Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14 -21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [

Regulation of messenger RNA is crucial in many contexts, including development, memory and cell growth. The 3' untranslated region is a rich repository of regulatory elements that bind proteins and microRNAs. Here we focus on PUF proteins, an important family of mRNA regulatory proteins crucial in stem-cell proliferation, pattern formation and synaptic plasticity. We show that two Caenorhabditis elegans PUF proteins, FBF and PUF-8, differ in RNA-binding specificity. FBF requires the presence of a single 'extra' nucleotide in the middle of an eight-nucleotide site, whereas PUF-8 requires its absence. A discrete protein segment is responsible for the difference. We propose that a structural distortion in the central region of FBF imposes the requirement for the additional nucleotide and that this mode of PUF specificity may be common. We suggest that new specificities can be designed and selected using the PUF scaffold.

Hypertension is a cardiovascular disease associated with increased plasma catecholamines, overactivation of the sympathetic nervous system, and increased vascular tone and total peripheral resistance. A key regulator of sympathetic nervous system function is the ␣ 1D -adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate ␣ 1D -ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, ␣ 1D -AR KO mice display a hypotensive phenotype and are resistant to salt-induced hypertension. Unfortunately, little information exists about how this important GPCR functions because of an inability to obtain functional expression in vitro. Here, we identified the dystrophin proteins, syntrophin, dystrobrevin, and utrophin as essential GPCR-interacting proteins for ␣ 1D -ARs. We found that dystrophins complex with ␣ 1D -AR both in vitro and in vivo to ensure proper functional expression. More importantly, we demonstrate that knock-out of multiple syntrophin isoforms results in the complete loss of ␣ 1D -AR function in mouse aortic smooth muscle cells and abrogation of ␣ 1D -AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with ␣ 1D

Background Posttraumatic stress disorder (PTSD) is a prevalent psychiatric disorder precipitated by exposure to extreme traumatic stress. Yet, most individuals exposed to traumatic stress do not develop PTSD and may be considered psychologically resilient. The neural circuits involved in susceptibility or resiliency to PTSD remain unclear, but clinical evidence implicates changes in the noradrenergic system. Methods An animal model of PTSD called Traumatic Experience with Reminders of Stress (TERS) was developed by exposing C57BL/6 mice to a single shock (2mA, 10sec) followed by exposure to six contextual1-minute reminders of the shock overa 25-dayperiod. Acoustic startle response (ASR) testing before the shock and after the last reminder allowed experimenters to separate the shocked mice into two cohorts: mice that developed a greatly increased ASR (TERS-susceptible mice) and mice that did not (TERS-resilient mice). Results Aggressive and social behavioral correlates of PTSD increased in TERS-susceptible mice but not in TERS-resilient mice or control mice. Characterization of c-Fos expression in stress-related brain regions revealed that TERS-susceptible and TERS-resilient mice displayed divergent brain activation following swim stress compared with control mice. Pharmacological activation of noradrenergic inhibitory autoreceptors or blockade of postsynaptic α1-adrenoreceptors normalized ASR, aggression, and social interaction in TERS-susceptible mice. The TERS-resilient, but not TERS-susceptible, mice showed a trend toward decreased behavioral responsiveness to noradrenergic autoreceptor blockade compared with control mice. Conclusions These data implicate the noradrenergic system as a possible site of pathological and perhaps also adaptive plasticity in response to traumatic stress.

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the ␣ 1A -AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of G␣ subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of G␣. Combining our data with a predictive Class A GPCR/G␣ model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of G␣. Such an interaction could promote opening of switch II of G␣ to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.A diverse network of signaling pathways have evolved within the hypothalamic-pituitary-gonadal axis to ensure precise neuroendocrine regulation of reproductive function in mammals (1). An essential feature of this physiological system is the pulsatile release of gonadotropin-releasing hormone from hypothalamic neurons, which subsequently initiates follicle-stimulating hormone and luteinizing hormone release from the pituitary and ultimately impinges on the gonads to elicit sex steroid secretion (2). Together, the components of the hypothalamic-pituitary-gonadal axis function with precise temporal and spatial accuracy to regulate the development and maintenance of proper reproductive function, including puberty onset and the estrous cycle (3). Thus, functional mutations in key elements of this critical physiological system can result in the development of various reproductive disorders. For example, idiopathic hypogonadotropic hypogonadism (IHH), 2 which is characterized by delayed or absent puberty, immature reproductive organs, low levels of sex steroids and infertility, is commonly associated with loss-of-function mutations in the gonadotropin-releasing hormone receptor (4, 5). More recently, IHH-causing mutations were identified in a relatively uncharacterized orphan G-protein-coupled recep...