Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

Dwivedi-Agnihotri, Hemlata; Chaturvedi, Madhu; Baidya, Mithu; Stępniewski, Tomasz Maciej; Pandey, Shubhi; Maharana, Jagannath; Srivastava, Ashish; Caengprasath, Natarin; Hanyaloglu, Aylin C.; Selent, Jana; Shukla, Arun K.

doi:10.1126/sciadv.abb8368

Cited by 63 publications

(64 citation statements)

References 47 publications

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“…Hence, these residues can serve as key phosphorylation sites that direct β-arrestin1 interaction and may serve as anchor points to switch on/off the recruitment of β-arrestin1. Recently, a single phosphorylation residue within the Vasopressin 2 receptor was found to serve as an anchor point for interaction with β-arrestin1 and 2 [ 58 ]. This key phosphorylation site was suggested to govern the stability of interaction with the β-arrestins regulating the interdomain rotation in β-arrestins [ 58 ].…”

Section: Discussionmentioning

confidence: 99%

“…Recently, a single phosphorylation residue within the Vasopressin 2 receptor was found to serve as an anchor point for interaction with β-arrestin1 and 2 [ 58 ]. This key phosphorylation site was suggested to govern the stability of interaction with the β-arrestins regulating the interdomain rotation in β-arrestins [ 58 ]. Another plausible explanation could be a hierarchical phosphorylation of ACKR3 C-tail, like CXCR4 [ 59 ], where phosphorylation of T 352 and S 355 would be indispensable for the phosphorylation of further residues directly responsible for β-arrestin1 interactions.…”

Section: Discussionmentioning

confidence: 99%

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Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization

Zarca

Pérez

Bor

et al. 2021

Cells

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Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization

Zarca

Pérez

Bor

et al. 2021

Cells

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“…In order to visualize βarr recruitment and trafficking for D6R and C5aR2, confocal microscopy was used following the protocol described previously ( 48 ). Briefly, HEK-293 cells were transfected with receptor (3.5 μg), βarr1-mYFP or βarr1-mYFP (3.5 μg).…”

Section: Methodsmentioning

confidence: 99%

Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

Pandey

Kumari

Baidya

et al. 2021

Preprint

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G protein-coupled receptors (GPCRs) are typically characterized by their seven transmembrane (7TM) architecture, and interaction with two universal signal-transducers namely, the heterotrimeric G-proteins and β-arrestins (βarrs). Synthetic ligands and receptor mutants have been designed to elicit transducer-coupling preferences and distinct downstream signaling outcomes for many GPCRs. This raises the question if some naturally-occurring 7TMRs may selectively engage one of these two signal-transducers, even in response to their endogenous agonists. Although there are scattered hints in the literature that some 7TMRs lack G-protein coupling but interact with βarrs, an in-depth understanding of their transducer-coupling preference, GRK-engagement, downstream signaling and structural mechanism remains elusive. Here, we use an array of cellular, biochemical and structural approaches to comprehensively characterize two non-canonical 7TMRs namely, the human decoy D6 receptor (D6R) and the human complement C5a receptor (C5aR2), in parallel with their canonical GPCR counterparts, CCR2 and C5aR1, respectively. We discover that D6R and C5aR2 couple exclusively to βarrs, exhibit distinct GRK-preference, and activate non-canonical downstream signaling partners. We also observe that βarrs, in complex with these receptors, adopt distinct conformations compared to their canonical GPCR counterparts despite being activated by a common natural agonist. Our study therefore establishes D6R and C5aR2 as bona-fide arrestin-coupled receptors (ACRs), and provides important insights into their regulation by GRKs and downstream signaling with direct implications for biased agonism.

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“…Additionally, ligand-dependent changes in these regions have been shown to lead to bias sampling of distinct arrestin conformations with different impacts on downstream signaling events. In particular, ligand-specific phosphorylation patterns of GPCRs, so-called 'phosphorylation barcodes', caused by selective recruitment of various GRKs that target different sets of phosphorylation sites, have been reported to favor certain arrestin conformations [164][165][166][167][168][169][170][171][172][173][174][175]. These phosphorylation-dependent structural alterations were initially demonstrated by using intramolecular bioluminescence resonance energy transfer (BRET) (reviewed in Refs [137,176]) and FRET biosensors of arrestins in whole cells [157,164,177].…”

Section: Conformational Dynamics Of Gpcrtransducer Signaling Complexesmentioning

confidence: 99%

The role of structural dynamics in GPCR‐mediated signaling

Hilger

2021

The FEBS Journal

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G protein-coupled receptors (GPCRs) play critical roles in the regulation of human physiology in response to a wide array of different extracellular stimuli and thus represent one of the largest groups of therapeutic drug targets. Recent advances in the structural characterization of GPCRs in different conformations and in complex with G proteins and arrestins have provided important insights into the mechanism and function of GPCRs. However, in order to truly understand the molecular basis of the functional versatility of GPCRs, the structural snapshots obtained by X-ray crystallography or cryo-EM need to be complimented with information about the conformational dynamics of receptors and their signaling complexes. In the last decade, a combination of biophysical approaches and computational studies has been utilized to examine the molecular motions of GPCRs and their transducer complexes and how they are regulated by ligands of different efficacy and bias. These studies revealed that GPCRs are highly dynamic allosteric proteins that can sample multiple conformational states. Ligands with distinct signaling profiles not only impact the conformational landscape of GPCRs but also of the receptor-engaged G proteins and arrestins. The conformational dynamics of GPCRs and their signaling complexes and the ligand-dependent bias sampling of distinct functional states are important underlying principles behind the complex signaling behavior of GPCRs.

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Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

Cited by 63 publications

References 47 publications

Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization

Differential Involvement of ACKR3 C-Tail in β-Arrestin Recruitment, Trafficking and Internalization

Intrinsic bias at non-canonical, β-arrestin-coupled seven transmembrane receptors

The role of structural dynamics in GPCR‐mediated signaling

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