Biochemical Pharmacology

The histamine H3 receptor (H3R) regulates as a presynaptic G protein-coupled receptor the release of histamine and other neurotransmitters in the brain, and is consequently a potential therapeutic target for neuronal disorders. The human H3R encodes for seven splice variants that vary in the length of intracellular loop 3 and/or the C-terminal tail but are all able to induce heterotrimeric Gi protein signaling. The last two decades H3R drug discovery and lead optimization has been exclusively focused on the 445 amino acids-long reference isoform H3R-445. In this study, we pharmacologically characterized for the first time all seven H3R isoforms by determining their binding affinities for reference histamine H3 receptor agonists and inverse agonists. The H3R-453, H3R-415, and H3R-413 isoforms display similar binding affinities for all ligands as the H3R-445. However, increased agonist binding affinities were observed for the three shorter isoforms H3R-329, H3R-365, and H3R-373, whereas inverse agonists such as the approved anti-narcolepsy drug pitolisant (Wakix(R)) displayed significantly decreased binding affinities for the latter two isoforms. This opposite change in binding affinity of agonist versus inverse agonists on H3R-365 and H3R-373 is associated with their higher constitutive activity in a cAMP biosensor assay as compared to the other 5 isoforms. The observed differences in pharmacology between longer and shorter H3R isoforms should be considered in future drug discovery programs.

Background and purposeMuscarinic acetylcholine receptors are key therapeutic targets, and ligands engaging both orthosteric and allosteric sites may offer improved selectivity and efficacy. The muscarinic antagonist KH-5 displays functional antagonistic potency exceeding its binding affinity, suggesting a non-classical mechanism of action. Here, we investigated whether KH-5 acts as a dualsteric antagonist and defined its mode of interaction with muscarinic receptors. Experimental approachFunctional responses at human M1 and M2 receptors expressed in CHO cells were assessed using inositol phosphate accumulation and [35S]GTP{gamma}S binding, respectively. Radioligand binding studies employed orthosteric antagonists and agonists in combination with KH-5 and classical allosteric modulators. Data were analysed using competitive, allosteric, and dualsteric binding and operational models. Molecular docking, molecular dynamics simulations, and site-directed mutagenesis were used to identify structural determinants of KH-5 binding. Key resultsKH-5 antagonised responses to multiple agonists in a saturable and probe-dependent manner consistent with an allosteric interaction. However, KH-5 did not decrease maximal response to agonists, contradicting simple allosteric antagonism. At M2 receptors, antagonism was largely competitive. Binding studies revealed transient enhancement of agonist binding at M1 receptors at nanomolar concentrations of KH-5, best described by a dualsteric binding model involving independent orthosteric and ectopic site interactions. KH-5 did not bind to the classical muscarinic allosteric site at the second extracellular loop but interacted with an extracellular vestibule site, supported by molecular modelling and mutation of key residues. Conclusions and implicationsThe simplest model explaining the KH-5 mechanism of action at muscarinic receptors combines two concurrent modes of interaction. From the allosteric site, it positively modulates functional responses to agonists. From the orthosteric site, it exerts competitive antagonism of functional responses. Additionally, molecules of KH-5 bound to allosteric and orthosteric sites exert positive cooperativity. HighlightsO_LIKH-5 antagonises muscarinic receptors with a potency exceeding its orthosteric binding affinity C_LIO_LIFunctional antagonism shows probe dependence, indicating an allosteric component C_LIO_LIBinding studies support independent interaction of KH-5 with orthosteric and ectopic sites C_LIO_LIKH-5 does not bind the classical muscarinic allosteric site C_LIO_LIExcept for xanomeline, the operational model of dualsterically modulated agonism explains the complex pharmacology of KH-5 at M1 receptors C_LI

IntroductionThe {beta}2-adrenoceptor ({beta}2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma, whereby {beta}2AR agonists relieve bronchoconstriction. The {beta}2AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the {beta}2AR is not understood. We hypothesized that the kinetics of ligand binding and GPCR-G protein interactions could play a role in ligand efficacy. We characterised the molecular pharmacology of a range of {beta}2AR agonists and examined the correlation between ligand and mini-Gs binding kinetics and efficacy. MethodsWe used a Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) based competition ligand binding assay to measure the affinity and residence times of a range of {beta}2AR agonists binding to the human {beta}2AR. TR-FRET between Lumi4-Tb3+ on the N terminus of the {beta}2AR and fluorescent CA200693 (S)-propranolol-green was measured using a PHERAstar FSX. The ability of these {beta}2AR agonists to activate the heterotrimeric Gs protein was measured using the CASE Gs protein biosensor. This assay senses a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the G subunit and Venus acceptor on the G{psi}, on receptor activation, quantified using the operational model of agonism. NanoBRET was also used to measure interactions between DDM solubilised {beta}2AR-nLuc and purified Venus-mini-Gs. A large excess of unlabelled mini-Gs was used to dissociate the {beta}2AR-nLuc: Venus-mini-Gs complex. ResultsCharacterisation of the molecular pharmacology of seven {beta}2AR agonists showed a broad range of ligand binding affinities (pKi = 4.4 {+/-} 0.09 to 9.2 {+/-} 0.08) and kinetics parameters. There was no correlation between ligand residence times and their ability (log{tau} ) to activate the Gs protein (R2=0.26, p=0.29). However, there were statistically significant differences in the association rate (kon (fast)) (3.36{+/-}0.64x105 to 9.19{+/-} 0.42x105) and affinity (Kd) values of mini-Gs binding to the agonist-{beta}2AR complex (pKd =6.0 to 6.7). Both an increase in ligand driven mini-Gs kon(fast) rate and associated increase in mini-Gs pKd for the receptor, were moderately correlated with efficacy (log{tau} ) (R2 =0.58 and R2 =0.50 respectively). ConclusionsThese data support a model in which agonists of increased efficacy cause the {beta}2AR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support a role for agonist binding kinetics in the molecular basis of efficacy.

The inhibitory analysis of intracellular signaling pathways is widely employed to gain insight into molecular mechanisms underlying diverse physiological processes. Unfortunately, the essential drawback of this basically effective methodology is that many, if not all, inhibitors, antagonists, modulators, and blockers can affect cellular functions not only acting through specified cellular targets, but also causing off-target effects. In particular, the class I phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and its PI3K-inactive structural analog LY303511 have been shown to affect agonist-induced Ca2+ signaling in cells of various types independently of PI3K activity. Here we studied serotonin-induced Ca2+ signaling in HEK293 cells expressing the recombinant mouse 5-HT2C receptor and analyzed the effects of LY294002 and LY303511 on cell responsiveness. As shown with Ca2+ imaging, both LY294002 and LY303511 affected intracellular Ca2+ but via distinct mechanisms. LY294002 suppressed responsiveness of assayed cells to serotonin in a manner suggesting that this substance acted as a competitive antagonist of the 5-HT2C receptor. In turn, LY303511 itself triggered Ca2+ transients in 5-HT2C-positive cells, exhibiting traits of a 5-HT2C agonist. In support of these findings, molecular docking and molecular dynamics simulations validated the binding of both LY294002 and LY303511 to the 5-HT2C receptor and occupying its orthosteric site. Altogether, physiological findings and computational data suggested that the observed effects of these compounds were most likely mediated by extracellular mechanisms associated with the direct interaction of both with the 5-HT2C receptor. This expands the list of non-specified cellular targets of LY294002 and LY303511 with 5-HT2C subtype of serotonin receptors.

Lysophosphatidic acid (LPA) and oleoyl-methoxy glycerophosphothionate (OMPT) increased LPA3 phosphorylation; OMPT being considerably more potent than LPA. OMPT was also more potent than LPA to activate ERK 1/2. In contrast, to increase intracellular calcium OMPT was less effective than LPA. LPA-induced LPA3-{beta}-arrestin 2 interaction was fast and robust, whereas that induced by OMPT was only detected at 60 min of incubation. LPA- and OMPT-induced receptor internalization was fast but that of OMPT was more marked. LPA-induced internalization was blocked by Pitstop 2, whereas OMPT-induced receptor internalization was partially inhibited by Pitstop 2 and Filipin and entirely by the combination of both. The data again indicate differences in the actions of these agonists. When LPA-stimulated cells were rechallenged with 1 {micro}M LPA, hardly any response was detected, i.e., a "refractory" state was induced. However, if OMPT was used as the second stimulus, a conspicuous and robust response was observed. These data again suggest the possibility that two binding sites for these agonists might exist in the LPA3 receptor, one showing a very high affinity for OMPT and another, likely shared by LPA and OMPT (structural analogs) with lower affinity. One sentence summaryOMPT, oleoyl-methoxy glycerophosphothionate, a biased agonits interacting with an additional binding site in LPA3 receptors.

Background & AimsIn intestinal smooth muscle cells, receptor-operated TRPC4 are responsible for the majority of muscarinic receptor cation current (mICAT), which initiates cholinergic excitation-contraction coupling. Our aim was to examine the effects of the TRPC4 inhibitor Pico145 on mICAT and Ca2+ signalling in mouse ileal myocytes, and on intestinal motility. MethodsIleal myocytes freshly isolated from two month-old male BALB/c mice were used for patch-clamp recordings of whole-cell currents and for intracellular Ca2+ imaging using Fura-2. Functional assessment of Pico145s effects was carried out by standard in vitro tensiometry, ex vivo video recordings and in vivo postprandial intestinal transit measurements using carmine red. ResultsCarbachol (50 {micro}M)-induced mICAT was strongly inhibited by Pico145 starting from 1 pM. The IC50 value for the inhibitory effect of Pico145 on this current evoked by intracellularly applied GTP{gamma}S (200 {micro}M), and thus lacking desensitisation, was found to be 3.1 pM, while carbachol-induced intracellular Ca2+ rises were inhibited with IC50 of 2.7 pM. In contrast, the current activated by direct TRPC4 agonist (-)-englerin A was less sensitive to the action of Pico145 that caused only [~]43% current inhibition at 100 pM. The inhibitory effect developed rather slowly and it was potentiated by membrane depolarisation. In functional assays, Pico145 produced concentration-dependent suppression of both spontaneous and carbachol-evoked intestinal smooth muscle contractions and delayed postprandial intestinal transit. ConclusionsPico145 is a potent GI-active small-molecule which completely inhibits mICAT at picomolar concentrations and which is as effective as trpc4 gene deficiency in in vivo intestinal motility tests.

The action of the antidepressants imipramine, amitriptyline, and paroxetine on LPA3 receptors was studied in cellulo, using receptor-transfected HEK 293 Flp-In TREx cells, and in silico, through docking simulations. These drugs showed a low affinity for LPA3 receptors with lesser efficacy than LPA (paroxetine {approx} 60% and imipramine and amitriptyline {approx} 30%). When LPA-treated cells (with the agonist present) were challenged with the antidepressants, paroxetine triggered a robust increase in intracellular calcium, whereas imipramine and amitriptyline decreased the calcium concentration below baseline values. For ERK 1/2 phosphorylation, imipramine induced a rapid and potent increase, whereas amitriptyline and paroxetine reduced ERK 1/2 phosphorylation below baseline. Similarly, imipramine produced rapid and robust ERK phosphorylation in LPA-stimulated cells, but amitriptyline decreased ERK 1/2 phosphorylation. Activation with the antidepressants leads to LPA3 internalization; dramatic morphological changes accompany these actions. Docking simulations showed these drugs interact with an LPA3 receptor pocket, denominated Upper Cavity. Although the agonist binding cavity was the same, the amino acids interacting with the various ligands were distinct due to their different chemical structure. The manuscript advances knowledge on the mechanisms of antidepressant effects on LPA3 receptors, which might have potential therapeutic implications.

The phosphoinositide 3-kinase (PI3K) is involved in regulation of multiple intracellular processes. Although the inhibitory analysis is generally employed for validating a physiological role of PI3K, increasing body of evidence suggests that PI3K inhibitors can exhibit PI3K-unrelated activity as well. Here we studied effects of PI3K inhibitor LY294002 and its inactive analogue LY303511 on Ca2+ and cAMP signals initiated by serotonin. In the present study several monoclonal HEK293 cell lines were used, in particular, monitoring of Ca2+ signals were carried out on Fura-2 loaded cells expressed recombinant serotonin 5-HT2C receptors, cAMP signals were studied on cells expressed the genetically encoded cAMP sensor Pink Flamindo and recombinant 5-HT4 receptors, for monitoring PI3K activity cells stably expressed the genetically encoded PIP3 sensor PH(Akt)-Venus were used. It turned out that LY294002 suppressed Ca2+ signals initiated by activation 5-HT2C receptors irrespectively of PI3K inhibition, but did not affect cAMP responses initiated by 5-HT4 receptors. In turn LY303511 suppressed cAMP signals initiated by 5-HT4 receptors, and elicited Ca2+ transients exclusively in cells expressed 5-HT2C receptors. Based on these facts and the results of the inhibitory analysis, we hypothesize that the described effects may be due to the activity of LY294002 and LY303511 on the serotonin 5-HT2C and 5-HT4 receptors.

The P2X3 receptor (P2X3R), an ATP-gated non-selective cation channel of the P2X receptor family, is expressed in sensory neurons and involved in nociception. P2X3R inhibition was shown to reduce chronic and neuropathic pain. In a previous screening of 2000 approved drugs, natural products and bioactive substances, various non-steroidal anti-inflammatory drugs (NSAIDs) were found to inhibit P2X3R-mediated currents. To investigate whether the inhibition of P2X receptors contributes to the analgesic effect of NSAIDs, we characterized the potency and selectivity of various NSAIDs at P2X3R and other P2XR subtypes using two-electrode voltage clamp electrophysiology. We identified diclofenac as a hP2X3R and hP2X2/3R antagonist with micromolar potency (with IC50 values of 138.2 {micro}M and 76.7 {micro}M, respectively). A weaker inhibition of hP2X1R, hP2X4R and hP2X7R by diclofenac was determined. Flufenamic acid (FFA) proved to inhibit hP2X3R, rP2X3R and hP2X7R (IC50 values of 221{micro}M, 264.1{micro}M and [~] 900{micro}M, respectively), questioning its widespread use as a nonselective ion channel blocker, when P2XR-mediated currents are under study. Inhibition of the hP2X3R or hP2X2/3R by diclofenac could be overcome by prolonged ATP-application or increasing concentrations of the agonist ,{beta}-meATP, respectively, indicating competition of diclofenac and the agonists. Molecular dynamics simulation showed that diclofenac largely overlaps with ATP bound to the open state of the hP2X3R. Our results strongly support a competitive antagonism through which diclofenac, by interacting with residues of the ATP-binding site, left flipper, and dorsal fin domains inhibits gating of P2X3R by conformational fixation of the left flipper and dorsal fin domains. In summary, we demonstrate the inhibition of the human P2X3 receptor by various NSAIDs. Diclofenac proved to be the most effective antagonist with a strong inhibition of hP2X3R and hP2X2/3R and a weaker inhibition of hP2X1R, hP2X4R and hP2X7R. Considering their involvement in nociception, inhibition of hP2X3R and hP2X2/3R by micromolar concentrations of diclofenac may contribute to the analgesic effect as well as the side effect of taste disturbances of diclofenac and represent an additional mode of action besides the well-known high potency COX inhibition.

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second-messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

In HEK-293T cells transiently transfected with the human histamine H3 receptor (hH3R), we studied the effect of over-expressing the human RGS9-2 protein on H3R-mediated stimulation of [35S]-GTP{gamma}S binding and inhibition of forskolin-induced cAMP formation. Maximal specific binding (Bmax) of [3H]-N-methyl-histamine to cell membranes was 468 {+/-} 12 and 442 {+/-} 38 fmol/mg protein for HEK-293T-hH3R and HEK-293T-hH3R/hRGS9-2 cells, respectively, with dissociation constants (Kd) 2.57 nM and 3.38 nM. The H3R agonist immepip stimulated [35S]-GTP{gamma}S binding with similar potency and efficacy (Emax 146.3 {+/-} 4.4 % and 150.0 {+/-} 5.3 % of basal, pEC50 8.57 {+/-} 0.26 and 9.00 {+/-} 0.33, respectively), but was significantly less efficacious to inhibit forskolin-induced cAMP accumulation in HEK-293T-hH3R/hRGS9 cells (-19.2 {+/-} 5.3 versus -37.7 {+/-} 5.1 % in HEK-293T-hH3R cells) with no significant difference in potency (pEC50 9.60 {+/-} 0.14 and 9.07 {+/-} 0.29, respectively). These results indicate that in HEK-293T cells hRGS9-2 regulates hH3R445 signaling downstream G protein activation.

Aims/hypothesisAcute hypoglycemia promotes pro-inflammatory cytokine production, increasing risk for cardiovascular events in diabetes. AMP-activated protein kinase (AMPK) is regulated by and influences production of pro-inflammatory cytokines. We tested the mechanistic role of AMPK in low glucose induced changes in the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which is elevated in patients with diabetes. MethodsMacrophage cell line Raw264.7 cells, primary macrophage bone marrow derived macrophages obtained from wild type mice or AMPK {gamma}1 gain-of-function mice were utilized, as were AMPK1/2 knockout mouse embryonic fibroblasts (MEF). Allosteric AMPK activators PF-06409577 and BI-9774 were used, in conjunction with inhibitor SBI-0206965 were also used. We examined changes in protein phosphorylation/expression using western blotting, and protein localisation using immunofluorescence. Metabolic function was assessed using extracellular flux analyses and luciferase-based ATP assay. Cytokine release was quantified by ELISA. Oxidative stress was detected using a fluorescence-based ROS assay, and cell viability was examined using flow cytometry. ResultsMacrophages exposed to low glucose showed a transient and modest activation of AMPK and a metabolic shift towards increased oxidative phosphorylation. Low glucose induced oxidative stress and increased release of macrophage migration inhibitory factor (MIF). Pharmacological activation of AMPK by PF-06409577 and BI-9774 attenuated low glucose-induced MIF release, with a similar trend noted with genetic activation using AMPK{gamma}1 gain-of-function (D316A) mice, which produced a mild effect on low glucose-induced MIF release. Inhibition of NFB signalling diminished MIF release and AMPK activation modestly but significantly reduced low glucose-induced nuclear translocation of NFB. AMPK activation did not alter low glucose-induced oxidative stress in macrophages but application of AMPK inhibitor SBI-0206965 enhanced oxidative stress in macrophages and in AMPK knockout MEFs, suggesting an AMPK-independent mechanism Conclusions/interpretationTaken together, these data indicate that pharmacological AMPK activation suppresses release of MIF from macrophages. This is mediated by reduced activation of NFB signalling in response to low glucose-induced oxidative stress and suggests that pharmacological AMPK activation could be a useful strategy for mitigating hypoglycemia-induced inflammation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/562445v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1d3364borg.highwire.dtl.DTLVardef@1919395org.highwire.dtl.DTLVardef@704923org.highwire.dtl.DTLVardef@1d1cc59_HPS_FORMAT_FIGEXP M_FIG Tweet Low glucose induces pro-inflammatory MIF release from macrophages, and pharmacological AMPK activation suppresses the release of MIF. AMPK/ NFB signalling pathway is involved, which may be a new strategy to attenuate the pro-inflammatory response in hypoglycemia. C_FIG

Free fatty acid receptor 1 (FFA1 or GPR40), activated by medium- and long-chain fatty acids, amplifies glucose-stimulated insulin secretion, making it a promising target for type 2 diabetes. Radioligand studies revealed distinct binding sites for partial and full agonists, with full agonists showing positive cooperativity. Functional assays demonstrated positive cooperativity between agonists and varying interactions with the endogenous fatty acid DHA. These findings suggest three allosterically linked binding sites on FFA1, with activation influenced by key arginine residues. Potent ligands with strong cooperativity hold significant therapeutic potential.

Although being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1983, Black and Leff introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves. Functional analysis of OMA demonstrates that the slope factor implemented by Black and Leff affects relationships among parameters of the OMA. Fitting of the OMA with Black & Leff slope factor to concentration-response curves theoretical model-based data resulted in wrong estimates of operational efficacy and affinity. In contrast, fitting the OMA modified by the Hill coefficient to the same data resulted in correct estimates of operational efficacy and affinity. Therefore, OMA modified by the Hill coefficient should be preferred over the Black & Leff equation for ranking of agonism and subsequent analysis, like quantification of signalling bias, when concentration-response curves differ in the slope factor and mechanism of action is known. Otherwise. Black & Leff equation should be used with extreme caution acknowledging potential pitfalls.

Several studies indicated anti-cancer effects of metformin in liver cancer. This was attributed to the activation of LKB-AMPK axis, which is associated with anti-hyperglycaemic effect and cytotoxicity. However, despite lack of evidence on cytotoxic effect of physiological metformin concentrations and ability of cancer cells to survive under glucose-deprivation, no study has examined the glucose-independent effect of non-cytotoxic metformin or metabolic reprogramming associated with it. In addition, no study has ever been conducted on reversibility of anti-cancer effects of metformin. Here, the dose-dependent effects of metformin on HepG2 cells were examined in presence and absence of glucose. The longitudinal evolution of metabolome was analyzed along with gene and protein expression as well as their correlations with and reversibility of cellular phenotype and metabolic signatures. Metformin concentrations up to 2.5mM were found to be non-cytotoxic but anti-proliferative irrespective of presence of glucose. Apart from mitochondrial impairment, derangement of fatty acid desaturation, one-carbon, glutathione and polyamine metabolism were associated with non-cytotoxic metformin treatment irrespective of glucose supplementation. Depletion of pantothenic acid, downregulation of essential amino acid uptake, metabolism and purine salvage were identified as novel glucose-independent effects of metformin. These were significantly correlated with cMyc expression and reduction in proliferation. Rescue experiments established reversibility upon metformin withdrawal and tight association between proliferation, metabotype and cMyc expression. Taken together, derangement of novel glucose-independent metabolic pathways and concomitant cMyc downregulation co-ordinately contribute to anti-proliferative effect of metformin even at non-cytotoxic concentrations, which is reversible and may influence its therapeutic utility.

Serotonin or 5-hydroxytryptamine receptors type 3 (5-HT3) belong to the family of pentameric ligand-gated ion channels (pLGICs), which also includes other neurotransmitter-gated ion channels such as nicotinic acetylcholine receptors (nAChRs). pLGICs have been long-standing therapeutic targets for psychiatric disorders such as anxiety, schizophrenia, and addiction, and neurological diseases like Alzheimers and Parkinsons disease. Due to structural conservation and significant sequence similarities of pLGICs extracellular and transmembrane domains across the more than 40 subunits found in humans, clinical trials for drug candidates targeting these two domains have been largely hampered by undesired effects mediated by off-subunit modulation. With the present study, we explore the interaction interface of the 5-HT3A intracellular domain (ICD) with the resistance to inhibitors of choline esterase (RIC-3) protein. Previously, we have shown that RIC-3 directly interacts with the ICD of 5-HT3A subunits. Using a sequential deletion approach, we identified the L1-MX segment of the ICD fused to maltose-binding protein as sufficient for the interaction. For the present study, synthetic L1-MX-based peptides, Ala-scanning, and a pull-down assay identified positions W347, R349, and L353 as critical for binding to RIC-3. In complementary studies with full-length 5-HT3A subunits, the identified Ala substitutions reduced the modulation of functional surface expression by co-expression of RIC-3. Additionally, we found and characterized a duplication of the binding motif at the transition between the ICD MA-helix and transmembrane segment M4. Analogous Ala substitutions at W447, R449, and L454 disrupt MAM4-peptide RIC-3 interactions and reduce modulation of functional surface expression. In summary we identify two binding sites for RIC-3 with a shared duplicated motif in 5-HT3A subunits, one in the MX-helix and one at the MAM4-helix transition.

Signaling by formyl peptide receptor 1 (FPR1), the prototype G protein-coupled receptor (GPCR) expressed in neutrophil leukocytes, is initiated by an activation of a G protein containing a Gi subunit. FPR1 activation results in an increase in the cytosolic concentration of free calcium ions ([Ca2+]i), and an activation of the superoxide anion producing NADPH oxidase. Receptor downstream signals generated by the danger molecule ATP recognized by the purinergic receptor P2Y2 are transduced by a G protein containing a Gq subunit. The neutrophil response induced by ATP also includes a transient rise in [Ca2+]i, but the downstream signals do not activate the NADPH oxidase. ATP can, however, activate this enzyme system through a receptor transactivation mechanism dependent not only on the ATP receptor but also on the free fatty acid receptor FFA2R, provided that this receptor is allosterically modulated. This occurs through a novel mechanism whereby FFA2R is activated from the cytosolic side of the plasma membrane by Gq transduced signals generated by the ATP receptor. Furthermore, in neutrophils with a disrupted actin cytoskeleton, ATP (as well as platelet activating factor; recognized by the Gq-coupled PAFR) becomes a potent NADPH oxidase activating agonist. At high concentrations of the actin cytoskeleton disrupting drug latrunculin A the activation was only partly reduced by Gq inhibition. More importantly, this response was also partly inhibited by pertussis toxin. The effects on the ATP-induced NADPH oxidase activity, of the Gq inhibitor and pertussis toxin were more and less pronounced, respectively, when the concentration of latrunculin A was reduced. Taken together, we show that in primary human neutrophils the actin cytoskeleton is part of the regulatory machinery that determines the activation of NADPH oxidase activation and the G protein recruitment profile downstream of activated of Gq-coupled GPCRs. HighlightsO_LIATP is a biased signaling agonist unable to activate the neutrophil NADPH oxidase C_LIO_LIATP activates the NADPH oxidase through P2Y2R mediated transactivation of FFA2R C_LIO_LIActin cytoskeleton disruption enables ATP to activate the NADPH oxidase C_LIO_LICytoskeleton regulated NADPH oxidase activation depends on Gi and Gq signaling C_LIO_LIThe actin cytoskeleton regulates the G protein recruitment profile of P2Y2R C_LI

The endocannabinoid system comprises a series of ligands and receptors that have putative roles in regulating physiological and cognitive processes such as pre- and post-natal development, appetite, pain sensation, mood, and memory. Besides the endocannabinoids, these endogenous receptors are also capable of mediating the pharmacological effects of phytocannabinoids, which are derived from the cannabis plant. We sought to interrogate a panel of cell lines, representative of multiple human diseases, to characterize their cannabinoid-mediating gene expression. We found all lines expressed one or more gene product that rendered the cell potentially responsive to cannabinoids. Moreover, the expression profiles differed between normal and cancerous cells, as well as between cells derived from the brain, pancreas, and skin. Taken together, given the presence of one or more of these mediating gene products, our findings suggest a potential therapeutic role for phytocannabinoids.

The endocannabinoid (eCB) system regulates several brain functions and is implicated in neurological disorders. The pharmacological blockade of cannabinoid receptors has a therapeutic potential for various cognitive deficits, but also produces severe psychiatric side effects. Hence, new cannabinoid compounds that potentiate therapeutic effects, while minimizing toxicity, are required. In this study, we synthesized and characterized a novel antagonist/inverse agonist of CB1 receptors. UVI3502 showed affinity for two [3H]CP55,940 binding sites (IC50Hi 0.47 {+/-} 1.94 nM and IC50Lo 1470 {+/-} 1.80 nM). Subsequent binding assays performed in CB1 and CB2 overexpressing membranes determined that the low affinity binding site corresponded to CB1, but the high-affinity binding site of UVI3502 did not correspond to CB2 and the possibility of it corresponding to GPR55 was analyzed. The affinity of UVI3502 for CB1 receptors was further confirmed with neuroanatomical specificity by autoradiography in key brain areas, in which functional [35S]GTP{gamma}S assays demonstrated that UVI3502 behaved as an antagonist/inverse agonist of CB1 receptors, blocking the stimulation evoked by potent cannabinoid receptor agonist CP55,940 and decreasing basal [35S]GTP{gamma}S binding. The in silico characterization of the binding to CB1 receptor through molecular docking and molecular dynamics suggests that this activity is explained by the planar and rigid structure of UVI3502, which is optimal for interactions with the inactive state of the receptor. These results indicate that UVI3502 is a novel antagonist/inverse agonist of CB1 receptors, making it a compelling candidate for pharmacologically blocking cannabinoid receptors in the central nervous system. Significance StatementUVI3502 is a novel antagonist/inverse agonist of CB1 receptors, with almost no affinity for CB2 receptors and an additional high-affinity binding site for a third, cannabinoid-like receptor, potentially GPR55. In relevant brain areas for learning and memory processes with a high expression of CB1, UVI3502 blocks the stimulation evoked by the cannabinoid receptor agonist CP55,940, rendering it as an interesting compound for the pharmacological blockade of cannabinoid receptors in the central nervous system.

Ligand-gated ion channels open in response to the binding of an agonist. The agonist binding site is typically located tens of Angstroms away from the channel gate, which lies within the membrane and thus the gating mechanism is considered a classic example of allostery within proteins. The multi-subunit nature of these proteins also means that modulatory effects on the gating process can also be mediated by several other distinct regions - so called allosteric modulatory sites. One of the most well-studied channels in this regard, is the nicotinic acetylcholine receptor. Super-agonists are compounds that can produce a greater maximal response than the endogenous ligand (acetylcholine in this case). They are able to stabilize the open state of the ion channel and this can have important consequences for neuronal signaling. Some super-agonist effects can be mediated through the orthosteric binding site, but others can be mediated by alternative binding sites. The latter are often much harder to identify and can depend very precisely on the subunit composition of the receptor. In this work we sought to identify the mechanism by which TC-2559, a known super-agonist that acts as such only at one particular combination of neuronal nicotinic acetylcholine receptors - the high sensitivity receptor which is comprised of 2 alpha subunits and 3 beta subunits (as opposed to the low sensitivity receptor which has 3 alpha and 2 beta subunits). By using advanced computational methods, supported by two-voltage electrode clamp experiments, we were able to show that TC-2559 not only binds to the orthosteric but also binds to the unique b2-b2 interface of the HS receptor. The binding of TC-2559 to this interface exerts unique interactions that other agonists are not able to make, but more importantly it induces changes in the interface that support that concept of an allosteric gain in overall efficacy. Our results highlight how allosteric control exists to modulate receptor function.