Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways.

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.

The multifaceted functions of ß-arrestins and their therapeutic potential in neurodegenerative diseases.

Arrestins are multifunctional proteins that regulate G-protein-coupled receptor (GPCR) desensitization, signaling, and internalization. The arrestin family consists of four subtypes: visual arrestin1, ß-arrestin1, ß-arrestin2, and visual arrestin-4. Recent studies have revealed the multifunctional roles of ß-arrestins beyond GPCR signaling, including scaffolding and adapter functions, and physically interacting with non-GPCR receptors. Increasing evidence suggests that ß-arrestins are involved in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia (FTD), and Parkinson's disease (PD). ß-arrestins physically interact with γ-secretase, leading to increased production and accumulation of amyloid-beta in AD. Furthermore, ß-arrestin oligomers inhibit the autophagy cargo receptor p62/SQSTM1, resulting in tau accumulation and aggregation in FTD. In PD, ß-arrestins are upregulated in postmortem brain tissue and an MPTP model, and the ß2AR regulates SNCA gene expression. In this review, we aim to provide an overview of ß-arrestin1 and ß-arrestin2, and describe their physiological functions and roles in neurodegenerative diseases. The multifaceted roles of ß-arrestins and their involvement in neurodegenerative diseases suggest that they may serve as promising therapeutic targets.

Characterization of the real-time internalization of nine GPCRs reveals distinct dependence on arrestins and G proteins.

G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.

Snapshot of the cannabinoid receptor 1-arrestin complex unravels the biased signaling mechanism.

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.

Ready for the sheet: ß-strand folding of phosphorylation clusters guides GPCR binding to arrestin.

The molecular dynamics of arrestin binding to G protein-coupled receptors (GPCRs) are still poorly understood. In this issue of Structure, Guillien et al. show that negative charges in GPCR key phosphorylation clusters induce the formation of a transient ß-strand that participates in an intermolecular ß-sheet in the associated complex.

Amphetamine pretreatment blunts dopamine-induced D2/D3-receptor occupancy by an arrestin-mediated mechanism: A PET study in internalization compromised mice.

While all reversible receptor-targeting radioligands for positron emission tomography (PET) can be displaced by competition with an antagonist at the receptor, many radiotracers show limited occupancies using agonists even at high doses. [11C]Raclopride, a D2/D3 receptor radiotracer with rapid kinetics, can identify the direction of changes in the neurotransmitter dopamine, but quantitative interpretation of the relationship between dopamine levels and radiotracer binding has proven elusive. Agonist-induced receptor desensitization and internalization, a homeostatic mechanism to downregulate neurotransmitter-mediated function, can shift radioligand-receptor binding affinity and confound PET interpretations of receptor occupancy. In this study, we compared occupancies induced by amphetamine (AMP) in drug-naive wild-type (WT) and internalization-compromised ß-arrestin-2 knockout (KO) mice using a within-scan drug infusion to modulate the kinetics of [11C]raclopride. We additionally performed studies at 3 h following AMP pretreatment, with the hypothesis that receptor internalization should markedly attenuate occupancy on the second challenge, because dopamine cannot access internalized receptors. Without prior AMP treatment, WT mice exhibited somewhat larger binding potential than KO mice but similar AMP-induced occupancy. At 3 h after AMP treatment, WT mice exhibited binding potentials that were 15 % lower than KO mice. At this time point, occupancy was preserved in KO mice but suppressed by 60 % in WT animals, consistent with a model in which most receptors contributing to binding potential in WT animals were not functional. These results demonstrate that arrestin-mediated receptor desensitization and internalization produce large effects in PET [11C]raclopride occupancy studies using agonist challenges.

Cardioprotective efficacy of Xin-shu-bao tablet in heart failure with reduced ejection fraction by modulating THBD/ARRB1/FGF1/STIM1 signaling.

Traditional Chinese medicine offer unique advantages in mitigating and preventing early or intermediate stage for treating heart failure (HF). The purpose of this study was to assess the in vivo therapeutic efficacy of Xin-shu-bao (XSB) at different stages of HF following induction of a myocardial infarction (MI) in mice and use mass spectrometry-based proteomics to identify potential therapeutic targets for different stages of HF based on the molecular changes following XSB treatment. XSB had high cardioprotective efficacy in the pre-HF with reduced ejection fraction (HFrEF) stages, but had a weak or no effect in the post-HFrEF stages. This was supported by echocardiographic measurements showing that XSB decreased ejection fraction and fractional shortening in HF. XSB administration improved cardiac function in the pre- and post-HFrEF mouse model, ameliorated deleterious changes to the morphology and subcellular structure of cardiomyocytes, and reduced cardiac fibrosis. Proteomics analysis showed that XSB intervention exclusively targeted thrombomodulin (THBD) and stromal interaction molecule 1 (STIM1) proteins when administered to the mice for both 8 and 6 weeks. Furthermore, XSB intervention for 8, 6, and 4 weeks after MI induction increased the expression of fibroblast growth factor 1 (FGF1) and decreased arrestin ß1 (ARRB1), which are classic biomarkers of cardiac fibroblast transformation and collagen synthesis, respectively. Overall, the study suggests that early intervention with XSB could be an effective strategy for preventing HFrEF and highlights potential therapeutic targets for further investigation into HFrEF remediation strategies.

Conserved class B GPCR activation by a biased intracellular agonist.

Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1-5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.

Identification of a ß-arrestin-biased negative allosteric modulator for the ß2-adrenergic receptor.

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.

Stabilization of pre-existing neurotensin receptor conformational states by ß-arrestin-1 and the biased allosteric modulator ML314.

The neurotensin receptor 1 (NTS1) 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 NTS1 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 13CεH3-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. ß-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 ß-arrestin biased allosteric modulator transforms the NTS1: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.

Mechanisms of Arrestin-Mediated Signaling.

Arrestins were first discovered as proteins that selectively bind active phosphorylated GPCRs and suppress (arrest) their G protein-mediated signaling. Nonvisual arrestins are also recognized as signaling proteins regulating a variety of cellular pathways. Arrestins are highly flexible; they can assume many different conformations. In their receptor-bound conformation, arrestins have higher affinity for a subset of binding 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 regulate other signaling pathways and localize signaling proteins to particular subcellular compartments. Recent findings suggest that the two visuals, arrestin-1 and arrestin-4, which are expressed in photoreceptor cells, not only regulate signaling via binding to photopigments but also interact with several 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. © 2023 Wiley Periodicals LLC.

Functional Role of Arrestin-1 Residues Interacting with Unphosphorylated Rhodopsin Elements.

Arrestin-1, or visual arrestin, exhibits an exquisite selectivity for light-activated phosphorylated rhodopsin (P-Rh*) over its other functional forms. That selectivity is believed to be mediated by two well-established structural elements in the arrestin-1 molecule, the activation sensor detecting the active conformation of rhodopsin and the phosphorylation sensor responsive to the rhodopsin phosphorylation, which only active phosphorylated rhodopsin can engage simultaneously. However, in the crystal structure of the arrestin-1-rhodopsin complex there are arrestin-1 residues located close to rhodopsin, which do not belong to either sensor. Here we tested by site-directed mutagenesis the functional role of these residues in wild type arrestin-1 using a direct binding assay to P-Rh* and light-activated unphosphorylated rhodopsin (Rh*). We found that many mutations either enhanced the binding only to Rh* or increased the binding to Rh* much more than to P-Rh*. The data suggest that the native residues in these positions act as binding suppressors, specifically inhibiting the arrestin-1 binding to Rh* and thereby increasing arrestin-1 selectivity for P-Rh*. This calls for the modification of a widely accepted model of the arrestin-receptor interactions.

Divergent regulation of α-arrestin ARRDC3 function by ubiquitination.

The α-arrestin ARRDC3 is a recently discovered tumor suppressor in invasive breast cancer that functions as a multifaceted adaptor protein to control protein trafficking and cellular signaling. However, the molecular mechanisms that control ARRDC3 function are unknown. Other arrestins are known to be regulated by posttranslational modifications, suggesting that ARRDC3 may be subject to similar regulatory mechanisms. Here we report that ubiquitination is a key regulator of ARRDC3 function and is mediated primarily by two proline-rich PPXY motifs in the ARRDC3 C-tail domain. Ubiquitination and the PPXY motifs are essential for ARRDC3 function in regulating GPCR trafficking and signaling. Additionally, ubiquitination and the PPXY motifs mediate ARRDC3 protein degradation, dictate ARRDC3 subcellular localization, and are required for interaction with the NEDD4-family E3 ubiquitin ligase WWP2. These studies demonstrate a role for ubiquitination in regulating ARRDC3 function and reveal a mechanism by which ARRDC3 divergent functions are controlled.

Differential regulation of histamine H1 receptor-mediated ERK phosphorylation by Gq proteins and arrestins.

Gq protein-coupled histamine H1 receptors play crucial roles in allergic and inflammatory reactions, in which the phosphorylation of extracellular signal-regulated kinase (ERK) appears to mediate the production of inflammatory cytokines. ERK phosphorylation is regulated by G protein- and arrestin-mediated signal transduction pathways. Here, we aimed to explore how H1 receptor-mediated processes of ERK phosphorylation might be differentially regulated by Gq proteins and arrestins. For this purpose, we evaluated the regulatory mechanism(s) of H1 receptor-mediated ERK phosphorylation in Chinese hamster ovary cells expressing Gq protein- and arrestin-biased mutants of human H1 receptors, S487TR and S487A, in which the Ser487 residue in the C-terminal was truncated and mutated to alanine, respectively. Immunoblotting analysis indicated that histamine-induced ERK phosphorylation was prompt and transient in cells expressing Gq protein-biased S487TR, whereas it was slow and sustained in cells expressing arrestin-biased S487A. Inhibitors of Gq proteins (YM-254890) and protein kinase C (PKC) (GF109203X), and an intracellular Ca2+ chelator (BAPTA-AM) suppressed histamine-induced ERK phosphorylation in cells expressing S487TR, but not those expressing S487A. Conversely, inhibitors of G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), clathrin (hypertonic sucrose), Raf (LY3009120), and MEK (U0126) suppressed histamine-induced ERK phosphorylation in cells expressing S487A, but not those expressing S487TR. These results suggest that H1 receptor-mediated ERK phosphorylation might be differentially regulated by the Gq protein/Ca2+/PKC and GRK/arrestin/clathrin/Raf/MEK pathways to potentially determine the early and late phases of histamine-induced allergic and inflammatory responses, respectively.

Migration mediated by the oxysterol receptor GPR183 depends on arrestin coupling but not receptor internalization.

The chemotactic G protein-coupled receptor GPR183 and its most potent endogenous oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) are important for immune cell positioning in secondary lymphoid tissues. This receptor-ligand pair is associated with various diseases, in some cases contributing favorably and in other cases adversely, making GPR183 an attractive target for therapeutic intervention. We investigated the mechanisms underlying GPR183 internalization and the role of internalization in the main biological function of the receptor, chemotaxis. We found that the C terminus of the receptor was important for ligand-induced internalization but less so for constitutive (ligand-independent) internalization. ß-arrestin potentiated ligand-induced internalization but was not required for ligand-induced or constitutive internalization. Caveolin and dynamin were the main mediators of both constitutive and ligand-induced receptor internalization in a mechanism independent of G protein activation. Clathrin-mediated endocytosis also contributed to constitutive GPR183 internalization in a ß-arrestin-independent manner, suggesting the existence of different pools of surface-localized GPR183. Chemotaxis mediated by GPR183 depended on receptor desensitization by ß-arrestins but could be uncoupled from internalization, highlighting an important biological role for the recruitment of ß-arrestin to GPR183. The role of distinct pathways in internalization and chemotaxis may aid in the development of GPR183-targeting drugs for specific disease contexts.

Carfentanil is a ß-arrestin-biased agonist at the µ opioid receptor.

BACKGROUND AND PURPOSE: The illicit use of fentanyl-like drugs (fentanyls), which are µ opioid receptor agonists, and the many overdose deaths that result, has become a major problem. Fentanyls are very potent in vivo, leading to respiratory depression and death. However, the efficacy and possible signalling bias of different fentanyls is not clearly known. Here, we compared the relative efficacy and bias of a series of fentanyls. EXPERIMENTAL APPROACH: For agonist signalling bias and efficacy measurements, Bioluminescence Resonance Energy Transfer experiments were undertaken in HEK293T cells transiently transfected with µ opioid receptors, to assess Gi protein activation and ß-arrestin 2 recruitment. Agonist-induced cell surface receptor loss was assessed using an enzyme-linked immunosorbent assay, whilst agonist-induced G protein-coupled inwardly rectifying potassium channel current activation was measured electrophysiologically from rat locus coeruleus slices. Ligand poses in the µ opioid receptor were determined in silico using molecular dynamics simulations. KEY RESULTS: Relative to the reference ligand DAMGO, carfentanil was ß-arrestin-biased, whereas fentanyl, sufentanil and alfentanil did not display bias. Carfentanil induced potent and extensive cell surface receptor loss, whilst the marked desensitisation of G protein-coupled inwardly rectifying potassium channel currents in the continued presence of carfentanil in neurones was prevented by a GRK2/3 inhibitor. Molecular dynamics simulations suggested unique interactions of carfentanil with the orthosteric site of the receptor that could underlie the bias. CONCLUSIONS AND IMPLICATIONS: Carfentanil is a ß-arrestin-biased opioid drug at the µ receptor. It is uncertain how such bias influences in vivo effects of carfentanil relative to other fentanyls.

Endocytic proteins mediating GPR15 receptor internalization provide insight into the underlying mechanisms.

GPR15 is a G protein-coupled receptor involved in immune disorders such as human immunodeficiency virus-induced enteropathy, multiple sclerosis, and colitis. Yet, the important endocytosis mechanism of GPR15 remained unclear. This study determined the participation of endocytic machinery proteins, including Gα proteins, G protein-coupled receptor kinases (GRKs), protein kinase C, arrestins, clathrin, caveolin, and dynamin in GPR15 internalization. The results demonstrate that GPR15 internalization is moderately dependent on GRKs and clathrin, and highly dependent on caveolin and dynamin. Moreover, a bystander arrestin recruitment assay showed that GPR15 recruits arrestin-3 to the cell membrane upon agonist stimulation, although GPR15 internalizes in an arrestin-independent manner. Overall, our study provides novel insights into ß-arrestin recruitment and receptor internalization mechanisms for the recently deorphanized GPR15.

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias.

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and ß-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses.

Molecular mechanism of biased signaling at the kappa opioid receptor.

The κ-opioid receptor (KOR) has emerged as an attractive drug target for pain management without addiction, and biased signaling through particular pathways of KOR may be key to maintaining this benefit while minimizing side-effect liabilities. As for most G protein-coupled receptors (GPCRs), however, the molecular mechanisms of ligand-specific signaling at KOR have remained unclear. To better understand the molecular determinants of KOR signaling bias, we apply structure determination, atomic-level molecular dynamics (MD) simulations, and functional assays. We determine a crystal structure of KOR bound to the G protein-biased agonist nalfurafine, the first approved KOR-targeting drug. We also identify an arrestin-biased KOR agonist, WMS-X600. Using MD simulations of KOR bound to nalfurafine, WMS-X600, and a balanced agonist U50,488, we identify three active-state receptor conformations, including one that appears to favor arrestin signaling over G protein signaling and another that appears to favor G protein signaling over arrestin signaling. These results, combined with mutagenesis validation, provide a molecular explanation of how agonists achieve biased signaling at KOR.

Arrestin-dependent nuclear export of phosphodiesterase 4D promotes GPCR-induced nuclear cAMP signaling required for learning and memory.

G protein-coupled receptors (GPCRs) promote the expression of immediate early genes required for learning and memory. Here, we showed that ß2-adrenergic receptor (ß2AR) stimulation induced the nuclear export of phosphodiesterase 4D5 (PDE4D5), an enzyme that degrades the second messenger cAMP, to enable memory consolidation. We demonstrated that the endocytosis of ß2AR phosphorylated by GPCR kinases (GRKs) mediated arrestin3-dependent nuclear export of PDE4D5, which was critical for promoting nuclear cAMP signaling and gene expression in hippocampal neurons for memory consolidation. Inhibition of the arrestin3-PDE4D5 association prevented ß2AR-induced nuclear cAMP signaling without affecting receptor endocytosis. Direct PDE4 inhibition rescued ß2AR-induced nuclear cAMP signaling and ameliorated memory deficits in mice expressing a form of the ß2AR that could not be phosphorylated by GRKs. These data reveal how ß2AR phosphorylated by endosomal GRK promotes the nuclear export of PDE4D5, leading to nuclear cAMP signaling, changes in gene expression, and memory consolidation. This study also highlights the translocation of PDEs as a mechanism to promote cAMP signaling in specific subcellular locations downstream of GPCR activation.