Oxysterols direct B-cell migration through EBI2.

EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein-Barr-virus infection. Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism. The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors. Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro, 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo, EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7ß, 27-dihydroxycholesterol (7ß, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7ß, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7ß, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Global deletion of MGL in mice delays lipid absorption and alters energy homeostasis and diet-induced obesity.

Monoacylglycerol lipase (MGL) is a ubiquitously expressed enzyme that catalyzes the hydrolysis of monoacylglycerols (MGs) to yield FFAs and glycerol. MGL contributes to energy homeostasis through the mobilization of fat stores and also via the degradation of the endocannabinoid 2-arachidonoyl glycerol. To further examine the role of MG metabolism in energy homeostasis, MGL(-/-) mice were fed either a 10% (kilocalories) low-fat diet (LFD) or a 45% (kilocalories) high-fat diet (HFD) for 12 weeks. Profound increases of MG species in the MGL(-/-) mice compared with WT control mice were found. Weight gain over the 12 weeks was blunted in both diet groups. MGL(-/-) mice were leaner than WT mice at both baseline and after 12 weeks of LFD feeding. Circulating lipids were decreased in HFD-fed MGL(-/-) mice, as were the levels of several plasma peptides involved in glucose homeostasis and energy balance. Interestingly, MGL(-/-) mice had markedly reduced intestinal TG secretion following an oral fat challenge, suggesting delayed lipid absorption. Overall, the results indicate that global MGL deletion leads to systemic changes that produce a leaner phenotype and an improved serum metabolic profile.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-µM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.

Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (µ-TRTX-Hh2a).

Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity.

Structure-Based Optimization of Selective and Brain Penetrant CK1δ Inhibitors for the Treatment of Circadian Disruptions.

Neuropsychiatric disorders such as major depressive disorders and schizophrenia are often associated with disruptions to the normal 24 h sleep wake cycle. Casein kinase 1 (CK1δ) is an integral part of the molecular machinery that regulates circadian rhythms. Starting from a cluster of bicyclic pyrazoles identified from a virtual screening effort, we utilized structure-based drug design to identify and reinforce a unique "hinge-flip" binding mode that provides a high degree of selectivity for CK1δ versus the kinome. Pharmacokinetics, brain exposure, and target engagement as measured by ex vivo autoradiography are described for advanced analogs.

Identification and characterization of select oxysterols as ligands for GPR17.

BACKGROUND AND PURPOSE: G-protein coupled receptor 17 (GPR17) is an orphan receptor involved in the process of myelination, due to its ability to inhibit the maturation of oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes. Despite multiple claims that the biological ligand has been identified, it remains an orphan receptor. EXPERIMENTAL APPROACH: Seventy-seven oxysterols were screened in a cell-free [35 S]GTPγS binding assay using membranes from cells expressing GPR17. The positive hits were characterized using adenosine 3',5' cyclic monophosphate (cAMP), inositol monophosphate (IP1) and calcium mobilization assays, with results confirmed in rat primary oligodendrocytes. Rat and pig brain extracts were separated by high-performance liquid chromatography (HPLC) and endogenous activator(s) were identified in receptor activation assays. Gene expression studies of GPR17, and CYP46A1 (cytochrome P450 family 46 subfamily A member 1) enzymes responsible for the conversion of cholesterol into specific oxysterols, were performed using quantitative real-time PCR. KEY RESULTS: Five oxysterols were able to stimulate GPR17 activity, including the brain cholesterol, 24(S)-hydroxycholesterol (24S-HC). A specific brain fraction from rat and pig extracts containing 24S-HC activates GPR17 in vitro. Expression of Gpr17 during mouse brain development correlates with the expression of Cyp46a1 and the levels of 24S-HC itself. Other active oxysterols have low brain concentrations below effective ranges. CONCLUSIONS AND IMPLICATIONS: Oxysterols, including but not limited to 24S-HC, could be physiological activators for GPR17 and thus potentially regulate OPC differentiation and myelination through activation of the receptor.

Neuropeptide S stimulates dopaminergic neurotransmission in the medial prefrontal cortex.

Neuropeptide S (NPS) is known to produce anxiolytic-like effects and facilitate extinction of conditioned fear. Catecholaminergic neurotransmission in the medial prefrontal cortex (mPFC) has been suggested to be crucially involved in these brain functions. In the current study, we investigated the effect of NPS on the release of dopamine and serotonin in the mPFC by in vivo microdialysis in rats. Central administration of NPS dose-dependently enhanced extracellular levels of dopamine and its major metabolite 3,4-dihydroxyphenylacetic acid, with maximal effects lasting up to 120 min. In contrast, no effect on serotonergic neurotransmission was detected. Dopamine release in the mPFC has been previously linked to modulation of anxiety states and fear extinction. The present results may thus provide a physiological and anatomical basis for the reported effects of NPS on these behaviors.

Modulation of hippocampal theta oscillations and spatial memory by relaxin-3 neurons of the nucleus incertus.

Hippocampal theta rhythm is thought to underlie learning and memory, and it is well established that "pacemaker" neurons in medial septum (MS) modulate theta activity. Recent studies in the rat demonstrated that brainstem-generated theta rhythm occurs through a multisynaptic pathway via the nucleus incertus (NI), which is the primary source of the neuropeptide relaxin-3 (RLN3). Therefore, this study examined the possible contribution of RLN3 to MS activity, and associated hippocampal theta activity and spatial memory. In anesthetized and conscious rats, we identified the ability of intraseptal RLN3 signaling to modulate neuronal activity in the MS and hippocampus and promote hippocampal theta rhythm. Behavioral studies in a spontaneous alternation task indicated that endogenous RLN3 signaling within MS promoted spatial memory and exploratory activity significantly increased c-Fos immunoreactivity in RLN3-producing NI neurons. Anatomical studies demonstrated axons/terminals from NI/RLN3 neurons make close contact with septal GABAergic (and cholinergic) neurons, including those that project to the hippocampus. In summary, RLN3 neurons of the NI can modulate spatial memory and underlying hippocampal theta activity through axonal projections to pacemaker neurons of the MS. NI/RLN3 neurons are highly responsive to stress and express corticotropin-releasing factor type-1 receptors, suggesting that the effects observed could be an important component of memory processing associated with stress responses.

Blockade of orexin-1 receptors attenuates orexin-2 receptor antagonism-induced sleep promotion in the rat.

Orexins are peptides produced by lateral hypothalamic neurons that exert a prominent role in the maintenance of wakefulness by activating orexin-1 (OX1R) and orexin-2 (OX2R) receptor located in wake-active structures. Pharmacological blockade of both receptors by the dual OX1/2R antagonist (2R)-2-[(1S)-6,7-dimethoxy-1-{2-[4-(trifluoromethyl)phenyl]ethyl}-3,4-dihydroisoquinolin-2(1H)-yl]-N-methyl-2-phenylethanamide (almorexant) has been shown to promote sleep in animals and humans during their active period. However, the selective distribution of OX1R and OX2R in distinct neuronal circuits may result in a differential impact of these receptors in sleep-wake modulation. The respective role of OX1R and OX2R on sleep in correlation with monoamine release was evaluated in rats treated with selective antagonists alone or in combination. When administered in either phase of the light/dark cycle, the OX2R antagonist 1-(2,4-dibromophenyl)-3-[(4S,5S)-2,2-dimethyl-4-phenyl-1,3-dioxan-5-yl]urea (JNJ-10397049) decreased the latency for persistent sleep and increased nonrapid eye movement and rapid eye movement sleep time. Almorexant produced less hypnotic activity, whereas the OX1R antagonist 1-(6,8-difluoro-2-methylquinolin-4-yl)-3-[4-(dimethylamino)phenyl]urea (SB-408124) had no effect. Microdialysis studies showed that either OX2R or OX1/2R antagonism decreased extracellular histamine concentration in the lateral hypothalamus, whereas both OX1R and OX1/2R antagonists increased dopamine release in the prefrontal cortex. Finally, coadministration of the OX1R with the OX2R antagonist greatly attenuated the sleep-promoting effects of the OX2R antagonist. These results indicate that blockade of OX2R is sufficient to initiate and prolong sleep, consistent with the hypothesis of a deactivation of the histaminergic system. In addition, it is suggested that simultaneous inhibition of OX1R attenuates the sleep-promoting effects mediated by selective OX2R blockade, possibly correlated with dopaminergic neurotransmission.

Relaxin family peptide receptors--from orphans to therapeutic targets.

The relaxin family peptides have distinct expression profiles and physiological functions. Several of them are the cognate ligands for 4 G-protein-coupled relaxin family peptide receptors (RXFPs; formerly LGR7, LGR8, GPCR135, GPCR142). The relaxin/RXFP1 system has roles in reproductive physiology but is also involved in fibrosis, wound healing and responses to infarction. Relaxin has a potential use in congestive heart failure where fibrosis plays an important role in organ failure. The INSL3/RXFP2 system has biological roles in reproductive biology that may have limited therapeutic potential. However, the recently characterized relaxin-3/RXFP3 system is important in stress/anxiety and body composition. RXFP3 receptor antagonists are potentially novel anti-anxiety and anti-obesity drugs.

2-Aryloxymethylmorpholine histamine H(3) antagonists.

The synthesis and biological activity of a new series of 2-aryloxymethylmorpholine histamine H(3) antagonists is described. The new compounds are high affinity histamine H(3) ligands that penetrate the CNS and occupy the histamine H(3) receptor in rat brain.

Evolution of membrane oxygenator technology for utilization during pediatric cardiopulmonary bypass.

The development of the membrane oxygenator for pediatric cardiopulmonary bypass has been an incorporation of ideology and technological advancements with contributions by many investigators throughout the past two centuries. With the pursuit of this technological achievement, the ability to care for mankind in the areas of cardiac surgery has been made possible. Heart disease can affect anyone within the general population, but one such segment that it can affect from inception includes children. Currently, congenital heart defects are the most common birth defects nationally and worldwide. A large meta-analysis study from 1930 to 2010 was conducted in review of published medical literature totaling 114 papers with a study population of 24,091,867 live births, and divulged a staggering incidence of congenital heart disease involving 164,396 subjects with diverse cardiac illnesses. The prevalence of these diseases increased from 0.6 per 1,000 live births from 1930-1934 to 9.1 per 1,000 live births after 1995. These data reveal an emphasis on a growing public health issue regarding congenital heart disease. This discovery displays a need for heightened awareness in the scientific and medical industrial community to accelerate investigative research on emerging cardiovascular devices in an effort to confront congenital anomalies. One such device that has evolved over the past several decades is the pediatric membrane oxygenator. The pediatric membrane oxygenator, in conjunction with the heart lung machine, assists in the repair of most congenital cardiac defects. Numerous children born with congenital heart disease with or without congestive heart failure have experienced improved clinical outcomes in quality of life, survival, and mortality as a result of the inclusion of this technology during their cardiac surgical procedure. The purpose of this review is to report a summary of the published medical and scientific literature related to development of the pediatric membrane oxygenator from its conceptual evolutionary stages to artificially supporting whole body perfusion in the modern pediatric cardiac surgical setting.

Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO.

Nicotine exerts its behavioral and additive actions through a family of brain nicotinic acetylcholine receptors (nAChRs). Enhancing α7-type nAChR signaling improves symptoms in Alzheimer's disease and schizophrenia. The pharmaceutical study of α7 receptors is hampered because these receptors do not form their functional pentameric structure in cell lines, and mechanisms that underlie α7 receptor assembly in neurons are not understood. Here, a genomic screening strategy solves this long-standing puzzle and identifies NACHO, a transmembrane protein of neuronal endoplasmic reticulum that mediates assembly of α7 receptors. NACHO promotes α7 protein folding, maturation through the Golgi complex, and expression at the cell surface. Knockdown of NACHO in cultured hippocampal neurons or knockout of NACHO in mice selectively and completely disrupts α7 receptor assembly and abolishes α7 channel function. This work identifies NACHO as an essential, client-specific chaperone for nAChRs and has implications for physiology and disease associated with these widely distributed neurotransmitter receptors.

Recent progress in relaxin-3-related research.

Relaxin-3 (R3) is the latest member of the insulin (INSL) superfamily, which is composed of peptides with diverse sequences held together by characteristic disulfide links connecting A and B peptide chains. R3 has nearly exclusive expression in the brainstem and was demonstrated to be an additional ligand for LGR7. We recently identified R3 as a ligand for two orphan G-protein coupled receptors, GPCR135 and GPCR142. The predominant brain expression for both R3 and GPCR135, coupled with their high affinity interaction, strongly suggests that R3 is the endogenous ligand for GPCR135. Both R3 and GPCR135 from different species are highly conserved from genetic sequences to in vitro pharmacology. By contrast, GPCR142 is a pseudogene in the rat, and the mouse gene is less conserved with human GPCR142, suggesting that GPCR142 may have a diminished role as a receptor for R3 in the rodent. In addition, the tissue expression pattern of GPCR142, which is primarily in peripheral tissue, is drastically different from that of R3, suggesting that GPCR142 may have an endogenous ligand other than R3. Sequence analysis among INSL/relaxin family members shows that INSL5 is the closest member of R3. We were able to demonstrate that INSL-5 is an agonist for GPCR142 but not for GPCR135. We also showed that the mRNA expression pattern of INSL-5 overlaps with that of GPCR142. By substituting the A chain of R3 with the A chain of INSL-5, we devised a chimeric peptide (R3/I5) that is about 1000-fold selective for GPCR135 and GPCR142 over LGR7. Autoradiographic distribution of GPCR135 binding sites using [125I]R3/I5 in rat brain shows that GPCR135 receptor is most prominent in areas known for the processing of sensory signals.

Relaxin-3 receptor (Rxfp3) gene knockout mice display reduced running wheel activity: implications for role of relaxin-3/RXFP3 signalling in sustained arousal.

Anatomical and pharmacological evidence suggests the neuropeptide, relaxin-3, is the preferred endogenous ligand for the relaxin family peptide-3 receptor (RXFP3) and suggests a number of putative stress- and arousal-related roles for RXFP3 signalling. However, in vitro and in vivo evidence demonstrates exogenous relaxin-3 can activate other relaxin peptide family receptors, and the role of relaxin-3/RXFP3 signalling in specific brain circuits and associated behaviours in mice is not well described. In this study, we characterised the behaviour of cohorts of male and female Rxfp3 gene knockout (KO) mice (C57/B6J(RXFP3TM1/DGen)), relative to wild-type (WT) littermates to determine if this receptor KO strain has a similar phenotype to its ligand KO equivalent. Rxfp3 KO mice displayed similar performance to WT littermates in several acute behavioural paradigms designed to gauge motor coordination (rotarod test), spatial memory (Y-maze), depressive-like behaviour (repeat forced-swim test) and sensorimotor gating (prepulse inhibition of acoustic startle). Notably however, male and female Rxfp3 KO mice displayed robust and consistent (dark phase) hypoactivity on voluntary home-cage running wheels (∼20-60% less activity/h), and a small but significant decrease in anxiety-like behavioural traits in the elevated plus maze and light/dark box paradigms. Importantly, this phenotype is near identical to that observed in two independent lines of relaxin-3 KO mice, suggesting these phenotypes are due to the elimination of ligand or receptor and RXFP3-linked signalling. Furthermore, this behavioural characterisation of Rxfp3 KO mice identifies them as a useful experimental model for studying RXFP3-linked signalling and assessing the selectivity and/or potential off-target actions of RXFP3 agonists and antagonists, which could lead to an improved understanding of dysfunctional arousal in mental health disorders, including depression, anxiety, insomnia and neurodegenerative diseases.

Clinical benefits of leukocyte filtration during valve surgery.

BACKGROUND: The accumulation of activated leukocytes in the pulmonary circulation plays an important role in the pathogenesis of lung dysfunction associated with cardiopulmonary bypass (CPB). Patients undergoing valve surgery have prolonged CPB owing to the complexity of the surgery. The goal of this study is to determine if arterial leukocyte filters during CPB improve clinical outcomes after valve surgery. METHODS: A prospective analysis of all patients receiving only valve surgery with leukocyte arterial filters from June 1999 to June 2002 was compared with a case matched cohort during the same time period. Two hundred fifty patients were identified and compared with a cohort who did not have leukocyte filters used during CPB. The following study points were evaluated preoperatively and postoperatively: white blood cell count, platelet count, arterial blood gas, time to extubation, intensive care unit stay, and total length of hospital stay. RESULTS: There were 500 patients in the study. The following valve operations were performed: 92 mitral valve replacements, 168 aortic valve replacements, 152 mitral valve repairs, 80 combined valve repair/replacements, and 8 tricuspid valve repairs, all evenly divided between the two treatment limbs. Patients with leukocyte filters had the following findings compared with nonfilter patients: The time to extubation 10.3 versus 16.2 hours (P = 0.009), postoperative respiratory quotient 407 versus 320 (P = 0.02), total length of stay 5.4 versus 7.2 days (P = 0.04). CONCLUSIONS: The use of arterial leukocyte filters in patients undergoing valve surgery leads to earlier extubation, improved oxygenation, and a decreased length of stay. Leukocyte filters should be used during CPB for patients having valve surgery.

Involvement of ß-arrestin-2 and clathrin in agonist-mediated internalization of the human cannabinoid CB2 receptor.

The CB2 cannabinoid receptor is a promising therapeutic target for the treatment of inflammatory diseases, neuropathic pain, liver diseases, cancer and cardiovascular diseases. Obtaining detailed information on the internalization and trafficking of the human CB2 receptor in response to agonist will have a significant impact on drug discovery. Visualization and quantitative detection of EGFP-tagged CB2 receptor showed that, upon WIN55,212-2 stimulation, the CB2 receptor was rapidly internalized in a dose- and time-dependent manner from the cell membrane into the cytoplasm. Pretreatment with hypertonic sucrose, MDC clathrin inhibitor, or siRNA-mediated knock-down of clathrin heavy chain led to significant inhibition of agonist-induced CB2 internalization. Using the RNA interference method, we showed that knockdown of ß-arrestin2 expression significantly impaired receptor internalisation. Further investigation demonstrated that the internalized CB2 receptors were co-localized with the early endosome probe and were recycled to the cell surface after the removal of agonist, but treatment with specific cell-permeable proteasome inhibitor MG132 a inhibited the recycling of internalized CB2 receptor, suggesting that the proteasome-mediated degradation pathway may be involved in CB2 internalization. Moreover, the single residue Ser(352) and residue cluster S(335)S(336)T(338)T(340) at the C-terminal tail are shown to be essential for receptor phosphorylation and ß-arrestin2 association. These data provide new insights into the mechanisms regulating agonist-mediated internalization and trafficking of the human CB2 receptor.

In vitro pharmacological characterization of RXFP3 allosterism: an example of probe dependency.

Recent findings suggest that the relaxin-3 neural network may represent a new ascending arousal pathway able to modulate a range of neural circuits including those affecting circadian rhythm and sleep/wake states, spatial and emotional memory, motivation and reward, the response to stress, and feeding and metabolism. Therefore, the relaxin-3 receptor (RXFP3) is a potential therapeutic target for the treatment of various CNS diseases. Here we describe a novel selective RXFP3 receptor positive allosteric modulator (PAM), 3-[3,5-Bis(trifluoromethyl)phenyl]-1-(3,4-dichlorobenzyl)-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]urea (135PAM1). Calcium mobilization and cAMP accumulation assays in cell lines expressing the cloned human RXFP3 receptor show the compound does not directly activate RXFP3 receptor but increases functional responses to amidated relaxin-3 or R3/I5, a chimera of the INSL5 A chain and the Relaxin-3 B chain. 135PAM1 increases calcium mobilization in the presence of relaxin-3(NH2) and R3/I5(NH2) with pEC50 values of 6.54 (6.46 to 6.64) and 6.07 (5.94 to 6.20), respectively. In the cAMP accumulation assay, 135PAM1 inhibits the CRE response to forskolin with a pIC50 of 6.12 (5.98 to 6.27) in the presence of a probe (10 nM) concentration of relaxin-3(NH2). 135PAM1 does not compete for binding with the orthosteric radioligand, [(125)I] R3I5 (amide), in membranes prepared from cells expressing the cloned human RXFP3 receptor. 135PAM1 is selective for RXFP3 over RXFP4, which also responds to relaxin-3. However, when using the free acid (native) form of relaxin-3 or R3/I5, 135PAM1 doesn't activate RXFP3 indicating that the compound's effect is probe dependent. Thus one can exchange the entire A-chain of the probe peptide while retaining PAM activity, but the state of the probe's c-terminus is crucial to allosteric activity of the PAM. These data demonstrate the existence of an allosteric site for modulation of this GPCR as well as the subtlety of changes in probe molecules that can affect allosteric modulation of RXFP3.