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PloS One 2024Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), which are then sorted for recycling or degradation. We investigated...
Activated GPCRs are phosphorylated and internalized mostly via clathrin-mediated endocytosis (CME), which are then sorted for recycling or degradation. We investigated how differential activation of the same GPCR affects its endocytic trafficking in vivo using rhodopsin as a model in pupal photoreceptors of flies expressing mCherry-tagged rhodopsin 1 (Rh1-mC) or GFP-tagged arrestin 1 (Arr1-GFP). Upon blue light stimulation, activated Rh1 recruited Arr1-GFP to the rhabdomere, which became co-internalized and accumulated in cytoplasmic vesicles of photoreceptors. This internalization was eliminated in shits1 mutants affecting dynamin. Moreover, it was blocked by either rdgA or rdgB mutations affecting the PIP2 biosynthesis. Together, the blue light-initiated internalization of Rh1 and Arr1 belongs to CME. Green light stimulation also triggered the internalization and accumulation of activated Rh1-mC in the cytoplasm but with faster kinetics. Importantly, Arr1-GFP was also recruited to the rhabdomere but not co-internalized with Rh1-mC. This endocytosis was not affected in shits1 nor rdgA mutants, indicating it is not CME. We explored the fate of internalized Rh1-mC following CME and observed it remained in cytoplasmic vesicles following 30 min of dark adaptation. In contrast, in the non-CME Rh1-mC appeared readily recycled back to the rhabdomere within five min of dark treatment. This faster recycling may be regulated by rhodopsin phosphatase, RdgC. Together, we demonstrate two distinct endocytic and recycling mechanisms of Rh1 via two light stimulations. It appears that each stimulation triggers a distinct conformation leading to different phosphorylation patterns of Rh1 capable of recruiting Arr1 to rhabdomeres. However, a more stable interaction leads to the co-internalization of Arr1 that orchestrates CME. A stronger Arr1 association appears to impede the recycling of the phosphorylated Rh1 by preventing the recruitment of RdgC. We conclude that conformations of activated rhodopsin determine the downstream outputs upon phosphorylation that confers differential protein-protein interactions.
Topics: Rhodopsin; Animals; Endocytosis; Phosphorylation; Protein Transport; Light; Mutation; Photoreceptor Cells, Invertebrate; Drosophila melanogaster; Clathrin
PubMed: 38848405
DOI: 10.1371/journal.pone.0303882 -
BMC Musculoskeletal Disorders Jun 2024Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative joint disorder characterized by extracellular matrix degeneration and inflammatory response of...
OBJECTIVE
Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative joint disorder characterized by extracellular matrix degeneration and inflammatory response of condylar cartilage. β-arrestin2 is an important regulator of inflammation response, while its role in TMJOA remains unknown. The objective of this study was to investigate the role of β-arrestin2 in the development of TMJOA at the early stage and the underlying mechanism.
METHODS
A unilateral anterior crossbite (UAC) model was established on eight-week-old wild-type (WT) and β-arrestin2 deficiency mice to simulate the progression of TMJOA. Hematoxylin-eosin (HE) staining and microcomputed tomography (micro-CT) analysis were used for histological and radiographic assessment. Immunohistochemistry was performed to detect the expression of inflammatory and degradative cytokines, as well as autophagy related factors. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay was carried out to assess chondrocyte apoptosis.
RESULTS
The loss of β-arrestin2 aggravated cartilage degeneration and subchondral bone destruction in the model of TMJOA at the early stage. Furthermore, in UAC groups, the expressions of degradative (Col-X) and inflammatory (TNF-α and IL-1β) factors in condylar cartilage were increased in β-arrestin2 null mice compared with WT mice. Moreover, the loss of β-arrestin2 promoted apoptosis and autophagic process of chondrocytes at the early stage of TMJOA.
CONCLUSION
In conclusion, we demonstrated for the first time that β-arrestin2 plays a protective role in the development of TMJOA at the early stage, probably by inhibiting apoptosis and autophagic process of chondrocytes. Therefore, β-arrestin2 might be a potential therapeutic target for TMJOA, providing a new insight for the treatment of TMJOA at the early stage.
Topics: Animals; Osteoarthritis; beta-Arrestin 2; Cartilage, Articular; Mandibular Condyle; Mice; Mice, Knockout; Temporomandibular Joint Disorders; Disease Models, Animal; Chondrocytes; Mice, Inbred C57BL; Apoptosis; Temporomandibular Joint; Male; X-Ray Microtomography; Autophagy
PubMed: 38844905
DOI: 10.1186/s12891-024-07558-z -
European Journal of Pharmacology Aug 2024β-arrestin2 is a versatile protein for signaling transduction in brain physiology and pathology. Herein, we investigated the involvement of β-arrestin2 in...
β-arrestin2 is a versatile protein for signaling transduction in brain physiology and pathology. Herein, we investigated the involvement of β-arrestin2 in pharmacological effects of fluoxetine for depression. A chronic mild stress (CMS) model was established using wild-type (WT) and β-arrestin2 mice. Behavioral results demonstrated that CMS mice showed increased immobility time in the tail suspension test and forced swimming test, elevated concentrations of pro-inflammatory factors in peripheral blood, increased expression of pyroptosis-related proteins, and increased co-labeling of glial fibrillary acidic protein and Caspase1 p10 in the hippocampus compared to the CON group. Treatment with fluoxetine (FLX) ameliorated these conditions. However, compared with the β-arrestin2 CMS group, these results of the β-arrestin2 CMS + FLX group showed no significant changes. These results suggested that the above effects of FLX could be eliminated by knocking out β-arrestin2. Mass spectrometry implying that FLX promoted the binding of β-arrestin2 to the NLRP2 inflammasome of depressed mice. Subsequently, the results of the cellular experiments suggested that the 5HT receptor antagonist may attenuate L-kynurenine + ATP-induced cell pyroptosis by attenuating NLRP2 binding to β-arrestin2. We further found that the lack of β-arrestin2 eliminated the anti-pyroptosis effect of fluoxetine. In conclusion, β-arrestin2 is an essential protein for fluoxetine to alleviate pyroptosis in the hippocampal astrocytes of CMS mice. Mechanistically, we found that the 5-HTR-β-arrestin2-NLRP2 axis is vital for maintaining the antidepressant effects of fluoxetine.
Topics: Animals; Fluoxetine; Pyroptosis; Disease Models, Animal; beta-Arrestin 2; Mice; Depression; Stress, Psychological; Male; Antidepressive Agents; Astrocytes; Mice, Inbred C57BL; Hippocampus; Mice, Knockout; Behavior, Animal; Inflammasomes; Chronic Disease
PubMed: 38834095
DOI: 10.1016/j.ejphar.2024.176693 -
Research Square May 2024Δ-tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite...
Δ-tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite stimulation and sleep apnea relief. THC's psychoactive actions are mediated primarily by the cannabinoid receptor CB. Here, we determine the cryo-EM structure of HU210, a THC analog and widely used tool compound, bound to CB and its primary transducer, G. We leverage this structure for docking and 1,000 ns molecular dynamics simulations of THC and 10 structural analogs delineating their spatiotemporal interactions at the molecular level. Furthermore, we pharmacologically profile their recruitment of G and β-arrestins and reversibility of binding from an active complex. By combining detailed CB structural information with molecular models and signaling data we uncover the differential spatiotemporal interactions these ligands make to receptors governing potency, efficacy, bias and kinetics. This may help explain the actions of abused substances, advance fundamental receptor activation studies and design better medicines.
PubMed: 38826401
DOI: 10.21203/rs.3.rs-4277209/v1 -
ACS Central Science May 2024We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the...
We report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CBR) selective inverse agonists ()- and ()-, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the -dimethylheptyl side chain. Epimer ()- exhibits high affinity for CBR with = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CBR independent of linker and fluorophore substitution. Ligands ()-, ()-, and their derivatives act as inverse agonists in CBR-mediated cAMP as well as G protein recruitment assays and do not trigger β-arrestin-receptor association. Furthermore, no receptor activation was detected in live cell ERK phosphorylation and Ca-release assays. Confocal fluorescence imaging experiments with ()- (Alexa488) and ()- (Alexa647) probes employing BV-2 microglial cells visualized CBR expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp258 and thereby stabilize CBR in its inactive state.
PubMed: 38799662
DOI: 10.1021/acscentsci.3c01461 -
BioRxiv : the Preprint Server For... May 2024respond to mating pheromone through the GPCRs Ste2 and Ste3, which promote growth of a mating projection in response to ligand binding. This commitment to mating is...
UNLABELLED
respond to mating pheromone through the GPCRs Ste2 and Ste3, which promote growth of a mating projection in response to ligand binding. This commitment to mating is nutritionally and energetically taxing, and so we hypothesized that the cell may suppress mating signaling during starvation. We set out to investigate negative regulators of the mating pathway in nutritionally depleted environments. Here, we report that nutrient deprivation led to loss of Ste2 from the plasma membrane. Recapitulating this effect with nitrogen starvation led us to hypothesize that it was due to TORC1 signaling. Rapamycin inhibition of TORC1 impacted membrane levels of all yeast GPCRs. Inhibition of TORC1 also dampened mating pathway output. Deletion analysis revealed that TORC1 repression leads to α-arrestin-directed CME through TORC2-Ypk1 signaling. We then set out to determine whether major downstream effectors of the TOR complexes also downregulate pathway output during mating. We found that autophagy contributes to pathway downregulation through analysis of strains lacking . We also show that Ypk1 significantly reduced pathway output. Thus, both autophagy machinery and TORC2-Ypk1 signaling serve as attenuators of pheromone signaling during mating. Altogether, we demonstrate that the stress-responsive TOR complexes coordinate GPCR endocytosis and reduce the magnitude of pheromone signaling, in ligand-independent and ligand-dependent contexts.
ONE SENTENCE SUMMARY
TOR signaling regulates the localization of all GPCRs during starvation and suppress the mating pathway in the presence and absence of ligand.
PubMed: 38798445
DOI: 10.1101/2024.05.09.593412 -
International Journal of Molecular... May 2024Lysophosphatidic acid (LPA) type 3 (LPA) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids....
Lysophosphatidic acid (LPA) type 3 (LPA) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were expressed in T-REx HEK293 cells, and the consequences of the substitutions were analyzed employing different functional parameters. Agonist- and LPA-mediated receptor phosphorylation was diminished in the IL3 and Ctail mutants and essentially abolished in the IL3/Ctail mutant, confirming that the main phosphorylation sites are present in both domains and their role in receptor phosphorylation eliminated by substitution and distributed in both domains. The WT and mutant receptors increased intracellular calcium and ERK 1/2 phosphorylation in response to LPA and PMA. The agonist, Ki16425, diminished baseline intracellular calcium, which suggests some receptor endogenous activity. Similarly, baseline ERK1/2 phosphorylation was diminished by Ki16425. An increase in baseline ERK phosphorylation was detected in the IL3/Ctail mutant. LPA and PMA-induced receptor interaction with β-arrestin 2 and LPA internalization were severely diminished in cells expressing the mutants. Mutant-expressing cells also exhibit increased baseline proliferation and response to different stimuli, which were inhibited by the antagonist Ki16425, suggesting a role of LPA receptors in this process. Migration in response to different attractants was markedly increased in the Ctail mutant, which the Ki16425 antagonist also attenuated. Our data experimentally show that receptor phosphorylation in the distinct domains is relevant for LPA receptor function.
Topics: Humans; Phosphorylation; Receptors, Lysophosphatidic Acid; HEK293 Cells; Lysophospholipids; Signal Transduction; Calcium; Endocytosis; Mutation
PubMed: 38791546
DOI: 10.3390/ijms25105508 -
Biomolecules May 2024Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using...
Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.
Topics: Humans; Tetraspanin 30; Extracellular Vesicles; Tumor Suppressor Protein p53; HEK293 Cells; Cell Line, Tumor; Apoptosis; Cell Proliferation; Endosomal Sorting Complexes Required for Transport; Transcription Factors; DNA-Binding Proteins; RNA, Messenger; Lysosomal-Associated Membrane Protein 1
PubMed: 38785998
DOI: 10.3390/biom14050591 -
Cell Death & Disease May 2024Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix...
Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ET) and B (ET) receptors, ET-1 enables the recruitment of β-arrestin1 (β-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might "educate" human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETR/β-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin β1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETR or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETR antagonists.
Topics: Humans; Female; Ovarian Neoplasms; beta-Arrestin 1; Cancer-Associated Fibroblasts; Cell Line, Tumor; Podosomes; Endothelin-1; Neoplasm Metastasis; Receptor, Endothelin A; Signal Transduction; Extracellular Matrix; Cell Movement; Cell Proliferation; Animals; Fibroblasts; Neoplasm Invasiveness
PubMed: 38777849
DOI: 10.1038/s41419-024-06730-6 -
BioRxiv : the Preprint Server For... May 2024Rab GTPases act as molecular switches to regulate organelle homeostasis and membrane trafficking. Rab6 plays a central role in regulating cargo flux through the Golgi...
Rab GTPases act as molecular switches to regulate organelle homeostasis and membrane trafficking. Rab6 plays a central role in regulating cargo flux through the Golgi and is activated via nucleotide exchange by the Ric1-Rgp1 protein complex. Ric1-Rgp1 is conserved throughout eukaryotes but the structural and mechanistic basis for its function has not been established. Here we report the cryoEM structure of a Ric1-Rgp1-Rab6 complex representing a key intermediate of the nucleotide exchange reaction. This structure reveals the overall architecture of the complex and enabled us to identify interactions critical for proper recognition and activation of Rab6 on the Golgi membrane surface. Ric1-Rgp1 interacts with the nucleotide-binding domain of Rab6 using an uncharacterized helical domain, which we establish as a novel RabGEF domain by identifying residues required for Rab6 nucleotide exchange. Unexpectedly, the complex uses an arrestin fold to interact with the Rab6 hypervariable domain, indicating that interactions with the unstructured C-terminal regions of Rab GTPases may be a common specificity mechanism used by their activators. Collectively, our findings provide a detailed mechanistic understanding of regulated Rab6 activation at the Golgi.
PubMed: 38766083
DOI: 10.1101/2024.05.06.592747