Focusing on the 5F-MDMB-PICA, 4F-MDMB-BICA synthetic cannabinoids and their primary metabolites in analytical and pharmacological aspects.

Nowadays, more and more new synthetic cannabinoids (SCs) appearing on the illicit market present challenges to analytical, forensic, and toxicology experts. For a better understanding of the physiological effect of SCs, the key issue is studying their metabolomic and psychoactive properties. In this study, our validated targeted reversed phase UHPLC-MS/MS method was used for determination of urinary concentration of 5F-MDMB-PICA, 4F-MDMB-BICA, and their primary metabolites. The liquid-liquid extraction procedure was applied for the enrichment of SCs. The pharmacological characterization of investigated SCs were studied by radioligand competition binding and ligand stimulated [35S]GTPγS binding assays. For 5F-MDMB-PICA and 4F-MDMB-BICA, the median urinary concentrations were 0.076 and 0.312 ng/mL. For primary metabolites, the concentration range was 0.029-881.02* ng/mL for 5F-MDMB-PICA-COOH, and 0.396-4579* ng/mL for 4F-MDMB-BICA-COOH. In the polydrug aspect, the 22 urine samples were verified to be abused with 6 illicit drugs. The affinity of the metabolites to CB1R significantly decreased compared to the parent ligands. In the GTPγS functional assay, both 5F-MDMB-PICA and 4F-MDMB-BICA were acting as full agonists, while the metabolites were found as weak inverse agonists. Additionally, the G-protein stimulatory effects of the full agonist 5F-MDMB-PICA and 4F-MDMB-BICA were reduced by metabolites. These results strongly indicate the dose-dependent CB1R-mediated weak inverse agonist effects of the two butanoic acid metabolites. The obtained high concentration of main urinary metabolites of 5F-MDMB-PICA and 4F-MDMB-BICA confirmed the relevance of their routine analysis in forensic and toxicological practices. Based on in vitro binding assays, the metabolites presumably might cause a lower psychoactive effect than parent compounds.

Characterization and Seasonal Modulation of Adenosine A1 Receptors in the Arctic Ground Squirrel Brain.

Hibernation is an adaptation that allows animals such as the Arctic ground squirrel (AGS) to survive the absence of food or water during the winter season. Understanding mechanisms of metabolic suppression during hibernation torpor promises new therapies for critical care. The activation of the Adenosine A1 receptor (A1AR) has been shown to be necessary and sufficient for entrance into hibernation with a winter season sensitization to the agonist, but the role of the A1AR in seasonal sensitization is unknown. In the current study, we characterize the A1AR in the forebrain, hippocampus and hypothalamus of summer and torpid AGS. For the first time, we define the pharmacological characteristics of the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in the AGS brain. In addition, we test the hypothesis that increased A1AR agonist efficacy is responsible for sensitization of the A1AR during the torpor season. The resulting 35S-GTPγS binding data indicate an increase in agonist potency during torpor in two out of three brain regions. In addition to 35S-GTPγS binding, [3H]DPCPX saturation and competition assays establish for the first-time pharmacological characteristics for the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in AGS brain.

Telmisartan Is a Promising Agent for Managing Neuropathic Pain and Delaying Opioid Analgesic Tolerance in Rats.

Despite the large arsenal of analgesic medications, neuropathic pain (NP) management is not solved yet. Angiotensin II receptor type 1 (AT1) has been identified as a potential target in NP therapy. Here, we investigate the antiallodynic effect of AT1 blockers telmisartan and losartan, and particularly their combination with morphine on rat mononeuropathic pain following acute or chronic oral administration. The impact of telmisartan on morphine analgesic tolerance was also assessed using the rat tail-flick assay. Morphine potency and efficacy in spinal cord samples of treated neuropathic animals were assessed by [35S]GTPγS-binding assay. Finally, the glutamate content of the cerebrospinal fluid (CSF) was measured by capillary electrophoresis. Oral telmisartan or losartan in higher doses showed an acute antiallodynic effect. In the chronic treatment study, the combination of subanalgesic doses of telmisartan and morphine ameliorated allodynia and resulted in a leftward shift in the dose-response curve of morphine in the [35S]GTPγS binding assay and increased CSF glutamate content. Telmisartan delayed morphine analgesic-tolerance development. Our study has identified a promising combination therapy composed of telmisartan and morphine for NP and opioid tolerance. Since telmisartan is an inhibitor of AT1 and activator of PPAR-γ, future studies are needed to analyze the effect of each component.

Probing thermostability of detergent-solubilized CB2 receptor by parallel G protein-activation and ligand-binding assays.

G protein-coupled receptors (GPCRs) comprise a large class of integral membrane proteins involved in the regulation of a broad spectrum of physiological processes and are a major target for pharmaceutical drug development. Structural studies can help advance the rational design of novel specific pharmaceuticals that target GPCRs, but such studies require expression of significant quantities of these proteins in pure, homogenous, and sufficiently stable form. An essential precursor for these structural studies is an assessment of protein stability under experimental conditions. Here we report that solubilization of a GPCR, type II cannabinoid receptor CB2, in a Façade detergent enables radioligand thermostability assessments of this receptor with low background from nonspecific interactions with lipophilic cannabinoid ligand. Furthermore, this detergent is compatible with a [35S]GTPγS radionucleotide exchange assay measuring guanine exchange factor activity that can be applied after heat treatment to further assess receptor thermostability. We demonstrate that both assays can be utilized to determine differences in CB2 thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB2 variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB1 mutation. We conclude that both ligand-binding and activity-based assays under optimized detergent conditions can support selection of thermostable variants of experimentally demanding GPCRs.

Millisecond Allosteric Dynamics of Activated Ras Reproduced with a Slowly Hydrolyzable GTP Analogue.

The millisecond timescale dynamics of activated Ras transiently sample a low-populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small-molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non-hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP. It was found that GTPγS, in addition to its better mimicking of the exchange kinetics than the commonly used non-hydrolyzable analogues GppNHp and GppCH2 p, can rigorously reproduce the natural dynamics network in active Ras, thus indicating its fitness for use in the development of allosteric inhibitors.

Mu Opioids Induce Biased Signaling at the Full-Length Seven Transmembrane C-Terminal Splice Variants of the mu Opioid Receptor Gene, Oprm1.

The biased signaling has been extensively studied in the original mu opioid receptor (MOR-1), particularly through G protein and ß-arrestin2 signaling pathways. The concept that the G protein pathway is often linked to the therapeutic effect of the drug, while the ß-arrestin pathway is associated to the side effects has been proposed to develop biased analgesic compounds with limited side-effects associated with traditional opiates. The mu opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms that are conserved from rodent to human. One type of the Oprm1 splice variants are the full-length 7 transmembrane (7TM) C-terminal splice variants, which have identical receptor structures including entire binding pocket, but contain a different intracellular C-terminal tail resulted from 3' alternative splicing. Increasing evidence suggest that these full-length 7TM C-terminal variants play important roles in mu opioid pharmacology, raising questions regarding biased signaling at these multiple C-terminal variants. In the present study, we investigated the effect of different C-terminal variants on mu agonist-induced G protein coupling, ß-arrestin2 recruitment, and ultimately, signaling bias. We found that mu agonists produced marked differences in G protein activation and ß-arrestin2 recruitment among various C-terminal variants, leading to biased signaling at various level. Particularly, MOR-1O, an exon 7-associated variant, showed greater ß-arrestin2 bias for most mu agonists than MOR-1, an exon 4-associated variant. Biased signaling of G protein-coupled receptors has been defined by evidences that different agonists can produce divergent signaling transduction pathways through a single receptor. Our findings that a single mu agonist can induce differential signaling through multiple 7TM splice variants provide a new perspective on biased signaling at least for Oprm1, which perhaps is important for our understanding of the complex mu opioid actions in vivo where all the 7TM splice variants co-exist.

Protons as second messenger regulators of G protein signaling.

In response to environmental stress, cells often generate pH signals that serve to protect vital cellular components and reprogram gene expression for survival. A major barrier to our understanding of this process has been the identification of signaling proteins that detect changes in intracellular pH. To identify candidate pH sensors, we developed a computer algorithm that searches proteins for networks of proton-binding sidechains. This analysis indicates that Gα subunits, the principal transducers of G protein-coupled receptor (GPCR) signals, are pH sensors. Our structure-based calculations and biophysical investigations reveal that Gα subunits contain networks of pH-sensing sidechains buried between their Ras and helical domains. Further, we show that proton binding induces changes in conformation that promote Gα phosphorylation and suppress receptor-initiated signaling. Together, our computational, biophysical, and cellular analyses reveal an unexpected function for G proteins as mediators of stress-response signaling.

The N termini of the inhibitory γ-subunits of phosphodiesterase-6 (PDE6) from rod and cone photoreceptors differentially regulate transducin-mediated PDE6 activation.

Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory γ-subunit (Pγ). Rhodopsin-catalyzed activation of the G protein transducin relieves this inhibition and enhances PDE6 catalysis. We hypothesized that amino acid sequence differences between rod- and cone-specific Pγs underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone Pγ sequences from all major vertebrate and cyclostome lineages and found that rod Pγ loci are far more conserved than cone Pγ sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (Pαß) with various rod or cone Pγ variants and analyzed PDE6 activation upon addition of the activated transducin α-subunit (Gtα*-GTPγS). This analysis revealed a rod-specific Pγ motif (amino acids 9-18) that reduces the ability of Gtα*-GTPγS to activate the reconstituted PDE6. In cone Pγ, Asn-13 and Gln-14 significantly enhanced Gtα*-GTPγS activation of cone Pγ truncation variants. Moreover, we observed that the first four amino acids of either rod or cone Pγ contribute to Gtα*-GTPγS-mediated activation of PDE6. We conclude that physiological differences between rod and cone photoreceptor light responsiveness can be partially ascribed to ancient, highly conserved amino acid differences in the N-terminal regions of Pγ isoforms, demonstrating for the first time a functional role for this region of Pγ in the differential activation of rod and cone PDE6 by transducin.

Lysophosphatidic Acid Receptor Agonism: Discovery of Potent Nonlipid Benzofuran Ethanolamine Structures.

Lysophosphatidic acid (LPA) is the natural ligand for two phylogenetically distinct families of receptors (LPA1-3, LPA4-6) whose pathways control a variety of physiologic and pathophysiological responses. Identifying the benefit of balanced activation/repression of LPA receptors has always been a challenge because of the high lability of LPA and the limited availability of selective and/or stable agonists. In this study, we document the discovery of small benzofuran ethanolamine derivatives (called CpX and CpY) behaving as LPA1-3 agonists. Initially found as rabbit urethra contracting agents, their elusive receptors were identified from [35S]GTPγS-binding and ß-arrestin2 recruitment investigations and then confirmed by [3H]CpX binding studies (urethra, hLPA1-2 membranes). Both compounds induced a calcium response in hLPA1-3 cells within a range of 0.4-1.5-log lower potency as compared with LPA. The contractions of rabbit urethra strips induced by these compounds perfectly matched binding affinities with values reaching the two-digit nanomolar level. The antagonist, KI16425, dose-dependently antagonized CpX-induced contractions in agreement with its affinity profile (LPA1≥LPA3>>LPA2). The most potent agonist, CpY, doubled intraurethral pressure in anesthetized female rats at 3 µg/kg i.v. Alternatively, CpX was shown to inhibit human preadipocyte differentiation, a process totally reversed by KI16425. Together with original molecular docking data, these findings clearly established these molecules as potent agonists of LPA1-3 and consolidated the pivotal role of LPA1 in urethra/prostate contraction as well as in fat cell development. The discovery of these unique and less labile LPA1-3 agonists would offer new avenues to investigate the roles of LPA receptors. SIGNIFICANCE STATEMENT: We report the identification of benzofuran ethanolamine derivatives behaving as potent selective nonlipid LPA1-3 agonists and shown to alter urethra muscle contraction or preadipocyte differentiation. Unique at this level of potency, selectivity, and especially stability, compared with lysophosphatidic acid, they represent more appropriate tools for investigating the physiological roles of lysophosphatidic acid receptors and starting point for optimization of drug candidates for therapeutic applications.

Structure optimization of positive allosteric modulators of GABAB receptors led to the unexpected discovery of antagonists/potential negative allosteric modulators.

Positive allosteric modulators (PAMs) of GABAB receptor represent an interesting alternative to receptor agonists such as baclofen, as they act on the receptor in a more physiological way and thus are devoid of the side effects typically exerted by the agonists. Based on our interest in the identification of new GABAB receptor PAMs, we followed a merging approach to design new chemotypes starting from selected active compounds, such as GS39783, rac-BHFF, and BHF177, and we ended up with the synthesis of four different classes of compounds. The new compounds were tested alone or in the presence of 10 µM GABA using [35S]GTPγS binding assay to assess their functionality at the receptor. Unexpectedly, a number of them significantly inhibited GABA-stimulated GTPγS binding thus revealing a functional switch with respect to the prototype molecules. Further studies on selected compounds will clarify if they act as negative modulators of the receptor or, instead, as antagonists at the orthosteric binding site.

Nucleotide-dependent farnesyl switch orchestrates polymerization and membrane binding of human guanylate-binding protein 1.

Dynamin-like proteins (DLPs) mediate various membrane fusion and fission processes within the cell, which often require the polymerization of DLPs. An IFN-inducible family of DLPs, the guanylate-binding proteins (GBPs), is involved in antimicrobial and antiviral responses within the cell. Human guanylate-binding protein 1 (hGBP1), the founding member of GBPs, is also engaged in the regulation of cell adhesion and migration. Here, we show how the GTPase cycle of farnesylated hGBP1 (hGBP1F) regulates its self-assembly and membrane interaction. Using vesicles of various sizes as a lipid bilayer model, we show GTP-dependent membrane binding of hGBP1F In addition, we demonstrate nucleotide-dependent tethering ability of hGBP1F Furthermore, we report nucleotide-dependent polymerization of hGBP1F, which competes with membrane binding of the protein. Our results show that hGBP1F acts as a nucleotide-controlled molecular switch by modulating the accessibility of its farnesyl moiety, which does not require any supportive proteins.

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes.

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes.

Although multiple roles of dopamine through D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via Gs/olf or Gi/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gαq activation in rat brain membranes was investigated. Agonist-induced Gαq activation was assessed by increase in guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding to Gαq determined by [35S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gαq functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D1-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D2-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D1-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT2A receptor, but not by dopamine D1-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D1-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT2A receptor has been mentioned.

Sex differences in the effectiveness of buprenorphine to decrease rates of responding in rhesus monkeys.

Sex differences in µ-opioid receptor (MOR) agonist-induced antinociception have been reported in nonhuman primates. The degree to which µ-opioid receptor agonist sex differences in nonhuman primates extend to other behavioral endpoints remains unknown. The present study compared the behavioral effects of three MOR ligands (fentanyl, buprenorphine, and naltrexone) that varied in efficacy to stimulate [S]-GTPγS binding (from highest to lowest: fentanyl, buprenorphine, and naltrexone) in male and female rhesus monkeys. Male (n=3) and female (n=3) monkeys were trained to respond under a fixed-ratio 10 schedule of food presentation during daily sessions consisting of multiple components. Once rates of responding were stable, cumulative dose-effect functions were determined for intramuscular fentanyl (0.00032-0.032 mg/kg), buprenorphine (0.001-1 mg/kg), and naltrexone (0.01-0.1 mg/kg). Fentanyl dose-dependently decreased rates of responding in both sexes and the corresponding ED50 values were not significantly different. Buprenorphine dose-dependently decreased rates of responding in females, but not males. Naltrexone did not significantly alter behavior in either females or males. Overall, these results suggest that the expression of sex differences in MOR pharmacology depends upon both the efficacy of the MOR ligand and the behavioral endpoint.

Functional characterisation of G protein-coupled receptors.

Characterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either directly monitoring ligand itself with techniques such as fluorescence polarisation or indirectly via resonance energy transfer (fluorescence/Forster resonance energy transfer, FRET and bioluminescence resonance energy transfer, BRET). Label free techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are also increasingly being used. For GPCRs, the main measure of receptor activation is to investigate the association of the G protein with the receptor. The chief assay measures the receptor-stimulated binding of GTP or a suitable analogue to the receptor. The direct association of the G protein with the receptor has been investigated via resonance energy techniques. These have also been used to measure ligand-induced conformational changes within the receptor; a variety of experimental techniques are available to incorporate suitable donors and acceptors within the receptor.

Functional activation of Gαq/11 protein via α1 -adrenoceptor in rat cerebral cortical membranes.

Although it is recognized that α1 -adrenoceptors are coupled to diverse intracellular signalling pathways, its primary transduction mechanisms are evoked by activating phospholipase C in the cell membrane through Gαq/11 , resulting in production of inositol 1,4,5-trisphosphate and diacylglycerol. However, there have been few studies that indicate directly the involvement of Gαq/11 proteins in this signalling pathway in the central nervous system. In the current study, we tried to pharmacologically characterize (-)-adrenaline-stimulated [35 S]GTPγS binding to Gαq/11 in rat brain membranes. Functional activation of Gαq/11 coupled to α1 -adrenoceptor was investigated by using [35 S]GTPγS binding/immunoprecipitation assay in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. The specific [35 S]GTPγS binding to Gαq/11 was stimulated by (-)-adrenaline in a concentration-dependent and saturable manner in rat cerebral cortical membranes. In hippocampal or striatal membranes, the stimulatory effects of (-)-adrenaline were scarce. The effect of (-)-adrenaline was potently inhibited by prazosin, a potent and selective α1 -adrenoceptor antagonist, but not by yohimbine, a selective α2 -adrenoceptor antagonist. The response was mimicked by cirazoline, but not by R(-)-phenylephrine. Although oxymetazoline also stimulated the specific [35 S]GTPγS binding to Gαq/11 as an apparent "super-agonist", detailed pharmacological characterization revealed that its agonistic properties in this experimental system were derived from off-target effects on 5-HT2A receptors, but not via α1 -adrenoceptors. In conclusion, functional coupling of α1 -adrenoceptors to Gαq/11 proteins are detectable in rat brain membranes by means of [35 S]GTPγS binding/immunoprecipitation assay. It is necessary to interpret the experimental data with caution when oxymetazoline is included as an agonist at α1 -adrenoceptors.

Direct regulation of p190RhoGEF by activated Rho and Rac GTPases.

Rho family GTPases regulate a wide range of cellular processes. This includes cellular dynamics where three subfamilies, Rho, Rac, and Cdc42, are known to regulate cell shape and migration though coordinate action. Activation of Rho proteins largely depends on Rho Guanine nucleotide Exchange Factors (RhoGEFs) through a catalytic Dbl homology (DH) domain linked to a pleckstrin homology (PH) domain that subserves various functions. The PH domains from Lbc RhoGEFs, which specifically activate RhoA, have been shown to bind to activated RhoA. Here, p190RhoGEF is shown to also bind Rac1·GTP. Crystal structures reveal that activated Rac1 and RhoA use their effector-binding surfaces to associate with the same hydrophobic surface on the PH domain. Both activated RhoA and Rac1 can stimulate exchange of nucleotide on RhoA by localization of p190RhoGEF to its substrate, RhoA·GDP, in vitro. The binding of activated RhoA provides a mechanism for positive feedback regulation as previously proposed for the family of Lbc RhoGEFs. In contrast, the novel interaction between activated Rac1 and p190RhoGEF reveals a potential mechanism for cross-talk regulation where Rac can directly effect stimulation of RhoA. The greater capacity of Rac1 to stimulate p190RhoGEF among the Lbc RhoGEFs suggests functional specialization.

ABIN-1 Negatively Regulates µ-Opioid Receptor Function.

The µ-opioid receptor (MOR) is a Gi/o protein-coupled receptor that mediates analgesic, euphoric, and reward effects. Using a bacterial two-hybrid screen, we reported that the carboxyl tail of the rat MOR associates with A20-binding inhibitor of nuclear factor κB (ABIN-1). This interaction was confirmed by direct protein-protein binding and coimmunoprecipitation of MOR and ABIN-1 proteins in cell lysates. Saturation binding studies showed that ABIN-1 had no effect on MOR binding. However, the interaction of ABIN-1 and MOR inhibited the activation of G proteins induced by DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin). MOR phosphorylation, ubiquitination, and internalization induced by DAMGO were decreased in Chinese hamster ovary cells that coexpressed MOR and ABIN-1. The suppression of forskolin-stimulated adenylyl cyclase by DAMGO was also inhibited by the interaction of ABIN-1 with MOR. In addition, extracellular signal-regulated kinase activation was also negatively regulated by overexpression of ABIN-1. These data suggest that ABIN-1 is a negative coregulator of MOR activation, phosphorylation, and internalization in vitro. ABIN-1 also inhibited morphine-induced hyperlocomotion in zebrafish larvae (AB strain). By utilization of an antisense morpholino oligonucleotide (MO) gene knockdown technology, the ABIN-1 MO-injected zebrafish larvae showed a significant increase (approximately 60%) in distance moved compared with control MO-injected larvae after acute morphine treatment (P < 0.01). Taken together, ABIN-1 negatively regulates MOR function in vitro and in vivo.

A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid.

The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin ß superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle.

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells.

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H3 receptor (hH3R445 and hH3R365) on [35S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca2+ ions ([Ca2+]i) and depolarization-evoked [3 H]-dopamine release. Maximal specific binding (Bmax) of [3 H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells, respectively, with similar dissociation constants (Kd, 0.86 nM and 0.81 nM). The mRNA of the hH3R365 isoform was 40.9 ± 7.9% of the hH3R445 isoform. No differences in receptor affinity were found for the H3R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [35S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH3R445 cells ([35S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hH3R365 cells; cAMP accumulation, -74.0 ± 4.9% versus -43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [3 H]-dopamine release evoked by 100 mM K+ (-18.9 ± 3.0% and -20.5 ± 3.3%, for SH-SY5Y-hH3R445 and SH-SY5Y-hH3R365 cells), or the inhibition of depolarization-induced increase in [Ca2+]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hH3R445 cells 0.639 ± 0.049, SH-SY5Y-hH3R365 cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hH3R445 and hH3R365 isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.