G Protein–Coupled Receptor Endocytosis Generates Spatiotemporal Bias in β-Arrestin Signaling

Medicine Basic Medical Sciences Cell Biology Biochemistry Life Sciences
G protein–coupled receptor β-arrestin endocytosis signal bias functional selectivity agonist efficacy receptor signaling

Academic News Report: The Temporal and Spatial Bias in β-arrestin Signal Transduction Mediated by G Protein-Coupled Receptor Endocytosis

Research Background

Among cell surface receptor families, G protein-coupled receptors (GPCRs) constitute the largest family. Upon activation by ligands, they interact with various signaling proteins to trigger intracellular signal transduction. This activation can be both balanced and selective, termed as biased signaling or functional selectivity. The significance of this phenomenon lies in the potential to develop biased drugs that achieve therapeutic effects while avoiding adverse side effects. For instance, Angiotensin II type 1 receptor (AT1R) is one of the most studied GPCRs. Its endogenous ligand Angiotensin II (AngII) acts as a full agonist for AT1R, but AngII derivatives lacking certain aromatic amino acids preferentially activate the β-arrestin pathway rather than G protein signaling. Additionally, AT1R agonists show multifunctional effects across different G protein and GPCR kinase (GRK) subtypes, and specific ligand biases correlate with particular in vivo effects, such as the β-arrestin biased agonist TRV120027 which has entered clinical trials.

Although there are theories suggesting that the cellular effects of biased ligands are achieved through stabilizing different receptor conformations, supported to some extent by crystal structure studies, the clinical application of these theories in relation to the temporal and spatial aspects of receptor signaling remains an unresolved issue. Recent advancements in live-cell sensors and gene-edited cell lines have significantly enhanced our understanding of signaling time courses, emphasizing the importance of “temporal bias” and “spatial bias.” Building on this research background, Tóth and colleagues conducted a systematic study of signal transduction bias in AT1R agonists, revealing the crucial regulatory role of receptor endocytosis.

Research Origin

This paper, authored by András D. Tóth, Bence Szalai, Orsolya T. Kovács, Dániel Garger, Susanne Prokop, Eszter Soltész-Katona, András Balla, Asuka Inoue, Péter Várnai, Gábor Turu, and László Hunyady, comes from the Institute of Molecular Life Sciences at the Hungarian Academy of Sciences, the Department of Physiology at Semmelweis University, Helmholtz Munich Center for Computational Health, the Molecular Physiology Laboratory of the Hungarian Research Network, and Tohoku University in Japan. The paper was published in the June 25, 2024 issue of “Scientific Signaling.”

Research Procedure

Subjects and Methods

The research employed a series of dynamic kinetic experiments and mathematical modeling to explore the temporal and spatial biases in the β-arrestin signaling pathway of AT1R agonists. The specific methods were as follows: 1. Dynamic Kinetic Experiments: - Used nine AT1R peptide ligands, each exhibiting significantly different affinity and signaling bias characteristics. - Applied bioluminescence resonance energy transfer (BRET) technology to measure the interaction between AT1R and β-arrestin. - Measured G protein activation by monitoring the dissociation of labeled G protein subunits.

  1. Inhibition of Endocytosis:

    • Used a dominant-negative form of dynamin, Dyn-K44A, to block receptor endocytosis.
    • Inhibited PtdIns(4,5)P2 degradation and used high-sucrose solution to inhibit receptor endocytosis.

  2. Recruitment of β-arrestin in Endosomes:

    • Designed biosensors localized to cell regions to monitor the transfer of β-arrestin between the plasma membrane and early endosomes.
    • Used confocal fluorescence microscopy and machine learning algorithms to quantify β-arrestin vesicle formation within cells.

  3. Mathematical Modeling:

    • Constructed a kinetic model simulating Gq protein activity and β-arrestin binding.
    • Conducted numerical simulations to analyze the impact of different ligand dissociation rates on β-arrestin recruitment.

Experimental Design and Innovations

The study introduced several innovative methods: 1. Designed BRET sensor systems targeted to various cell compartments to precisely monitor the intracellular dynamics of β-arrestin. 2. Utilized mathematical modeling to deeply analyze receptor-β-arrestin interaction kinetics under multi-factor regulation. 3. Developed a new machine learning algorithm for automated analysis of fluorescence microscopy images, improving the efficiency and accuracy of experimental data processing.

Major Results

Impact of Endocytosis on β-arrestin Interaction

The study found that AT1R endocytosis significantly influences β-arrestin recruitment: - Inhibiting receptor endocytosis led to consistent ligand-specific β-arrestin binding curves, indicating endocytosis’s crucial role in β-arrestin interaction. - Different ligands exhibited significant differences in their ability to recruit β-arrestin in endosomes, with stronger agonists forming more stable receptor-β-arrestin complexes in endosomes. - Observations with confocal microscopy confirmed the BRET measurement results: β-arrestin recruitment in endosomes is a uniquely regulated factor.

Impact of Ligand Dissociation Rate on β-arrestin Interaction

Further research revealed: - The ligand dissociation rate (koff_lr) directly affected the rate of β-arrestin2 dissociation from the receptor, subsequently impacting the total amount of β-arrestin signaling. - Potency analysis showed an inverse correlation between higher ligand dissociation rates and lower β-arrestin recruitment efficacy, though G protein activity in comparable periods mainly promoted the formation of receptor-β-arrestin complexes.

Broad Applicability of Endocytosis

The study also indicated: - Another class B receptor, V2 receptor (V2R), displayed endocytosis-dependent β-arrestin recruitment similar to AT1R. - The β2AR did not recruit β-arrestin in endosomes, but through genetic engineering to convert it into a class B receptor, it showed marked ligand dependence and enhanced endocytosis effects.

Mathematical Modeling Supports Experimental Results

Model simulation results were consistent with experimental findings: - After incorporating the endocytosis process, the β-arrestin recruitment effect of ligands with different koff_lr values in endosomes was significant. - Intracellular signal transduction could coordinate ligand temporal and spatial bias effects through endocytosis, achieving differential functional selectivity.

Conclusion and Significance

Research Conclusions and Value

The study demonstrated: - Endocytosis plays a key regulatory role in the temporal and spatial bias of GPCR signaling. - The multifactorial regulatory mechanisms during the endocytosis process are essential for the functional selectivity of biased ligands, especially the profound impacts of ligand dissociation rates and G protein activity on receptor-β-arrestin interactions. - These findings advance the understanding of GPCR signaling bias mechanisms and provide new directions for drug design, particularly in developing selective drugs that may avoid side effects.

Research Highlights

  • The study comprehensively revealed the crucial regulatory role of endocytosis in GPCR biased signaling for the first time.
  • By delving into the mechanisms, it uncovered the specific influences of ligand dissociation rates and G protein activity.
  • The consistency between mathematical modeling and experimental results enhanced the credibility and broad applicability of the research.

This study underscores the importance of combining empirical and intelligent data processing methods, opening new avenues and perspectives for future drug development and signal transduction research.