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BioRxiv : the Preprint Server For... Apr 2024Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7 leading cause of premature death. Despite this,...
Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7 leading cause of premature death. Despite this, there are currently only 3 FDA approved pharmacological treatments for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant misuse. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces binge-like ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances.
PubMed: 38645173
DOI: 10.1101/2024.04.10.588903 -
Cellular Signalling Jul 2024G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular...
G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the β-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with β-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for β-arrestin-2 binding and to investigate the role of β-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to β-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to β-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the β-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the β-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.
Topics: beta-Arrestin 2; Humans; HEK293 Cells; Protein Binding; Receptors, G-Protein-Coupled; Animals; Receptors, Peptide; Signal Transduction; Binding Sites; Phosphorylation; Receptors, Gastrointestinal Hormone
PubMed: 38631405
DOI: 10.1016/j.cellsig.2024.111175 -
Hypertension (Dallas, Tex. : 1979) Jun 2024ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the ATR [angiotensin type 1...
BACKGROUND
ANG (angiotensin II) elicits dipsogenic and pressor responses via activation of the canonical Gαq (G-protein component of the ATR [angiotensin type 1 receptor])-mediated ATR in the subfornical organ. Recently, we demonstrated that ARRB2 (β-arrestin 2) global knockout mice exhibit a higher preference for salt and exacerbated pressor response to deoxycorticosterone acetate salt. However, whether ARRB2 within selective neuroanatomical nuclei alters physiological responses to ANG is unknown. Therefore, we hypothesized that ARRB2, specifically in the subfornical organ, counterbalances maladaptive dipsogenic and pressor responses to the canonical ATR signaling.
METHODS
Male and female mice received intracerebroventricular injection of either adeno-associated virus (AAV)-Cre-GFP (green fluorescent protein) to induce brain-specific deletion of ARRB2 (). mice receiving ICV-AAV-GFP were used as control (). Infection with ICV-AAV-Cre primarily targeted the subfornical organ with few off targets. Fluid intake was evaluated using the 2-bottle choice paradigm with 1 bottle containing water and 1 containing 0.15 mol/L NaCl.
RESULTS
mice exhibited a greater pressor response to acute ICV-ANG infusion. At baseline conditions, mice exhibited a significant increase in saline intake compared with controls, resulting in a saline preference. Furthermore, when mice were subjected to water-deprived or sodium-depleted conditions, which would naturally increase endogenous ANG levels, mice exhibited elevated saline intake.
CONCLUSIONS
Overall, these data indicate that ARRB2 in selective cardiovascular nuclei in the brain, including the subfornical organ, counterbalances canonical ATR responses to both exogenous and endogenous ANG. Stimulation of the ATR/ARRB axis in the brain may represent a novel strategy to treat hypertension.
Topics: Animals; Subfornical Organ; Mice; Blood Pressure; Male; Homeostasis; beta-Arrestin 2; Female; Mice, Knockout; Angiotensin II; Brain; Receptor, Angiotensin, Type 1
PubMed: 38629290
DOI: 10.1161/HYPERTENSIONAHA.124.22874 -
Molecular and Cellular Endocrinology Aug 2024Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the...
Luteinizing hormone (LH) is essential for reproduction, controlling ovulation and steroidogenesis. Its receptor (LHR) recruits various transducers leading to the activation of a complex signaling network. We recently identified iPRC1, the first variable fragment from heavy-chain-only antibody (VHH) interacting with intracellular loop 3 (ICL3) of the follicle-stimulating hormone receptor (FSHR). Because of the high sequence similarity of the human FSHR and LHR (LHCGR), here we examined the ability of the iPRC1 intra-VHH to modulate LHCGR activity. In this study, we demonstrated that iPRC1 binds LHCGR, to a greater extent when the receptor was stimulated by the hormone. In addition, it decreased LH-induced cAMP production, cAMP-responsive element-dependent transcription, progesterone and testosterone production. These impairments are not due to Gs nor β-arrestin recruitment to the LHCGR. Consequently, iPRC1 is the first intra-VHH to bind and modulate LHCGR biological activity, including steroidogenesis. It should help further understand signaling mechanisms elicited at this receptor and their outcomes on reproduction.
Topics: Receptors, LH; Humans; Signal Transduction; Luteinizing Hormone; Animals; Cyclic AMP; Protein Binding; Progesterone; Receptors, FSH; Testosterone; HEK293 Cells; GTP-Binding Proteins; Steroids
PubMed: 38621656
DOI: 10.1016/j.mce.2024.112235 -
Biochemical Pharmacology Jun 2024Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can...
Arrestins are key negative regulators of G Protein-Coupled Receptors (GPCRs) through mediation of G protein desensitisation and receptor internalisation. Arrestins can also contribute to signal transduction by scaffolding downstream signalling effectors for activation. GPCR kinase (GRK) enzymes phosphorylate the intracellular C-terminal domain, or intracellular loop regions of GPCRs to promote arrestin interaction. There are seven different GRK subtypes, which may uniquely phosphorylate the C-terminal tail in a type of 'phosphorylation barcode,' potentially differentially contributing to arrestin translocation and arrestin-dependent signalling. Such contributions may be exploited to develop arrestin-biased ligands. Here, we examine the effect of different GRK subtypes on the ability to promote translocation of arrestin-2 and arrestin-3 to the cannabinoid CB receptor (CB) with a range of ligands. We find that most GRK subtypes (including visual GRK1) can enhance arrestin-2 and -3 translocation to CB, and that GRK-dependent changes in arrestin-2 and arrestin-3 translocation were broadly shared for most agonists tested. GRK2/3 generally enhanced arrestin translocation more than the other GRK subtypes, with some small differences between ligands. We also explore the interplay between G protein activity and GRK2/3-dependent arrestin translocation, highlighting that high-efficacy G protein agonists will cause GRK2/3 dependent arrestin translocation. This study supports the hypothesis that arrestin-biased ligands for CB must engage GRK5/6 rather than GRK2/3, and G protein-biased ligands must have inherently low efficacy.
Topics: Humans; Receptor, Cannabinoid, CB1; Signal Transduction; HEK293 Cells; Arrestins; Protein Transport; GTP-Binding Proteins; G-Protein-Coupled Receptor Kinases; Animals; beta-Arrestin 2
PubMed: 38604257
DOI: 10.1016/j.bcp.2024.116190 -
Nature May 2024The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better...
The µ-opioid receptor (µOR) is an important target for pain management and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of β-arrestin-1 with the μOR core binding site appears less specific and occurs with much lower affinity than binding of G.
Topics: Humans; beta-Arrestin 1; Binding Sites; Fluorescence Resonance Energy Transfer; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Diphosphate; Ligands; Models, Molecular; Protein Binding; Protein Conformation; Receptors, Opioid, mu; Single Molecule Imaging
PubMed: 38600384
DOI: 10.1038/s41586-024-07295-2 -
Cell Communication and Signaling : CCS Apr 2024Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The...
BACKGROUND
Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of β-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D-like receptors (DR, DR, DR), have suggested the existence of other desensitization mechanisms. The present study employed DR/DR variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT.
RESULTS
AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin β1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of β-Arr2. The deubiquitinated β-Arr2 then forms a complex with Gβγ, which serves as a biomarker for GPCRs desensitization. Like in DR, ubiquitination of AKT is also involved in the desensitization of β adrenoceptors.
CONCLUSION
Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.
Topics: TNF Receptor-Associated Factor 6; Proto-Oncogene Proteins c-akt; Receptors, G-Protein-Coupled; Ubiquitination; Phosphorylation; Karyopherins
PubMed: 38566235
DOI: 10.1186/s12964-024-01592-z -
BioRxiv : the Preprint Server For... Mar 2024Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the μ-opioid receptor. Opioid use is limited by...
Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the μ-opioid receptor. Opioid use is limited by side effects, including significant risk of opioid use disorder. Improvement of the effect/side effect profile of opioid medications is a key pursuit of opioid research, yet there is no consensus on how to achieve this goal. One hypothesis is that the degree of arrestin-3 recruitment to the μ-opioid receptor impacts therapeutic utility. However, it is not clear whether increased or decreased interaction of the μ-opioid receptor with arrestin-3 would reduce compulsive drug-seeking. To examine this question, we utilized three genotypes of mice with varying abilities to recruit arrestin-3 to the μ-opioid receptor in response to morphine in a novel longitudinal operant self-administration model. We demonstrate that arrestin-3 knockout and wild type mice have highly variable drug-seeking behavior with few genotype differences. In contrast, in mice where the μ-opioid receptor strongly recruits arrestin-3, drug-seeking behavior is much less varied. We created a quantitative method to define compulsivity in drug-seeking and found that mice lacking arrestin-3 were more likely to meet the criteria for compulsivity whereas mice with enhanced arrestin-3 recruitment did not develop a compulsive phenotype. Our data suggest that opioids that engage both G protein and arrestin-3, recapitulating the endogenous signaling pattern, will reduce abuse liability.
PubMed: 38562752
DOI: 10.1101/2023.03.30.534994 -
European Journal of Pharmacology May 2024Synthetic cannabinoid receptor agonists (SCRAs) remain one the largest classes of new psychoactive substances, and are increasingly associated with severe adverse...
Synthetic cannabinoid receptor agonists (SCRAs) remain one the largest classes of new psychoactive substances, and are increasingly associated with severe adverse effects and death compared to the phytocannabinoid Δ-tetrahydrocannabinol (THC). In the attempt to circumvent the rapid emergence of novel SCRAs, several nations have implemented 'generic' legislations, or 'class-wide' bans based on common structural scaffolds. However, this has only encouraged the incorporation of new chemical entities, including distinct core and linker structures, for which there is a dearth of pharmacological data. The current study evaluated five emergent OXIZID SCRAs for affinity and functional activity at the cannabinoid CB receptor (CB) in HEK 293 cells, as well as pharmacological equivalence with THC in drug discrimination in mice. All OXIZID compounds behaved as agonists in Gα protein activation and β-arrestin 2 translocation assays, possessing low micromolar affinity at CB. All ligands also substituted for THC in drug discrimination, where potencies broadly correlated with in vitro activity, with the methylcyclohexane analogue BZO-CHMOXIZID being the most potent. Notably, MDA-19 (BZO-HEXOXIZID) exhibited partial efficacy in vitro, generating an activity profile most similar to that of THC, and partial substitution in vivo. Overall, the examined OXIZIDs were comparatively less potent and efficacious than previous generations of SCRAs. Further toxicological data will elucidate whether the moderate cannabimimetic activity for this series of SCRAs will translate to severe adverse health effects as seen with previous generations of SCRAs.
Topics: Humans; Mice; Animals; Cannabinoid Receptor Agonists; HEK293 Cells; Receptors, Cannabinoid; Ligands; Protein Processing, Post-Translational; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2
PubMed: 38561104
DOI: 10.1016/j.ejphar.2024.176549 -
International Journal of Molecular... Mar 2024Arrestins 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...
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 H receptor-mediated activation of ERK is dually regulated by G proteins and arrestins. In this study, we investigated the roles of G proteins and arrestins in the H receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H receptors, the G 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 H receptors (ketotifen and diphenhydramine), G proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), β-arrestin2 (β-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H receptor-mediated phosphorylation of JNK is regulated by G-protein/Ca/PKC-dependent but GRK/arrestin/clathrin-independent pathways.
Topics: Animals; Cricetinae; Humans; Arrestin; Arrestins; beta-Arrestins; CHO Cells; Clathrin; Cricetulus; Extracellular Signal-Regulated MAP Kinases; G-Protein-Coupled Receptor Kinases; GTP-Binding Proteins; Histamine; Phosphorylation; Protein Kinase C; Receptors, Histamine H1; Signal Transduction
PubMed: 38542369
DOI: 10.3390/ijms25063395