Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Changxiu; Gong, Zheng; Liu, Hongda; Li, Fahui; Wang, Hongmei; He, Dongfang; Song, Chen; Tian, Changlin; Xiao, Kunhong; Wang, Jiangyun; Sun, Junying

doi:10.1038/ncomms9202

Cited by 172 publications

(188 citation statements)

References 63 publications

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“…PKC-phosphorylation was sufficient to induce conformational changes in β-arrestin2 N-domain. This role of K11,12 is in good agreement with the results of a previous study, where phosphopeptide-induced β-arrestin1 conformational shifts were investigated (55). Interestingly, the C-domain sensors (F225, F263) in arrestin did not respond to the recruitment upon PKC-phosphorylation of the receptor or in the absence of stability lock, indicating that both stability lock and receptor activity are required for the full set of structural rearrangements in the Cdomain.…”

Section: O N F I D E N T I a L Heterologous Regulation Of Inactive supporting

confidence: 78%

“…the canonical homologous and the novel heterologous activation, induce β-arrestin binding to the same receptor in different conformations in a physiologically relevant setting. Previous studies suggested that the multiple active conformations of β-arrestins have different functions (55,57,58). This may explain our finding that the PKC-induced β-arrestin2 recruitment directed the receptor to Rab7-enriched late endosomes to a lesser extent than AngII.…”

Section: O N F I D E N T I a L Heterologous Regulation Of Inactive supporting

confidence: 66%

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Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

Tóth

Prokop

Gyombolai

et al. 2018

Journal of Biological Chemistry

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β-arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptorsand 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. Since 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 q/11-coupled GPCR or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT1 angiotensin receptor (AT1R). 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 Ndomain. Using improved FlAsH-based 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 AT1R 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.The family of G protein-coupled receptors (GPCRs) consists of ~800 members in humans and about 30% of modern drugs target these molecules (1). GPCRs respond to a wide variety of endogenous ligands, including hormones, neurotransmitters, and lipids. Despite their huge diversity, the signal transduction mechanisms of GPCRs share several common features: agonist binding is followed by the activation of a relatively small number of heterotrimeric G proteins, which initiate complex intracellular signaling cascades. Receptor responsiveness to further stimulation is attenuated by a multistep process, called desensitization (2). In case of homologous desensitization, active GPCRs are phosphorylated by GPCR kinases (GRKs) followed by the recruitment of β-arrestin proteins (β-arrestin1 and JBC C o n f i d e n t i a lHeterologous regulation of inactive receptors via β-arrestin 2 β-arrestin2, also known as arrestin-2 and arrestin-3, respectively). β-arrestins uncouple the receptors from G proteins and initiate receptor internalization (3), thereby serving as the key regulators of GPCRs' function. In contrast, heterologous desensitization is mediated by second-messenger activated kinases, such as protein kinase C (PKC), which can phosphorylate active and inactive receptors. Heterologous desensitization was originally thought to be independent of β-arrestins, however, some data have challenged this concept (4-6). In addition to their rol...

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Section: O N F I D E N T I a L Heterologous Regulation Of Inactive supporting

confidence: 78%

Section: O N F I D E N T I a L Heterologous Regulation Of Inactive supporting

confidence: 66%

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

Tóth

Prokop

Gyombolai

et al. 2018

Journal of Biological Chemistry

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“…The scaffolding function of βarrs, as signal transducers, has been characterized for multiple signaling proteins, including c-Src (18,19). Formation of GPCR-βarr1-c-Src ternary complexes has been demonstrated to regulate multiple cellular functions downstream of various GPCRs (20).…”

Section: Resultsmentioning

confidence: 99%

Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

Cahill

Thomsen

Tarrasch

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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313

308

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β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-βarr complexes: the "tail" conformation, with βarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, βarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of βarrs is unknown. Here, we created a mutant form of βarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and βarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-βarr conformations can carry out distinct functions.O ver the past decade, significant efforts have been made to understand the molecular properties and regulatory mechanisms that control the function of β-arrestin (βarr) interactions with G protein-coupled receptors (GPCRs) (1, 2). Once activated, GPCRs initiate a highly conserved signaling and regulatory cascade marked by interactions with: (i) heterotrimeric G proteins, which mediate their actions largely by promoting second-messenger generation (3); (ii) GPCR kinases (GRKs), which phosphorylate activated conformations of receptors (4); and (iii) βarrs, which bind to the phosphorylated receptors to mediate desensitization of G protein signaling and receptor internalization (5, 6). In addition to their canonical function of desensitization and internalization, βarrs have been appreciated as independent signaling units by virtue of their crucial role as both adaptors and scaffolds for an increasing number of signaling pathways (7-11).There are two driving forces that mediate βarr interactions with an activated GPCR: phosphorylation of the C-terminal tail of the receptor by GRKs and/or binding to the transmembrane core of the receptor. How each of these interactions contributes to βarr functionality remains unclear. Moreover, GPCRs tend to either interact with βarr transiently, termed "class A" GPCRs [e.g., β 2 -adrenergic receptor (β 2 AR)], or tightly, known as "class B" GPCRs [e.g., vasopressin type 2 receptor (V 2 R)]. For the current study, we use a previously described chimeric β 2 V 2 R construct, which comprises the β 2 AR with its C-terminal tail exchanged with the V 2 R C-terminal tail (12-14). The β 2 V 2 R construct provides an ideal system for studying a GPCR-βarr complex in vitro, because it maintains identical pharmacological properties to the WT β 2 AR and has a robustly increased class B affinity for βarr1, which allows stable β 2 V 2 R-βarr complexes to be formed and purified.Structural insights have shed some light onto the complexity of the interaction between GPCRs and βarrs. A recent struc...

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“…For example, ␤arr1 can bind between blades 1 and 2 of the clathrin ␤-propeller via an intrinsically disordered clathrin-binding box, but can also interact with a binding pocket formed by blades 4 and 5 of clathrin via an 8-amino acid splice loop found only in the long ␤arr1 isoform (99). Further insights into the allosteric regulation of ␤arr signaling have recently been provided by an NMR study that used 19 F probes in ␤arr1 to probe changes in its structure induced by different phosphopeptides derived from the V2R C terminus (100). Although all the phosphopeptides interacted with the phosphate sensor to induce changes in the finger and middle loops, there were also distinct phospho-interaction patterns that were related to the spacing of the multiple ␤arr phospho-binding sites.…”

Section: Signal Transduction To Effectorsmentioning

confidence: 99%

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors

Smith¹,

Rajagopal

2016

Journal of Biological Chemistry

266

222

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The ␤-arrestins (␤arrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, ␤arrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that ␤arrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for ␤arrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. ␤-Arrestins (␤arrs)2 are ubiquitously expressed proteins that were first described for their role in desensitizing G proteincoupled receptors (GPCRs) (1). We now appreciate that these proteins are multifunctional adapter proteins that regulate a vast array of cellular functions. ␤arrs were identified through their sequence homology to visual arrestin (arrestin-1), so named because of its ability to "arrest" rhodopsin signaling in the retina (2). There are two ␤arr isoforms, ␤-arrestin1 and ␤-arrestin2 (also denoted as arrestin-2 and arrestin-3, respectively). Both are expressed ubiquitously and share 78% sequence homology (3). ␤arrs are highly conserved across species, with ϳ50% sequence homology between vertebrates and invertebrates. The other arrestins are expressed in the eye: arrestin-1 (visual arrestin) and arrestin-4 (cone arrestin) (4). There are other proteins, termed ␣-arrestins or arrestin domain-containing proteins, that share the arrestin structural fold and are involved in receptor endocytosis, although the full breadth of their functions is still emerging (5). Similar to arrestin's function in the visual system, ␤arrs were first identified for their capacity to desensitize ␤2 adrenergic receptor (␤2AR) G protein signaling following agonist stimulation (1). Through a number of investigations, it became apparent that the two ␤arr isoforms shared the capability to interact with activated GPCRs, but that they differed in terms of their expression patterns, their specificity for different GPCRs, and their functional effects (6). We now appreciate that the ␤arrs regulate a diverse array of cellular processes including MAPK signaling, receptor transactivation, receptor trafficking, and transcriptional regulation in addition to the canonical roles of GPCR desensitization and internalization (7,8). These studies have revealed the current spectrum of ␤arr-mediated cell processes downstream of GPCRs (Fig. 1). Distinct and Overlapping Roles for the ␤arrs␤arr1 and ␤arr2 knockouts are phenotypically normal and produce viable progeny, but these mice display abnormal responses to physiologic stresses (9, 10). This suggests a compensatory ability for each isoform. Nevertheless, important differences between ␤arr isoforms are present (11). Although both accumulate in the cytoplasm following overexpression, ␤arr1, but not...

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Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

Cited by 172 publications

References 63 publications

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors

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