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International Journal of Molecular... Jun 2024The first member of the arrestin family, visual arrestin-1, was discovered in the late 1970s. Later, the other three mammalian subtypes were identified and cloned. The... (Review)
Review
The first member of the arrestin family, visual arrestin-1, was discovered in the late 1970s. Later, the other three mammalian subtypes were identified and cloned. The first described function was regulation of G protein-coupled receptor (GPCR) signaling: arrestins bind active phosphorylated GPCRs, blocking their coupling to G proteins. It was later discovered that receptor-bound and free arrestins interact with numerous proteins, regulating GPCR trafficking and various signaling pathways, including those that determine cell fate. Arrestins have no enzymatic activity; they function by organizing multi-protein complexes and localizing their interaction partners to particular cellular compartments. Today we understand the molecular mechanism of arrestin interactions with GPCRs better than the mechanisms underlying other functions. However, even limited knowledge enabled the construction of signaling-biased arrestin mutants and extraction of biologically active monofunctional peptides from these multifunctional proteins. Manipulation of cellular signaling with arrestin-based tools has research and likely therapeutic potential: re-engineered proteins and their parts can produce effects that conventional small-molecule drugs cannot.
Topics: Humans; Animals; Arrestins; Signal Transduction; Receptors, G-Protein-Coupled; Protein Binding; Phosphorylation
PubMed: 38892473
DOI: 10.3390/ijms25116284 -
Structure (London, England : 1993) Jun 2024Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in...
Arrestins interact with phosphorylated G protein-coupled receptors (GPCRs) and regulate the homologous desensitization and internalization of GPCRs. The gate loop in arrestins is a critical region for both stabilization of the basal state and interaction with phosphorylated receptors. We investigated the roles of specific residues in the gate loop (K292, K294, and H295) using β-arrestin-1 and phosphorylated C-tail peptide of vasopressin receptor type 2 (V2Rpp) as a model system. We measured the binding affinity of V2Rpp and analyzed conformational dynamics of β-arrestin-1. Our results suggest that K294 plays a critical role in the interaction with V2Rpp without influencing the overall conformation of the V2Rpp-bound state. The residues K292 and H295 contribute to the stability of the polar core in the basal state and form a specific conformation of the finger loop in the V2Rpp-bound state.
PubMed: 38889722
DOI: 10.1016/j.str.2024.05.014 -
The Journal of Neuroscience : the... Jun 2024During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its...
During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src-family-kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that β-arrestin1 and β-arrestin2 (β-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that β-arrestins are expressed in rat commissural neurons. We also found that Smo, β-arrestins and SFKs form a tripartite complex, with the complex formation dependent on β-arrestins. β-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that β-arrestins are required to activate Src kinase downstream of Shh. β-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant negative β-arrestins, β-arrestin1 V53D which blocks the internalization of Smo and β-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo the expression of these dominant negative β-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that β-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance. The correct guidance of axons is important for the formation of the nervous system. Sonic hedgehog (Shh)-mediated axon guidance relies on the activation of Src family kinases (SFKs) downstream of the atypical G protein-coupled receptor (GPCR) Smoothened (Smo). How SFKs are activated downstream of Smo was unknown. In this study, we found that β-arrestin1 and 2 (β-arrestins) serve as scaffolding proteins between Smo and SFKs. We also found that β-arrestins are required for the activation of SFKs. Knocking down β-arrestins or expressing dominant negative β-arrestins caused loss of Shh-mediated attraction of commissural axons. In vivo, the expression of dominant negative β-arrestins caused commissural axon guidance defects. Our work identifies for the first time a role for β-arrestins in axon guidance.
PubMed: 38886055
DOI: 10.1523/JNEUROSCI.0261-24.2024 -
Fungal Genetics and Biology : FG & B Jun 2024In the filamentous fungus Aspergillus oryzae, large amounts of amylolytic enzymes are inducibly produced by isomaltose, which is converted from maltose incorporated via...
Glucose-induced endocytic degradation of the maltose transporter MalP is mediated through ubiquitination by the HECT-ubiquitin ligase HulA and its adaptor CreD in Aspergillus oryzae.
In the filamentous fungus Aspergillus oryzae, large amounts of amylolytic enzymes are inducibly produced by isomaltose, which is converted from maltose incorporated via the maltose transporter MalP. In contrast, the preferred sugar glucose strongly represses the expression of both amylolytic and malP genes through carbon catabolite repression. Simultaneously, the addition of glucose triggers the endocytic degradation of MalP on the plasma membrane. In budding yeast, the signal-dependent ubiquitin modification of plasma membrane transporters leads to selective endocytosis into the vacuole for degradation. In addition, during glucose-induced MalP degradation, the homologous of E6AP C-terminus-type E3 ubiquitin ligase (HulA) is responsible for the ubiquitin modification of MalP, and the arrestin-like protein CreD is required for HulA targeting. Although CreD-mediated MalP internalization occurs in response to glucose, the mechanism by which CreD regulates HulA-dependent MalP ubiquitination remains unclear. In this study, we demonstrated that three (P/L)PxY motifs present in the CreD protein are essential for functioning as HulA adaptors so that HulA can recognize MalP in response to glucose stimulation, enabling MalP internalization. Furthermore, four lysine residues (three highly conserved among Aspergillus species and yeast and one conserved among Aspergillus species) of CreD were found to be necessary for its ubiquitination, resulting in efficient glucose-induced MalP endocytosis. The results of this study pave the way for elucidating the regulatory mechanism of MalP endocytic degradation through ubiquitination by the HulA-CreD complex at the molecular level.
PubMed: 38885923
DOI: 10.1016/j.fgb.2024.103909 -
Journal of Medicinal Chemistry Jun 2024The human orphan G protein-coupled receptor GPR18, activated by Δ-tetrahydrocannabinol (THC), constitutes a promising drug target in immunology and cancer. However,...
The human orphan G protein-coupled receptor GPR18, activated by Δ-tetrahydrocannabinol (THC), constitutes a promising drug target in immunology and cancer. However, studies on GPR18 are hampered by the lack of suitable tool compounds. In the present study, potent and selective GPR18 agonists were developed showing low nanomolar potency at human and mouse GPR18, determined in β-arrestin recruitment assays. Structure-activity relationships were analyzed, and selectivity versus cannabinoid (CB) and CB-like receptors was assessed. Compound (PSB-KK1415, EC 19.1 nM) was the most potent GPR18 agonist showing at least 25-fold selectivity versus CB receptors. The most selective GPR18 agonist (PSB-KK1445, EC 45.4 nM) displayed >200-fold selectivity versus both CB receptor subtypes, GPR55, and GPR183. The new GPR18 agonists showed minimal species differences, while THC acted as a weak partial agonist at the mouse receptor. The newly discovered compounds represent the most potent and selective GPR18 agonists reported to date.
Topics: Receptors, G-Protein-Coupled; Humans; Animals; Structure-Activity Relationship; Mice; Neoplasms; HEK293 Cells; Receptors, Cannabinoid; Dronabinol
PubMed: 38885438
DOI: 10.1021/acs.jmedchem.3c02423 -
IScience Jun 2024Oxytocin plays critical roles in the brain as a neuromodulator, regulating social and other affective behavior. However, the regulatory mechanisms controlling oxytocin...
Oxytocin plays critical roles in the brain as a neuromodulator, regulating social and other affective behavior. However, the regulatory mechanisms controlling oxytocin receptor (OXTR) signaling in neurons remain unexplored. In this study, we have identified robust and rapid-onset desensitization of OXTR response in multiple regions of the mouse brain. Both cell autonomous spiking response and presynaptic activation undergo similar agonist-induced desensitization. G-protein-coupled receptor kinases (GRK) GRK2, GRK3, and GRK6 are recruited to the activated OXTR in neurons, followed by recruitment of β-arrestin-1 and -2. Neuronal OXTR desensitization was impaired by suppression of GRK2/3/6 kinase activity but remained unaltered with double knockout of β-arrestin-1 and -2. Additionally, we observed robust agonist-induced internalization of neuronal OXTR and its Rab5-dependent recruitment to early endosomes, which was impaired by GRK2/3/6 inhibition. This work defines distinctive aspects of the mechanisms governing OXTR desensitization and internalization in neurons compared to prior studies in heterologous cells.
PubMed: 38883814
DOI: 10.1016/j.isci.2024.110047 -
Archives of Toxicology Jun 20242-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of...
2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on μ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (β-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.
PubMed: 38877156
DOI: 10.1007/s00204-024-03774-7 -
Nature Metabolism Jun 2024Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes. Yet both activation and inhibition of the glucose-dependent insulinotropic...
Incretin-based therapies are highly successful in combatting obesity and type 2 diabetes. Yet both activation and inhibition of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in combination with glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) activation have resulted in similar clinical outcomes, as demonstrated by the GIPR-GLP-1R co-agonist tirzepatide and AMG-133 (ref. ) combining GIPR antagonism with GLP-1R agonism. This underlines the importance of a better understanding of the GIP system. Here we show the necessity of β-arrestin recruitment for GIPR function, by combining in vitro pharmacological characterization of 47 GIPR variants with burden testing of clinical phenotypes and in vivo studies. Burden testing of variants with distinct ligand-binding capacity, Gs activation (cyclic adenosine monophosphate production) and β-arrestin 2 recruitment and internalization shows that unlike variants solely impaired in Gs signalling, variants impaired in both Gs and β-arrestin 2 recruitment contribute to lower adiposity-related traits. Endosomal Gs-mediated signalling of the variants shows a β-arrestin dependency and genetic ablation of β-arrestin 2 impairs cyclic adenosine monophosphate production and decreases GIP efficacy on glucose control in male mice. This study highlights a crucial impact of β-arrestins in regulating GIPR signalling and overall preservation of biological activity that may facilitate new developments in therapeutic targeting of the GIPR system.
PubMed: 38871982
DOI: 10.1038/s42255-024-01061-4 -
European Journal of Pharmacology Jun 2024The Ca-sensing receptor (CaSR) is a G-protein-coupled receptor activated by elevated concentrations of extracellular Ca, and was initially known for its regulation of... (Review)
Review
The Ca-sensing receptor (CaSR) is a G-protein-coupled receptor activated by elevated concentrations of extracellular Ca, and was initially known for its regulation of parathyroid hormone (PTH) release. Ubiquitous expression of CaSR in different tissues and organs was later noted and CaSR participation in various physiological functions was demonstrated. Accumulating evidence has suggested that CaSR functionally interacts with transient receptor potential (TRP) channels, which are mostly non-selective cation channels involved in sensing temperature, pain and stress. This review describes the interactions of CaSR with TRP channels in diverse cell types to trigger a variety of biological responses. CaSR has been known to interact with different types of G proteins. Possible involvements of G proteins, other signaling and scaffolding protein intermediates in CaSR-TRP interaction are discussed. In addition, an attempt will be made to extend the current understanding of biased agonism of CaSR.
PubMed: 38857682
DOI: 10.1016/j.ejphar.2024.176717 -
Bioorganic Chemistry May 2024Protease-activated receptor 2 (PAR2) has garnered attention as a potential therapeutic target in breast cancer. PAR2 is implicated in the activation of extracellular...
Protease-activated receptor 2 (PAR2) has garnered attention as a potential therapeutic target in breast cancer. PAR2 is implicated in the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) via G protein and beta-arrestin pathways, contributing to the proliferation and metastasis of breast cancer cells. Despite the recognized role of PAR2 in breast cancer progression, clinically effective PAR2 antagonists remain elusive. To address this unmet clinical need, we synthesized and evaluated a series of novel compounds that target the orthosteric site of PAR2. Using in silico docking simulations, we identified compound 9a, an optimized derivative of compound 1a ((S)-N-(1-(benzylamino)-1-oxo-3-phenylpropan-2-yl)benzamide), which exhibited enhanced PAR2 antagonistic activity. Subsequent molecular dynamics simulations comparing 9a with the partial agonist 9d revealed that variations in ligand-induced conformational changes and interactions dictated whether the compound acted as an antagonist or agonist of PAR2. The results of this study suggest that further development of 9a could contribute to the advancement of PAR2 antagonists as potential therapeutic agents for breast cancer.
PubMed: 38850590
DOI: 10.1016/j.bioorg.2024.107496