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Science Advances Dec 2023The metabotropic glutamate receptors (mGluRs) are family C, dimeric G protein-coupled receptors (GPCRs), which play critical roles in synaptic transmission. Despite an...
The metabotropic glutamate receptors (mGluRs) are family C, dimeric G protein-coupled receptors (GPCRs), which play critical roles in synaptic transmission. Despite an increasing appreciation of the molecular diversity of this family, how distinct mGluR subtypes are regulated remains poorly understood. We reveal that different group II/III mGluR subtypes show markedly different beta-arrestin (β-arr) coupling and endocytic trafficking. While mGluR2 is resistant to internalization and mGluR3 shows transient β-arr coupling, which enables endocytosis and recycling, mGluR8 and β-arr form stable complexes, which leads to efficient lysosomal targeting and degradation. Using chimeras and mutagenesis, we pinpoint carboxyl-terminal domain regions that control β-arr coupling and trafficking, including the identification of an mGluR8 splice variant with impaired internalization. We then use a battery of high-resolution fluorescence assays to find that heterodimerization further expands the diversity of mGluR regulation. Together, this work provides insight into the relationship between GPCR/β-arr complex formation and trafficking while revealing diversity and intricacy in the regulation of mGluRs.
Topics: beta-Arrestins; Receptors, Metabotropic Glutamate
PubMed: 38055809
DOI: 10.1126/sciadv.adi8076 -
Clinical Science (London, England :... Dec 2023Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently,...
Angiotensin receptor blockers (ARBs) are the first-line treatment for hypertension; they act by inhibiting signaling through the angiotensin 1 receptor (AT1R). Recently, a novel biased AT1R agonist, TRV120027 (TRV), which selectively activates the β-arrestin cascade and blocks the G-protein-coupled receptor pathway has been proposed as a potential blood pressure medication. Here, we explored the effects of TRV and associated β-arrestin signaling in podocytes, essential cells of the kidney filter. We used human podocyte cell lines to determine β-arrestin's involvement in calcium signaling and cytoskeletal reorganization and Dahl SS rats to investigate the chronic effects of TRV administration on glomerular health. Our experiments indicate that the TRV-activated β-arrestin pathway promotes the rapid elevation of intracellular Ca2+ in a dose-dependent manner. Interestingly, the amplitude of β-arrestin-mediated Ca2+ influx was significantly higher than the response to similar Ang II concentrations. Single-channel analyses show rapid activation of transient receptor potential canonical (TRPC) channels following acute TRV application. Furthermore, the pharmacological blockade of TRPC6 significantly attenuated the β-arrestin-mediated Ca2+ influx. Additionally, prolonged activation of the β-arrestin pathway in podocytes resulted in pathological actin cytoskeleton rearrangements, higher apoptotic cell markers, and augmented glomerular damage. TRV-activated β-arrestin signaling in podocytes may promote TRPC6 channel-mediated Ca2+ influx, foot process effacement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment requires further investigation to assess the balance of the benefits versus possible deleterious effects and off-target damage.
Topics: Rats; Animals; Humans; Podocytes; TRPC6 Cation Channel; Calcium; beta-Arrestins; Angiotensin Receptor Antagonists; Rats, Inbred Dahl; Angiotensin-Converting Enzyme Inhibitors; Kidney Diseases; Hypertension; TRPC Cation Channels
PubMed: 38051199
DOI: 10.1042/CS20230313 -
Molecular Biomedicine Dec 2023G protein-coupled receptors (GPCRs) are versatile and vital proteins involved in a wide array of physiological processes and responses, such as sensory perception (e.g.,... (Review)
Review
G protein-coupled receptors (GPCRs) are versatile and vital proteins involved in a wide array of physiological processes and responses, such as sensory perception (e.g., vision, taste, and smell), immune response, hormone regulation, and neurotransmission. Their diverse and essential roles in the body make them a significant focus for pharmaceutical research and drug development. Currently, approximately 35% of marketed drugs directly target GPCRs, underscoring their prominence as therapeutic targets. Recent advances in structural biology have substantially deepened our understanding of GPCR activation mechanisms and interactions with G-protein and arrestin signaling pathways. This review offers an in-depth exploration of both traditional and recent methods in GPCR structure analysis. It presents structure-based insights into ligand recognition and receptor activation mechanisms and delves deeper into the mechanisms of canonical and noncanonical signaling pathways downstream of GPCRs. Furthermore, it highlights recent advancements in GPCR-related drug discovery and development. Particular emphasis is placed on GPCR selective drugs, allosteric and biased signaling, polyphamarcology, and antibody drugs. Our goal is to provide researchers with a thorough and updated understanding of GPCR structure determination, signaling pathway investigation, and drug development. This foundation aims to propel forward-thinking therapeutic approaches that target GPCRs, drawing upon the latest insights into GPCR ligand selectivity, activation, and biased signaling mechanisms.
PubMed: 38047990
DOI: 10.1186/s43556-023-00156-w -
Modulating signalling lifetime to optimise a prototypical animal opsin for optogenetic applications.Pflugers Archiv : European Journal of... Dec 2023Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal...
Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal opsins offer excellent photosensitivity, but their temporal resolution can be limited by long photoresponse duration when expressed outside their native cellular environment. Here, we explore methods for addressing this limitation for a prototypical animal opsin (human rod opsin) in HEK293T cells. We find that the application of the canonical rhodopsin kinase (GRK1)/visual arrestin signal termination mechanism to this problem is complicated by a generalised suppressive effect of GRK1 expression. This attenuation can be overcome using phosphorylation-independent mutants of arrestin, especially when these are tethered to the opsin protein. We further show that point mutations targeting the Schiff base stability of the opsin can also reduce signalling lifetime. Finally, we apply one such mutation (E122Q) to improve the temporal fidelity of restored visual responses following ectopic opsin expression in the inner retina of a mouse model of retinal degeneration (rd1). Our results reveal that these two strategies (targeting either arrestin binding or Schiff-base hydrolysis) can produce more time-delimited opsin signalling under heterologous expression and establish the potential of this approach to improve optogenetic performance.
Topics: Animals; Mice; Humans; Rod Opsins; Opsins; Optogenetics; HEK293 Cells; Arrestins
PubMed: 38036775
DOI: 10.1007/s00424-023-02879-9 -
Nature Communications Nov 2023β-Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well...
β-Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in βarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of βarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of βarr1 during activation using F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different βarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of βarr1 that can be intricately modulated during signaling.
Topics: beta-Arrestin 1; beta-Arrestins; Signal Transduction; Receptors, G-Protein-Coupled; beta-Arrestin 2; Phosphorylation
PubMed: 38030602
DOI: 10.1038/s41467-023-43694-1 -
Annual Review of Physiology Feb 2024The harmful side effects of opioid drugs such as respiratory depression, tolerance, dependence, and abuse potential have limited the therapeutic utility of opioids for... (Review)
Review
The harmful side effects of opioid drugs such as respiratory depression, tolerance, dependence, and abuse potential have limited the therapeutic utility of opioids for their entire clinical history. However, no previous attempt to develop effective pain drugs that substantially ameliorate these effects has succeeded, and the current opioid epidemic affirms that they are a greater hindrance to the field of pain management than ever. Recent attempts at new opioid development have sought to reduce these side effects by minimizing engagement of the regulatory protein arrestin-3 at the mu-opioid receptor, but there is significant controversy around this approach. Here, we discuss the ongoing effort to develop safer opioids and its relevant historical context. We propose a new model that reconciles results previously assumed to be in direct conflict to explain how different signaling profiles at the mu-opioid receptor contribute to opioid tolerance and dependence. Our goal is for this framework to inform the search for a new generation of lower liability opioid analgesics.
Topics: Humans; Analgesics, Opioid; Drug Tolerance; Signal Transduction
PubMed: 38029388
DOI: 10.1146/annurev-physiol-042022-015914 -
Frontiers in Pharmacology 2023Modern methods for quantifying signaling bias at G protein-coupled receptors (GPCRs) rely on using a single β-arrestin isoform. However, it is increasingly appreciated...
Modern methods for quantifying signaling bias at G protein-coupled receptors (GPCRs) rely on using a single β-arrestin isoform. However, it is increasingly appreciated that the two β-arrestin isoforms have unique roles, requiring the ability to assess β-arrestin isoform preference. Thus, methods are needed to efficiently screen the recruitment of both β-arrestin isoforms as they compete for a target GPCR in cells. We used molecular cloning to develop fusion proteins of the δ-opioid receptor (δOR), β-arrestin 1, and β-arrestin 2 to fragments of click beetle green and click beetle red luciferases. In this assay architecture, recruitment of either β-arrestin 1 or 2 to the δOR generates a spectrally distinct bioluminescent signal, allowing us to co-transfect all three constructs into cells prior to agonist challenge. We demonstrate that our new assay, named "ClickArr," is a live-cell assay that simultaneously reports the recruitment of both β-arrestin isoforms as they compete for interaction with the δOR. We further find that the partial δOR agonist TAN67 has a significant efficacy bias for β-arrestin 2 over β-arrestin 1 when recruitment is normalized to the reference agonist leu-enkephalin. We confirm that ClickArr reports this bias when run either as a high-throughput endpoint or high-throughput kinetic assay, and cross-validate this result using the PathHunter assay, an orthogonal commercial assay for reporting β-arrestin recruitment to the δOR. Our results suggest that agonist:GPCR complexes can have relative β-arrestin isoform bias, a novel signaling bias that may potentially open up a new dimension for drug development.
PubMed: 38027002
DOI: 10.3389/fphar.2023.1295518 -
Pharmacology Research & Perspectives Dec 2023The rapid structural evolution and emergence of novel synthetic cannabinoid receptor agonists (SCRAs) in the recreational market remains a key public health concern....
The rapid structural evolution and emergence of novel synthetic cannabinoid receptor agonists (SCRAs) in the recreational market remains a key public health concern. Despite representing one of the largest classes of new psychoactive substances, pharmacological data on new SCRAs is limited, particularly at the cannabinoid CB receptor (CB ). Hence, the current study aimed to characterize the molecular pharmacology of a structurally diverse panel of SCRAs at CB , including 4-cyano MPP-BUT7AICA, 4F-MDMB-BUTINACA, AMB-FUBINACA, JWH-018, MDMB-4en-PINACA, and XLR-11. The activity of SCRAs was assessed in a battery of in vitro assays in CB -expressing HEK 293 cells: G protein activation (Gα and Gα ), phosphorylation of ERK1/2, and β-arrestin 1/2 translocation. The activity profiles of the ligands were further evaluated using the operational analysis to identify ligand bias. All SCRAs activated the CB signaling pathways in a concentration-dependent manner, although with varying potencies and efficacies. Despite the detection of numerous instances of statistically significant bias, compound activities generally appeared only subtly distinct in comparison with the reference ligand, CP55940. In contrast, the phytocannabinoid THC exhibited an activity profile distinct from the SCRAs; most notably in the translocation of β-arrestins. These findings demonstrate that CB is able to accommodate a structurally diverse array of SCRAs to generate canonical agonist activity. Further research is required to elucidate whether the activation of CB contributes to the toxicity of these compounds.
Topics: Humans; Cannabinoid Receptor Agonists; Receptors, Cannabinoid; Ligands; HEK293 Cells; Cannabinoids
PubMed: 38018694
DOI: 10.1002/prp2.1157 -
BioRxiv : the Preprint Server For... Oct 2023Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G...
Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein-coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen-binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands. The neurokinin-1 receptor (NK1R), a GPCR targeted for the treatment of pain, is activated by peptide agonists such as Substance P (SP) and neurokinin A (NKA), which induce signaling through multiple pathways (G, G and β-arrestin). In this study, we investigated whether conjugating NK1R ligands with Nbs that bind to a separate location on the receptor would provide chimeric compounds with distinctive signaling properties. We employed sortase A-mediated ligation to generate several conjugates consisting of Nbs linked to NK1R ligands. Many of these conjugates exhibited divergent and unexpected signaling properties and transcriptional outputs. For example, some Nb-NKA conjugates showed enhanced receptor binding capacity, high potency partial agonism, prolonged cAMP production, and an increase in transcriptional output associated with G signaling; whereas other conjugates were virtually inactive. Nanobody conjugation caused only minor alterations in ligand-induced upstream G signaling with unexpected enhancements in transcriptional (downstream) responses. Our findings underscore the potential of nanobody conjugation for providing compounds with advantageous properties such as biased agonism, prolonged duration of action, and enhanced transcriptional responses. These compounds hold promise not only for facilitating fundamental research on GPCR signal transduction mechanisms but also for the development of more potent and enduring therapeutics.
PubMed: 37986858
DOI: 10.1101/2023.10.08.561411 -
The Journal of Biological Chemistry Dec 2023G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3...
G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3 ubiquitin ligases. E3 ligases have been linked to agonist-stimulated ubiquitination of GPCRs via simultaneous binding to βarrestins. In addition, βarrestins have been suggested to assist E3 ligases for ubiquitination of key effector molecules, yet mechanistic insight is lacking. Here, we developed an in vitro reconstituted system and show that βarrestin1 (βarr1) serves as an adaptor between the effector protein signal-transducing adaptor molecule 1 (STAM1) and the E3 ligase atrophin-interacting protein 4. Via mass spectrometry, we identified seven lysine residues within STAM1 that are ubiquitinated and several types of ubiquitin linkages. We provide evidence that βarr1 facilitates the formation of linear polyubiquitin chains at lysine residue 136 on STAM1. This lysine residue is important for stabilizing the βarr1:STAM1 interaction in cells following GPCR activation. Our study identifies atrophin-interacting protein 4 as only the second E3 ligase known to conjugate linear polyubiquitin chains and a possible role for linear ubiquitin chains in GPCR signaling and trafficking.
Topics: Lysine; Polyubiquitin; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; beta-Arrestin 1
PubMed: 37981209
DOI: 10.1016/j.jbc.2023.105474