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1.
Cell ; 186(10): 2238-2255.e20, 2023 05 11.
Article in English | MEDLINE | ID: mdl-37146613

ABSTRACT

ß-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-ß-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in ß-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that ß-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes ß-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of ß-arrestin function at the plasma membrane, revealing a critical role for ß-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.


Subject(s)
Receptors, G-Protein-Coupled , Signal Transduction , beta-Arrestins , beta-Arrestins/metabolism , Cell Membrane/metabolism , Clathrin/metabolism , Endocytosis , Lipid Bilayers , Receptors, G-Protein-Coupled/metabolism , Molecular Dynamics Simulation
2.
Annu Rev Pharmacol Toxicol ; 60: 73-87, 2020 01 06.
Article in English | MEDLINE | ID: mdl-31539479

ABSTRACT

G protein-coupled receptors (GPCRs) mediate the effects of numerous hormones and neurotransmitters and are major pharmacological targets. Classical studies with crude cell lysates or membrane preparations have identified the main biochemical steps involved in GPCR signaling. Moreover, recent studies on purified proteins have provided astounding details at the atomic level of the 3-D structures of receptors in multiple conformations, including in complex with G proteins and ß-arrestins. However, several fundamental questions remain regarding the highly specific effects and rapid nature of GPCR signaling. Recent developments in single-molecule microscopy are providing important contributions to answering these questions. Overall, single-molecule studies have revealed unexpected levels of complexity, with receptors existing in different conformations and dynamically interacting among themselves, their signaling partners, and structural elements of the plasma membrane to produce highly localized signals in space and time. These findings may provide a new basis to develop innovative strategies to modulate GPCR function for pharmacological purposes.


Subject(s)
GTP-Binding Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , beta-Arrestins/metabolism , Cell Membrane/metabolism , Humans , Receptors, G-Protein-Coupled/chemistry , Signal Transduction/physiology , Single Molecule Imaging/methods
3.
PLoS Biol ; 17(8): e3000097, 2019 08.
Article in English | MEDLINE | ID: mdl-31430273

ABSTRACT

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.


Subject(s)
Glucagon-Like Peptide-1 Receptor/metabolism , Insulin-Secreting Cells/metabolism , Animals , CHO Cells , Cell Membrane/metabolism , Cluster Analysis , Cricetulus , Cyclic AMP/metabolism , Diabetes Mellitus, Type 2 , Endocytosis/drug effects , Glucagon-Like Peptide 1/agonists , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide-1 Receptor/physiology , HEK293 Cells , Humans , Insulin/metabolism , Insulin Secretion/physiology , Insulin-Secreting Cells/physiology , Lipoylation , Signal Transduction/drug effects
4.
Entropy (Basel) ; 23(8)2021 Aug 13.
Article in English | MEDLINE | ID: mdl-34441183

ABSTRACT

In this article, we introduce a new method to detect transient trapping events within a single particle trajectory, thus allowing the explicit accounting of changes in the particle's dynamics over time. Our method is based on new measures of a smoothed recurrence matrix. The newly introduced set of measures takes into account both the spatial and temporal structure of the trajectory. Therefore, it is adapted to study short-lived trapping domains that are not visited by multiple trajectories. Contrary to most existing methods, it does not rely on using a window, sliding along the trajectory, but rather investigates the trajectory as a whole. This method provides useful information to study intracellular and plasma membrane compartmentalisation. Additionally, this method is applied to single particle trajectory data of ß2-adrenergic receptors, revealing that receptor stimulation results in increased trapping of receptors in defined domains, without changing the diffusion of free receptors.

5.
Nat Commun ; 13(1): 7109, 2022 11 19.
Article in English | MEDLINE | ID: mdl-36402762

ABSTRACT

Carvedilol is among the most effective ß-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of ß1-adrenoceptors, arrestin-biased signalling via ß2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol's cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through ß2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the ß-adrenoceptor system.


Subject(s)
Adrenergic beta-Antagonists , Myocardial Infarction , Humans , Carvedilol/pharmacology , Adrenergic beta-Antagonists/pharmacology , Receptors, Adrenergic, beta-2/genetics , Myocardial Infarction/drug therapy
6.
Pharmacol Res Perspect ; 7(6): e00547, 2019 12.
Article in English | MEDLINE | ID: mdl-31832205

ABSTRACT

Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by Mrgprb2. MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC50 ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg9-amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2.


Subject(s)
Anaphylaxis/chemically induced , Mast Cells/drug effects , Nerve Tissue Proteins/agonists , Peptide Fragments/adverse effects , Receptors, G-Protein-Coupled/agonists , Receptors, Neuropeptide/agonists , Vancomycin/adverse effects , Anaphylaxis/immunology , Animals , Cell Degranulation/drug effects , Cell Degranulation/immunology , Cell Line, Tumor , Cell-Penetrating Peptides/administration & dosage , Cell-Penetrating Peptides/adverse effects , Disease Models, Animal , Drug Evaluation, Preclinical/adverse effects , HEK293 Cells , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Humans , Mast Cells/immunology , Mast Cells/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Peptide Fragments/administration & dosage , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/immunology , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/immunology , Receptors, Neuropeptide/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Syndrome , Vancomycin/administration & dosage , p-Methoxy-N-methylphenethylamine/pharmacology
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