Mia C. DeFino

Long-Acting κ Opioid Antagonists Disrupt Receptor Signaling And Produce Noncompetitive Effects By Activating C-Jun N-Terminal Kinase

Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective κ 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 [3H]U69,593 to KOR showed that Kd and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK.

Disease-causing mutation in GPR54 reveals the importance of the second intracellular loop for class A G-protein-coupled receptor function.

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 α1A-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 Gα subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Gα. Combining our data with a predictive Class A GPCR/Gα model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Gα. Such an interaction could promote opening of switch II of Gα to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Blood pressure is regulated by an α1D-adrenergic receptor/dystrophin signalosome

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 α1D-adrenergic receptor (AR), which belongs to the adrenergic family of G-protein-coupled receptors (GPCRs). Endogenous catecholamines norepinephrine and epinephrine activate α1D-ARs on vascular smooth muscle to stimulate vasoconstriction, which increases total peripheral resistance and mean arterial pressure. Indeed, α1D-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 α1D-ARs. We found that dystrophins complex with α1D-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 α1D-AR function in mouse aortic smooth muscle cells and abrogation of α1D-AR-mediated increases in blood pressure. Our findings demonstrate that syntrophin and utrophin associate with α1D-ARs to create a functional signalosome, which is essential for α1D-AR regulation of vascular tone and blood pressure.

The Role of Norepinephrine in Differential Response to Stress in an Animal Model of Posttraumatic Stress Disorder

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 (2 mA, 10 sec) followed by exposure to six contextual 1-minute reminders of the shock over a 25-day period. 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 α(1)-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.

Differential regulation of GPR54 transcription by specificity protein-1 and partial estrogen response element in mouse pituitary cells

Precise spatial and temporal expression of the recently identified G-protein coupled receptor GPR54 is critical for proper reproductive function and metastasis suppression. However, regulatory factors that control GPR54 expression remain unknown. Thus, the identification of these cis-acting DNA elements can provide insight into the role of GPR54 in reproduction and cancer. Using luciferase reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we demonstrate that three SP1 sites and a partial estrogen response element modulate mouse GPR54 (mGPR54) promoter activity. Supporting experiments show transcription factor SP1 binds directly to the mGPR54 promoter region and activates gene expression. In conclusion, these novel findings now identify factors that regulate activity of the mGPR54 promoter, and these factors are highly conserved across multiple mammalian species.

Syntrophin isoforms play specific functional roles in the α1D-adrenergic receptor/DAPC signalosome

α(1D)-Adrenergic receptors, key regulators of cardiovascular system function, are organized as a multi-protein complex in the plasma membrane. Using a Type-I PDZ-binding motif in their distal C-terminal domain, α(1D)-ARs associate with syntrophins and dystrophin-associated protein complex (DAPC) members utrophin, dystrobrevin and α-catulin. Three of the five syntrophin isoforms (α, β(1) and β(2)) interact with α(1D)-ARs and our previous studies suggest multiple isoforms are required for proper α(1D)-AR function in vivo. This study determined the contribution of each specific syntrophin isoform to α(1D)-AR function. Radioligand binding experiments reveal α-syntrophin enhances α(1D)-AR binding site density, while phosphoinositol and ERK1/2 signaling assays indicate β(2)-syntrophin augments full and partial agonist efficacy for coupling to downstream signaling mechanisms. The results of this study provide clear evidence that the cytosolic components within the α(1D)-AR/DAPC signalosome significantly alter the pharmacological properties of α(1)-AR ligands in vitro.