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American Journal of Physiology. Cell... Jun 2024G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon... (Review)
Review
G protein-coupled receptors (GPCRs) are ubiquitously expressed cell surface receptors that mediate numerous physiological responses and are highly druggable. Upon activation, GPCRs rapidly couple to heterotrimeric G proteins and are then phosphorylated and internalized from the cell surface. Recent studies indicate that GPCRs not only localize at the plasma membrane but also exist in intracellular compartments where they are competent to signal. Intracellular signaling by GPCRs is best described to occur at endosomes. Several studies have elegantly documented endosomal GPCR-G protein and GPCR-β-arrestin signaling. Besides phosphorylation, GPCRs are also posttranslationally modified with ubiquitin. GPCR ubiquitination has been studied mainly in the context of receptor endosomal-lysosomal trafficking. However, new studies indicate that ubiquitination of endogenous GPCRs expressed in endothelial cells initiates the assembly of an intracellular p38 mitogen-activated kinase signaling complex that promotes inflammatory responses from endosomes. In this mini-review, we discuss emerging discoveries that provide critical insights into the function of ubiquitination in regulating GPCR inflammatory signaling at endosomes.
Topics: Endosomes; Humans; Receptors, G-Protein-Coupled; Signal Transduction; Animals; Ubiquitination; Inflammation; Ubiquitin; Phosphorylation
PubMed: 38646783
DOI: 10.1152/ajpcell.00161.2024 -
BioRxiv : the Preprint Server For... Apr 2024Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7 leading cause of premature death. Despite this,...
Alcohol use disorders (AUDs) impose an enormous societal and financial burden, and world-wide, alcohol misuse is the 7 leading cause of premature death. Despite this, there are currently only 3 FDA approved pharmacological treatments for the treatment of AUDs in the United States. The neurotensin (Nts) system has long been implicated in modulating behaviors associated with alcohol misuse. Recently, a novel compound, SBI-553, that biases the action of Nts receptor 1 (NTSR1) activation, has shown promise in preclinical models of psychostimulant misuse. Here we investigate the efficacy of this compound to alter ethanol-mediated behaviors in a comprehensive battery of experiments assessing ethanol consumption, behavioral responses to ethanol, sensitivity to ethanol, and ethanol metabolism. Additionally, we investigated behavior in avoidance and cognitive assays to monitor potential side effects of SBI-553. We find that SBI-553 reduces binge-like ethanol consumption in mice without altering avoidance behavior or novel object recognition. We also observe sex-dependent differences in physiological responses to sequential ethanol injections in mice. In rats, we show that SBI-553 attenuates sensitivity to the interoceptive effects of ethanol (using a Pavlovian drug discrimination task). Our data suggest that targeting NTSR1 signaling may be promising to attenuate alcohol misuse, and adds to a body of literature that suggests NTSR1 may be a common downstream target involved in the psychoactive effects of multiple reinforcing substances.
PubMed: 38645173
DOI: 10.1101/2024.04.10.588903 -
Biochemical Pharmacology Apr 2024Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features...
Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features but apparently differ in severity. The two known carriers of the Met374Arg variant had early childhood disease onset and more severe motor, cognitive, and neuropsychiatric deficits than any known carriers of the Ile212Phe variant, whose symptoms were first apparent in adolescence. Here, we evaluated if differences in the function of the two variants in cultured cells could explain differing pathogenicity. Both variants were expressed less abundantly than the wild type receptor and exhibited loss of agonist-induced arrestin binding, but differences in expression and arrestin binding between the variants were minor. Basal and agonist-induced activation of heterotrimeric G proteins, however, showed clear differences; agonists were generally more potent at Met374Arg than at the Ile212Phe or wild type variants. Furthermore, all Gα subtypes tested were constitutively activated more by Met374Arg than by Ile212Phe. Met374Arg produced greater constitutive inhibition of cyclic AMP accumulation than Ile212Phe or the wild type D2 receptor. Met374Arg and Ile212Phe were more sensitive to thermal inactivation than the wild type D2 receptor, as reported for other constitutively active receptors, but Ile212Phe was affected more than Met374Arg. Additional pharmacological characterization suggested that the mutations differentially affect the shape of the agonist binding pocket and the potency of dopamine, norepinephrine, and tyramine. Molecular dynamics simulations provided a structural rationale for enhanced constitutive activation and agonist potency. Enhanced constitutive and agonist-induced G protein-mediated signaling likely contributes to the pathogenicity of these novel variants.
PubMed: 38643909
DOI: 10.1016/j.bcp.2024.116228 -
Trends in Biochemical Sciences Jun 2024G protein-coupled receptors (GPCRs) located at the cell surface bind extracellular ligands and convey intracellular signals via activation of heterotrimeric G proteins.... (Review)
Review
G protein-coupled receptors (GPCRs) located at the cell surface bind extracellular ligands and convey intracellular signals via activation of heterotrimeric G proteins. Traditionally, G protein signaling was viewed to occur exclusively at this subcellular region followed by rapid desensitization facilitated by β-arrestin (βarr)-mediated G protein uncoupling and receptor internalization. However, emerging evidence over the past 15 years suggests that these βarr-mediated events do not necessarily terminate receptor signaling and that some GPCRs continue to activate G proteins after having been internalized into endosomes. Here, we review the recently elucidated mechanistic basis underlying endosomal GPCR signaling and discuss physiological implications and pharmacological targeting of this newly appreciated signaling mode.
Topics: Endosomes; Signal Transduction; Receptors, G-Protein-Coupled; Humans; Animals; beta-Arrestins
PubMed: 38643023
DOI: 10.1016/j.tibs.2024.03.006 -
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