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Journal of Molecular Biology Apr 2022Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that...
Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. β-arrestin1 and β-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.
Topics: Mitogen-Activated Protein Kinase 1; Protein Binding; Scattering, Small Angle; X-Ray Diffraction; beta-Arrestin 1; beta-Arrestin 2
PubMed: 35077767
DOI: 10.1016/j.jmb.2022.167465 -
Progress in Molecular Biology and... 2018Arrestins were discovered as proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) and block their interactions with G proteins, i.e., for their... (Review)
Review
Arrestins were discovered as proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) and block their interactions with G proteins, i.e., for their role in homologous desensitization of GPCRs. Mammals express only four arrestin subtypes, two of which are largely restricted to the retina. Two nonvisual arrestins are ubiquitous and interact with hundreds of different GPCRs and dozens of other binding partners. Changes of just a few residues on the receptor-binding surface were shown to dramatically affect GPCR preference of inherently promiscuous nonvisual arrestins. Mutations on the cytosol-facing side of arrestins modulate their interactions with individual downstream signaling molecules. Thus, it appears feasible to construct arrestin mutants specifically linking particular GPCRs with signaling pathways of choice or mutants that sever the links between selected GPCRs and unwanted pathways. Signaling-biased "designer arrestins" have the potential to become valuable molecular tools for research and therapy.
Topics: Animals; Arrestins; Humans; Models, Molecular; Phosphorylation; Signal Transduction
PubMed: 30470292
DOI: 10.1016/bs.pmbts.2018.07.007 -
Proceedings of the National Academy of... Oct 2023β-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor...
β-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. β-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. β-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, β-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by β-arrestins. Here, we demonstrate that β-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that β-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which β-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state β-arrestin 2 is more robust than by active-state β-arrestin 1, highlighting differential capacities of β-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which β-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.
Topics: beta-Arrestins; beta-Arrestin 1; Arrestins; Allosteric Regulation; Signal Transduction; Receptors, G-Protein-Coupled; Phosphorylation; beta-Arrestin 2
PubMed: 37844230
DOI: 10.1073/pnas.2303794120 -
PloS One 2023G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins....
G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins. [Sarcosine1Ile4Ile8]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in physiologically relevant models. Despite this, SII and several derivatives induce cellular signaling outcomes through β-arrestin-2-dependent mechanisms. However, studies reliant on exogenous AT1R overexpression indicate that SII is a partial agonist for G protein signaling and lacks β-arrestin-exclusive functional specificity. We investigated this apparent discrepancy by profiling changes in functional specificity at increasing expression levels of AT1R using a stably integrated tetracycline-titratable expression system stimulated with AngII, SII, and four other AngII analogs displaying different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all tested receptor concentrations. In contrast, β-arrestin-biased ligands induced dose-dependent calcium signaling only at higher AT1R overexpression levels. Using inhibitors of G proteins, we demonstrated that both Gi and Gq/11 mediated overexpression-dependent calcium signaling by β-arrestin-biased ligands. Regarding β-arrestin-mediated cellular events, the β-arrestin-biased ligand TRV026 induced receptor internalization at low physiological receptor levels insufficient for it to initiate calcium signaling. In contrast, unbiased AngII exhibited no relative preference between these outcomes under such low receptor conditions. However, with high receptor overexpression, TRV026 lost its functional selectivity. These results suggest receptor overexpression misleadingly distorts the bias of AT1R ligands and highlight the risks of using overexpressed systems to infer the signaling bias of GPCR ligands in physiologically relevant contexts.
Topics: Humans; Signal Transduction; Receptors, G-Protein-Coupled; Angiotensin II; Ligands; beta-Arrestins; GTP-Binding Proteins; beta-Arrestin 1; Receptor, Angiotensin, Type 1; HEK293 Cells
PubMed: 36961836
DOI: 10.1371/journal.pone.0283477 -
International Journal of Molecular... Jan 2022β-arrestins were initially identified to desensitize and internalize G-protein-coupled receptors (GPCRs). Receptor-bound β-arrestins also initiate a second wave of... (Review)
Review
β-arrestins were initially identified to desensitize and internalize G-protein-coupled receptors (GPCRs). Receptor-bound β-arrestins also initiate a second wave of signaling by scaffolding mitogen-activated protein kinase (MAPK) signaling components, MAPK kinase kinase, MAPK kinase, and MAPK. In particular, β-arrestins facilitate ERK1/2 or JNK3 activation by scaffolding signal cascade components such as ERK1/2-MEK1-cRaf or JNK3-MKK4/7-ASK1. Understanding the precise molecular and structural mechanisms of β-arrestin-mediated MAPK scaffolding assembly would deepen our understanding of GPCR-mediated MAPK activation and provide clues for the selective regulation of the MAPK signaling cascade for therapeutic purposes. Over the last decade, numerous research groups have attempted to understand the molecular and structural mechanisms of β-arrestin-mediated MAPK scaffolding assembly. Although not providing the complete mechanism, these efforts suggest potential binding interfaces between β-arrestins and MAPK signaling components and the mechanism for MAPK signal amplification by β-arrestin-mediated scaffolding. This review summarizes recent developments of cellular and molecular works on the scaffolding mechanism of β-arrestin for MAPK signaling cascade.
Topics: Animals; Humans; MAP Kinase Signaling System; Models, Molecular; Protein Binding; Protein Conformation; Receptors, G-Protein-Coupled; beta-Arrestins
PubMed: 35055186
DOI: 10.3390/ijms23021000 -
Current Protocols in Pharmacology Dec 2014Arrestins are characterized by their ability to selectively bind active, phosphorylated GPCRs and suppress (arrest) receptor coupling to G proteins. Nonvisual arrestins... (Review)
Review
Arrestins are characterized by their ability to selectively bind active, phosphorylated GPCRs and suppress (arrest) receptor coupling to G proteins. Nonvisual arrestins are also signaling proteins in their own right, activating a variety of cellular pathways. Arrestins are highly flexible proteins that can assume many distinct conformations. In their receptor-bound conformation, arrestins have higher affinity for a subset of partners. This explains how receptor activation regulates certain branches of arrestin-dependent signaling via arrestin recruitment to GPCRs. However, free arrestins are also active molecular entities that act in other pathways and localize signaling proteins to particular subcellular compartments, such as cytoskeleton. These functions are regulated by the enhancement or reduction of arrestin affinity for target proteins by other binding partners and by proteolytic cleavage. Recent findings suggest that the two visual arrestins, arrestin-1 and arrestin-4, which are expressed in photoreceptor cells, do not regulate signaling solely via binding to photopigments but also interact with a variety of nonreceptor partners, critically affecting the health and survival of photoreceptor cells. Detailed in this overview are GPCR-dependent and independent modes of arrestin-mediated regulation of cellular signaling pathways.
Topics: Animals; Arrestins; Humans; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 25446289
DOI: 10.1002/0471141755.ph0210s67 -
International Journal of Molecular... Jan 2022The two β-arrestins (β-arrestin-1 and -2; alternative names: arrestin-2 and -3, respectively) are well known for their ability to inhibit signaling via G... (Review)
Review
The two β-arrestins (β-arrestin-1 and -2; alternative names: arrestin-2 and -3, respectively) are well known for their ability to inhibit signaling via G protein-coupled receptors. However, β-arrestins can also act as signaling molecules in their own right. Although the two proteins share a high degree of sequence and structural homology, early studies with cultured cells indicated that β-arrestin-1 and -2 are not functionally redundant. Recently, the in vivo metabolic roles of the two β-arrestins have been studied using mutant mice selectively lacking either β-arrestin-1 or -2 in cell types that are of particular relevance for regulating glucose and energy homeostasis. These studies demonstrated that the β-arrestin-1 and -2 mutant mice displayed distinct metabolic phenotypes in vivo, providing further evidence for the functional heterogeneity of these two highly versatile signaling proteins.
Topics: Animals; Diabetes Mellitus; Disease Models, Animal; Energy Metabolism; Glucose; Mice; Obesity; Receptors, G-Protein-Coupled; Signal Transduction; beta-Arrestin 1; beta-Arrestin 2
PubMed: 35008921
DOI: 10.3390/ijms23010495 -
Nature Mar 2020Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and...
Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human β-arrestin 1 (βarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with βarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.
Topics: Cryoelectron Microscopy; Humans; Models, Molecular; Phosphorylation; Protein Stability; Protein Structure, Quaternary; Receptors, Neurotensin; beta-Arrestin 1
PubMed: 31945771
DOI: 10.1038/s41586-020-1953-1 -
Nature Communications Jun 2023The neurotensin receptor 1 (NTS) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and...
The neurotensin receptor 1 (NTS) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used CH-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor's intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs - without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.
Topics: Receptors, Neurotensin; beta-Arrestin 1; Receptors, G-Protein-Coupled; beta-Arrestins; GTP-Binding Proteins; Arrestin
PubMed: 37286565
DOI: 10.1038/s41467-023-38894-8 -
Neuropharmacology Jul 2017Arrestins play a prominent role in shutting down signaling via G protein-coupled receptors. In recent years, a signaling role for arrestins independent of their function...
Arrestins play a prominent role in shutting down signaling via G protein-coupled receptors. In recent years, a signaling role for arrestins independent of their function in receptor desensitization has been discovered. Two ubiquitously expressed arrestin isoforms, arrestin-2 and arrestin-3, perform similarly in the desensitization process and share many signaling functions, enabling them to substitute for one another. However, signaling roles specific to each isoform have also been described. Mice lacking arrestin-3 (ARR3KO) were reported to show blunted acute responsiveness to the locomotor stimulatory effect of amphetamine (AMPH). It has been suggested that mice with deletion of arrestin-2 display a similar phenotype. Here we demonstrate that the AMPH-induced locomotion of male ARR3KO mice is reduced over the 7-day treatment period and during AMPH challenge after a 7-day withdrawal. The data are consistent with impaired locomotor sensitization to AMPH and suggest a role for arrestin-3-mediated signaling in the sensitization process. In contrast, male ARR2KO mice showed enhanced early responsiveness to AMPH and the lack of further sensitization, suggesting a role for impaired receptor desensitization. The comparison of mice possessing one allele of arrestin-3 and no arrestin-2 with ARR2KO littermates revealed reduced activity of the former line, consistent with a contribution of arrestin-3-mediated signaling to AMPH responses. Surprisingly, ARR3KO mice with one arrestin-2 allele showed significantly reduced locomotor responses to AMPH combined with lower novelty-induced locomotion, as compared to the ARR3KO line. These data suggest that one allele of arrestin-2 is unable to support normal locomotor behavior due to signaling and/or developmental defects.
Topics: Amphetamine; Analysis of Variance; Animals; Arrestins; Central Nervous System Stimulants; Locomotion; Mice; Mice, Inbred C57BL; Mice, Knockout; Time Factors; beta-Arrestin 1
PubMed: 28419873
DOI: 10.1016/j.neuropharm.2017.04.021