β-arrestin1 and 2 exhibit distinct phosphorylation-dependent conformations when coupling to the same GPCR in living cells

Haider, Raphael; Matthees, Edda S F; Drube, Julia; Reichel, Mona; Zabel, Ulrike; Inoue, Asuka; Chevigné, Andy; Krasel, Cornelius; Deupí, Xavier; Hoffmann, Carsten

doi:10.1038/s41467-022-33307-8

Cited by 33 publications

(39 citation statements)

References 60 publications

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“…These data led to the conclusion that phosphorylation of the ICL3 promotes a conformation of β-arrestin that selectively targets the ERK1/2 signaling pathway. Similar results were recently found by Haider et al (2022) who showed that different phosphorylation sites on the parathyroid hormone 1 receptor were involved in regulating ERK1/2 activation and receptor internalization, which are both β-arrestin-mediated processes [ 52 ].…”

Section: Discussionsupporting

confidence: 88%

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating β-Arrestin Binding and Activation

Moritz

Madaras

Rankin

et al. 2023

IJMS

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The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein β-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting β-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

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Section: Discussionsupporting

confidence: 88%

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating β-Arrestin Binding and Activation

Moritz

Madaras

Rankin

et al. 2023

IJMS

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“…Activation also includes change of the angle between the N and C domains of β-arrestin-2 (74, 75). Additionally, the C-edge loops of arrestins can act as a membrane anchor to stabilize the PAR1-β-arrestin complex (77). Activated β-arrestin-2 can have different conformations, which in part are mediated by phosphorylation of at least six residues including Thr178, Ser194, Ser267, Ser268, Ser281, Ser361 and Thr383 (76).…”

Section: The Model Of Apc-mediated Par1 Biased Signaling Complexmentioning

confidence: 99%

The structural basis of the EPCR-APC complex induced biased PAR1 signaling

Iakhiaev

2023

Preprint

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Activated Protein C (APC) is an effector enzyme of the natural anticoagulant pathway. In addition to its anticoagulant function, endothelial protein C receptor (EPCR)-bound APC induces biased protease-activated receptor type 1 (PAR1)-mediated signaling. Despite intensive investigation, the mechanism of biased signaling is not completely clear. To gain new insights into APC-induced PAR1-biased signaling we reviewed the published data and created three-dimensional models of the proteins and their complexes involved in the early stages of PAR1 signaling. A comparative study of models related to canonical and biased signaling demonstrated that interactions between APC, EPCR, PAR1, and Caveolin-1 (Cav1) can provide plausible explanations for the differences between the two types of PAR1 signaling. The model suggests that the interaction of the PAR1 peptide 22-ARTRARRPESK-32 with 162-helix of APC positions the PAR1 N-terminus for the preferential cleavage at R46. By contrast, the hirudin-like sequence of PAR1 is involved in the positioning of the N-terminus of PAR1 for cleavage at R41 by thrombin in canonical signaling. The model and molecular dynamics (MD) simulations of the tethered ligand (TL) interaction with APC suggest that the TL facilitates direct interaction of the EPCR transmembrane (TM) domain with the PAR1 TM helices 6 and 7 by transient binding to the light chain of APC and keeping EPCR-APC in close proximity to PAR1. The biased signaling paradigm considers the ligand-induced conformational changes in PAR1 as solely being responsible for the biased signaling. Our models suggest that Cav1, EPCR, and PAR1 interactions can provide a selective advantage to biased signaling over canonical signaling. First, the complex comprised of caveolin-1 oligomer-EPCR-APC-PAR1 positions EPCR-APC and PAR1 at a distance favorable for PAR1 activation. Second, the Cav1 presence favors selectivity for the PAR1 bound β-arrestin-2, not the PAR1-bound G protein alpha (Gα) subunit. The potential reason for β-arrestin-2 selectivity includes Gα binding to the Cav1 and its immobilization resulting in the inability of PAR1-bound Gα to periodically interact with the plasma membrane required for its function. MD simulations of the PAR1-EPCR-β-arrestin-2 complex demonstrated that one of the mechanisms of the APC-induced PAR1-biased signaling is the interaction of the EPCR TM domain with the PAR1-bound β-arrestin-2, leading to the stabilization of the PAR1-β-arrestin-2 complex and activation of β-arrestin-2. Thus, models suggest that Cav1 and EPCR-APC mediated interactions provide a selective advantage for the β-arrestin-2 dependent biased signaling, not the G proteins mediated canonical signaling by the PAR1 receptor.

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“…Along with their signaling functions, β-arrestins are able to modulate the interaction of GPCRs with different types of G-proteins, as was shown for type 1 parathyroid hormone receptor (PTH1R) coupled to both Gs-and Gq/11proteins [29]. It is important to point out that the functions of different types of arrestins, β1 and β2, in parathyroid hormone-induced signaling differ significantly, which is due to differences in their conformations when binding to an activated PTH1R [29].…”

Section: Introductionmentioning

confidence: 96%

“…Being in the "tail" conformation, β-arrestins preferentially interact with the intracellular C-terminal domain of GPCR, which is phosphorylated by specific G protein-coupled receptor kinases (GRKs), while in the "core" conformation they are capable of interact with the transmembrane domain (TMD) of the receptor [25][26][27][28]. Along with their signaling functions, β-arrestins are able to modulate the interaction of GPCRs with different types of G-proteins, as was shown for type 1 parathyroid hormone receptor (PTH1R) coupled to both Gs-and Gq/11proteins [29]. It is important to point out that the functions of different types of arrestins, β1 and β2, in parathyroid hormone-induced signaling differ significantly, which is due to differences in their conformations when binding to an activated PTH1R [29].…”

Section: Introductionmentioning

confidence: 99%

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

Ao¹

2023

Preprint

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A separate group consists of compounds that are able to simultaneously interact with both orthosteric and allosteric sites, which are classified as bitopic GPCR ligands [74, 76-81]. They have two pharmacophores, one of which binds with high affinity to the orthosteric site, while the other, with lower affinity, binds to the allosteric site. If these sites are spatially separated in the receptor, then the pharmacophores in the bitopic ligand must be connected with a flexible linker, the length of which exactly corresponds to the distance between the orthosteric and allosteric sites. At the same time, it is important that the linker does not significantly affect the conformational rearrangements in the receptor caused by its activation by orthosteric and (or) allosteric agonists [74, 79].

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β-arrestin1 and 2 exhibit distinct phosphorylation-dependent conformations when coupling to the same GPCR in living cells

Cited by 33 publications

References 60 publications

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating β-Arrestin Binding and Activation

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating β-Arrestin Binding and Activation

The structural basis of the EPCR-APC complex induced biased PAR1 signaling

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

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