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The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of β-arrestins (βarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on βarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated βarrs in complex with distinct phosphorylation patterns derived from the carboxyl terminus of different GPCRs. These structures help identify a P-X-P-P type phosphorylation motif in GPCRs that interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of βarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in βarr activation is demonstrated by targeted mutagenesis experiments combined with an intrabody-based conformational sensor. Taken together, our findings provide important structural insights into the ability of distinct GPCRs to activate βarrs through a significantly conserved mechanism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

β-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-β-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 β-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that β-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 β-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 β-arrestin function at the plasma membrane, revealing a critical role for β-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to β-arrestins (βarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Kleist et al. combine NMR spectroscopy and residue contact network analysis to identify a potential allosteric network in CXCR7, a β-arrestin-biased chemokine receptor, which links the extracellular ligand-binding pocket and the intracellular transducer-coupling region through the receptor transmembrane core., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V 2 R), agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V 2 R (i.e., V 2 R T360A ) results in near-complete loss of βarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated βarr1, efficiently rescues the endosomal trafficking of βarr1 and ERK1/2 activation for V 2 R T360A . Molecular dynamics simulations reveal that Ib30 enriches active-like βarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances βarr1-β 2 -adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery., (© 2022. The Author(s).)

Agonist-induced recruitment of β-arrestins (βarrs) to G protein-coupled receptors (GPCRs) plays a central role in regulating the spatio-temporal aspects of GPCR signaling. Several recent studies have provided novel structural and functional insights into our understanding of GPCR-βarr interaction, subsequent βarr activation and resulting functional outcomes. In this review, we discuss these recent advances with a particular emphasis on recognition of receptor-bound phosphates by βarrs, the emerging concept of spatial positioning of key phosphorylation sites, the conformational transition in βarrs during partial to full-engagement, and structural differences driving functional outcomes of βarr isoforms. We also highlight the key directions that require further investigation going forward to fully understand the structural mechanisms driving βarr activation and functional responses., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

GPCRs modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This 'biased signalling' paradigm requires that we now characterize physiological signalling not just by receptors but by ligand-receptor pairs. Ligands eliciting biased signalling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease-relevant in vivo models., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Selective engagement of signal transducers such as G proteins and β-arrestins with GPCRs upon stimulation with biased agonists is thought to be due to distinct receptor conformations. Kawakami et al. propose an additional mechanism whereby activation of Gq determines GPCR kinase subtype selectivity to the activated angiotensin receptor, leading to distinct binding modalities of β-arrestins and functional outcomes.

Reversible protein-protein interaction in cells is an integral and central aspect of intracellular signaling mechanisms. This allows distinct signaling cascades to become active upon stimulation with external signal resulting in cellular and physiological responses. Several distinct methods are currently available and utilized routinely to monitor protein-protein interactions including co-immunoprecipitation (co-IP). An inherent limitation associated with co-IP assay however is the inability to efficiently capture transient and short-lived interactions in cells. Chemical cross-linking of such transient interactions in cellular context using cell permeable reagents followed by co-IP overcomes this limitation, and allows a simplified approach without requiring any sophisticated instrumentation. In this chapter, we present a step-by-step protocol for monitoring protein-protein interaction by combining chemical cross-linking and co-immunoprecipitation using GPCR-β-arrestin complex as a case example. This protocol is based on previously validated method that can potentially be adapted to capture and visualize transient protein-protein interactions in general., (Copyright © 2022 Elsevier Inc. All rights reserved.)

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and β-arrestins (βarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with βarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to βarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that βarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as βarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism., Competing Interests: Declaration of interests The authors have no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

In this issue of Molecular Cell, Su et al. (2020) report a cryo-EM structure of the β1-adrenergic receptor (β1AR) in complex with a heterotrimeric Gs protein, which offers novel insights into receptor activation and provides a structural framework to better understand the transducer-coupling mechanism for adrenergic receptors., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The wave of resolution revolution in cryo-EM has touched, and made a significant impact on, the structural biology of GPCRs. High-resolution structures of several GPCR-G-protein complexes are now determined by cryo-EM and they illuminate fine structural details of this central macromolecular complex involved in cellular signaling., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biased agonism at G-protein-coupled receptors is generally conceptualized as the ability of certain stimuli to trigger downstream signaling exclusively through one of two effectors. Recent studies reveal that signaling downstream of the β1 adrenergic receptor and the angiotensin II type 1 receptor induced by biased stimuli actually involves both effectors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

G protein-coupled receptors (GPCRs) are major signal recognition and transmission units in the plasma membrane. The interaction of activated and phosphorylated GPCRs with the multifunctional adaptor proteins β-arrestins (βarrs) is crucial for regulation of their signaling and functional outcomes. Over the past few years, a range of structural, biochemical, and cellular studies have revealed novel insights into GPCR-βarr interaction and signaling. Some of these findings have come as a surprise and therefore have the potential to significantly refine the conceptual framework of the GPCR-βarr system. Here we discuss these recent advances with particular emphasis on biphasic GPCR-βarr interaction, the formation of GPCR-G-protein-βarr supercomplexes, and receptor-specific conformational signatures in βarrs. We also underline the emerging research areas that are likely to be at the center stage of investigations in the coming years., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

G Protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors and drug targets. GPCR signalling and desensitization is critically regulated by β-arrestins (βarr). GPCR-βarr interaction is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N-domain of βarr first and then seven transmembrane core of the receptor engages with βarr. It is currently unknown whether fully engaged GPCR-βarr complex is essential for functional outcomes or partially engaged complex can also be functionally competent. Here we assemble partially and fully engaged complexes of a chimeric β 2 V 2 R with βarr1, and discover that the core interaction is dispensable for receptor endocytosis, ERK MAP kinase binding and activation. Furthermore, we observe that carvedilol, a βarr biased ligand, does not promote detectable engagement between βarr1 and the receptor core. These findings uncover a previously unknown aspect of GPCR-βarr interaction and provide novel insights into GPCR signalling and regulatory paradigms.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

β-arrestin-dependent activation of the ERK MAP kinase downstream of GPCRs typically originates from internalized receptor-β-arrestin-ERK complexes. A new study now reports that β-arrestin 2 is sufficiently 'primed' after 'kissing' the β1-adrenergic receptor to initiate ERK activation even in the absence of formation of the receptor-β-arrestin-ERK complex and receptor internalization., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The discovery of biased signaling at G protein-coupled receptors (GPCRs), the largest class of cell surface receptors and primary drug targets for numerous human diseases, has redefined the classical concepts of receptor pharmacology. It not only highlights the depth of signaling diversity within the GPCR system, but also offers possibilities for novel and more-effective therapeutics. Here, we highlight the recent biophysical and structural advances in our understanding of ligand-receptor interactions and conformational changes in the receptors, which provide novel mechanistic insights into biased GPCR signaling. We also underline key aspects of GPCR-biased signaling that remain to be investigated in greater detail to develop a complete molecular understanding of this process and overall GPCR signaling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The discovery of "functional selectivity" or "biased signaling" through G protein-coupled receptors (GPCRs) has redefined the classical GPCR signaling paradigm. Moreover, the therapeutic potential of biased signaling by and biased ligands for GPCRs is changing the landscape of GPCR drug discovery. The concept of biased signaling has primarily been developed and discussed in the context of ligands that bind to the extracellular regions of GPCRs. However, two recent reports demonstrate that it is also possible to bias GPCR signaling from inside the cell by targeting intracellular regions of these receptors. These findings present a novel handle for delineating the functional outcomes of biased signaling by GPCRs. Moreover, these approaches also uncover a previously unexplored framework for biasing GPCR signaling for drug discovery.

The concept of biased agonism has recently come to the fore with the realization that seven-transmembrane receptors (7TMRs, also known as G protein-coupled receptors, or GPCRs) activate complex signaling networks and can adopt multiple active conformations upon agonist binding. As a consequence, the "efficacy" of receptors, which was classically considered linear, is now recognized as pluridimensional. Biased agonists selectively stabilize only a subset of receptor conformations induced by the natural "unbiased" ligand, thus preferentially activating certain signaling mechanisms. Such agonists thus reveal the intriguing possibility that one can direct cellular signaling with unprecedented precision and specificity and support the notion that biased agonists may identify new classes of therapeutic agents that have fewer side effects. This review focuses on one particular class of biased ligands that has the ability to alter the balance between G protein-dependent and β-arrestin-dependent signal transduction.

β-Arrestins, originally discovered to desensitize activated seven transmembrane receptors (7TMRs; also known as G-protein-coupled receptors, GPCRs), are now well established mediators of receptor endocytosis, ubiquitylation and G protein-independent signaling. Recent global analyses of β-arrestin interactions and β-arrestin-dependent phosphorylation events have uncovered several previously unanticipated roles of β-arrestins in a range of cellular signaling events. These findings strongly suggest that the functional roles of β-arrestins are much broader than currently understood. Biophysical studies aimed at understanding multiple active conformations of the 7TMRs and the β-arrestins have begun to unravel the mechanistic basis for the diverse functional capabilities of β-arrestins in cellular signaling., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Phosphorylation of G protein-coupled receptors (GPCRs, which are also known as seven-transmembrane spanning receptors) by GPCR kinases (GRKs) plays essential roles in the regulation of receptor function by promoting interactions of the receptors with β-arrestins. These multifunctional adaptor proteins desensitize GPCRs, by reducing receptor coupling to G proteins and facilitating receptor internalization, and mediate GPCR signaling through β-arrestin-specific pathways. Detailed mapping of the phosphorylation sites on GPCRs targeted by individual GRKs and an understanding of how these sites regulate the specific functional consequences of β-arrestin engagement may aid in the discovery of therapeutic agents targeting individual β-arrestin functions. The β(2)-adrenergic receptor (β(2)AR) has many serine and threonine residues in the carboxyl-terminal tail and the intracellular loops, which are potential sites of phosphorylation. We monitored the phosphorylation of the β(2)AR at specific sites upon stimulation with an agonist that promotes signaling by both G protein-mediated and β-arrestin-mediated pathways or with a biased ligand that promotes signaling only through β-arrestin-mediated events in the presence of the full complement of GRKs or when either GRK2 or GRK6 was depleted. We correlated the specific and distinct patterns of receptor phosphorylation by individual GRKs with the functions of β-arrestins and propose that the distinct phosphorylation patterns established by different GRKs establish a "barcode" that imparts distinct conformations to the recruited β-arrestin, thus regulating its functional activities.

Beta-arrestins are multifunctional adaptors that mediate the desensitization, internalization, and some signaling functions of seven-transmembrane receptors (7TMRs). Agonist-stimulated ubiquitination of beta-arrestin2 mediated by the E3 ubiquitin ligase Mdm2 is critical for rapid beta(2)-adrenergic receptor (beta(2)AR) internalization. We now report the discovery that the deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) binds beta-arrestin2 and leads to the deubiquitination of beta-arrestins. USP33 and Mdm2 function reciprocally and favor respectively the stability or lability of the receptor beta-arrestin complex, thus regulating the longevity and subcellular localization of receptor signalosomes. Receptors such as the beta(2)AR, previously shown to form loose complexes with beta-arrestin ("class A") promote a beta-arrestin conformation conducive for binding to the deubiquitinase, whereas the vasopressin V2R, which forms tight beta-arrestin complexes ("class B"), promotes a distinct beta-arrestin conformation that favors dissociation of the enzyme. Thus, USP33-beta-arrestin interaction is a key regulatory step in 7TMR trafficking and signal transmission from the activated receptors to downstream effectors.

β-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of β-arrestins pertinent to these functions has been documented. The discovery of several novel functions of β-arrestins stimulated us to perform a global proteomics analysis of β-arrestin-interacting proteins (interactome) as modulated by a model seven- transmembrane receptor, the angiotensin II type la receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS. 71 proteins interacted with β-arrestin 1, 164 interacted with β-arrestin 2, and 102 interacted with both β-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the β-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with β-arrestins. The β-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified β-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind β-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology. [ABSTRACT FROM AUTHOR]