Next on the Dr. GPCR Podcast, we have Dr. Nicola Smith, Molecular Pharmacologist, lab head, and senior lecturer at UNSW Sydney. Subscribe to the podcast today and get notified when new episodes are released! Please share! https://www.ecosystem.drgpcr.com/dr-gpcr-podcast/ #gpcr #drgpcr #podcast

Nanobody binding stabilizes G-protein-coupled receptors (GPCR) in a fully active state and modulates their affinity for bound ligands. However, the atomic-level basis for this allosteric regulation remains elusive. Here, we investigate the conformational changes induced by the binding of a nanobody (Nb80) on the active-like β2 adrenergic receptor (β2AR) via enhanced sampling molecular dynamics simulations. Dimensionality reduction analysis shows that Nb80 stabilizes structural features of the β2AR with an ∼14 Å outward movement of transmembrane helix 6 and a close proximity of transmembrane (TM) helices 5 and 7, and favors the fully active-like conformation of the receptor, independent of ligand binding, in contrast to the conditions under which no intracellular binding partner is bound, in which case the receptor is only stabilized in an intermediate-active state. This activation is supported by the residues located at hotspots located on TMs 5, 6, and 7, as shown by supervised machine learning methods. Besides, ligand-specific subtle differences in the conformations assumed by intracellular loop 2 and extracellular loop 2 are captured from the trajectories of various ligand-bound receptors in the presence of Nb80. Dynamic network analysis further reveals that Nb80 binding triggers tighter and stronger local communication networks between the Nb80 and the ligand-binding sites, primarily involving residues around ICL2 and the intracellular end of TM3, TM5, TM6, as well as ECL2, ECL3, and the extracellular ends of TM6 and TM7. In particular, we identify unique allosteric signal transmission mechanisms between the Nb80-binding site and the extracellular domains in conformations modulated by a full agonist, BI167107, and a G-protein-biased partial agonist, salmeterol, involving mainly TM1 and TM2, and TM5, respectively. Altogether, our results provide insights into the effect of intracellular binding partners on the GPCR activation mechanism, which should be taken into account in structure-based drug discovery. Read full article

Bursicon receptor gene HLGR2 as a potential RNA interference target for control of the fall webworm Hyphantria cunea Background: Insect G protein-coupled receptors (GPCRs) have been identified as a new generation of attractive targets for RNA interference (RNAi)-based pest control. A functional study of the leucine-rich repeat-containing (LGR2) gene in Hyphantria cunea (HLGR2) was performed to examine whether it can be used in the molecular control of this notorious pest. Results: The complementary DNA (cDNA) sequence and deduced amino acids of HLGR2 were obtained and analyzed in the present study. HLGR2 is a typical GPCR and shows high structural and sequence similarity with other insect LGR2 proteins. The spatiotemporal expression profiles of HLGR2 showed that HLGR2 was highly expressed at the egg stage and tissues of head and silk gland. After RNAi of HLGR2, distinct phenotypes were observed when HLGR2 expression was suppressed, indicating that HLGR2 is essential in pupation and eclosion. HLGR2 RNAi led to a low pupation rate (45.00%), body malformation, abnormal wing expansion, failed cuticle melanization (63.33%), and high mortality rate (48.33%). Furthermore, we identified eight genes that are regulated by HLGR2. The expression of these eight genes was induced by the HLGR2 signaling pathway and correlated well with cuticle sclerotization. Unlike LGR2 in other insect species, HLGR2 was found to play a crucial role in the control of H. cunea during ecdysis and postecdysial stages. Conclusion: HLGR2 is essential for the growth and development and wing expansion and maturation in H. cunea, suggesting HLGR2 is a promising candidate for application in RNAi-based control of this notorious agriculture-forest pest. Read full articles

Intracellular calcium ([Ca2+]i) signaling is a critical regulator of chondrogenesis, chondrocyte differentiation, and cartilage development. Calcium (Ca2+) signaling is known to direct processes that govern chondrocyte gene expression, protein synthesis, cytoskeletal remodeling, and cell fate. Control of chondrocyte/chondroprogenitor Ca2+ signaling has been attempted through mechanical and/or pharmacological activation of endogenous Ca2+ signaling transducers; however, such approaches can lack specificity and/or precision regarding Ca2+ activation mechanisms. Synthetic signaling platforms permitting precise and selective Ca2+ signal transduction can improve dissection of the roles that [Ca2+]i signaling plays in chondrocyte behavior. One such platform is the chemogenetic DREADD (designer receptor exclusively activated by designer drugs) hM3Dq, which activates [Ca2+]i signaling via the Gαq-PLCβ-IP3-ER pathway upon clozapine N-oxide (CNO) administration. We previously demonstrated hM3Dq's ability to precisely and synthetically initiate robust [Ca2+]i transients and oscillatory [Ca2+]i signaling in chondrocyte-like ATDC5 cells. Here, we investigate the effects that long-term CNO stimulatory culture have on hM3Dq [Ca2+]i signaling dynamics, proliferation, and protein deposition in 2D ATDC5 cultures. Long-term culturing under repeated CNO stimulation modified the temporal dynamics of hM3Dq [Ca2+]i signaling, increased cell proliferation, and enhanced matrix production in a CNO dose- and frequency-dependent manner, and triggered the formation of cell condensations that developed aligned, anisotropic neotissue structures rich in cartilaginous proteoglycans and collagens, all in the absence of differentiation inducers. This study demonstrated Gαq-G-protein coupled receptor (GPCR)-mediated [Ca2+]i signaling involvement in chondroprogenitor proliferation and cartilage-like matrix production, and it established hM3Dq as a powerful tool for elucidating the role of GPCR-mediated Ca2+ signaling in chondrogenesis and chondrocyte differentiation. Read full article

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumour in adults. Treatments include surgical resection, radiotherapy, and chemotherapy. Despite this, the prognosis remains poor, with an impacted quality of life during treatment coupled with brain tumour recurrence; thus, new treatments are desperately needed. In this review, we focus on recent advances in G-protein-coupled receptor (GPCR) targets. To date, the most promising targets are the chemokine, cannabinoid, and dopamine receptors, but future work should further examine the melanocortin receptor-4 (MC4R), adhesion, lysophosphatidic acid (LPA) and smoothened (Smo) receptors to initiate new drug-screening strategies and targeted delivery of safe and effective GBM therapies. Read full article

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates. Read full article

A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein-coupled receptors (GPCRs). Starting from vercirnon, an intracellular C-C chemokine receptor type 9 (CCR9) antagonist and previous phase III clinical candidate for the treatment of Crohn's disease, we developed a chemical biology toolbox targeting the IABS of CCR9. We first synthesized a fluorescent ligand enabling equilibrium and kinetic binding studies via NanoBRET as well as fluorescence microscopy. Applying this molecular tool in a membrane-based setup and in living cells, we discovered a 4-aminopyrimidine analogue as a new intracellular CCR9 antagonist with improved affinity. To chemically induce CCR9 degradation, we then developed the first PROTAC targeting the IABS of GPCRs. In a proof-of-principle study, we succeeded in showing that our CCR9-PROTAC is able to reduce CCR9 levels, thereby offering an unprecedented approach to modulate GPCR activity. Read full article

Rapid flip-flop of phospholipids across the two leaflets of biological membranes is crucial for many aspects of cellular life. The transport proteins that facilitate this process are classified as pump-like flippases and floppases and channel-like scramblases. Unexpectedly, Class A G protein-coupled receptors (GPCRs), a large class of signaling proteins exemplified by the visual receptor rhodopsin and its apoprotein opsin, are constitutively active as scramblases in vitro. In liposomes, opsin scrambles lipids at a unitary rate of >100,000 per second. Atomistic molecular dynamics simulations of opsin in a lipid membrane reveal conformational transitions that expose a polar groove between transmembrane helices 6 and 7. This groove enables transbilayer lipid movement, conceptualized as the swiping of a credit card (lipid) through a card reader (GPCR). Conformational changes that facilitate scrambling are distinct from those associated with GPCR signaling. In this review, we discuss the physiological significance of GPCR scramblase activity and the modes of its regulation in cells. Expected final online publication date for the Annual Review of Biophysics , Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates. Read full article

Recurrent high-impact mutations at cognate structural positions in class A G protein-coupled receptors expressed in tumors G protein-coupled receptors (GPCRs) are the largest family of human proteins. They have a common structure and, signaling through a much smaller set of G proteins, arrestins, and effectors, activate downstream pathways that often modulate hallmark mechanisms of cancer. Because there are many more GPCRs than effectors, mutations in different receptors could perturb signaling similarly so as to favor a tumor. We hypothesized that somatic mutations in tumor samples may not be enriched within a single gene but rather that cognate mutations with similar effects on GPCR function are distributed across many receptors. To test this possibility, we systematically aggregated somatic cancer mutations across class A GPCRs and found a nonrandom distribution of positions with variant amino acid residues. Individual cancer types were enriched for highly impactful, recurrent mutations at selected cognate positions of known functional motifs. We also discovered that no single receptor drives this pattern, but rather multiple receptors contain amino acid substitutions at a few cognate positions. Phenotypic characterization suggests these mutations induce perturbation of G protein activation and/or β-arrestin recruitment. These data suggest that recurrent impactful oncogenic mutations perturb different GPCRs to subvert signaling and promote tumor growth or survival. The possibility that multiple different GPCRs could moonlight as drivers or enablers of a given cancer through mutations located at cognate positions across GPCR paralogs opens a window into cancer mechanisms and potential approaches to therapeutics. Read full article

In presynaptic terminals, membrane-delimited Gi/o-mediated presynaptic inhibition is ubiquitous and acts through Gβγ to inhibit Ca2+ entry, or directly at SNARE complexes to inhibit Ca2+-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals 5-HT1B and GABAB receptors colocalize. GABAB receptors inhibit Ca2+ entry, whereas 5-HT1B receptors target SNARE complexes. We demonstrate in male and female rats that GABAB receptors receptors alter Pr, whereas 5-HT1B receptors reduce evoked cleft glutamate concentrations allowing differential inhibition of AMPA and NMDA receptor EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR).Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of Pr changes, consistent with GABAB receptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT1B receptor-mediated inhibition are well-fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provide synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca2+ accumulation forces frequency-dependent recovery of neurotransmission during 5-HT1B receptor activation. This is consistent with competition between Ca2+-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT1B receptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABAB receptors which alter presynaptic Ca2+ In combination these pathways allow complex presynaptic integration. SIGNIFICANCE STATEMENT Two G protein coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gβγ, but one - a GABAB receptor inhibits Ca2+ entry while another - a 5-HT1B receptor competes with Ca2+-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABAB receptors alter Pr leaving synaptic properties unchanged, while 5-HT1B receptors fundamentally change properties of synaptic transmission, modifying AMPA receptor but sparing NMDA receptor responses. Co-activation of these receptors allows synaptic integration because of convergence of GABAB receptor alteration on Ca2+ and the effect of this altered Ca2+ signal on 5-HT1B receptor signaling. This presynaptic convergence provides a novel form of synaptic integration. Read full article

Prostaglandin (PG) signaling regulates a wide variety of physiological and pathological processes, including body temperature, cardiovascular homeostasis, reproduction, and inflammation. Recent studies have revealed that PGs play pivotal roles in embryo development, ciliogenesis, and organ formation. Prostaglandin E2 (PGE2) and its receptor EP4 modulate ciliogenesis by increasing the anterograde intraflagellar transport. Many G-protein-coupled receptors (GPCRs) including EP4 are localized in cilia for modulating cAMP signaling under various conditions. During development, PGE2 signaling regulates embryogenesis, hepatocyte differentiation, hematopoiesis, and kidney formation. Prostaglandins are also essential for skeletal muscle repair. This review outlines recent advances in understanding the functions and mechanisms of prostaglandin signaling in ciliogenesis, embryo development, and organ formation. Read full article

The hepatitis B virus X protein (HBx) is involved in the process of hepatocellular carcinoma via the activation of various oncogenes. Our previous study indicated that ARBB1 (arrestin beta 1) promotes hepatocellular carcinogenesis (HCC). However, the role of ARRB1 in HBx-related HCC remains unclear. Herein, we identified that ARRB1 was upregulated by HBx in vivo and in vitro. Arrb1 deficiency suppressed HBx-induced hepatocellular carcinogenesis in several mouse models. Furthermore, knockdown of ARRB1 blocked HBx-induced macroautophagic/autophagic flux and disrupted the formation of autophagosomes. ARRB1 interacted with HBx, and the autophagic core protein MAP1LC3/LC3, a scaffolding protein, was essential for complete autophagy. Inhibition of autophagy by 3-methyladenine or interference of ATG5 or ATG7 attenuated HBx-induced cell cycle acceleration and the subsequent proliferative response via the induction of G1/S arrest. The absence of autophagy abolished the phosphorylation of CDK2 and the activity of the CDK2-CCNE1 complex. Our results demonstrate that ARRB1 plays a critical role in HBV-related HCC via modulating autophagy and the CDKN1B-CDK2-CCNE1-E2F1 axis and indicate that ARRB1 may be a potential therapeutic target for HCC. Abbreviations: ARRB1: arrestin beta 1; ACTB: actin beta; AMPK: adenosine monophosphate (AMP)-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf A1: bafilomycin A1; CDK2: cyclin dependent kinase 2; CDKN1B/p27Kip1: cyclin dependent kinase inhibitor 1B; CQ: chloroquine; E2F1: E2F transcription factor 1; FBS: fetal bovine serum; GPCRs: G protein-coupled receptors; GST: glutathione S-transferase; HCC: hepatocellular carcinoma; HBV: hepatitis B virus; HBx: hepatitis B virus X protein; HMGB1: high mobility group box 1; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; IHC: immunohistochemistry; JAK1: Janus kinase 1; LOX: lysyl oxidase; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MKI67: marker of proliferation Ki-67; MTOR: mechanistic target of rapamycin kinase; MAPK: mitogen-activated protein kinase; 3-MA: 3-methyladenine; NFKB/NF-κB: nuclear factor kappa B; PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PHHs: primary human hepatocytes; RB1: RB transcriptional corepressor 1; SQSTM1/p62: sequestosome 1; STAT: signal transducer and activator of transcription; TACR1/NK1R: tachykinin receptor 1. Read full article

Electron cryo-microscopy (cryo-EM) is a powerful technique for the structural characterization of biological macromolecules, enabling high-resolution analysis of targets once inaccessible to structural interrogation. In recent years, pharmaceutical companies have begun to utilize cryo-EM for structure-based drug design. Structural analysis of integral membrane proteins, which comprise a large proportion of druggable targets and pose particular challenges for X-ray crystallography, by cryo-EM has enabled insights into important drug target families such as G protein-coupled receptors (GPCRs), ion channels, and solute carrier (SLCs) proteins. Structural characterization of biologics, such as vaccines, viral vectors, and gene therapy agents, has also become significantly more tractable. As a result, cryo-EM has begun to make major impacts in bringing critical therapeutics to market. In this review, we discuss recent instructive examples of impacts from cryo-EM in therapeutics design, focusing largely on its implementation at Pfizer. We also discuss the opportunities afforded by emerging technological advances in cryo-EM, and the prospects for future development of the technique. Read full article

The physiological mechanisms driving synapse formation are elusive. Although numerous signals are known to regulate synapses, it remains unclear which signaling mechanisms organize initial synapse assembly. Here, we describe new tools, referred to as "SynTAMs" for synaptic targeting molecules, that enable localized perturbations of cAMP signaling in developing postsynaptic specializations. We show that locally restricted suppression of postsynaptic cAMP levels or of cAMP-dependent protein-kinase activity severely impairs excitatory synapse formation without affecting neuronal maturation, dendritic arborization, or inhibitory synapse formation. In vivo, suppression of postsynaptic cAMP signaling in CA1 neurons prevented formation of both Schaffer-collateral and entorhinal-CA1/temporoammonic-path synapses, suggesting a general principle. Retrograde trans-synaptic rabies virus tracing revealed that postsynaptic cAMP signaling is required for continuous replacement of synapses throughout life. Given that postsynaptic latrophilin adhesion-GPCRs drive synapse formation and produce cAMP, we suggest that spatially restricted postsynaptic cAMP signals organize assembly of postsynaptic specializations during synapse formation. Read full article

GPCRs Are Optimal Regulators of Complex Biological Systems and Orchestrate the Interface between Health and Disease GPCRs arguably represent the most effective current therapeutic targets for a plethora of diseases. GPCRs also possess a pivotal role in the regulation of the physiological balance between healthy and pathological conditions; thus, their importance in systems biology cannot be underestimated. The molecular diversity of GPCR signaling systems is likely to be closely associated with disease-associated changes in organismal tissue complexity and compartmentalization, thus enabling a nuanced GPCR-based capacity to interdict multiple disease pathomechanisms at a systemic level. GPCRs have been long considered as controllers of communication between tissues and cells. This communication involves the ligand-mediated control of cell surface receptors that then direct their stimuli to impact cell physiology. Given the tremendous success of GPCRs as therapeutic targets, considerable focus has been placed on the ability of these therapeutics to modulate diseases by acting at cell surface receptors. In the past decade, however, attention has focused upon how stable multiprotein GPCR superstructures, termed receptorsomes, both at the cell surface membrane and in the intracellular domain dictate and condition long-term GPCR activities associated with the regulation of protein expression patterns, cellular stress responses and DNA integrity management. The ability of these receptorsomes (often in the absence of typical cell surface ligands) to control complex cellular activities implicates them as key controllers of the functional balance between health and disease. A greater understanding of this function of GPCRs is likely to significantly augment our ability to further employ these proteins in a multitude of diseases. Read full article

G-protein coupled receptors (GPCRs) play a paramount role in diverse brain functions. Almost 20 years ago, GPCR activity was shown to be regulated by membrane potential in vitro, but whether the voltage dependence of GPCRs contributes to neuronal coding and behavioral output under physiological conditions in vivo has never been demonstrated. Here we show that muscarinic GPCR mediated neuronal potentiation in vivo is voltage dependent. This voltage dependent potentiation is abolished in mutant animals expressing a voltage independent receptor. Depolarization alone, without a muscarinic agonist, results in a nicotinic ionotropic receptor potentiation that is mediated by muscarinic receptor voltage dependency. Finally, muscarinic receptor voltage independence causes a strong behavioral effect of increased odor habituation. Together, this study identifies a physiological role for the voltage dependency of GPCRs by demonstrating crucial involvement of GPCR voltage dependence in neuronal plasticity and behavior. Thus, this study suggests that GPCR voltage dependency plays a role in many diverse neuronal functions including learning and memory. Read full article

Opioid receptors are G-protein-coupled receptors (GPCRs) part of cell signaling paths of direct interest to treat pain. Pain may associate with inflamed tissue characterized by acidic pH. The potentially low pH at tissue targeted by opioid drugs in pain management could impact drug binding to the opioid receptor, because opioid drugs typically have a protonated amino group that contributes to receptor binding, and the functioning of GPCRs may involve protonation change. In this review, we discuss the relationship between structure, function, and dynamics of opioid receptors from the perspective of the usefulness of computational studies to evaluate protonation-coupled opioid-receptor interactions. Read full article

Arrestins regulate a wide range of signaling events, most notably when bound to active G protein-coupled receptors (GPCRs). Among the known effectors recruited by GPCR-bound arrestins are Src family kinases, which regulate cellular growth and proliferation. Here, we focus on arrestin-3 interactions with Fgr kinase, a member of the Src family. Previous reports demonstrated that Fgr exhibits high constitutive activity, but can be further activated by both arrestin-dependent and arrestin-independent pathways. We report that arrestin-3 modulates Fgr activity with a hallmark bell-shaped concentration-dependence, consistent with a role as a signaling scaffold. We further demonstrate using NMR spectroscopy that a polyproline motif within arrestin-3 interacts directly with the SH3 domain of Fgr. To provide a framework for this interaction, we determined the crystal structure of the Fgr SH3 domain at 1.9 Å resolution and developed a model for the GPCR-arrestin-3-Fgr complex that is supported by mutagenesis. This model suggests that Fgr interacts with arrestin-3 at multiple sites and is consistent with the locations of disease-associated Fgr mutations. Collectively, these studies provide a structural framework for arrestin-dependent activation of Fgr. Read full article

The sixth transmembrane region of a pheromone G-protein coupled receptor, Map3, is implicated in discrimination of closely related pheromones in Schizosaccharomyces pombe Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycete fungi including yeasts, mating between cells of opposite mating type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focused on the stringency of pheromone receptors. The fission yeast Schizosaccharomyces pombe has two mating types, Plus (P) and Minus (M). Here, we investigated the stringency of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the activation by the pheromone. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are key residues important for discrimination of closely related M-factors. Thus, the differences in these two GPCRs might reflect the significantly distinct stringency/flexibility of their respective pheromone/receptor systems; nevertheless, species-specific pheromone recognition remains incomplete. Read full article

Engineered G protein-coupled receptors (GPCRs) are commonly used in chemogenetics as designer receptors exclusively activated by designer drugs (DREADDs). Although several GPCRs have been studied in astrocytes using a chemogenetic approach, the functional role of the astrocytic Gi pathway is not clear, as the literature is conflicting depending on the brain regions or behaviors investigated. In this study, we evaluated the role of the astrocytic Gi pathway in neuroinflammation using a Gi -coupled DREADD (hM4Di). Gi -DREADD was expressed in hippocampal astrocytes of a lipopolysaccharide (LPS)-induced neuroinflammation mouse model using adeno-associated viruses. We found that astrocyte Gi -DREADD stimulation using clozapine N-oxide (CNO) inhibits neuroinflammation, as characterized by decreased levels of proinflammatory cytokines, glial activation, and cognitive impairment in mice. Subsequent experiments using primary astrocyte cultures revealed that Gi -DREADD stimulation significantly downregulated LPS-induced expression of Nos2 mRNA and nitric oxide production. Similarly, in vitro calcium imaging showed that activation of the astrocytic Gi pathway attenuated intracellular calcium transients triggered by LPS treatment, suggesting a positive correlation between enhanced calcium transients and the inflammatory phenotype of astrocytes observed in the inflamed brain. Taken together, our results indicate that the astrocytic Gi pathway plays an inhibitory role in neuroinflammation, providing an opportunity to identify potential cellular and molecular targets to control neuroinflammation. Read full article

β-arrestins (βarrs) play multifaceted roles in the function of G protein-coupled receptors (GPCRs). βarrs typically interact with phosphorylated C-terminal tail (C tail) and transmembrane core (TM core) of GPCRs. However, the effects of the C tail- and TM core-mediated interactions on the conformational activation of βarrs have remained elusive. Here, we show the conformational changes for βarr activation upon the C tail- and TM core-mediated interactions with a prototypical GPCR by nuclear magnetic resonance (NMR) spectroscopy. Our NMR analyses demonstrated that while the C tail-mediated interaction alone induces partial activation, in which βarr exists in equilibrium between basal and activated conformations, the TM core- and the C tail-mediated interactions together completely shift the equilibrium toward the activated conformation. The conformation-selective antibody, Fab30, promotes partially activated βarr into the activated-like conformation. This plasticity of βarr conformation in complex with GPCRs engaged in different binding modes may explain the multifunctionality of βarrs. Read full article

Constitutive, Basal, and β-Alanine-Mediated Activation of the Human Mas-Related G Protein-Coupled Receptor D Induces Release of the Inflammatory Cytokine IL-6 and Is Dependent on NF-κB Signaling G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist β-alanine, we observed a release of IL-6. β-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for β-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay. Read full article

G protein-coupled receptor interactions and modification of signalling involving the ghrelin receptor, GHSR1a The growth hormone secretagogue receptor 1a (GHSR1a) is intriguing because of its potential as a therapeutic target and its diverse molecular interactions. Initial studies of the receptor focused on the potential therapeutic ability for growth hormone (GH) release to reduce wasting in aging individuals, as well as food intake regulation for treatment of cachexia. Known roles of GHSR1a now extend to regulation of neurogenesis, learning and memory, gastrointestinal motility, glucose/lipid metabolism, the cardiovascular system, neuronal protection, motivational salience, and hedonic feeding. Ghrelin, the endogenous agonist of GHSR1a, is primarily located in the stomach and is absent from the central nervous system (CNS), including the spinal cord. However, ghrelin in the circulation does have access to a small number of CNS sites, including the arcuate nucleus, which is important in feeding control. At some sites, such as at somatotrophs, GHSR1a has high constitutive activity. Typically, ghrelin-dependent and constitutive GHSR1a activation occurs via Gαq/11 pathways. In vitro and in vivo data suggest that GHSR1a heterodimerises with multiple G protein-coupled receptors (GPCRs), including dopamine D1 and D2, serotonin 2C, orexin, oxytocin and melanocortin 3 receptors (MCR3), as well as the MCR3 accessory protein, MRAP2, providing possible mechanisms for its many physiological effects. In all cases, the receptor interaction changes downstream signalling and the responses to receptor agonists. This review discusses the signalling mechanisms of GHSR1a alone and in combination with other GPCRs, and explores the physiological consequences of GHSR1a coupling with other GPCRs. Read full article

The mouse cytomegalovirus G protein-coupled receptor homolog, M33, coordinates key features of in vivo infection via distinct components of its signalling repertoire Common to all cytomegalovirus (CMV) genomes analysed to date is the presence of G protein-coupled receptors (GPCR). Animal models of CMV provide insights into their role in viral fitness. The mouse cytomegalovirus (MCMV) GPCR, M33, facilitates dendritic cell (DC)-dependent viremia, the extravasation of blood-borne infected DC to the salivary gland and the frequency of reactivation events from latently-infected tissue explants. Constitutive G protein-coupled M33 signalling is required for these phenotypes, although the contribution of distinct biochemical pathways activated by M33 is unknown. M33 engages Gq/11 to constitutively activate phospholipase C β (PLCβ) and downstream cyclic AMP response-element binding protein (CREB) in vitro . Identification of a MCMV M33 mutant (M33ΔC38) for which CREB signalling was disabled, but PLCβ activation was preserved, provided the opportunity to investigate their relevance in vivo . Following intranasal infection with MCMV M33ΔC38, the absence of M33 CREB Gq/11-dependent signalling correlated with reduced mobilisation of lytically-infected DC to draining lymph node high endothelial venules (HEVs) and reduced viremia compared with wild type MCMV. In contrast, M33ΔC38-infected DC within the vascular compartment extravasated to the salivary glands via a pertussis toxin-sensitive, Gi/o-dependent and CREB-independent mechanism. In the context of MCMV latency, spleen explants from M33ΔC38-infected mice were markedly attenuated for reactivation. Taken together, these data demonstrate that key features of the MCMV lifecycle are coordinated in diverse tissues by distinct pathways of the M33 signalling repertoire. IMPORTANCE G protein-coupled receptors (GPCRs) act as cell surface molecular "switches" which regulate the cellular response to environmental stimuli. All cytomegalovirus (CMV) genomes analysed to date possess GPCR homologs with phylogenetic evidence for independent gene capture events, signifying important in vivo roles. The mouse CMV (MCMV) GPCR homolog, designated M33, is important for cell-associated virus spread and for the establishment and/or reactivation of latent MCMV infection. The signalling repertoire of M33 is distinct from cellular GPCRs and little is known of the relevance of component signalling pathways for in vivo M33 function. In this report, we show temporal and tissue-specific M33 signalling is required facilitating in vivo infection. Understanding the relevance of the viral GPCR signalling profiles for in vivo function will provide opportunities for future targeted interventions. Read full article

Alterations in the expression and/or activity of brain G-protein-coupled receptors (GPCRs) such as dopamine D1R, D2LR, D3R, and D4R, vasopressin V1AR, and serotonin 5-HT1AR are noted in various neurodegenerative diseases (NDDs). Since studies have indicated that flavonoids can target brain GPCRs and provide neuroprotection via inhibition of monoamine oxidases (hMAOs), our study explored the functional role of kurarinone, an abundant lavandulated flavonoid in Sophora flavescens , on dopamine receptor subtypes, V1AR, 5-HT1AR, and hMAOs. Radioligand binding assays revealed considerable binding of kurarinone on D1R, D2LR, and D4R. Functional GPCR assays unfolded the compound's antagonist behavior on D1R (IC50 42.1 ± 0.35 μM) and agonist effect on D2LR and D4R (EC50 22.4 ± 3.46 and 71.3 ± 4.94 μM, respectively). Kurarinone was found to inhibit hMAO isoenzymes in a modest and nonspecific manner. Molecular docking displayed low binding energies during the intermolecular interactions of kurarinone with the key residues of the deep orthosteric binding pocket and the extracellular loops of D1R, D2LR, and D4R, validating substantial binding affinities to these prime targets. With appreciable D2LR and D4R agonism and D1R antagonism, kurarinone might be a potential compound that can alleviate clinical symptoms of Parkinson's disease and other NDDs. Read full article

G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine-tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAMs tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface. Read full article

Objective: The goal of this study was to determine the glucometabolic effects of acute activation of Gs signaling in skeletal muscle (SKM) in vivo and its contribution to whole-body glucose homeostasis. Methods: To address this question, we studied mice that express a Gs-coupled designer G protein-coupled receptor (Gs-DREADD or GsD) selectively in skeletal muscle. We also identified two Gs-coupled GPCRs that are endogenously expressed by SKM at relatively high levels (β2-adrenergic receptor and CRF2 receptor) and studied the acute metabolic effects of activating these receptors in vivo by highly selective agonists (clenbuterol and urocortin 2 (UCN2), respectively). Results: Acute stimulation of GsD signaling in SKM impaired glucose tolerance in lean and obese mice by decreasing glucose uptake selectively into SKM. The acute metabolic effects following agonist activation of β2-adrenergic and, potentially, CRF2 receptors appear primarily mediated by altered insulin release. Clenbuterol injection improved glucose tolerance by increasing insulin secretion in lean mice. In SKM, clenbuterol stimulated glycogen breakdown. UCN2 injection resulted in decreased glucose tolerance associated with lower plasma insulin levels. The acute metabolic effects of UCN2 were not mediated by SKM Gs signaling. Conclusions: Selective activation of Gs signaling in SKM causes an acute increase in blood glucose levels. However, acute in vivo stimulation of endogenous Gs-coupled receptors enriched in SKM has only a limited impact on whole-body glucose homeostasis, most likely due to the fact that these receptors are also expressed by pancreatic islets where they modulate insulin release. Read full article

Lysophosphatidic Acid and Several Neurotransmitters Converge on Rho-Kinase 2 Signaling to Manage Motoneuron Excitability Intrinsic membrane excitability (IME) sets up neuronal responsiveness to synaptic drive. Several neurotransmitters and neuromodulators, acting through G-protein-coupled receptors (GPCRs), fine-tune motoneuron (MN) IME by modulating background K+ channels TASK1. However, intracellular partners linking GPCRs to TASK1 modulation are not yet well-known. We hypothesized that isoform 2 of rho-kinase (ROCK2), acting as downstream GPCRs, mediates adjustment of MN IME via TASK1. Electrophysiological recordings were performed in hypoglossal MNs (HMNs) obtained from adult and neonatal rats, neonatal knockout mice for TASK1 ( task1 -/-) and TASK3 ( task3 -/-, the another highly expressed TASK subunit in MNs), and primary cultures of embryonic spinal cord MNs (SMNs). Small-interfering RNA (siRNA) technology was also used to knockdown either ROCK1 or ROCK2. Furthermore, ROCK activity assays were performed to evaluate the ability of various physiological GPCR ligands to stimulate ROCK. Microiontophoretically applied H1152, a ROCK inhibitor, and siRNA-induced ROCK2 knockdown both depressed AMPAergic, inspiratory-related discharge activity of adult HMNs in vivo , which mainly express the ROCK2 isoform. In brainstem slices, intracellular constitutively active ROCK2 (aROCK2) led to H1152-sensitive HMN hyper-excitability. The aROCK2 inhibited pH-sensitive and TASK1-mediated currents in SMNs. Conclusively, aROCK2 increased IME in task3 -/-, but not in task1 -/- HMNs. MN IME was also augmented by the physiological neuromodulator lysophosphatidic acid (LPA) through a mechanism entailing Gαi/o-protein stimulation, ROCK2, but not ROCK1, activity and TASK1 inhibition. Finally, two neurotransmitters, TRH, and 5-HT, which are both known to increase MN IME by TASK1 inhibition, stimulated ROCK2, and depressed background resting currents via Gαq/ROCK2 signaling. These outcomes suggest that LPA and several neurotransmitters impact MN IME via Gαi/o/Gαq-protein-coupled receptors, downstream ROCK2 activation, and subsequent inhibition of TASK1 channels. Read full article