G-protein Activation Research Articles

Caffeine – treat or trigger? Disparate behavioral and long-term dopaminergic changes in control and schizophrenia-like Wisket rats

The influence of caffeine on behavioral functions in both healthy and schizophrenic subjects is controversial. Here we aimed to reveal the effects of repeated caffeine pre- and post-training treatments on motor and exploratory activities and cognitive functions in a reward-based test (Ambitus) along with a brain region-specific dopamine D2 receptor profile in control and schizophrenia-like WISKET model rats. In the control animals, pre-treatment caused temporary enhancement in motor activity, while permanent improvement in learning function was detected in the WISKET animals. Post-treatment produced significant impairments in both groups. Caffeine caused short-lasting hyperactivity followed by a rebound in the inactive phase determined in undisturbed circumstance. Caffeine treatment substantially enhanced the dopamine D2 receptor mediated G-protein activation in the prefrontal cortex and olfactory bulb of both groups, while it increased in the dorsal striatum and cerebral cortex only in the WISKET animals. Caffeine enhanced the maximal binding capacity in the hippocampus and cerebral cortex of WISKET animals, but it decreased in the prefrontal cortex of the control animals. Regarding the dopamine D2 receptor mRNA expression, caffeine treatment caused significant enhancement in the prefrontal cortex of WISKET animals, while it increased the hippocampal dopamine D2 receptor protein amount in both groups. This study highlights the disparate effects of caffeine pre- versus post-training treatments on behavioral parameters in both control and schizophrenia-like animals and the prolonged changes in the dopaminergic system. It is supposed that the delayed depressive effects of caffeine might be compensated by frequent coffee intake, as observed in schizophrenic patients.

Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.

Ligand-Induced GPR110 Activation Facilitates Axon Growth after Injury.

Recovery from axonal injury is extremely difficult, especially for adult neurons. Here, we demonstrate that the activation of G-protein coupled receptor 110 (GPR110, ADGRF1) is a mechanism to stimulate axon growth after injury. N-docosahexaenoylethanolamine (synaptamide), an endogenous ligand of GPR110 that promotes neurite outgrowth and synaptogenesis in developing neurons, and a synthetic GPR110 ligand stimulated neurite growth in axotomized cortical neurons and in retinal explant cultures. Intravitreal injection of GPR110 ligands following optic nerve crush injury promoted axon extension in adult wild-type, but not in gpr110 knockout, mice. In vitro axotomy or in vivo optic nerve injury rapidly induced the neuronal expression of gpr110. Activating the developmental mechanism of neurite outgrowth by specifically targeting GPR110 that is upregulated upon injury may provide a novel strategy for stimulating axon growth after nerve injury in adults.

Mechanistic diversity involved in the desensitization of G protein-coupled receptors.

The desensitization of G protein-coupled receptors (GPCRs), which involves rapid loss of responsiveness due to repeated or chronic exposure to agonists, can occur through various mechanisms at different levels of signaling pathways. In this review, the mechanisms of GPCR desensitization are classified according to their occurrence at the receptor level and downstream to the receptor. The desensitization at the receptor level occurs in a phosphorylation-dependent manner, wherein the activated receptors are phosphorylated by GPCR kinases (GRKs), thereby increasing their affinities for arrestins. Arrestins bind to receptors through the cavity on the cytoplasmic region of heptahelical domains and interfere with the binding and activation of G-protein. Diverse mechanisms are involved in the desensitization that occurs downstream of the receptor. Some of these include the sequestration of G proteins, such as Gq and Gi/o by GRK2/3 and deubiquitinated arrestins, respectively. Mechanistically, GRK2/3 attenuates GPCR signaling by sequestering the Gα subunits of the Gq family and Gβγ via regulators of G protein signaling and pleckstrin homology domains, respectively. Moreover, studies on Gi/o-coupled D2-like receptors have reported that arrestins are deubiquitinated under desensitization condition and form a stable complex with Gβγ, thereby preventing them from coupling with Gα and the receptor, eventually leading to receptor signaling inhibition. Notably, the desensitization mechanism that involves arrestin deubiquitination is interesting; however, this is a new mechanism and needs to be explored further.

Structural insights into the lipid and ligand regulation of serotonin receptors.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtypefamily of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Chronic phenmetrazine treatment promotes D2 dopaminergic and α2-adrenergic receptor desensitization and alters phosphorylation of signaling proteins and local cerebral glucose metabolism in the rat brain

Phenmetrazine (PHEN) is a putative treatment for cocaine and psychostimulant recidivism; however, neurochemical changes underlying its activity have not been fully elucidated. We sought to characterize brain homeostatic adaptations to chronic PHEN, specifically on functional brain activity (local cerebral glucose utilization), G-Protein Coupled Receptor-stimulated G-protein activation, and phosphorylation of ERK1/2Thr202/Tyr204, GSK3βTyr216, and DARPP-32Thr34. Male Sprague-Dawley rats were implanted with sub-cutaneous minipumps delivering either saline (vehicle), acute (2-day) or chronic (14-day) low dose (25 mg/kg/day) or high dose (50 mg/kg/day) PHEN. Acute administration of high dose PHEN increased local cerebral glucose utilization measured by 2-[14C]-deoxyglucose uptake in basal ganglia and motor-related regions of the rat brain. However, chronically treated animals developed tolerance to these effects. To identify the neurochemical changes associated with PHEN’s activity, we performed [35S]GTPγS binding assays on unfixed and immunohistochemistry on fixed coronal brain sections. Chronic PHEN treatment dose-dependently attenuated D2 dopamine and α2-adrenergic, but not 5-HT1A, receptor-mediated G-protein activation. Two distinct patterns of effects on pERK1/2 and pDARPP-32 were observed: 1) chronic low dose PHEN decreased pERK1/2, and also significantly increased pDARPP-32 levels in some regions; 2) acute and chronic PHEN increased pERK1/2, but chronic high dose PHEN treatment tended to decrease pDARPP-32. Chronic low dose, but not high dose, PHEN significantly reduced pGSK3β levels in several regions. Our study provides definitive evidence that extended length PHEN dosage schedules elicit distinct modes of neuronal acclimatization in cellular signaling. These pharmacodynamic modifications should be considered in drug development for chronic use.

Activation of G-protein coupled estradiol receptor 1 in the dorsolateral striatum attenuates preference for cocaine and saccharin in male but not female rats

There are sex differences in the response to psychomotor stimulants, where females exhibit a greater response than males, due to the presence of the gonadal hormone estradiol (E2). Extensive research has shown that E2 enhances drug-seeking and the rewarding properties of cocaine for females. The role of E2 in male drug-seeking, however, is not well understood. The current study investigated pharmacological manipulation of E2 receptors in the dorsolateral striatum (DLS) on preference for cocaine in gonad-intact male and female rats. In males, activation of G-protein coupled E2 receptor 1 (GPER1), via administration of ICI 182,780 or G1, attenuated conditioned place preference for 10 mg/kg cocaine, while inhibition of GPER1, via G15, enhanced preference at a 5 mg/kg cocaine dose. Similarly, GPER1 activation, via G1, prevented males from forming a preference for 0.1% saccharin (SACC) versus plain water. Surprisingly, activation of GPER1 did not alter preference for cocaine or SACC in females. These studies also examined the quantity of E2 receptor mRNA in the dorsal striatum, using qPCR. No sex differences in relative mRNA expression of ERα, ERβ, and GPER1 were observed. However, there was greater GPER1 mRNA, relative to ERα and ERβ, in both males and females. The results presented here indicate that E2, acting via GPER1, may be protective against drug preference in male rats.

The IgG3 subclass of β1-adrenergic receptor autoantibodies is an endogenous biaser of β1AR signaling.

Dysregulation of immune responses has been linked to the generation of immunoglobulin G (IgG) autoantibodies that target human β1ARs and contribute to deleterious cardiac outcomes. Given the benefits of β-blockers observed in patients harboring the IgG3 subclass of autoantibodies, we investigated the role of these autoantibodies in human β1AR function. Serum and purified IgG3(+) autoantibodies from patients with onset of cardiomyopathy were tested using human embryonic kidney (HEK) 293 cells expressing human β1ARs. Unexpectedly, pretreatment of cells with IgG3(+) serum or purified IgG3(+) autoantibodies impaired dobutamine-mediated adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) generation while enhancing biased β-arrestin recruitment and Extracellular Regulated Kinase (ERK) activation. In contrast, the β-blocker metoprolol increased AC activity and cAMP in the presence of IgG3(+) serum or IgG3(+) autoantibodies. Because IgG3(+) autoantibodies are specific to human β1ARs, non–failing human hearts were used as an endogenous system to determine their ability to bias β1AR signaling. Consistently, metoprolol increased AC activity, reflecting the ability of the IgG3(+) autoantibodies to bias β-blocker toward G-protein coupling. Importantly, IgG3(+) autoantibodies are specific toward β1AR as they did not alter β2AR signaling. Thus, IgG3(+) autoantibody biases β-blocker toward G-protein coupling while impairing agonist-mediated G-protein activation but promoting G-protein–independent ERK activation. This phenomenon may underlie the beneficial outcomes observed in patients harboring IgG3(+) β1AR autoantibodies.

Suppression of delayed rectifier K+ channels by gentamicin induces membrane hyperexcitability through JNK and PKA signaling pathways in vestibular ganglion neurons

Aminoglycoside antibiotics, such as gentamicin, are known to have vestibulotoxic effects, including ataxia and disequilibrium. To date, however, the underlying cellular and molecular mechanisms are still unclear. In this study, we determined the role of gentamicin in regulating the sustained delayed rectifier K+ current (IDR) and membrane excitability in vestibular ganglion (VG) neurons in mice. Our results showed that the application of gentamicin to VG neurons decreased the IDR in a concentration-dependent manner, while the transient outward A-type K+ current (IA) remained unaffected. The decrease in IDR induced by gentamicin was independent of G-protein activity and led to a hyperpolarizing shift of the inactivation Vhalf. The analysis of phospho-c-Jun N-terminal kinase (p-JNK) revealed that gentamicin significantly stimulated JNK, while p-ERK and p-p38 remained unaffected. Blocking Kv1 channels with α-dendrotoxin or pretreating VG neurons with the JNK inhibitor II abrogated the gentamicin-induced decrease in IDR. Antagonism of JNK signaling attenuated the gentamicin-induced stimulation of PKA activity, whereas PKA inhibition prevented the IDR response induced by gentamicin. Moreover, gentamicin significantly increased the number of action potentials fired in both phasic and tonic firing type neurons; pretreating VG neurons with the JNK inhibitor II and the blockade of the IDR abolished this effect. Taken together, our results demonstrate that gentamicin decreases the IDR through a G-protein-independent but JNK and PKA-mediated signaling pathways. This gentamicin-induced IDR response mediates VG neuronal hyperexcitability and might contribute to its pharmacological vestibular effects.

The negative charge of the 343 site is essential for maintaining physiological functions of CXCR4

BackgroundWarts, hypogammaglobulinemia, recurrent bacterial infections and myelokathexis (WHIM) syndrome is a primary immunodeficiency disease (PID) usually caused by autosomal dominant mutations in the chemokine receptor CXCR4 gene. To date, a total of nine different mutations including eight truncation mutations and one missense mutation (E343K, CXCR4E343K) distributed in the C-terminus of CXCR4 have been identified in humans. Studies have clarified that the loss of phosphorylation sites in the C-terminus of truncated CXCR4 impairs the desensitization process, enhances the activation of G-protein, prolongs downstream signaling pathways and introduces over immune responses, thereby causing WHIM syndrome. So far, there is only one reported case of WHIM syndrome with a missense mutation, CXCR4E343K, which has a full length of C-terminus with entire phosphorylation sites, no change in all potential phosphorylation sites. The mechanism of the missense mutation (CXCR4E343K) causing WHIM syndrome is unknown. This study aimed to characterize the effect of mutation at the 343 site of CXCR4 causing the replacement of arginine/E with glutamic acid/K on the receptor signal transduction, and elucidate the mechanism underling CXCR4E343K causing WHIM in the reported family.ResultsWe completed a series of mutagenesis to generate different mutations at the 343 site of CXCR4 tail, and established a series of HeLa cell lines stably expressing CXCR4WT or CXCR4E343D (glutamic acid/E replaced with aspartic acid/D) or CXCR4E343K (glutamic acid/E replaced with lysine/K) or CXCR4E343R (glutamic acid/E replaced with arginine/R) or CXCR4E343A (glutamic acid/E replaced with alanine/A) and then systematically analyzed functions of the CXCR4 mutants above. Results showed that the cells overexpressing of CXCR4E343D had no functional changes with comparison that of wild type CXCR4. However, the cells overexpressing of CXCR4E343K or CXCR4E343R or CXCR4E343A had enhanced cell migration, prolonged the phosphorylation of ERK1/2, p38, JNK1/2/3, aggravated activation of PI3K/AKT/NF-κB signal pathway, introduced higher expression of TNFa and IL6, suggesting over immune response occurred in CXCR4 mutants with charge change at the 343 site of receptor tail, as a result, causing WHIM syndrome. Biochemical analysis of those mutations at the 343 site of CXCR4 above shows that CXCR4 mutants with no matter positive or neutral charge have aberrant signal pathways downstream of activated mutated CXCR4, only CXVR4 with negative charge residues at the site shows normal signal pathway post activation with stromal-derived factor (SDF1, also known as CXCL12).ConclusionTaken together, our results demonstrated that the negative charge at the 343 site of CXCR4 plays an essential role in regulating the down-stream signal transduction of CXCR4 for physiological events, and residue charge changes, no matter positive or neutral introduce aberrant activities and functions of CXCR4, thus consequently lead to WHIM syndrome.

Computer simulation to identify selective inhibitor for human phosphodiesterase10A

Phosphodiesterases (PDEs) play an important function in transduction of cellular signals by modulating the activation of G-proteins by hydrolyzing cAMP and cGMP. PDE10A has emerged as an inhibitory target for development of effective therapeutics against various disorders such as schizophrenia, psychosis, Huntington disease, and other disorders related to the central nervous system. We utilized various computational methods such as molecular docking, molecular dynamic simulations, and MM-PBSA to find a more potent and selective PDE10A inhibitor compared to papaverine, which is a known standard inhibitor of PDE10A. Molecular docking of papaverine and twenty-eight in house synthesized 3-Methylenisoindolin-1-one based molecules provided two potential lead molecules (molecule #3 and molecule #28), which could be developed as potent PDE10A inhibitors. However, molecule #28 was excluded from further studies due to its cross reactivity with other PDE isoforms. Molecular dynamics and MM-PBSA techniques were used to further analyze the molecular docking results. Molecule #3 binds to the conserved substrate recognizing residue (Gln726) and also fits into the selectivity pocket by anchoring to the hydrophobic residue Tyr693. This study provides a promising candidate molecule that could be developed as a more potent and selective inhibitor of PDE10A.

ARL3 activation requires the co-GEF BART and effector-mediated turnover.

The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here, we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.

Drug repurposing for opioid use disorders: integration of computational prediction, clinical corroboration, and mechanism of action analyses

Morbidity and mortality from opioid use disorders (OUD) and other substance use disorders (SUD) is a major public health crisis, yet there are few medications to treat them. There is an urgency to accelerate SUD medication development. We present an integrated drug repurposing strategy that combines computational prediction, clinical corroboration using electronic health records (EHRs) of over 72.9 million patients and mechanisms of action analysis. Among top-ranked repurposed candidate drugs, tramadol, olanzapine, mirtazapine, bupropion, and atomoxetine were associated with increased odds of OUD remission (adjusted odds ratio: 1.51 [1.38–1.66], 1.90 [1.66–2.18], 1.38 [1.31–1.46], 1.37 [1.29–1.46], 1.48 [1.25–1.76], p value < 0.001, respectively). Genetic and functional analyses showed these five candidate drugs directly target multiple OUD-associated genes including BDNF, CYP2D6, OPRD1, OPRK1, OPRM1, HTR1B, POMC, SLC6A4 and OUD-associated pathways, including opioid signaling, G-protein activation, serotonin receptors, and GPCR signaling. In summary, we developed an integrated drug repurposing approach and identified five repurposed candidate drugs that might be of value for treating OUD patients, including those suffering from comorbid conditions.

A long isoform of GIV/Girdin contains a PDZ-binding module that regulates localization and G-protein binding

PDZ domains are one of the most abundant protein domains in eukaryotes and are frequently found on junction-localized scaffold proteins. Various signaling molecules bind to PDZ proteins via PDZ-binding motifs (PBM) and fine-tune cellular signaling. However, how such interaction affects protein function is difficult to predict and must be solved empirically. Here we describe a long isoform of the guanine nucleotide exchange factor GIV/Girdin (CCDC88A) that we named GIV-L, which is conserved throughout evolution, from invertebrates to vertebrates, and contains a PBM. Unlike GIV, which lacks PBM and is cytosolic, GIV-L localizes onto cell junctions and has a PDZ interactome (as shown through annotating Human Cell Map and BioID-proximity labeling studies), which impacts GIV-L's ability to bind and activate trimeric G-protein, Gαi, through its guanine-nucleotide exchange modulator (GEM) module. This GEM module is found exclusively in vertebrates. We propose that the two functional modules in GIV may have evolved sequentially: the ability to bind PDZ proteins via the PBM evolved earlier in invertebrates, whereas G-protein binding and activation may have evolved later only among vertebrates. Phenotypic studies in Caco-2 cells revealed that GIV and GIV-L may have antagonistic effects on cell growth, proliferation (cell cycle), and survival. Immunohistochemical analysis in human colon tissues showed that GIV expression increases with a concomitant decrease in GIV-L during cancer initiation. Taken together, these findings reveal how regulation in GIV/CCDC88A transcript helps to achieve protein modularity, which allows the protein to play opposing roles either as a tumor suppressor (GIV-L) or as an oncogene (GIV).

Effects of inflammatory pain on CB1 receptor in the midbrain periaqueductal gray.

The periaqueductal gray (PAG) mediates the antinociceptive properties of analgesics, including opioids and cannabinoids. Administration of either opioids or cannabinoids into the PAG induces antinociception. However, most studies characterizing the antinociceptive properties of cannabinoids in the PAG have been conducted in naive animals. Few studies have reported on the role of CB1 receptors in the PAG during conditions which would prompt the administration of analgesics, namely, during pain states. To examine inflammatory pain-induced changes in CB1 receptor expression and function in the midbrain periaqueductal gray. In this study, we used the Complete Freund Adjuvant model to characterize CB1 receptor expression and G-protein coupling during persistent inflammatory pain. Inflammatory pain induced an upregulation in the expression of synaptic CB1 receptors in the PAG. Despite this pain-induced change in CB1 expression, there was no corresponding upregulation of CB1 mRNA after the induction of inflammatory pain, suggesting a pain-induced recruitment of CB1 receptors to the synaptic sites within PAG neurons or increased coupling efficiency between the receptor and effector systems. Inflammatory pain also enhanced ventrolateral PAG CB1 receptor activity, as there was an increase in CP55,940-stimulated G-protein activation compared with pain-naïve control animals. These findings complement a growing body of evidence which demonstrate pain-induced changes in brain regions that are responsible for both the analgesic and rewarding properties of analgesic pharmacotherapies. Because much of our understanding of the pharmacology of cannabinoids is based on studies which use largely pain-naïve male animals, this work fills in important gaps in the knowledge base by incorporating pain-induced adaptations and cannabinoid pharmacology in females.

Optogenetic Potentials of Diverse Animal Opsins: Parapinopsin, Peropsin, LWS Bistable Opsin.

Animal opsin-based pigments are light-activated G-protein-coupled receptors (GPCRs), which drive signal transduction cascades via G-proteins. Thousands of animal opsins have been identified, and molecular phylogenetic and biochemical analyses have revealed the unexpected diversity in selectivity of G-protein activation and photochemical property. Here we discuss the optogenetic potentials of diverse animal opsins, particularly recently well-characterized three non-canonical opsins, parapinopsin, peropsin, and LWS bistable opsin. Unlike canonical opsins such as vertebrate visual opsins that have been conventionally used for optogenetic applications, these opsins are bistable; opsin-based pigments do not release the chromophore retinal after light absorption, and the stable photoproducts revert to their original dark states upon subsequent light absorption. Parapinopsins have a "complete photoregeneration ability," which allows a clear color-dependent regulation of signal transductions. On the other hand, peropsins serve as a "dark-active and light-inactivated" GPCR to regulate signal transductions in the opposite way compared with usual opsins. In addition, an LWS bistable opsin from a butterfly was revealed to be the longest wavelength-sensitive animal opsin with its absorption maximum at ~570nm. The property-dependent optical regulations of signal transductions were demonstrated in mammalian cultured cells, showing potentials of new optogenetic tools.

Synthesis and Pharmacology of a Novel μ-δ Opioid Receptor Heteromer-Selective Agonist Based on the Carfentanyl Template.

In this work, we studied a series of carfentanyl amide-based opioid derivatives targeting the mu opioid receptor (μOR) and the delta opioid receptor (δOR) heteromer as a credible novel target in pain management therapy. We identified a lead compound named MP135 that exhibits high G-protein activity at μ-δ heteromers compared to the homomeric δOR or μOR and low β-arrestin2 recruitment activity at all three. Furthermore, MP135 exhibits distinct signaling profile, as compared to the previously identified agonist targeting μ-δ heteromers, CYM51010. Pharmacological characterization of MP135 supports the utility of this compound as a molecule that could be developed as an antinociceptive agent similar to morphine in rodents. In vivo characterization reveals that MP135 maintains untoward side effects such as respiratory depression and reward behavior; together, these results suggest that optimization of MP135 is necessary for the development of therapeutics that suppress the classical side effects associated with conventional clinical opioids.

Metabotropic glutamate 2,3 receptor stimulation desensitizes agonist activation of G-protein signaling and alters transcription regulators in mesocorticolimbic brain regions.

Metabotropic glutamate (mGlu) receptors are regulators of glutamate release and targets for development of therapies for hyperactive glutamatergic signaling. However, the effects of long-term stimulation of mGlu receptors on cellular signaling in the brain have not been described. This study investigated the effects of 2-day and 14-day osmotic mini-pump administration of the mGlu2,3 agonist LY379268 (3.0mgkg-1 day-1 ) to rats on receptor-mediated G-protein activation and signaling in mesocorticolimbic regions in rat brain sections. A significant reduction in LY379268-stimulated [35 S]GTPγS binding was observed in the 14-day group in some cortical regions, prefrontal cortex, nucleus accumbens, and ventral pallidum. The 14-day LY379268 treatment group exhibited mGlu2 mRNA levels significantly lower in hippocampus, nucleus accumbens, caudate, and ventral pallidum. In both 2-day and 14-day treatment groups immunodetectable phosphorylated cAMP Response Element-Binding protein (CREB) was significantly reduced across all brain regions. In the 2-day group, we observed significantly lower immunodetectable CREB protein across all brain regions, which was subsequently increased in the 14-day group but failed to achieve control values. Neither immunodetectable extracellular signal-regulated kinase (ERK) protein nor phosphorylated ERK from 2-day or 14-day treatment groups differed significantly from control across all brain regions. However, the ratio of phosphorylated ERK to total ERK protein was significantly greater in the 14-day treatment group compared with the control. These results identify compensatory changes to mGlu2,3 signal transduction in rat brains after chronic systemic administration of agonist, which could be predictive of the mechanism of action in human pharmacotherapies.

Modulation of G-protein-coupled receptor 55-mediated signaling by regulator of G-protein signaling 2

Activation of seven-transmembrane G-protein coupled receptor (GPCR) mediates extracellular signals into intracellular responses. G-protein coupled receptor 55 (GPR55) is one of GPCRs and activated by endogenous cannabinoids. A family of regulators of G-protein signaling (RGS) stimulates GTP hydrolysis of alpha subunit of G-protein (Gα) and inhibits GPCR/Gα-mediated signaling. RGS2 is member of R4 RGS family and mainly attenuates GPCR/Gαq signaling. Although RGS2 is known to modulate some GPCR signaling, the specific effects of RGS2 on GPR55-mediated signaling are not fully understood at present. Previously, we reported some RGS proteins interact with protease-activated receptors, one of GPCRs, and modulate their functions. Here, we investigated whether GPR55 interacts with RGS2, employing bioluminescence resonance energy transfer and co-immunoprecipitation analyses. Interestingly, GPR55 interacted with RGS2 alone and also formed a ternary complex with RGS2 and either Gαq or Gα12. In the presence of GPR55 alone and together with Gαq or Gα12, RGS2 translocated from the cytoplasm to plasma membrane while RGS1 remained in the cytoplasm. GPR55 activation significantly induced ERK phosphorylation and intracellular calcium mobilization, which were markedly inhibited by RGS2 in HCT116 colon cancer cell line. Furthermore, GPR55-mediated cell proliferation and migration of HCT116 cells, was significantly attenuated by RGS2. Our collective findings highlight a novel physiological function of RGS2, supporting its utility as a therapeutic target to control GPR55-induced pathophysiology.

Non-genomic actions of sex hormones on pregnant uterine contractility in rats: An in vitro study at term

AimsThe non-genomic (prompt) actions of sex steroids on pregnant uterine contractility are not fully explored yet, the aim of our study was to clarify such effects of 17-β estradiol (E2), progesterone (P4) and testosterone (T) on late (22-day) pregnant uterine contractions together with the signaling pathways in rats in vitro. MethodsThe uterine effects of sex steroids on KCl-stimulated contractions were examined in the presence of genomic pathway blocker actinomycin D and cycloheximide, sex hormone receptor antagonists (flutamide, fulvestrant, mifepristone) and also after removing the endometrium. The modifications in uterine G-protein activation and cAMP levels were also detected. ResultsT and E2 both relaxed the uterine contractions in the concentration range of 10−8–10−3 M with an increase in the activated G-protein and cAMP levels of the uterus, while P4 was ineffective. Cycloheximide, actinomycin D, antagonist for T and E2 were not able to modify the responses along with the endothelium removal. Mifepristone blocked the relaxing effects of T and E2 and reduced the activation of G-protein and the formation of cAMP. SignificanceT and E2 can inhibit KCl-stimulated contractions in the late pregnant uterus in high concentrations and in a non-genomic manner. Their actions are mediated by a G-protein coupled receptor that can be blocked by mifepristone. A single and high dose of T or E2 might be considered in premature contractions, however, further preclinical and clinical studies are required for the approval of such a therapeutic intervention.