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Decoding β-Arrestins: from Structure to function

Fine-tuning GPCR signaling: conformational dynamics and intracellular responses

GPCR signaling is a complex process modulated by protein conformational states. Following extracellular stimulus detection, receptor activation initiates conformational changes, exposing an intracellular cavity (Kang, Y. et al. 2015, Chen, Q. et al. 2021), that allow interaction with trimeric G proteins, which regulate second messengers like cAMP or Ca2+. While some receptors selectively activate specific G protein families, others are more versatile, yielding diverse responses based on cell-specific G protein expression. Apart from G proteins, GPCRs engage other effectors for signaling modulation. GPCR kinases (GRKs) and β-arrestins are activated by agonist-bound GPCRs and interact with the receptor cavity. Originally recognized for inhibiting G protein signaling, they also influence specific pathways such as MAPK signaling (Song, X. et al. 2009, Coffa, S. et al. 2011). Notably, GRKs phosphorylate active GPCRs, enabling high-affinity arrestin binding, which is crucial for receptor internalization. β-Arrestins facilitate this process by interacting with adapter protein 2 (AP-2) and clathrin. The diversity in GPCR signaling regulation suggests an individualized control mechanism. The "barcode hypothesis" proposes that β-arrestins decode distinct GPCR phosphorylation patterns, which influence their conformational states and functions (Matthees, E. S. F et al. 2021, Chen, H. et al. 2022). Recently, Maharana et al. determined multiple structures of activated b-arrestins in complex with the carboxyl terminus phosphopeptides of different GPCRs using cryo-EM, and discovered a significantly conserved phosphorylation motif in GPCRs that drives b-arrestin interaction and activation (Maharana et al. 2023).

While GPCR signaling predominantly occurs at the plasma membrane, certain receptors retain their active conformation during internalization and intracellular trafficking, enabling endocytic signaling. The hypothesis arises that GPCR and β-arrestin-centered effector complexes vary based on subcellular localization, potentially scaffolding distinct signaling platforms. Consequently, understanding dynamic interactions between effectors during trafficking becomes crucial. Despite multifocal signaling, recent studies indicate that signaling occurs within a 100 nm range from the point of origin, suggesting the formation of active "nano domains" in specific membrane niches (Anton, S. E. et al. 2022).

Probing GPCR and β-Arrestin conformational states: methods and implications

The formation of functional complexes involving GPCRs and β-arrestins hinges on their specific conformational states, influenced by their intricate three-dimensional structures. X-ray protein crystallography yields high-resolution protein structures, illuminating side chain orientations and overall conformational states (Kang, Y. et al. 2015). While it provides precise information, it requires significant protein amounts and suitable crystallization conditions. Cryo-EM, on the other hand, requires less protein and has evolved to achieve resolutions comparable to X-ray crystallography (García-Nafría, J., & Tate, C. G. 2021). Recent years have seen cryo-EM dominate new GPCR structure determinations, offering insight into GPCR-effector complexes. However, both methods struggle with flexible or dynamic regions. Other techniques include nuclear magnetic resonance (NMR) spectroscopy for conformational dynamics analysis context (Park, S. H., & Lee, J. H. 2020, Casiraghi, M. et al. 2019), double electron-electron resonance (DEER) spectroscopy for high-resolution conformational state determination (Wingler, L. M. et al. 2019), and hydrogen-deuterium exchange (HDX) mass spectrometry for time-dependent conformational insights (Komolov, K. E. et al. 2017). Despite their contributions, these methods often lack cellular auxiliary structures and proteins. Emerging strategies incorporate unnatural amino acids and crosslinking for structural data inference in cellular environments. This approach reveals binding interfaces and interactions between GPCRs and β-arrestins, paving the way for capturing challenging protein complexes (Böttke, T., et al. 2020, Aydin, Y. et al. 2023). Moreover, Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), and multi-color fluorescence microscopy assays employ conformation-selective probes for monitoring activation-induced changes in cellular environments (Irannejad, R., et al. 2013). The combination of these diverse methods contributes to a holistic understanding of the intricate dynamics and functions of these signaling proteins.

GPCR–β-Arrestin complexes: versatile scaffolding platforms

β-arrestins exhibit interactions with over 100 diverse proteins, presenting an array of effectors that could be recruited to GPCR–β-arrestin complexes (Xiao, K et al. 2017). This includes proteins like AP-2 and clathrin, vital for internalization, as well as MAPK cascade kinases which can be activated through specific β-arrestin conformations (Song, X. et al. 2008, Coffa, S. et al. 2011). Notably, β-arrestin isoforms interact distinctively with certain signaling kinases, emphasizing their role as hubs for increasing local effector concentrations near active GPCRs (Perry-Hauser, N. A. et al. 2022). This indicates that the scaffolding role of β-arrestins depends on their conformational state, rearranging to expose specific binding interfaces.

Uncovering these interacting effector proteins requires comprehensive and unbiased proteomic datasets. Nevertheless, the diverse functionalities of various GPCRs, coupled with the fluctuating expression of proteins in distinct tissues and cellular environments, introduce challenges to this endeavor. Furthermore, the temporal dynamics of β-arrestin subcellular localization, leading to variations in potential interaction partners, further complicates the comprehensive elucidation of the entire β-arrestin interactome. Recent advancements in experimental and bioinformatics tools offer the potential to explore larger portions of the β-arrestin interactome (Crépieux, P. et al. 2017) although there are several challenges to uncover β-arrestin interactome in all its dimensions. For example, the binding sites for many β-arrestin interaction partners remain elusive, and certain effector proteins with overlapping sites may experience mutually exclusive binding due to steric hindrance (Crépieux, P. et al. 2017). In addition, the question of whether β-arrestin1 and 2 serve overlapping or distinct functions remains, raising the need for more comprehensive protein-protein interaction analyses to uncover connections between the binding partners and diverse cellular processes.

Knockout studies in mice suggest that β-arrestin isoforms possess some level of functional redundancy, as depleting one isoform results leads to relatively mild effects (Conner, D. A., et al. 1997, Bohn, L. M. et al. 1999), while dual knockout is lethal (Schmid, C. L., & Bohn, L. M. 2009). However, the evolutionary conservation of both isoforms from fish to mammals indicates their non-redundant roles (Gurevich, E. V., & Gurevich, V. V. 2006). Additionally, not all receptors recruit both isoforms equally and arrestins can undergo different conformational changes for the binding to the same GPCR to mediate differential regulatory effects (Haider, R. S. et al. 2022). Thus, exploring large-scale protein-protein interaction datasets could shed light on connections between β-arrestin isoforms and various cellular processes.

Tissue expression levels of β-Arrestins and their implications in cancer

In addition to molecular interactions, protein expression levels play a crucial role in determining the formation of protein complexes and their functions within specific tissues (Matthees, E. S. F. et al. 2021). Fine-tuned systems of protein expression regulate various processes, and their imbalance is linked to pathological conditions, including cancer (Gros, R. et al. 2000, Sun, W.-Y. et al 2018). Changes in the availability of regulatory proteins like GRKs can impact GPCR phosphorylation and subsequent β-arrestin binding. Such alterations can also extend to β-arrestin expression levels, potentially affecting the composition and prevalence of GPCR–β-arrestin–effector complexes. By comparing β-arrestin expression data in healthy tissues from the Genotype-Tissue Expression (GTEx) database with data from cancer samples in The Cancer Genome Atlas (TCGA) through the Gene Expression Profiling Interactive Analysis (GEPIA) tool, it is evident that β-arrestin expression is dysregulated in many cancer types. However, β-arrestins do not consistently act as oncogenes or tumor suppressors. In certain cancer types, only one β-arrestin isoform's expression may be altered, while the other remains unchanged. The observed variations in β-arrestin expression likely impact the composition and function of GPCR-mediated downstream signaling complexes, contributing to the complex landscape of cancer-related signaling pathways.

Future perspectives

In recent years, remarkable strides have been taken in unraveling the intricacies of GPCR signaling and the formation of effector complexes. Innovative structural biology techniques, pharmacological methods, state-of-the-art biosensors, supplementary analyses, including interactome studies and evaluations of tissue-specific expression, have played a pivotal role in identifying essential effector proteins that modulate GPCR functions across both physiological and pathological states.

Nonetheless, there is an ongoing need to delve deeper into the intricate mechanisms governing GPCR activation and the assembly of effector complexes. Understanding how specific active conformational states influence the interaction or dissociation of crucial effector proteins within distinct subcellular locations remains a fundamental research question. Ultimately, these efforts hold the promise of unveiling novel insights into cellular signaling and its implications for health and disease. Pinpointing signaling pathways governed by β-arrestins, which exhibit increased or reduced activity within distinct cell types or tissues, will undoubtedly provide valuable guidance for advancing such therapeutic interventions.

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