Development of Macrocyclic Neurotensin Receptor Type 2 (NTS2) Opioid-Free Analgesics.

The opioid crisis has highlighted the urgent need to develop non-opioid alternatives for managing pain, with an effective, safe, and non-addictive pharmacotherapeutic profile. Using an extensive structure-activity relationship approach, here we have identified a new series of highly selective neurotensin receptor type 2 (NTS2) macrocyclic compounds that exert potent, opioid-independent analgesia in various experimental pain models. To our knowledge, the constrained macrocycle in which the Ile12 residue of NT(7-12) was substituted by cyclopentylalanine, Pro7 and Pro10 were replaced by allyl-glycine followed by side-chain to side-chain cyclization is the most selective analog targeting NTS2 identified to date (Ki 2.9 nM), showing 30,000-fold selectivity over NTS1. Of particular importance, this macrocyclic analog is also able to potentiate the analgesic effects of morphine in a dose- and time-dependent manner. Exerting complementary analgesic actions via distinct mechanisms of nociceptive transmission, NTS2-selective macrocycles can therefore be exploited as opioid-free analgesics or as opioid-sparing therapeutics, offering superior pain relief with reduced adverse effects to pain patients.

Assessing Protein-Protein Docking Protocols: Case Studies of G-Protein-Coupled Receptor Interactions.

The interactions of G-protein-coupled receptors (GPCRs) with other proteins are critical in several cellular processes but resolving their structural dynamics remains challenging. An increasing number of GPCR complexes have been experimentally resolved but others including receptor variants are yet to be characterized, necessitating computational predictions of their interactions. Although integrative approaches with multi-scale simulations would provide rigorous estimates of their conformational dynamics, protein-protein docking remains a first tool of choice of many researchers due to the availability of open-source programs and easy to use web servers with reasonable predictive power. Protein-protein docking algorithms have limited ability to consider protein flexibility, environment effects, and entropy contributions and are usually a first step towards more integrative approaches. The two critical steps of docking: the sampling and scoring algorithms have improved considerably and their performance has been validated against experimental data. In this chapter, we provide an overview and generalized protocol of a few docking protocols using GPCRs as test cases. In particular, we demonstrate the interactions of GPCRs with extracellular protein ligands and an intracellular protein effectors (G-protein) predicted from docking approaches and test their limitations. The current chapter will help researchers critically assess docking protocols and predict experimentally testable structures of GPCR complexes.

Effects of regulator of G protein signaling 2 (RGS2) overexpression in the paraventricular nucleus on blood pressure in rats with angiotensin II-induced hypertension.

The hypothalamic paraventricular nucleus (PVN) regulates sympathetic activity and blood pressure. The regulator of G protein signaling 2 (RGS2) is a negative G protein regulator, which selectively regulates G⍺q signaling, a potential cause of hypertension. This study aimed to examine angiotensin II (ANG II)-G protein-RGS2 signaling on the central mechanisms of blood pressure control, sympathetic activation, and kidney function. The Sprague Dawley rats were infused with ANG II (200 ng/kg/min) via osmotic mini pump to induce hypertension. Adenovirus (AV) vectors encoding RGS2 was transfected into the PVN in vivo. By radio telemetry measurements, we found AV-RGS2 transfection to the PVN significantly attenuated the increase of mean arterial pressure in ANG II infusion rats from days 2-7 of the 2-week experiment (Day 7: ANG II + AV-RGS2 141.3 ± 10.0 mmHg vs. ANG II 166.9 ± 9.3 mmHg, p < 0.05). AV-RGS2 transfection significantly reduced the serum norepinephrine level and acute volume reflex and increased daily urine volume and sodium excretion in ANG II-infused hypertensive rats. AV-RGS2 transfection significantly reduced G⍺q and PKC protein expressions within the PVN in ANG II infusion rats. In cultured mouse hypothalamic cells, real-time PCR study showed ANG II treatment increased mRNA expression of G⍺q, G⍺s, and RGS2, and AV-RGS2 treatment decreased ANG II-induced mRNA expression of G⍺q and G⍺s. Using confocal imagery, we found that AV-RGS2 attenuated the increase of calcium influx in ANG II-treated cells. Our results suggest that central overexpression of RGS2 in the PVN attenuated the increase of blood pressure and sympathetic outflow, and improves kidney excretory function in hypertensive rats. This may be via the alteration of ANG II-G-protein-RGS2 signaling in the central nervous system.

Neuron specific quantitation of Gαolf expression and signaling in murine brain tissue.

The heterotrimeric G-protein α subunit, Gαolf, acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Gαolf, have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Gαolf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS. Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Gαolf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL, with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Gαolf signaling in vivo in neurons that highly express Gnal.

Smooth operator(s): dialing up and down neurotransmitter responses by G-protein regulators.

G-protein-coupled receptors (GPCRs) are essential mediators of neuromodulation and prominent pharmacological targets. While activation of heterotrimeric G-proteins (GαßÉ£) by GPCRs is essential in this process, much less is known about the postreceptor mechanisms that influence G-protein activity. Neurons express G-protein regulators that shape the amplitude and kinetics of GPCR-mediated synaptic responses. Although many of these operate by directly altering how G-proteins handle guanine-nucleotides enzymatically, recent discoveries have revealed alternative mechanisms by which GPCR-stimulated G-protein responses are modulated at the synapse. In this review, we cover the molecular basis for, and consequences of, the action of two G-protein regulators that do not affect the enzymatic activity of G-proteins directly: Gα inhibitory interacting protein (GINIP), which binds active Gα subunits, and potassium channel tetramerization domain-containing 12 (KCTD12), which binds active Gßγ subunits.

G-Protein Coupled Receptor 1 Is Involved in Tetrachlorobisphenol A-Induced Inflammatory Response in Jurkat Cells.

Estrogens can affect the immune inflammatory response through estrogen receptor alpha (ERα), but the specific role of estrogen member receptor G-protein coupled receptor 1 (GPER1) in this process remains unclear. In this study, we evaluated the effects of tetrachlorobisphenol A (TCBPA), which has estrogen activity, on immune inflammatory-related indicators of Jurkat cells, as well as investigated the role of GPER1 in these effects. The results showed that TCBPA at lower concentrations significantly promoted the viability of Jurkat cells, whereas higher concentrations decreased cell viability. TCBPA at concentrations ranging from 1 to 25 µM increased the intracellular reactive oxygen species (ROS) levels. Additionally, treatment with 10 µM TCBPA increased the protein expression of ERα and GPER1, elevated the phosphorylation of protein kinase B (p-Akt), and upregulated the mRNA levels of GPER1, Akt, and phosphoinositide 3-kinase (PI3K) genes. Treatment with 10 µM TCBPA also upregulated the protein or gene expression of pro-inflammatory cytokines, such as interleukins (IL1ß, IL2, IL6, IL8, IL12α) and tumor necrosis factor alpha (TNFα) in Jurkat cells. Furthermore, pretreatment with a GPER1 inhibitor G15 significantly reduced the mRNA levels of Akt induced by 10 µM TCBPA. Moreover, the upregulation of mRNA expression of RelA (p65), TNFα, IL6, IL8, and IL12α induced by 10 µM TCBPA was also significantly attenuated after G15 pretreatment. These findings suggest that TCBPA upregulates the expression of genes related to inflammatory responses by activating the GPER1-mediated PI3K/Akt signaling pathway. This study provides new insights into the mechanism of TCBPA-induced inflammatory response.

Advances in the pathophysiology, diagnosis and management of chronic diarrhoea from bile acid malabsorption: a systematic review.

Bile acid malabsorption (BAM) is an important disorder of digestive pathophysiology as it generates chronic diarrhoea. This condition originates from intricate pathways involving bile acid synthesis and metabolism in the liver and gut, the composition of gut microbiota, enterohepatic circulation and key receptors as farnesoid X receptor (FXR), fibroblast growth factor receptor 4 (FGFR4), and the G-protein bile acid receptor-1 (GPBAR-1). Although symptoms can resemble those related to disorders of gut brain interaction, accurate diagnosis of BAM may greatly benefit the patient. The empiric diagnosis of BAM is primarily based on the clinical response to bile acid sequestrants. Specific tests including the 48-hour fecal bile acid test, serum levels of 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19 (FGF19), and the 75Selenium HomotauroCholic Acid Test (SeHCAT) are not widely available. Nevertheless, lack of diagnostic standardization of BAM may account for poor recognition and delayed management. Beyond bile acid sequestrants, therapeutic approaches include the use of FXR agonists, FGF19 analogues, glucagon-like peptide-1 (GLP-1) receptor agonists, and microbiota modulation. These novel agents can best make their foray into the therapeutic armamentarium if BAM does not remain a diagnosis of exclusion. Ignoring BAM as a specific condition may continue to contribute to increased healthcare costs and reduced quality of life. Here, we aim to provide a comprehensive review of the pathophysiology, diagnosis, and management of BAM.

A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells.

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

E3 ligase DECREASED GRAIN SIZE 1 promotes degradation of a G-protein subunit and positively regulates grain size in rice.

Grain size is one of the most important traits determining crop yield. However, the mechanism controlling grain size remains unclear. Here, we confirmed the E3 ligase activity of DECREASED GRAIN SIZE 1 (DGS1) in positive regulation of grain size in rice (Oryza sativa) suggested in a previous study. Rice G-protein subunit gamma 2 (RGG2), which negatively regulates grain size, was identified as an interacting protein of DGS1. Biochemical analysis suggested that DGS1 specifically interacts with canonical Gγ subunits (rice G-protein subunit gamma 1 [RGG1] and rice G-protein subunit gamma 2 [RGG2]) rather than non-canonical Gγ subunits (DENSE AND ERECT PANICLE 1 [DEP1], rice G-protein gamma subunit type C 2 [GCC2], GRAIN SIZE 3 [GS3]). We also identified the necessary domains for interaction between DGS1 and RGG2. As an E3 ligase, DGS1 ubiquitinated and degraded RGG2 via a proteasome pathway in several experiments. DGS1 also ubiquitinated RGG2 by its K140, K145 and S147 residues. Thus, this work identified a substrate of the E3 ligase DGS1 and elucidated the post transcriptional regulatory mechanism of the G-protein signalling pathway in the control of grain size.

Differential requirement of Formyl Peptide Receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection.

Formyl peptide receptors (FPR), part of the G-protein coupled receptor superfamily, are pivotal in directing phagocyte migration towards chemotactic signals from bacteria and host tissues. Although their roles in acute bacterial infections are well-documented, their involvement in immunity against tuberculosis (TB) remains unexplored. This study investigates the functions of Fpr1 and Fpr2 in defense against Mycobacterium tuberculosis (Mtb), the causative agent of TB. Elevated levels of Fpr1 and Fpr2 were found in the lungs of mice, rabbits and peripheral blood of humans infected with Mtb, suggesting a crucial role in the immune response. The effects of Fpr1 and Fpr2 deletion on bacterial load, lung damage, and cellular inflammation were assessed using a TB model of hypervirulent strain of Mtb from the W-Beijing lineage. While Fpr2 deletion showed no impact on disease outcome, Fpr1-deficient mice demonstrated improved bacterial control, especially by macrophages. Bone marrow-derived macrophages from these Fpr1 -/- mice exhibited an enhanced ability to contain bacterial growth over time. Contrarily, treating genetically susceptible mice with Fpr1-specific inhibitors caused impaired early bacterial control, corresponding with increased bacterial persistence in necrotic neutrophils. Furthermore, ex vivo assays revealed that Fpr1 -/- neutrophils were unable to restrain Mtb growth, indicating a differential function of Fpr1 among myeloid cells. These findings highlight the distinct and complex roles of Fpr1 in myeloid cell-mediated immunity against Mtb infection, underscoring the need for further research into these mechanisms for a better understanding of TB immunity.

Clinical and Molecular Profiling in GNAO1 Permits Phenotype-Genotype Correlation.

BACKGROUND: Defects in GNAO1, the gene encoding the major neuronal G-protein Gαo, are related to neurodevelopmental disorders, epilepsy, and movement disorders. Nevertheless, there is a poor understanding of how molecular mechanisms explain the different phenotypes. OBJECTIVES: We aimed to analyze the clinical phenotype and the molecular characterization of GNAO1-related disorders. METHODS: Patients were recruited in collaboration with the Spanish GNAO1 Association. For patient phenotyping, direct clinical evaluation, analysis of homemade-videos, and an online questionnaire completed by families were analyzed. We studied Gαo cellular expression, the interactions of the partner proteins, and binding to guanosine triphosphate (GTP) and G-protein-coupled receptors (GPCRs). RESULTS: Eighteen patients with GNAO1 genetic defects had a complex neurodevelopmental disorder, epilepsy, central hypotonia, and movement disorders. Eleven patients showed neurological deterioration, recurrent hyperkinetic crisis with partial recovery, and secondary complications leading to death in three cases. Deep brain stimulation improved hyperkinetic crisis, but had inconsistent benefits in dystonia. The molecular defects caused by pathogenic Gαo were aberrant GTP binding and hydrolysis activities, an inability to interact with cellular binding partners, and reduced coupling to GPCRs. Decreased localization of Gαo in the plasma membrane was correlated with the phenotype of "developmental and epileptic encephalopathy 17." We observed a genotype-phenotype correlation, pathogenic variants in position 203 were related to developmental and epileptic encephalopathy, whereas those in position 209 were related to neurodevelopmental disorder with involuntary movements. Milder phenotypes were associated with other molecular defects such as del.16q12.2q21 and I344del. CONCLUSION: We highlight the complexity of the motor phenotype, which is characterized by fluctuations throughout the day, and hyperkinetic crisis with a distinct post-hyperkinetic crisis state. We confirm a molecular-based genotype-phenotype correlation for specific variants. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Structural Features Influencing the Bioactive Conformation of Angiotensin II and Angiotensin A: Relationship between Receptor Desensitization, Addiction, and the Blood-Brain Barrier.

The N-terminal portion of the octapeptide angiotensin II (DRVYIHPF; AngII), a vasopressor peptide that favorably binds to, and activates, AngII type 1 receptor (AT1R), has an important role in maintaining bioactive conformation. It involves all three charged groups, namely (i) the N-terminal amino group cation, (ii) the Asp sidechain anion and (iii) the Arg guanidino cation. Neutralization of any one of these three charged groups results in a substantial reduction (<5%) in bioactivity, implicating a specialized function for this cluster. In contrast, angiotensin A (ARVYIHPF; AngA) has reduced bioactivity at AT1R; however, replacement of Asp in AngII with sarcosine (N-methyl-glycine) not only restores bioactivity but increases the activity of agonist, antagonist, and inverse agonist analogues. A bend produced at the N-terminus by the introduction of the secondary amino acid sarcosine is thought to realign the functional groups that chaperone the C-terminal portion of AngII, allowing transfer of the negative charge originating at the C-terminus to be transferred to the Tyr hydroxyl-forming tyrosinate anion, which is required to activate the receptor and desensitizes the receptor (tachyphylaxis). Peptide (sarilesin) and nonpeptide (sartans) moieties, which are long-acting inverse agonists, appear to desensitize the receptor by a mechanism analogous to tachyphylaxis. Sartans/bisartans were found to bind to alpha adrenergic receptors resulting in structure-dependent desensitization or resensitization. These considerations have provided information on the mechanisms of receptor desensitization/tolerance and insights into possible avenues for treating addiction. In this regard sartans, which appear to cross the blood-brain barrier more readily than bisartans, are the preferred drug candidates.

Role of iRhom2 in Olfaction: Implications for Odorant Receptor Regulation and Activity-Dependent Adaptation.

The cell surface metalloprotease ADAM17 (a disintegrin and metalloprotease 17) and its binding partners iRhom2 and iRhom1 (inactive Rhomboid-like proteins 1 and 2) modulate cell-cell interactions by mediating the release of membrane proteins such as TNFα (Tumor necrosis factor α) and EGFR (Epidermal growth factor receptor) ligands from the cell surface. Most cell types express both iRhoms, though myeloid cells exclusively express iRhom2, and iRhom1 is the main iRhom in the mouse brain. Here, we report that iRhom2 is uniquely expressed in olfactory sensory neurons (OSNs), highly specialized cells expressing one olfactory receptor (OR) from a repertoire of more than a thousand OR genes in mice. iRhom2-/- mice had no evident morphological defects in the olfactory epithelium (OE), yet RNAseq analysis revealed differential expression of a small subset of ORs. Notably, while the majority of ORs remain unaffected in iRhom2-/- OE, OSNs expressing ORs that are enriched in iRhom2-/- OE showed fewer gene expression changes upon odor environmental changes than the majority of OSNs. Moreover, we discovered an inverse correlation between the expression of iRhom2 compared to OSN activity genes and that odor exposure negatively regulates iRhom2 expression. Given that ORs are specialized G-protein coupled receptors (GPCRs) and many GPCRs activate iRhom2/ADAM17, we investigated if ORs could activate iRhom2/ADAM17. Activation of an olfactory receptor that is ectopically expressed in keratinocytes (OR2AT4) by its agonist Sandalore leads to ERK1/2 phosphorylation, likely via an iRhom2/ADAM17-dependent pathway. Taken together, these findings point to a mechanism by which odor stimulation of OSNs activates iRhom2/ADAM17 catalytic activity, resulting in downstream transcriptional changes to the OR repertoire and activity genes, and driving a negative feedback loop to downregulate iRhom2 expression.

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the ßγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

A universal method for surface-based binding assays by preparing immobilized ß2-adrenergic receptor stationary phase using solid binding peptide as a fusion tag.

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize ß2-adrenergic receptor (ß2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4-1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged ß2-adrenoceptor (ß2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, ß2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on ß2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M-1 respectively, which were two orders of magnitude higher than those on the Halo-ß2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.

The ric-8b protein (resistance to inhibitors of cholinesterase 8b) is key to preserving contractile function in the adult heart.

Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function, but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis, and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodeling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the ß-adrenergic pathways. Using fluorescence resonance energy transfer-based assays, we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gαs) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gαs gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.

Redox-modulated SNX25 as a novel regulator of GPCR-G protein signaling from endosomes.

GPCR-G protein signaling from endosomes plays a crucial role in various physiological and pathological processes. However, the mechanism by which endosomal G protein signaling is terminated remains largely unknown. In this study, we aimed to investigate the regulatory mechanisms involved in terminating the signaling of Gα subunits from endosomes. Through structural analysis and cell-based assays, we have discovered that SNX25, a protein that targets endosomes via its PXA or PXC domain, interacts with regulator of G protein signaling (RGS) proteins (including RGS2, RGS4, RGS8, and RGS17) in a redox-regulated manner. The interaction between SNX25 and these RGS proteins enhances their GTPase-accelerating activity towards Gαi/q and their ability to bind GDP-bound (inactive form) Gαi/q. As a result, SNX25 recruits these RGS proteins to endosomes, leading to the termination of endosomal Gαi/q signaling. Furthermore, we have found that the SNX25/RGS complex also exerts a negative regulatory effect on Gαi/q signaling from the plasma membrane. This is achieved by recruiting Gαi/q to endosomes and preventing its activation on the plasma membrane. Our findings shed light on the previously unknown role of redox-modulated SNX25 in inhibiting Gαi/q signaling, thereby uncovering a novel mechanism for terminating Gαi/q signaling from endosomes. Importantly, this study expands our understanding of the regulation of GPCR-Gαi/q signaling beyond the plasma membrane.

GPCR Function in Autophagy Control: A Systematic Approach of Chemical Intervention.

Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.

Difluorinated thromboxane A2 reveals crosstalk between platelet activatory and inhibitory pathways by targeting both the TP and IP receptors.

BACKGROUND AND PURPOSE: Thromboxane A2 (TXA2) is a prostanoid produced during platelet activaton, important in enhancing platelet reactivity by activation of TP receptors. However, due to the short half-life, studying TXA2 signalling is challenging. To enhance our understanding of TP receptor-mediated platelet biology, we therefore synthesised mono and difluorinated TXA2 analogues and explored their pharmacology on heterologous and endogenously expressed TP receptor function. EXPERIMENTAL APPROACH: Platelet functional and signalling responses were studied using aggregometry, Ca2+ mobilisation experiments and immunoblotting and compared with an analogue of the TXA2 precursor prostaglandin H2, U46619. Gαq/Gαs receptor signalling was determined using a bioluminescence resonance energy transfer (BRET) assay in a cell line overexpression system. KEY RESULTS: BRET studies revealed that F-TXA2 and F2-TXA2 promoted receptor-stimulated TP receptor G-protein activation similarly to U46619. Unexpectedly, F2-TXA2 caused reversible aggregation in platelets, whereas F-TXA2 and U46619 induced sustained aggregation. Blocking the IP receptor switched F2-TXA2-mediated reversible aggregation into sustained aggregation. Further BRET studies confirmed F2-TXA2-mediated IP receptor activation. F2-TXA2 rapidly and potently stimulated platelet TP receptor-mediated protein kinase C/P-pleckstrin, whereas IP-mediated protein kinase A/P-vasodilator-stimulated phosphoprotein was more delayed. CONCLUSION AND IMPLICATIONS: F-TXA2 is a close analogue to TXA2 used as a selective tool for TP receptor platelet activation. In contrast, F2-TXA2 acts on both TP and IP receptors differently over time, resulting in an initial wave of TP receptor-mediated platelet aggregation followed by IP receptor-induced reversibility of aggregation. This study reveals the potential difference in the temporal aspects of stimulatory and inhibitory pathways involved in platelet activation.

Isothermal Titration Calorimetry for Fragment-Based Analysis of Ion Channel Interactions.

Ion channels are membrane proteins that may also have intracellular and extracellular domains that interact with other ligands. In many cases, these interaction sites are highly mobile and may undergo changes in the configuration upon binding with regulatory signaling molecules. Isothermal titration calorimetry (ITC) is a powerful technique to quantify protein-ligand interactions of purified samples in solution. This chapter describes a fragment-based analysis method using ITC to quantify the interactions between a domain of the voltage-gated Kv7 channel and the calcium-regulated protein calmodulin. This example can be used to quantify the interactions between specific domains of other ion channels and their regulatory signaling proteins.