, we investigated the binding mode and interfaces between arrestin-3 and JNK3 using hydrogen/deuterium exchange mass spectrometry, 19F-NMR, and tryptophan-induced Atto 655 fluorescence-quenching techniques. Results suggested that the ß1 strand of arrestin-3 is the major and potentially only interaction site with JNK3. The results also suggested that C-lobe regions near the activation loop of JNK3 form the potential binding interface, which is variable depending on the ATP binding status. Because the ß1 strand of arrestin-3 is buried by the C-terminal strand in its basal state, C-terminal truncation (i.e., pre-activation) of arrestin-3 facilitates the arrestin-3/JNK3 interaction.

Critical role of the finger loop in arrestin binding to the receptors.

We tested the interactions with four different G protein-coupled receptors (GPCRs) of arrestin-3 mutants with substitutions in the four loops, three of which contact the receptor in the structure of the arrestin-1-rhodopsin complex. Point mutations in the loop at the distal tip of the N-domain (Glu157Ala), in the C-loop (Phe255Ala), back loop (Lys313Ala), and one of the mutations in the finger loop (Gly65Pro) had mild variable effects on receptor binding. In contrast, the deletion of Gly65 at the beginning of the finger loop reduced the binding to all GPCRs tested, with the binding to dopamine D2 receptor being affected most dramatically. Thus, the presence of a glycine at the beginning of the finger loop appears to be critical for the arrestin-receptor interaction.

Methods to Investigate Arrestins in Complex with Phosphodiesterases.

The many functions of ß-arrestin proteins in the desensitization of G-protein-coupled receptors have been well characterized; however, the discovery that this scaffold protein could actually recruit phosphodiesterases (PDEs) to the site of cAMP synthesis changed the way researchers thought about the static nature of precisely localized cAMP hydrolysis by anchored PDEs. Before this discovery, the compartmentalization of cAMP gradients formed by the activation of specific receptors was generally understood to be underpinned by highly localized pools of specific PDEs that were anchored by large static anchors such as A-kinase-anchoring proteins (AKAPs). Such anchors acted to position cAMP effector proteins such as protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) in places that would allow cAMP concentrations to breach their activation threshold only when a specific receptor activation occurred. In this arrangement PDEs acted as local "sinks" for cAMP and this enforced receptor-specific function by allowing the correct activation of a distinct pool of cAMP effectors in precise localizations. The discovery that ß-arrestin could shuttle cAMP hydrolyzing activity to the membrane shortly after receptor activation added to the complexity of this process by restricting cAMP diffusion into the cell interior for some receptors. This chapter describes the methods used to identify, confirm, and test the function of PDE-ß-arrestin complexes.

Arrestin-1 engineering facilitates complex stabilization with native rhodopsin.

Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC50) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin-arrestin-1 complex. The achieved stabilization of the active rhodopsin-arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).