RESUMEN
ß-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-ß-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 ß-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that ß-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 ß-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 ß-arrestin function at the plasma membrane, revealing a critical role for ß-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.
Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , beta-Arrestinas , beta-Arrestinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitosis , Membrana Dobles de Lípidos , Receptores Acoplados a Proteínas G/metabolismo , Simulación de Dinámica MolecularRESUMEN
Cannabis activates the cannabinoid receptor 1 (CB1), which elicits analgesic and emotion regulation benefits, along with adverse effects, via Gi and ß-arrestin signaling pathways. However, the lack of understanding of the mechanism of ß-arrestin-1 (ßarr1) coupling and signaling bias has hindered drug development targeting CB1. Here, we present the high-resolution cryo-electron microscopy structure of CB1-ßarr1 complex bound to the synthetic cannabinoid MDMB-Fubinaca (FUB), revealing notable differences in the transducer pocket and ligand-binding site compared with the Gi protein complex. ßarr1 occupies a wider transducer pocket promoting substantial outward movement of the TM6 and distinctive twin toggle switch rearrangements, whereas FUB adopts a different pose, inserting more deeply than the Gi-coupled state, suggesting the allosteric correlation between the orthosteric binding pocket and the partner protein site. Taken together, our findings unravel the molecular mechanism of signaling bias toward CB1, facilitating the development of CB1 agonists.
Asunto(s)
Arrestina , Receptor Cannabinoide CB1 , Transducción de Señal , Arrestina/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismo , Microscopía por Crioelectrón , Receptor Cannabinoide CB1/metabolismo , Humanos , Animales , Línea CelularRESUMEN
ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.
Asunto(s)
Fosfopéptidos , Receptores Acoplados a Proteínas G , Fosfopéptidos/metabolismo , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Binding of arrestin to phosphorylated G protein-coupled receptors (GPCRs) is crucial for modulating signaling. Once internalized, some GPCRs remain complexed with ß-arrestins, while others interact only transiently; this difference affects GPCR signaling and recycling. Cell-based and in vitro biophysical assays reveal the role of membrane phosphoinositides (PIPs) in ß-arrestin recruitment and GPCR-ß-arrestin complex dynamics. We find that GPCRs broadly stratify into two groups, one that requires PIP binding for ß-arrestin recruitment and one that does not. Plasma membrane PIPs potentiate an active conformation of ß-arrestin and stabilize GPCR-ß-arrestin complexes by promoting a fully engaged state of the complex. As allosteric modulators of GPCR-ß-arrestin complex dynamics, membrane PIPs allow for additional conformational diversity beyond that imposed by GPCR phosphorylation alone. For GPCRs that require membrane PIP binding for ß-arrestin recruitment, this provides a mechanism for ß-arrestin release upon translocation of the GPCR to endosomes, allowing for its rapid recycling.
Asunto(s)
Arrestinas , Fosfatidilinositoles , beta-Arrestinas/metabolismo , Fosfatidilinositoles/metabolismo , Arrestinas/metabolismo , beta-Arrestina 1/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Biasing G protein-coupled receptor signaling with ligands that promote select pathways is emerging as a powerful approach for therapeutic drug discovery. In this issue of Cell, Slosky et al. report a ß-arrestin-biased neurotensin receptor ligand that may curtail drug abuse without the side effects induced by G protein signaling.
Asunto(s)
Conducta Adictiva , Receptores de Neurotensina , Ligandos , Neurotensina , Receptores de Neurotensina/metabolismo , beta-Arrestina 1 , beta-ArrestinasRESUMEN
ß-arrestins (ßarrs) play multifaceted roles in the signaling and regulation of G-protein-coupled receptors (GPCRs) including their desensitization and endocytosis. Recently determined cryo-EM structures of two different GPCRs in complex with ßarr1 provide the first glimpse of GPCR-ßarr engagement and a structural framework to understand their interaction.
Asunto(s)
Receptores Acoplados a Proteínas G/ultraestructura , beta-Arrestinas/metabolismo , beta-Arrestinas/ultraestructura , Arrestinas/metabolismo , Endocitosis/fisiología , Proteínas de Unión al GTP/metabolismo , Humanos , Fosforilación , Unión Proteica , Isoformas de Proteínas/ultraestructura , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Relación Estructura-Actividad , beta-Arrestina 1/metabolismo , Arrestina beta 2/metabolismoRESUMEN
Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.
Asunto(s)
Conducta Adictiva/metabolismo , Receptores de Neurotensina/metabolismo , beta-Arrestinas/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Conducta Adictiva/tratamiento farmacológico , Línea Celular , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.
Asunto(s)
Angiotensinas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina/metabolismo , Arrestinas/metabolismo , Línea Celular , Humanos , Ligandos , Conformación Proteica , Receptores de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Espectroscopía de Pérdida de Energía de Electrones/métodos , beta-Arrestinas/metabolismoRESUMEN
The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs. The peptide engages the AT1R through an extensive interface spanning from the receptor core to its extracellular face and N terminus, remodeling the ligand-binding cavity. Remarkably, the mechanism used to propagate conformational changes through the receptor diverges from other GPCRs at several key sites, highlighting the diversity of allosteric mechanisms among GPCRs. Our structure provides insight into how AngII and its analogs stimulate full or biased signaling, respectively.
Asunto(s)
Receptor de Angiotensina Tipo 1/metabolismo , Anticuerpos de Dominio Único/farmacología , Angiotensina II , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Arrestinas/metabolismo , Células HEK293 , Humanos , Fragmentos de Inmunoglobulinas/farmacología , Conformación Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Anticuerpos de Dominio Único/metabolismo , beta-Arrestinas/metabolismoRESUMEN
The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor whose disruption causes obesity. We functionally characterized 61 MC4R variants identified in 0.5 million people from UK Biobank and examined their associations with body mass index (BMI) and obesity-related cardiometabolic diseases. We found that the maximal efficacy of ß-arrestin recruitment to MC4R, rather than canonical Gαs-mediated cyclic adenosine-monophosphate production, explained 88% of the variance in the association of MC4R variants with BMI. While most MC4R variants caused loss of function, a subset caused gain of function; these variants were associated with significantly lower BMI and lower odds of obesity, type 2 diabetes, and coronary artery disease. Protective associations were driven by MC4R variants exhibiting signaling bias toward ß-arrestin recruitment and increased mitogen-activated protein kinase pathway activation. Harnessing ß-arrestin-biased MC4R signaling may represent an effective strategy for weight loss and the treatment of obesity-related cardiometabolic diseases.
Asunto(s)
Mutación con Ganancia de Función/genética , Obesidad/patología , Receptor de Melanocortina Tipo 4/genética , Transducción de Señal , Adulto , Anciano , Índice de Masa Corporal , Enfermedad de la Arteria Coronaria/complicaciones , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , AMP Cíclico/metabolismo , Bases de Datos Factuales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Obesidad/metabolismo , Polimorfismo de Nucleótido Simple , Receptor de Melanocortina Tipo 4/química , Receptor de Melanocortina Tipo 4/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit ßarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that ßarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/efectos adversos , Receptores Opioides mu/agonistas , Animales , Fentanilo/administración & dosificación , Proteínas de Unión al GTP/metabolismo , Ratones , Morfina/administración & dosificación , Receptores Opioides mu/química , Sistema Respiratorio/efectos de los fármacos , Transducción de Señal , beta-Arrestinas/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.
Asunto(s)
Ascorbato Peroxidasas/química , Receptor de Angiotensina Tipo 1/análisis , Receptor de Angiotensina Tipo 1/metabolismo , Transducción de Señal , Coloración y Etiquetado/métodos , Ascorbato Peroxidasas/metabolismo , Biotina/química , Proteínas de Unión al GTP/análisis , Células HEK293 , Humanos , Oligopéptidos/farmacología , Ingeniería de Proteínas , Receptor de Angiotensina Tipo 1/agonistas , beta-Arrestinas/químicaRESUMEN
Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-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, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-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 ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , beta-Arrestinas/metabolismo , Transferencia de Energía por Resonancia de Bioluminiscencia , AMP Cíclico/metabolismo , Endosomas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Células HEK293 , Humanos , Microscopía Confocal , Microscopía Electrónica , Complejos Multiproteicos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , beta-Arrestinas/químicaRESUMEN
The two non-visual arrestins, arrestin2 and arrestin3, bind hundreds of GPCRs with different phosphorylation patterns, leading to distinct functional outcomes. Structural information on these interactions is available only for very few GPCRs. Here, we have characterized the interactions between the phosphorylated human CC chemokine receptor 5 (CCR5) and arrestin2. We identified several new CCR5 phosphorylation sites necessary for stable arrestin2 complex formation. Structures of arrestin2 in the apo form and complexes with CCR5 C-terminal phosphopeptides, together with NMR, biochemical, and functional assays, revealed three phosphoresidues in a pXpp motif that are essential for arrestin2 binding and activation. The identified motif appears responsible for robust arrestin2 recruitment in many other GPCRs. An analysis of receptor sequences and available structural and functional information provides hints on the molecular basis of arrestin2/arrestin3 isoform specificity. Our findings demonstrate how multi-site phosphorylation controls GPCRâ arrestin interactions and provide a framework to probe the intricate details of arrestin signaling.
Asunto(s)
Fosfopéptidos , Receptores CCR5 , Humanos , Fosforilación , beta-Arrestinas/metabolismo , Fosfopéptidos/metabolismo , Receptores CCR5/metabolismo , Línea CelularRESUMEN
Agonist-induced GPCR phosphorylation is a key determinant for the binding and activation of ß-arrestins (ßarrs). However, it is not entirely clear how different GPCRs harboring divergent phosphorylation patterns impart converging active conformation on ßarrs leading to broadly conserved functional responses such as desensitization, endocytosis, and signaling. Here, we present multiple cryo-EM structures of activated ßarrs 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 ßarrs. Sequence analysis of the human GPCRome reveals the presence of this phosphorylation pattern in a large number of receptors, and its contribution in ßarr 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 ßarrs through a significantly conserved mechanism.
Asunto(s)
Endocitosis , Transducción de Señal , Humanos , beta-Arrestinas/metabolismo , Fosforilación , Transducción de Señal/fisiología , Dominios Proteicos , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Kleist et al. combine NMR spectroscopy and residue contact network analysis to identify a potential allosteric network in CXCR7, a ß-arrestin-biased chemokine receptor, which links the extracellular ligand-binding pocket and the intracellular transducer-coupling region through the receptor transmembrane core.
Asunto(s)
Transducción de Señal , Ligandos , beta-ArrestinasRESUMEN
G protein-coupled receptors (GPCRs) generally accommodate specific ligands in the orthosteric-binding pockets. Ligand binding triggers a receptor allosteric conformational change that leads to the activation of intracellular transducers, G proteins and ß-arrestins. Because these signals often induce adverse effects, the selective activation mechanism for each transducer must be elucidated. Thus, many orthosteric-biased agonists have been developed, and intracellular-biased agonists have recently attracted broad interest. These agonists bind within the receptor intracellular cavity and preferentially tune the specific signalling pathway over other signalling pathways, without allosteric rearrangement of the receptor from the extracellular side1-3. However, only antagonist-bound structures are currently available1,4-6, and there is no evidence to support that biased agonist binding occurs within the intracellular cavity. This limits the comprehension of intracellular-biased agonism and potential drug development. Here we report the cryogenic electron microscopy structure of a complex of Gs and the human parathyroid hormone type 1 receptor (PTH1R) bound to a PTH1R agonist, PCO371. PCO371 binds within an intracellular pocket of PTH1R and directly interacts with Gs. The PCO371-binding mode rearranges the intracellular region towards the active conformation without extracellularly induced allosteric signal propagation. PCO371 stabilizes the significantly outward-bent conformation of transmembrane helix 6, which facilitates binding to G proteins rather than ß-arrestins. Furthermore, PCO371 binds within the highly conserved intracellular pocket, activating 7 out of the 15 class B1 GPCRs. Our study identifies a new and conserved intracellular agonist-binding pocket and provides evidence of a biased signalling mechanism that targets the receptor-transducer interface.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs , Imidazolidinas , Receptores Acoplados a Proteínas G , Humanos , Regulación Alostérica , beta-Arrestinas/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Desarrollo de Medicamentos , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/ultraestructura , Imidazolidinas/química , Imidazolidinas/farmacología , Ligandos , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/clasificación , Receptores Acoplados a Proteínas G/ultraestructura , Transducción de SeñalRESUMEN
Here, we talk to first author Shubhi Pandey about her paper, "Intrinsic bias at non-canonical, ß-arrestin-coupled seven transmembrane receptors," and group leader Arun K. Shukla about the research in his lab, the evolving research landscape in India, and his wish to motivate young Indian researchers abroad to return to India.
Asunto(s)
beta-Arrestinas , Femenino , HumanosRESUMEN
G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, 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 ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs 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 ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.
Asunto(s)
Membrana Celular/metabolismo , Conformación Proteica , Transducción de Señal , beta-Arrestinas/química , Animales , Proteínas de Unión al GTP/química , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Transporte de Proteínas , Receptor de Anafilatoxina C5a/metabolismoRESUMEN
GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the ß-arrestin pathways through the µ-opioid receptor (µOR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific µOR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists trigger µOR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair ß-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.