10 research outputs found
Single particle tracking of G protein-coupled receptor â beta-arrestin interactions
Following activation by an agonist, G protein-coupled receptors (GPCRs) recruit b-arrestins, which mediate rapid signal desensitisation and receptor internalisation. b-arrestins can also activate non-classical signalling pathways, distinct from those triggered by G proteins. The mechanisms that govern receptorâb-arrestin interactions in the complexity of a living cell, however, remain poorly understood.
Classically, b-arrestins are thought to exist in the cytoplasm and form stable complexes with activated GPCRs at the plasma membrane, diffusing together with GPCRs to clathrin-coated pits (CCPs). Recent biophysical data has brought this model into disrepute, suggesting that transient interactions govern signalling, to meet the everchanging needs of the cell.
Here, a dynamic characterisation of GPCRâb-arrestin interactions is presented, at the plasma membrane of living cells with single-molecule resolution. b-adrenergic receptors were used as a prototypical GPCR family, given their crucial roles in cardiac physiology and pathologies dominated by aberrant b-arrestin signalling. bArr2 was shown to spontaneously bind to the plasma membrane, where it transiently interacts with b2ARs via lateral diffusion. Following receptor interaction, the plasma membrane stabilises bArr2 in a membrane-bound, active-like conformation, allowing it to reach clathrin-coated pits (CCPs) without the activating receptor.
A major lipid-anchoring site in the C-edge of bArr2 and a novel role of the finger loop region were identified â independent of a bound receptor. These results shed new light on the complex sequence of events involved in b-arrestin interaction with GPCRs and its activation, revealing a critical role for b-arrestin interactions with the lipid bilayer.
Given the importance of b-adrenergic receptors in the development and treatment of chronic heart failure, single particle tracking methodologies were then used to generate assays which would enable the imaging of endogenous receptors and their signalling proteins in physiologically relevant cells, where they exert their effects. Fluorescent ligands were employed to image adult mouse cardiac myocytes, so that the diffusion and spatial localisation of GPCRs could be observed in a complex cell model. This pushes the capabilities of single-molecule microscopy techniques to their current limits, providing insights into endogenous plasma membrane receptors in live, beating heart cells
How Carvedilol activates ÎČ<sub>2</sub>-adrenoceptors
Carvedilol is among the most effective ÎČ-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of ÎČ(1)-adrenoceptors, arrestin-biased signalling via ÎČ(2)-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilolâs 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 ÎČ(2)ARs. 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 ÎČ-adrenoceptor system
Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells
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
Agonist-induced membrane nanodomain clustering drives GLP-1 receptor responses in pancreatic beta cells
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
Multiple-bias analysis as a technique to address systematic error in measures of abortion-related mortality
Detecting transient trapping from a single trajectory:a structural approach
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 particleâs 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 ÎČ2-adrenergic receptors, revealing that receptor stimulation results in increased trapping of receptors in defined domains, without changing the diffusion of free receptors
Plasma membrane preassociation drives ÎČ-arrestin coupling to receptors and activation
beta-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-b-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 b-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that b 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 b-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 b-arrestin function at the plasma membrane, revealing a critical role for b-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation