Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

Zhuo, Ya; Vishnivetskiy, Sergey A.; Zhan, Xiaoli; Gurevich, Vsevolod V.; Klug, Candice S.

doi:10.1074/jbc.m114.560680

Cited by 64 publications

(84 citation statements)

References 68 publications

(101 reference statements)

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“…In the inactive state of β-arrestins these lysines are shielded by the β-arrestin C-terminus. Receptor docking of arrestin induces the disruption of the "polar core" and displaces the β-arrestin C-terminus (44,45). Thereafter, K11 and K12 can interact with phosphorylated residues of the receptor C-tail, thereby completing and maintaining the C-terminal swap, or rebind the…”

Section: Discussionmentioning

confidence: 99%

“…The altered basal conformation of K2A-β-arrestin2 was expected, since it has been shown previously that K2 region binds the C-terminal part of arrestins to the N-domain in the inactive state (33,43). Activation of β-arrestin2 results in the release of the C-terminus (44,45), which might be reflected by BRET ratio change with F410 ( Figure 6a), and also by altered basal BRET ratio with F410-K2A ( Figure S3c). Strikingly, when we stimulated the AT 1R co-expressing cells with AngII, a similar pattern of structural rearrangements was observed to that of AT1R-TSTS/A and wild type β-arrestin2: F154 and F225 signals were missing, F410 was reversed and F263 signal was abolished ( Figure 6d).…”

Section: O N F I D E N T I a Lmentioning

confidence: 99%

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

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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: Discussionmentioning

confidence: 99%

Section: O N F I D E N T I a Lmentioning

confidence: 99%

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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“…It is possible that different phosphorylation barcodes mediate different arrestin conformations and that this results in different arrestin-dependent signaling (Nobles et al, 2011). Although it is now clear that arrestin adopts an active conformation on interaction with receptors (Zhuo et al, 2014) and that a recent crystal structure of arrestin in complex with a phosphorylated C-terminal tail peptide of a GPCR provides atomic-level detail of the active arrestin conformation (Shukla et al, 2013), it is not yet clear whether differential phosphorylation can result in different active conformational states. Our data do, however, support the hypothesis that differential receptor phosphorylation can result in different signaling outcomes.…”

Section: Phosphorylation Profile Of Mffa4 Determines Signalingmentioning

confidence: 99%

Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein–Coupled Receptor 120

et al. 2016

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It is established that long-chain free fatty acids including v-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here, we employed mass spectrometry to determine that phosphorylation of mouse (m) FFAR4 occurs at five serine and threonine residues clustered in two separable regions of the C-terminal tail, designated cluster 1 (Thr 347 , Thr 349 , and Ser 350 ) and cluster 2 (Ser 357 and Ser 361 ). Mutation of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of G q / 11 proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but instead significantly compromised receptor internalization and arrestin 3 recruitment. Distinctly, mutation of the phosphoacceptor sites within cluster 1 had no effect on receptor internalization and had a less extensive effect on arrestin 3 recruitment but significantly uncoupled the receptor from Akt activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode) at the C terminus of the receptor.

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“…Interestingly, the C-tail in the basal conformation of all arrestins is anchored to the N-domain, 12–16 whereas receptor binding triggers its release. 17–19 The expression of separated arrestin C-tail carrying these sites inhibits GPCR internalization, apparently by winning the competition with the arrestin–receptor complexes for clathrin and AP2. 20 This finding provided the first clear evidence of functional significance of shielding of the arrestin C-tail in the basal conformation and its release upon receptor binding.…”

Section: Non-visual Arrestins Mediate Gpcr Internalization Via Coamentioning

confidence: 99%

Arrestins

Gurevich

2015

Progress in Molecular Biology and Translational Science

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Arrestins specifically bind active phosphorylated G protein-coupled receptors (GPCRs). Receptor binding induces the release of the arrestin C-tail, which in non-visual arrestins contains high-affinity binding sites for clathrin and its adaptor AP2. Thus, serving as a physical link between the receptor and key components of the internalization machinery of the coated pit is the best-characterized function of non-visual arrestins in GPCR trafficking. However, arrestins also regulate GPCR trafficking less directly by orchestrating their ubiquitination and deubiquitination. Several reports suggest that arrestins play additional roles in receptor trafficking. Non-visual arrestins appear to be required for the recycling of internalized GPCRs, and the mechanisms of their function in this case remain to be elucidated. Moreover, visual and non-visual arrestins were shown to directly bind N-ethylmaleimide-sensitive factor, an important ATPase involved in vesicle trafficking, but neither molecular details nor the biological role of these interactions is clear. Considering how many different proteins arrestins appear to bind, we can confidently expect the elucidation of additional trafficking-related functions of these versatile signaling adaptors.

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Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

Cited by 64 publications

References 68 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 Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein–Coupled Receptor 120

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