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Theranostics 2024Neurons in the ventral tegmental area (VTA) are sensitive to stress and their maladaptation have been implicated in the psychiatric disorders such as anxiety and...
Neurons in the ventral tegmental area (VTA) are sensitive to stress and their maladaptation have been implicated in the psychiatric disorders such as anxiety and addiction, . The cellular properties of the VTA neurons in response to different stressors related to different emotional processing remain to be investigated. By combining immediate early gene (IEG)-dependent labeling, rabies virus tracing, ensemble-specific transcriptomic analysis and fiber photometry recording in the VTA of male mice, the spatial distribution, brain-wide connectivity and cellular signaling pathways in the VTA neuronal ensembles in response to morphine (Mor-Ens) or foot shock (Shock-Ens) stimuli were investigated. Optogenetic activation of the Mor-Ens drove approach behavior, whereas chemogenetic activation of the Shock-Ens increased the anxiety level in mice. Mor-Ens were clustered and enriched in the ventral VTA, contained a higher proportion of dopaminergic neurons, received more inputs from the dorsal medial striatum and the medial hypothalamic zone, and exhibited greater axonal arborization in the zona incerta and ventral pallidum. Whereas Shock-Ens were more dispersed, contained a higher proportion of GABAergic neurons, and received more inputs from the ventral pallidum and the lateral hypothalamic area. The downstream targets of the G protein and β-arrestin pathways, PLCβ3 and phosphorylated AKT1, were relatively enriched in the Mor-Ens and Shock-Ens, respectively. Cariprazine, the G-protein-biased agonist for the dopamine D2 receptor, increased the response of Mor-Ens to sucrose water and decreased the anxiety-like behavior during morphine withdrawal, whereas the β-arrestin-biased agonist UNC9994 decreased the response of Shock-Ens to tail suspension. Taken together, these findings reveal the heterogeneous connectivity and signaling pathways of the VTA neurons in response to morphine and foot shock, providing new insights for development of specific interventions for psychiatric disorders caused by various stressors associated with different VTA neuronal functions.
Topics: Humans; Male; Animals; Mice; Ventral Tegmental Area; Dopaminergic Neurons; Signal Transduction; beta-Arrestins; Morphine Derivatives
PubMed: 38250036
DOI: 10.7150/thno.90792 -
Marine Drugs Dec 2023NMR and MS/MS-based metabolomics of a cyanobacterial extract from Piti Bomb Holes, Guam, indicated the presence of unique enyne-containing halogenated fatty acid amides....
Chlorinated Enyne Fatty Acid Amides from a Marine Cyanobacterium: Discovery of Taveuniamides L-M and Pharmacological Characterization of Taveuniamide F as a GPCR Antagonist with CNR1 Selectivity.
NMR and MS/MS-based metabolomics of a cyanobacterial extract from Piti Bomb Holes, Guam, indicated the presence of unique enyne-containing halogenated fatty acid amides. We isolated three new compounds of this class, taveuniamides L-N (-) along with the previously reported taveuniamide F (), which was the most abundant analog. The planar structures of the new compounds were established using 1D and 2D NMR as well as mass spectrometry. We established the configuration of this chemical class to be at C-8 via Mosher's analysis of after reduction of the carboxamide group. Our biological investigations with revealed that the compound binds to the cannabinoid receptor CNR1, acting as an antagonist/inverse agonist in the canonical G-protein signaling pathways. In selectivity profiling against 168 GPCR targets using the β-arrestin functional assay, we found that antagonizes GPR119, NPSR1b, CCR9, CHRM4, GPR120, HTR2A, and GPR103, in addition to CNR1. Interestingly, showed a 6.8-fold selectivity for CNR1 over CNR2. The binding mode of to CNR1 was investigated using docking and molecular dynamics simulations with both natural and unnatural stereoisomers, revealing important CNR1 residues for the interaction and also providing a possible reasoning for the observed CNR1/CNR2 selectivity.
Topics: Drug Inverse Agonism; Tandem Mass Spectrometry; Cyanobacteria; Amides; Fatty Acids
PubMed: 38248654
DOI: 10.3390/md22010028 -
Cells Jan 2024The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in a wide range of pathological conditions including primary nephrotic syndromes and...
The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in a wide range of pathological conditions including primary nephrotic syndromes and acute kidney injuries. suPAR can trigger transduction cascades in podocytes by outside-in activation of αVβ3-integrin, but there is evidence that the functional cell surface response element is actually a complex of different types of receptors, which may also include the receptor for advanced glycation end-products (RAGE) and formyl peptide receptors (FPRs). Here we observed that ROS accumulation and Src activation could be evoked by continuous 24 h exposure to either suPAR or the FPR agonist fMLF. Responses to suPAR and fMLF were completely blocked by either the FPR antagonist WRW4 or by the αV-integrin inhibitor cilengitide. Moreover, endogenous podocyte mouse Fpr1 co-immunoprecipitates with β3-integrin, suggesting that these receptors occur as a complex on the cell surface. suPAR- and fMLF-evoked activation of Src and ROS differed in time course. Thus, robust pertussis toxin (PTX)-sensitive responses were evoked by 60 min exposures to fMLF but not to suPAR. By contrast, responses to 24 h exposures to either suPAR or fMLF were PTX-resistant and were instead abolished by knockdown of β-arrestin-1 (BAR1). FPRs, integrins, and RAGE (along with various Toll-like receptors) can all function as pattern-recognition receptors that respond to "danger signals" associated with infections and tissue injury. The fact that podocytes express such a wide array of pattern-recognition receptors suggests that the glomerular filter is designed to change its function under certain conditions, possibly to facilitate clearance of toxic macromolecules.
Topics: Animals; Mice; beta-Arrestin 1; Integrin beta3; Podocytes; Reactive Oxygen Species; Receptor for Advanced Glycation End Products; Receptors, Formyl Peptide; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Integrin alpha5
PubMed: 38247863
DOI: 10.3390/cells13020172 -
Cell Communication and Signaling : CCS Jan 2024The human CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) interacting with multiple chemokines (i.e., CXC chemokine ligands CXCL1-3 and CXCL5-8)....
BACKGROUND
The human CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) interacting with multiple chemokines (i.e., CXC chemokine ligands CXCL1-3 and CXCL5-8). It is involved in inflammatory diseases as well as cancer. Consequently, much effort is put into the identification of CXCR2 targeting drugs. Fundamental research regarding CXCR2 signaling is mainly focused on CXCL8 (IL-8), which is the first and best described high-affinity ligand for CXCR2. Much less is known about CXCR2 activation induced by other chemokines and it remains to be determined to what extent potential ligand bias exists within this signaling system. This insight might be important to unlock new opportunities in therapeutic targeting of CXCR2.
METHODS
Ligand binding was determined in a competition binding assay using labeled CXCL8. Activation of the ELR + chemokine-induced CXCR2 signaling pathways, including G protein activation, β-arrestin1/2 recruitment, and receptor internalization, were quantified using NanoBRET-based techniques. Ligand bias within and between these pathways was subsequently investigated by ligand bias calculations, with CXCL8 as the reference CXCR2 ligand. Statistical significance was tested through a one-way ANOVA followed by Dunnett's multiple comparisons test.
RESULTS
All chemokines (CXCL1-3 and CXCL5-8) were able to displace CXCL8 from CXCR2 with high affinity and activated the same panel of G protein subtypes (Gα, Gα, Gα, Gα, Gα, and Gα) without any statistically significant ligand bias towards any one type of G protein. Compared to CXCL8, all other chemokines were less potent in β-arrestin1 and -2 recruitment and receptor internalization while equivalently activating G proteins, indicating a G protein activation bias for CXCL1,-2,-3,-5,-6 and CXCL7. Lastly, with CXCL8 used as reference ligand, CXCL2 and CXCL6 showed ligand bias towards β-arrestin1/2 recruitment compared to receptor internalization.
CONCLUSION
This study presents an in-depth analysis of signaling bias upon CXCR2 stimulation by its chemokine ligands. Using CXCL8 as a reference ligand for bias index calculations, no ligand bias was observed between chemokines with respect to activation of separate G proteins subtypes or recruitment of β-arrestin1/2 subtypes, respectively. However, compared to β-arrestin recruitment and receptor internalization, CXCL1-3 and CXCL5-7 were biased towards G protein activation when CXCL8 was used as reference ligand.
Topics: Humans; Receptors, Interleukin-8B; beta-Arrestins; Ligands; Chemokines; GTP-Binding Proteins
PubMed: 38233929
DOI: 10.1186/s12964-023-01460-2 -
Experimental & Molecular Medicine Feb 2024Arrestins are multifunctional proteins that regulate G-protein-coupled receptor (GPCR) desensitization, signaling, and internalization. The arrestin family consists of... (Review)
Review
Arrestins are multifunctional proteins that regulate G-protein-coupled receptor (GPCR) desensitization, signaling, and internalization. The arrestin family consists of four subtypes: visual arrestin1, β-arrestin1, β-arrestin2, and visual arrestin-4. Recent studies have revealed the multifunctional roles of β-arrestins beyond GPCR signaling, including scaffolding and adapter functions, and physically interacting with non-GPCR receptors. Increasing evidence suggests that β-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). β-arrestins physically interact with γ-secretase, leading to increased production and accumulation of amyloid-beta in AD. Furthermore, β-arrestin oligomers inhibit the autophagy cargo receptor p62/SQSTM1, resulting in tau accumulation and aggregation in FTD. In PD, β-arrestins are upregulated in postmortem brain tissue and an MPTP model, and the β2AR regulates SNCA gene expression. In this review, we aim to provide an overview of β-arrestin1 and β-arrestin2, and describe their physiological functions and roles in neurodegenerative diseases. The multifaceted roles of β-arrestins and their involvement in neurodegenerative diseases suggest that they may serve as promising therapeutic targets.
Topics: Humans; beta-Arrestins; Arrestin; Frontotemporal Dementia; Neurodegenerative Diseases; Receptors, G-Protein-Coupled; Alzheimer Disease
PubMed: 38212557
DOI: 10.1038/s12276-023-01144-4 -
International Journal of Molecular... Dec 2023G protein-coupled receptors (GPCRs) represent promising therapeutic targets due to their involvement in numerous physiological processes mediated by downstream G...
G protein-coupled receptors (GPCRs) represent promising therapeutic targets due to their involvement in numerous physiological processes mediated by downstream G protein- and β-arrestin-mediated signal transduction cascades. Although the precise control of GPCR signaling pathways is therapeutically valuable, the molecular details for governing biased GPCR signaling remain elusive. The Angiotensin II type 1 receptor (AT1R), a prototypical class A GPCR with profound implications for cardiovascular functions, has become a focal point for biased ligand-based clinical interventions. Herein, we used single-molecule live-cell imaging techniques to evaluate the changes in stoichiometry and dynamics of AT1R with distinct biased ligand stimulations in real time. It was revealed that AT1R existed predominantly in monomers and dimers and underwent oligomerization upon ligand stimulation. Notably, β-arrestin-biased ligands induced the formation of higher-order aggregates, resulting in a slower diffusion profile for AT1R compared to G protein-biased ligands. Furthermore, we demonstrated that the augmented aggregation of AT1R, triggered by activation from each biased ligand, was completely abrogated in β-arrestin knockout cells. These findings furnish novel insights into the intricate relationship between GPCR aggregation states and biased signaling, underscoring the pivotal role of molecular behaviors in guiding the development of selective therapeutic agents.
Topics: Receptor, Angiotensin, Type 1; Ligands; Single Molecule Imaging; Signal Transduction; beta-Arrestin 1; GTP-Binding Proteins
PubMed: 38203545
DOI: 10.3390/ijms25010374 -
Communications Biology Jan 2024Patients with idiopathic pulmonary fibrosis show a strongly upregulated expression of chemokine CXCL14, whose target is still unknown. Screening of CXCL14 in a panel of...
Patients with idiopathic pulmonary fibrosis show a strongly upregulated expression of chemokine CXCL14, whose target is still unknown. Screening of CXCL14 in a panel of human G protein-coupled receptors (GPCRs) revealed its potent and selective activation of the orphan MAS-related GPCR X2 (MRGPRX2). This receptor is expressed on mast cells and - like CXCL14 - upregulated in bronchial inflammation. CXCL14 induces robust activation of MRGPRX2 and its putative mouse ortholog MRGPRB2 in G protein-dependent and β-arrestin recruitment assays that is blocked by a selective MRGPRX2/B2 antagonist. Truncation combined with mutagenesis and computational studies identified the pharmacophoric sequence of CXCL14 and its presumed interaction with the receptor. Intriguingly, C-terminal domain sequences of CXCL14 consisting of 4 to 11 amino acids display similar or increased potency and efficacy compared to the full CXCL14 sequence (77 amino acids). These results provide a rational basis for the future development of potential idiopathic pulmonary fibrosis therapies.
Topics: Animals; Humans; Mice; Amino Acids; Biological Assay; Chemokines; Chemokines, CXC; Idiopathic Pulmonary Fibrosis; Nerve Tissue Proteins; Receptors, G-Protein-Coupled; Receptors, Neuropeptide
PubMed: 38184723
DOI: 10.1038/s42003-023-05739-5 -
Molecular insights into atypical modes of β-arrestin interaction with seven transmembrane receptors.Science (New York, N.Y.) Jan 2024β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven...
β-arrestins (βarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of βarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the βarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of βarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of βarr2 from a β strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-βarr complexes with direct implications for exploring novel therapeutic avenues.
Topics: beta-Arrestins; Cryoelectron Microscopy; Receptors, G-Protein-Coupled; Signal Transduction; Protein Conformation, beta-Strand; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Humans
PubMed: 38175886
DOI: 10.1126/science.adj3347 -
Molecular Pharmacology Feb 2024Dualsteric G protein-coupled receptor (GPCR) ligands are a class of bitopic ligands that consist of an orthosteric pharmacophore, which binds to the pocket occupied by...
Dualsteric G protein-coupled receptor (GPCR) ligands are a class of bitopic ligands that consist of an orthosteric pharmacophore, which binds to the pocket occupied by the receptor's endogenous agonist, and an allosteric pharmacophore, which binds to a distinct site. These ligands have the potential to display characteristics of both orthosteric and allosteric ligands. To explore the signaling profiles that dualsteric ligands of the angiotensin II type 1 receptor (AT1R) can access, we ligated a 6e epitope tag-specific nanobody (single-domain antibody fragment) to angiotensin II (AngII) and analogs that show preferential allosteric coupling to Gq (TRV055, TRV056) or -arrestin (TRV027). While the nanobody itself acts as a probe-specific neutral or negative allosteric ligand of -terminally 6e-tagged AT1R, nanobody conjugation to orthosteric ligands had varying effects on Gq dissociation and -arrestin plasma membrane recruitment. The potency of certain AngII analogs was enhanced up to 100-fold, and some conjugates behaved as partial agonists, with up to a 5-fold decrease in maximal efficacy. Nanobody conjugation also biased the signaling of TRV055 and TRV056 toward Gq, suggesting that Gq bias at AT1R can be modulated through molecular mechanisms distinct from those previously elucidated. Both competition radioligand binding experiments and functional assays demonstrated that orthosteric antagonists (angiotensin receptor blockers) act as non-competitive inhibitors of all these nanobody-peptide conjugates. This proof-of-principle study illustrates the array of pharmacological patterns that can be achieved by incorporating neutral or negative allosteric pharmacophores into dualsteric ligands. Nanobodies directed toward linear epitopes could provide a rich source of allosteric reagents for this purpose. SIGNIFICANCE STATEMENT: Here we engineer bitopic (dualsteric) ligands for epitope-tagged angiotensin II type 1 receptor by conjugating angiotensin II or its biased analogs to an epitope-specific nanobody (antibody fragment). Our data demonstrate that nanobody-mediated interactions with the receptor -terminus endow angiotensin analogs with properties of allosteric modulators and provide a novel mechanism to increase the potency, modulate the maximal effect, or alter the bias of ligands.
Topics: Receptor, Angiotensin, Type 1; Angiotensin II; Ligands; beta-Arrestins; Epitopes; Allosteric Regulation
PubMed: 38164609
DOI: 10.1124/molpharm.123.000797 -
Pharmacological Research Jan 2024The 5-hydroxytryptamine 7 receptor (5-HT) is necessary for 5-HT to cause a concentration-dependent vascular relaxation and hypotension. 5-HT is recognized as having...
The 5-hydroxytryptamine 7 receptor (5-HT) is necessary for 5-HT to cause a concentration-dependent vascular relaxation and hypotension. 5-HT is recognized as having biased signaling, transduced through either Gs or β -arrestin. It is unknown whether 5-HT signals in a biased manner to cause vasorelaxation/hypotension. We used the recently described β-arrestin selective 5-HT receptor agonist serodolin to test the hypothesis that 5-HT activation does not cause vascular relaxation or hypotension via the β -arrestin pathway. Isolated abdominal aorta (no functional 5-HT) and vena cava (functional 5-HT) from male Sprague Dawley rats were used in isometric contractility studies. Serodolin (1 nM - 10 μM) did not change baseline tone of isolated tissues and did not relax the endothelin-1 (ET-1)-contracted vena cava or aorta. In the aorta, serodolin acted as a 5-HT receptor antagonist, evidenced by a rightward shift in 5-HT-induced concentration response curve [pEC 5-HT [M]: Veh = 5.2 +/- 0.15; Ser (100 nM) = 4.49 +/- 0.08; p < 0.05]. In the vena cava, serodolin acted as a 5-HT receptor antagonist, shifting the concentration response curve to 5-HT left and upward (%10 μM NE contraction; Veh = 3.2 +/- 1.7; Ser (10 nM) = 58 +/- 11; p < 0.05) and blocking relaxation of pre-contracted tissue to the 5-HT agonist 5-carboxamidotryptamine. In anesthetized rats, 5-HT or serodolin was infused at 5, 25 and 75 μg/kg/min, iv. Though 5-HT caused concentration-dependent depressor responses, serodolin caused an insignificant small depressor responses at all three infusion rates. With the final dose of serodolin on board, 5-HT was unable to reduce blood pressure. Collectively the data indicate that serodolin functions as a 5-HT antagonist with additional 5-HT blocking properties. 5-HT activation does not cause vascular relaxation or hypotension via the β -arrestin pathway.
Topics: Rats; Animals; Male; Serotonin; Serotonin Receptor Agonists; beta-Arrestins; Rats, Sprague-Dawley; Hypotension
PubMed: 38157998
DOI: 10.1016/j.phrs.2023.107047