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Journal of Molecular Endocrinology Jul 2024The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer....
The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding the spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, β-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE), which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs-biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane-initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This, in turn, could highlight important considerations for future selective targeting of EP2 signalling pathways.
Topics: Humans; Receptors, Prostaglandin E, EP2 Subtype; Signal Transduction; Female; Pregnancy; Cyclic AMP; GTP-Binding Proteins; Endosomes; Protein Transport; Myometrium; Alprostadil; HEK293 Cells; Animals
PubMed: 38639976
DOI: 10.1530/JME-23-0153 -
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 -
The Journal of Pharmacology and... May 2024opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical...
opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[()--((2,5)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-,-diethylbenzamide (SNC80)/4-[(*)--((2*,5*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-,-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a -arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious -arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.
Topics: Receptors, Opioid, delta; Animals; Mice; Male; Humans; Spiro Compounds; Piperazines; Mice, Inbred C57BL; Molecular Docking Simulation; Benzamides; Cricetulus; beta-Arrestins; HEK293 Cells; CHO Cells
PubMed: 38621994
DOI: 10.1124/jpet.123.001735 -
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 -
Journal of Oral Biosciences Jun 2024Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling...
OBJECTIVES
Local anesthetics act on G protein-coupled receptors (GPCRs); thus, their potential as allosteric modulators of GPCRs has attracted attention. Intracellular signaling via GPCRs involves both G-protein- and β-arrestin-mediated pathways. To determine the effects of local anesthetics on muscarinic acetylcholine receptors (mAChR), a family of GPCRs, we analyzed the effects of local anesthetics on mAChR-mediated Ca responses and formation of receptor-β-arrestin complexes in the HSY human parotid cell line.
METHODS
Ca responses were monitored by fura-2 spectrofluorimetry. Ligand-induced interactions between mAChR and β-arrestin were examined using a β-arrestin GPCR assay kit.
RESULTS
Lidocaine reduced mAChR-mediated Ca responses but did not change the intracellular Ca concentration in non-stimulated cells. The membrane-impermeant lidocaine analog QX314 and procaine inhibited mAChR-mediated Ca responses, with EC values of 48.0 and 20.4 μM, respectively, for 50 μM carbachol-stimulated Ca responses. In the absence of extracellular Ca, the pretreatment of cells with QX314 reduced carbachol-induced Ca release, indicating that QX314 reduced Ca release from intracellular stores. Lidocaine and QX314 did not affect store-operated Ca entry as they did not alter the thapsigargin-induced Ca response. QX314 and procaine reduced the carbachol-mediated recruitment of β-arrestin, and administration of procaine suppressed pilocarpine-induced salivary secretion in mice.
CONCLUSION
Local anesthetics, including QX314, act on mAChR to reduce carbachol-induced Ca release from intracellular stores and the recruitment of β-arrestin. These findings support the notion that local anesthetics and their derivatives are starting points for the development of functional allosteric modulators of mAChR.
Topics: Humans; Anesthetics, Local; beta-Arrestins; Calcium; Receptors, Muscarinic; Animals; Mice; Parotid Gland; Lidocaine; Cell Line; Carbachol; Calcium Signaling; Procaine
PubMed: 38614428
DOI: 10.1016/j.job.2024.04.002 -
Free Radical Biology & Medicine Jul 2024Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting...
Bupivacaine (BUP) is an anesthetic commonly used in clinical practice that when used for spinal anesthesia, might exert neurotoxic effects. Thioredoxin-interacting protein (TXNIP) is a member of the α-arrestin protein superfamily that binds covalently to thioredoxin (TRX) to inhibit its function, leading to increased oxidative stress and activation of apoptosis. The role of TXNIP in BUP-induced oxidative stress and apoptosis remains to be elucidated. In this context, the present study aimed to explore the effects of TXNIP knockdown on BUP-induced oxidative stress and apoptosis in the spinal cord of rats and in PC12 cells through the transfection of adeno-associated virus-TXNIP short hairpin RNA (AAV-TXNIP shRNA) and siRNA-TXNIP, respectively. In vivo, a rat model of spinal neurotoxicity was established by intrathecally injecting rats with BUP. The BUP + TXNIP shRNA and the BUP + Control shRNA groups of rats were injected with an AAV carrying the TXNIP shRNA and the Control shRNA, respectively, into the subarachnoid space four weeks prior to BUP treatment. The Basso, Beattie & Bresnahan (BBB) locomotor rating score, % MPE of TFL, H&E staining, and Nissl staining analyses were conducted. In vitro, 0.8 mM BUP was determined by CCK-8 assay to establish a cytotoxicity model in PC12 cells. Transfection with siRNA-TXNIP was carried out to suppress TXNIP expression prior to exposing PC12 cells to BUP. The results revealed that BUP effectively induced neurological behavioral dysfunction and neuronal damage and death in the spinal cord of the rats. Similarly, BUP triggered cytotoxicity and apoptosis in PC12 cells. In addition, treated with BUP both in vitro and in vivo exhibited upregulated TXNIP expression and increased oxidative stress and apoptosis. Interestingly, TXNIP knockdown in the spinal cord of rats through transfection of AAV-TXNIP shRNA exerted a protective effect against BUP-induced spinal neurotoxicity by ameliorating behavioral and histological outcomes and promoting the survival of spinal cord neurons. Similarly, transfection with siRNA-TXNIP mitigated BUP-induced cytotoxicity in PC12 cells. In addition, TXNIP knockdown mitigated the upregulation of ROS, MDA, Bax, and cleaved caspase-3 and restored the downregulation of GSH, SOD, CAT, GPX4, and Bcl2 induced upon BUP exposure. These findings suggested that TXNIP knockdown protected against BUP-induced spinal neurotoxicity by suppressing oxidative stress and apoptosis. In summary, TXNIP could be a central signaling hub that positively regulates oxidative stress and apoptosis during neuronal damage, which renders TXNIP a promising target for treatment strategies against BUP-induced spinal neurotoxicity.
Topics: Animals; Rats; Oxidative Stress; Bupivacaine; PC12 Cells; Apoptosis; Spinal Cord; RNA, Small Interfering; Carrier Proteins; Gene Knockdown Techniques; Male; Thioredoxins; Injections, Spinal; Rats, Sprague-Dawley; Cell Cycle Proteins; Neurotoxicity Syndromes; Neurons
PubMed: 38614227
DOI: 10.1016/j.freeradbiomed.2024.04.223 -
British Journal of Pharmacology Apr 2024Pancreatic islets are modulated by cross-talk among different cell types and paracrine signalling plays important roles in maintaining glucose homeostasis. Urocortin 3...
BACKGROUND AND PURPOSE
Pancreatic islets are modulated by cross-talk among different cell types and paracrine signalling plays important roles in maintaining glucose homeostasis. Urocortin 3 (UCN3) secreted by pancreatic β cells activates the CRF receptor (CRFR) and downstream pathways mediated by different G protein or arrestin subtypes in δ cells to cause somatostatin (SST) secretion, and constitutes an important feedback circuit for glucose homeostasis.
EXPERIMENTAL APPROACH
Here, we used Arrb1, Arrb2, Gs and Gq knockout mice, the G-shRNA-GFP lentivirus, as well as functional assays and pharmacological characterization to study how the coupling of G, G and β-arrestin1 to CRFR contributed to UCN3-induced SST secretion in pancreatic δ cells.
KEY RESULTS
Our study showed that CRFR coupled to a panel of G protein and arrestin subtypes in response to UCN3 engagement. While RyR3 phosphorylation by PKA at the S156, S2706 and S4697 sites may underlie the Gs-mediated UCN3- CRFR axis for SST secretion, the interaction of SYT1 with β-arrestin1 is also essential for efficient SST secretion downstream of CRFR. The specific expression of the transcription factor Stat6 may contribute to G expression in pancreatic δ cells. Furthermore, we found that different UCN3 concentrations may have distinct effects on glucose homeostasis, and these effects may depend on different CRFR downstream effectors.
CONCLUSIONS AND IMPLICATIONS
Collectively, our results provide a landscape view of signalling mediated by different G protein or arrestin subtypes downstream of paracrine UCN3- CRFR signalling in pancreatic β-δ-cell circuits, which may facilitate the understanding of fine-tuned glucose homeostasis networks.
PubMed: 38613153
DOI: 10.1111/bph.16351 -
Cell Chemical Biology Apr 2024Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand...
Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand binding liberates a conserved sequence that acts as an intramolecular, tethered agonist (TA), yet this model has not been evaluated systematically for all aGPCRs. Here, we assessed the TA-dependent activities of all 33 aGPCRs in a suite of transcriptional reporter, G protein activation, and β-arrestin recruitment assays using a new fusion protein platform. Strikingly, only ∼50% of aGPCRs exhibited robust TA-dependent activation, and unlike other GPCR families, aGPCRs showed a notable preference for G signaling. AlphaFold2 predictions assessing TA engagement in the predicted intramolecular binding pocket aligned with the TA dependence of the cellular responses. This dataset provides a comprehensive resource to inform the investigation of all human aGPCRs and for targeting aGPCRs therapeutically.
PubMed: 38608683
DOI: 10.1016/j.chembiol.2024.03.004 -
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