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

2020 ◽  
Vol 6 (37) ◽  
pp. eabb8368 ◽  
Author(s):  
Hemlata Dwivedi-Agnihotri ◽  
Madhu Chaturvedi ◽  
Mithu Baidya ◽  
Tomasz Maciej Stepniewski ◽  
Shubhi Pandey ◽  
...  
Keyword(s):  
G Protein ◽  
Phosphorylation Site ◽  
Dynamics Simulation ◽  
Phosphorylation Sites ◽  
Functional Responses ◽  
Biased Signaling ◽  
The Stability ◽  
G Protein Coupled ◽  
Structural Aspects

Agonist-induced phosphorylation of G protein–coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling.

scholarly journals Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site

2004 ◽  
Vol 379 (3) ◽  
pp. 573-585 ◽  
Author(s):  
Frank NEUSCHÄFER-RUBE ◽  
Ricardo HERMOSILLA ◽  
Mathias REHWALD ◽  
Lars RÖNNSTRAND ◽  
Ralf SCHÜLEIN ◽  
...  
Keyword(s):  
G Protein ◽  
Phosphorylation Site ◽  
Receptor Subtype ◽  
Receptor Complexes ◽  
Prostaglandin Receptor ◽  
Terminal Domain ◽  
Prostaglandin E2 Receptor ◽  
The Stability ◽  
Necessary And Sufficient ◽  
G Protein Coupled

hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a Gs-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced β-arrestin complex formation, which is stabilized by phosphorylation. Subsequently β-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with β-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370–382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389–Ser-390–Thr-391–Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of β-arrestin1. However, phosphorylation greatly enhanced the stability of the β-arrestin1–receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both β-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.

scholarly journals Evidence of a common mechanism of disassembly of adherens junctions through Gα13 targeting of VE-cadherin

2014 ◽  
Vol 211 (3) ◽  
pp. 579-591 ◽  
Author(s):  
Haixia Gong ◽  
Xiaopei Gao ◽  
Shaoting Feng ◽  
M. Rizwan Siddiqui ◽  
Alexander Garcia ◽  
...  
Keyword(s):  
G Protein ◽  
Adherens Junctions ◽  
Common Mechanism ◽  
Binding Motif ◽  
P120 Catenin ◽  
Unique Role ◽  
Proinflammatory Mediators ◽  
The Stability ◽  
G Protein Coupled

The heterotrimeric G protein Gα13 transduces signals from G protein–coupled receptors (GPCRs) to induce cell spreading, differentiation, migration, and cell polarity. Here, we describe a novel GPCR-independent function of Gα13 in regulating the stability of endothelial cell adherens junctions (AJs). We observed that the oxidant H2O2, which is released in response to multiple proinflammatory mediators, induced the interaction of Gα13 with VE-cadherin. Gα13 binding to VE-cadherin in turn induced Src activation and VE-cadherin phosphorylation at Tyr 658, the p120-catenin binding site thought to be responsible for VE-cadherin internalization. Inhibition of Gα13–VE-cadherin interaction using an interfering peptide derived from the Gα13 binding motif on VE-cadherin abrogated the disruption of AJs in response to inflammatory mediators. These studies identify a unique role of Gα13 binding to VE-cadherin in mediating VE-cadherin internalization and endothelial barrier disruption and inflammation.

scholarly journals Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization

2008 ◽  
Vol 295 (3) ◽  
pp. E665-E671 ◽  
Author(s):  
Susanne U. Miedlich ◽  
Abdul B. Abou-Samra
Keyword(s):  
Parathyroid Hormone ◽  
Phospholipase C ◽  
G Protein ◽  
Phosphorylation Site ◽  
Receptor Internalization ◽  
Phosphorylation Sites ◽  
G Protein Coupled Receptor ◽  
Parathyroid Hormone Receptor ◽  
Direct Binding ◽  
G Protein Coupled

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to Gq/11 proteins upon PTH stimulation predominantly caused by elimination of Ser491/492/493, Ser501, or Ser504. Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to Gq/11 proteins at least partially by direct binding to Gq/11 proteins.

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

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 618
Author(s):  
Aurélien Zarca ◽  
Claudia Perez ◽  
Jelle van den Bor ◽  
Jan Paul Bebelman ◽  
Joyce Heuninck ◽  
...  
Keyword(s):  
G Protein ◽  
Phosphorylation Site ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Vascular Diseases ◽  
Cell Types ◽  
Receptor Internalization ◽  
G Protein Coupled ◽  
Single Cluster

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.

Role of amino acid residues surrounding the phosphorylation site in peptide substrates of G protein-coupled receptor kinase 2 (GRK2)

Amino Acids ◽  
2016 ◽  
Vol 48 (12) ◽  
pp. 2875-2880 ◽  
Author(s):  
Daisuke Asai ◽  
Masaharu Murata ◽  
Riki Toita ◽  
Takahito Kawano ◽  
Hideki Nakashima ◽  
...  
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Phosphorylation Site ◽  
Amino Acid Residues ◽  
Receptor Kinase ◽  
G Protein Coupled Receptor ◽  
Peptide Substrates ◽  
G Protein Coupled

scholarly journals Computational Investigations on the Binding Mode of Ligands for the Cannabinoid-Activated G Protein-Coupled Receptor GPR18

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 686 ◽  
Author(s):  
Alexander Neumann ◽  
Viktor Engel ◽  
Andhika B. Mahardhika ◽  
Clara T. Schoeder ◽  
Vigneshwaran Namasivayam ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
G Protein ◽  
Phenyl Ring ◽  
Binding Mode ◽  
Dynamics Simulation ◽  
Simulation Studies ◽  
Binding Modes ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

GPR18 is an orphan G protein-coupled receptor (GPCR) expressed in cells of the immune system. It is activated by the cannabinoid receptor (CB) agonist ∆9-tetrahydrocannabinol (THC). Several further lipids have been proposed to act as GPR18 agonists, but these results still require unambiguous confirmation. In the present study, we constructed a homology model of the human GPR18 based on an ensemble of three GPCR crystal structures to investigate the binding modes of the agonist THC and the recently reported antagonists which feature an imidazothiazinone core to which a (substituted) phenyl ring is connected via a lipophilic linker. Docking and molecular dynamics simulation studies were performed. As a result, a hydrophobic binding pocket is predicted to accommodate the imidazothiazinone core, while the terminal phenyl ring projects towards an aromatic pocket. Hydrophobic interaction of Cys251 with substituents on the phenyl ring could explain the high potency of the most potent derivatives. Molecular dynamics simulation studies suggest that the binding of imidazothiazinone antagonists stabilizes transmembrane regions TM1, TM6 and TM7 of the receptor through a salt bridge between Asp118 and Lys133. The agonist THC is presumed to bind differently to GPR18 than to the distantly related CB receptors. This study provides insights into the binding mode of GPR18 agonists and antagonists which will facilitate future drug design for this promising potential drug target.

Marijuana and β-estradiol interactions on spatial learning and memory in young female rats: Lack of role of the G protein-coupled estrogen receptor (GPR30)

Life Sciences ◽  
2021 ◽  
pp. 119723
Author(s):  
Mohadeseh Chahkandi ◽  
Gholamreza Komeili ◽  
Gholamreza Sepehri ◽  
Mohammad Khaksari ◽  
Sedigheh Amiresmaili
Keyword(s):  
Estrogen Receptor ◽  
Learning And Memory ◽  
G Protein ◽  
Spatial Learning ◽  
Young Female ◽  
Female Rats ◽  
Spatial Learning And Memory ◽  
G Protein Coupled

Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

2021 ◽  
pp. 247255522097979
Author(s):  
Kyung-Soon Lee ◽  
Edelmar Navaluna ◽  
Nicole M. Marsh ◽  
Eric M. Janezic ◽  
Chris Hague
Keyword(s):  
G Protein ◽  
Adrenergic Receptor ◽  
Near Infrared ◽  
Human Cells ◽  
Receptor Subtypes ◽  
G Protein Coupled Receptor ◽  
Functional Responses ◽  
Epitope Tag ◽  
Nir Imaging ◽  
G Protein Coupled

We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives ( t1/2) using LICOR NIR imaging–polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous β-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous β2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.

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