Inhibition of sequestration of human B-2 bradykinin receptor by phenylarsine oxide or sucrose allows determination of a receptor affinity shift and ligand dissociation in intact cells

Depending on their interaction with intracellular proteins, G proteincoupled receptors (GPCR) often display different affinities for agonists at 37degreesC. Determining the affinity at that temperature is often difficult in intact cells as most GPCRs are internalized after activation. When sequestration of the B-2 bradykinin receptor (B2R) was inhibited by either 0.5 M sucrose or phenylarsine oxide (PAO), a shift in the affinity was detected when the incubation temperature was raised from 4degreesC to 37degreesC or lowered from 37degreesC to 4degreesC. In contrast, binding of the antagonist {[}H-3]NPC 17731 was temperatureindependent. B2R mutants displayed different affinity shifts allowing conclusions on the role of the involved amino acids. By inhibiting receptor sequestration it was possible to determine also dissociation of {[}H-3]BK and of {[}H-3]NPC 17731 from intact cells at 37degreesC. Surprisingly, both dissociation rates were markedly enhanced by the addition of unlabeled ligand, most likely via prevention of reassociation of dissociated {[}H-3]ligand. This suggests that dissociated {[}H-3]ligand cannot move freely away from the receptor. In summary, our data demonstrate that inhibition of receptor internalization either by PAO or sucrose provides an excellent method to study receptor function and the effects of mutations in intact cells

G Protein α subunits activate tubulin GTPase and modulate microtubule polymerization dynamics

G proteins serve many functions involving the transfer of signals from cell surface receptors to intracellular effector molecules. Considerable evidence suggests that there is an interaction between G proteins and the cytoskeleton. In this report, G protein α subunits Gi1α, Gsα, and Goα are shown to activate the GTPase activity of tubulin, inhibit microtubule assembly, and accelerate microtubule dynamics. Giα inhibited polymerization of tubulin-GTP into microtubules by 80-90% in the absence of exogenous GTP. Addition of exogenous GTP, but not guanylylimidodiphosphate, which is resistant to hydrolysis, overcame the inhibition. Analysis of the dynamics of individual microtubules by video microscopy demonstrated that Gi1α increases the catastrophe frequency, the frequency of transition from growth to shortening. Thus, Gα may play a role in modulating microtubule dynamic instability, providing a mechanism for the modification of the cytoskeleton by extracellular signals

Brain-derived neurotrophic factor association with amygdala response in major depressive disorder

Background: Brain-derived neurotrophic factor (BDNF) has an essential role in synaptic plasticity and neurogenesis. BDNF mediates amygdala-dependent learning for both aversive and appetitive emotional memories. The expression of BDNF in limbic regions is posited to contribute the development of depression, and amygdala responsivity is a potential marker of depressive state. Methods: The present study examined the relationship between platelet BDNF levels and amygdala volume and function in major depressive disorder (MDD). Participants were 23 MDD (mean age 38.9 years) and 23 healthy controls (mean age 38.8 years). All participants were recruited from the community. MDD participants were in a current depressive episode of moderate severity and medication-free. Amygdala responses were acquired during a functional MRI task of implicit emotional processing with sad facial expressions. Results: Significant correlation was observed between platelet BDNF levels and left amygdala responses, but no significant correlations were found with right amygdala responses or with amygdala volumes. Limitations: Interactions with neuroprotective as well as neurotoxic metabolites in the kyneurenine pathway were not examined. Conclusions: Relationship between BDNF levels and amygdala responsivity to emotionally salient stimuli in MDD could reflect the importance of BDNF in amygdala-dependent learning with clinical implications for potential pathways for treatment

Linoleic acid: Is this the key that unlocks the quantum brain? Insights linking broken symmetries in molecular biology, mood disorders and personalistic emergentism

In this paper we present a mechanistic model that integrates subneuronal structures, namely ion channels, membrane fatty acids, lipid rafts, G proteins and the cytoskeleton in a dynamic system that is finely tuned in a healthy brain. We also argue that subtle changes in the composition of the membrane's fatty acids may lead to down-stream effects causing dysregulation of the membrane, cytoskeleton and their interface. Such exquisite sensitivity to minor changes is known to occur in physical systems undergoing phase transitions, the simplest and most studied of them is the so-called Ising model, which exhibits a phase transition at a finite temperature between an ordered and disordered state in 2- or 3-dimensional space. We propose this model in the context of neuronal dynamics and further hypothesize that it may involve quantum degrees of freedom dependent upon variation in membrane domains associated with ion channels or microtubules. Finally, we provide a link between these physical characteristics of the dynamical mechanism to psychiatric disorders such as major depression and antidepressant action

Structural model of a complex between the heterotrimeric G protein, Gsα, and tubulin

AbstractA number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The α subunits of some heterotrimeric G proteins, including Gsα, have been shown to interact strongly with tubulin. Binding of Gα to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsα via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsα was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, α2–β4 (the region between switch II and switch III) and α3–β5 (just distal to the switch III region) domains of Gsα are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gβγ interaction regions of Gsα and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Gα and the ability of Gα to activate tubulin GTPase

Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein Gs and an orphan member of the G protein–coupled receptor family, GPR3. To determine whether GPR3 activates Gs, the localization of Gαs in follicle-enclosed oocytes from Gpr3+/+ and Gpr3−/− mice was compared by using immunofluorescence and GαsGFP. GPR3 decreased the ratio of Gαs in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Gαs in the oocyte. Both of these properties indicate that GPR3 activates Gs. The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent Gs activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity

Multimodal functional and structural neuroimaging investigation of major depressive disorder following treatment with duloxetine

Background: Longitudinal neuroimaging studies of major depressive disorder (MDD) have most commonly assessed the effects of antidepressants from the serotonin reuptake inhibitor class and usually reporting a single measure. Multimodal neuroimaging assessments were acquired from MDD patients during an acute depressive episode with serial measures during a 12-week treatment with the serotonin-norepinephrine reuptake inhibitor (SNRI) duloxetine. Methods: Participants were medication-free MDD patients (n = 32; mean age 40.2 years) in an acute depressive episode and healthy controls matched for age, gender, and IQ (n = 25; mean age 38.8 years). MDD patients received treatment with duloxetine 60 mg daily for 12 weeks with an optional dose increase to 120 mg daily after 8 weeks. All participants had serial imaging at weeks 0, 1, 8, and 12 on a 3 Tesla magnetic resonance imaging (MRI) scanner. Neuroimaging tasks included emotional facial processing, negative attentional bias (emotional Stroop), resting state functional MRI and structural MRI. Results: A significant group by time interaction was identified in the anterior default mode network in which MDD patients showed increased connectivity with treatment, while there were no significant changes in healthy participants. In the emotional Stroop task, increased posterior cingulate activation in MDD patients normalized following treatment. No significant group by time effects were observed for happy or sad facial processing, including in amygdala responsiveness, or in regional cerebral volumes. Reduced baseline resting state connectivity within the orbitofrontal component of the default mode network was predictive of clinical response. An early increase in hippocampal volume was predictive of clinical response. Conclusions: Baseline resting state functional connectivity was predictive of subsequent clinical response. Complementary effects of treatment were observed from the functional neuroimaging correlates of affective facial expressions, negative attentional bias, and resting state. No significant effects were observed in affective facial processing, while the interaction effect in negative attentional bias and individual group effects in resting state connectivity could be related to the SNRI class of antidepressant medication. The specificity of the observed effects to SNRI pharmacological treatments requires further investigation. Trial registration: Registered at clinicaltrials.gov (NCT01051466)

SDF1-Induced Antagonism of Axonal Repulsion Requires Multiple G-Protein Coupled Signaling Components That Work in Parallel

SDF1 reduces the responsiveness of axonal growth cones to repellent guidance cues in a pertussis-toxin-sensitive, cAMP-dependent manner. Here, we show that SDF1's antirepellent effect can be blocked in embryonic chick dorsal root ganglia (DRGs) by expression of peptides or proteins inhibiting either Gαi, Gαq, or Gβγ. SDF1 antirepellent activity is also blocked by pharmacological inhibition of PLC, a common effector protein for Gαq. We also show that SDF1 antirepellent activity can be mimicked by overexpression of constitutively active Gαi, Gαq, or Gαs. These results suggest a model in which multiple G protein components cooperate to produce the cAMP levels required for SDF1 antirepellent activity

Human depression: a new approach in quantitative psychiatry

The biomolecular approach to major depression disorder is explained by the different steps that involve cell membrane viscosity, Gsα protein and tubulin. For the first time it is hypothesised that a biomolecular pathway exists, moving from cell membrane viscosity through Gsα protein and Tubulin, which can condition the conscious state and is measurable by electroencephalogram study of the brain's γ wave synchrony