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February 2022 "We are proud to announce the success of our IPO! Thank you to our shareholders, both historical and new, for their trust. The €25 million fundraising gives us the means to achieve our ambitions: to become a leading player in the field of brain diseases! #IPO #biotech #innovation #traitements #addiction #cannabis #Trisomie21 #Downsyndrome" Read more at the source #DrGPCR #GPCR #IndustryNews

February 2022 Trevena Announces Submission of New Drug Application in China for OLINVYK® by its Partner Jiangsu Nhwa Pharmaceutical "Submission supported by data from a Phase 3 bridging study of oliceridine injection compared to IV morphine, conducted in China by Nhwa Trevena is eligible to receive future success payments upon approval and commercialization milestones, as well as a 10% royalty on net sales in China CHESTERBROOK, Pa., Jan. 27, 2022 A biopharmaceutical company focused on the development and commercialization of novel medicines for patients with central nervous system (CNS) disorders, today announced that China’s National Medical Products Administration (NMPA) has accepted submission of a New Drug Application (NDA) for oliceridine injection. The NDA was submitted by Trevena’s partner, Jiangsu Nhwa Pharmaceutical, and follows completion by Nhwa of a Phase 3 bridging trial for OLINVYK (oliceridine) injection, a novel IV analgesic that has been approved in the United States by the Food and Drug Administration (FDA) for use in adults for the management of acute pain severe enough to require an intravenous opioid analgesic and for whom alternative treatments are inadequate." Read more at the source #DrGPCR #GPCR #IndustryNews

February 2022 Read more at the source #DrGPCR #GPCR #IndustryNews

Integration and Spatial Organization of Signaling by G Protein-Coupled Receptor Homo- and Heterodimer Information flow from a source to a receiver becomes informative when the recipient can process the signal into a meaningful form. Information exchange and interpretation is essential in biology and understanding how cells integrate signals from a variety of information-coding molecules into complex orchestrated responses is a major challenge for modern cell biology. In complex organisms, cell to cell communication occurs mostly through neurotransmitters and hormones, and receptors are responsible for signal recognition at the membrane level and information transduction inside the cell. The G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, with nearly 800 genes coding for these proteins. The recognition that GPCRs may physically interact with each other has led to the hypothesis that their dimeric state can provide the framework for temporal coincidence in signaling pathways. Furthermore, the formation of GPCRs higher order oligomers provides the structural basis for organizing distinct cell compartments along the plasma membrane where confined increases in second messengers may be perceived and discriminated. Here, we summarize evidence that supports these conjectures, fostering new ideas about the physiological role played by receptor homo- and hetero-oligomerization in cell biology. Read full article

Episode 55 of the Dr. GPCR podcast is now available! Our guest is Mark Schmeizl! He is a pro at Matchmaking the top talent in the life sciences with the industry’s leading companies. Listen where you get your podcasts https://www.ecosystem.drgpcr.com/dr-gpcr-podcast/ #drgpcr #gpcr #podcast

January 2022 "We are an experienced, innovative, global team on a bold mission to improve health by creating and developing life-changing medicines for people around the world." Read more at the source #DrGPCR #GPCR #IndustryNews

January 2022 "The Neurocrine Biosciences story puts into words what makes the work and people at Neurocrine Biosciences so special. It captures our values and vision for the future, our culture, and our unique approach to science. Simply put, it represents what we do and why we do it. Hear the story from our colleagues in this video." Read more at the source #DrGPCR #GPCR #IndustryNews

A role for BET proteins in regulating basal, dopamine-induced and cAMP/PKA-dependent transcription in rat striatal neurons The activity of striatal medium-spiny projection neurons is regulated by D1 and D2 dopamine receptors. The D1 receptor (D1R) is a Gαs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Upon activation via D1R, PKA can translocate to the nucleus to regulate transcription through the phosphorylation of various targets. One candidate effector of PKA-dependent transcriptional regulation is the BET protein Brd4. It is known that when Brd4 is activated by phosphorylation, it binds more readily to acetylated histones at promoters and enhancers; moreover, in non-neuronal cells, PKA signalling has been shown to increase recruitment of Brd4 to chromatin. However, it is unknown whether BET proteins, or Brd4 specifically, are involved in transcriptional activation by cAMP/PKA in neurons. Here, we demonstrate that in adult rats, inhibition of BET proteins with the bromodomain inhibitor JQ1 suppressed the expression of ~25% of D1R-upregulated genes, while also increasing the expression of a subset of immediate-early genes. We further found that cAMP/PKA signalling promotes Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown of Brd4 attenuates D1R-induced gene expression. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify the BET protein family, and Brd4 in particular, as novel regulators of basal and D1R-dependent transcription in rat striatal neurons, and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription. Read full article

Characterization of a new WHIM syndrome mutant reveals mechanistic differences in regulation of the chemokine receptor CXCR4 WHIM syndrome is a rare immunodeficiency disorder that is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. While several gain-of-function mutations that lead to C-terminal truncations, frame shifts and point mutations in the chemokine receptor CXCR4 have been identified in WHIM syndrome patients, the functional effect of these mutations are not fully understood. Here, we report on a new WHIM syndrome mutation that results in a frame shift within the codon for Ser339 (S339fs5) and compare the properties of S339fs5 with wild type CXCR4 and a previously identified WHIM syndrome mutant, R334X. The S339fs5 and R334X mutants exhibited significantly increased signaling compared to wild type CXCR4 including agonist-promoted calcium flux and extracellular signal-regulated kinase activation. This increase is at least partially due to a significant decrease in agonist-promoted phosphorylation, β-arrestin binding, and endocytosis of S339fs5 and R334X compared to wild type CXCR4. Interestingly, there were also significant differences in receptor degradation, with S339fs5 having a very high basal level of degradation compared to that of R334X and wild type CXCR4. In contrast to wild type CXCR4, both R334X and S339fs5 were largely insensitive to CXCL12-promoted degradation. Moreover, while basal and agonist-promoted degradation of wild type CXCR4 was effectively inhibited by the CXCR4 antagonist TE-14016, this had no effect on the degradation of the WHIM mutants. Taken together, these studies identify a new WHIM syndrome mutant, CXCR4-S339fs5, that promotes enhanced signaling, reduced phosphorylation, β-arrestin binding and endocytosis, and a very high basal rate of degradation that is not protected by antagonist treatment. Read full article

Retinitis pigmentosa (RP) is a group of hereditary degenerative diseases affecting 1 of 4000 people worldwide and being the most prevalent cause of visual handicap among working populations in developed countries. These disorders are mainly related to the abnormalities in the rod G protein-coupled receptor (GPCR), rhodopsin reflected in the dysregulated membrane trafficking, stability and phototransduction processes that lead to progressive loss of retina function and eventually blindness. Currently, there is no cure for RP, and the therapeutic options are limited. Targeting rhodopsin with small molecule chaperones to improve the folding and stability of the mutant receptor is one of the most promising pharmacological approaches to alleviate the pathology of RP. This review provides an update on the current knowledge regarding small molecule compounds that have been evaluated as rhodopsin modulators to be considered as leads for the development of novel therapies for RP. Read full article

Self-docking and cross-docking simulations of G protein-coupled receptor-ligand complexes: Impact of ligand type and receptor activation state G protein-coupled receptors (GPCR) are the largest family of cell surface receptors in vertebrates. Their abundance and role in nearly all physiological systems make GPCR the largest protein family targeted for development of pharmaceuticals. Ligand discovery aimed at identification of chemical tools and drug leads is aided by molecular docking simulations that allow critical analysis of the potential interactions between small molecules and proteins in resulting complexes. However, blind assessments of ligand pose quality and affinity prediction have thus far not provided broadly generalizable performance expectations for docking into experimentally-characterized GPCR targets. Likewise, the relative importance of receptor activation state and ligand function differences have also not been systematically assessed. This study compares performance when docking ligands of varied function into varied GPCR activation states in the absence of extensive resampling of the input GPCR structure, and only limited sidechain flexibility after ligand placement. Simulations were performed using 37 experimental structures of 11 Class A GPCR crystallized in multiple activation states (giving rise to 37 self-docking and 68 cross docking simulations). Our results show that one specific subset of cross-docking simulations gave results of similar quality to self-docking. Median ligand RMSD values for top-scored poses were 1.2 Å and 2.0 Å for self-docking and StateMatch/FunctionMatch cross-docking, respectively. The distributions of ligand RMSD values were not statistically different for these two conditions, according to a Kolmogorov-Smirnov test. Therefore, docking performance against GPCR targets can be estimated in advance based on docking target structure activation states, with higher accuracy expected when docking agonists into active state structures and inverse agonists or antagonists into inactive state structures. Receptor conformational sampling in advance of docking or receptor conformational adjustment after docking are more likely to produce substantial improvements for other pairings of receptor activation state and ligand function. Read full article

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

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

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

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

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

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

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 STATEMENTTwo 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

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 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

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