CODA-RET reveals functional selectivity as a result of GPCR heteromerization

Here we present a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(-)-10,11-dihydroxy-N-n-propylnoraporphine (NPA) to change the Gα(i) conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity in which a drug acts differently at a G protein-coupled receptor (GPCR) protomer depending on the identity of the second protomer participating in the formation of the signaling unit--opening the door to enhancing pharmacological specificity by targeting differences between homomeric and heteromeric signaling.     

The Effects of a 6-Week Strength Training on Critical Velocity,Anaerobic Running Distance, 30-M Sprint and Yo-Yo Intermittent Running Test Performances in Male Soccer Players

The objectives of this study were to examine the effects of a moderate intensity strength training on changes in critical velocity (CV), anaerobic running distance (D''), sprint performance and Yo-Yo intermittent running test (Yo-Yo IR1) performances. Methods: two recreational soccer teams were divided in a soccer training only group (SO; n = 13) and a strength and soccer training group (ST; n = 13). Both groups were tested for values of CV, D'', Yo-Yo IR1 distance and 30-m sprint time on two separate occasions (pre and post intervention). The ST group performed a concurrent 6-week upper and lower body strength and soccer training, whilst the SO group performed a soccer only training. Results: after the re-test of all variables, the ST demonstrated significant improvements for both, YoYo IR1 distance (p = 0.002) and CV values (p<0.001) with no significant changes in the SO group. 30-m sprint performance were slightly improved in the ST group with significantly decreased performance times identified in the SO group (p<0.001). Values for D'' were slightly reduced in both groups (ST -44.5 m, 95% CI = -90.6 to 1.6; SO -42.6 m, 95% CI = -88.7 to 3.5). Conclusions: combining a 6-week moderate strength training with soccer training significantly improves CV, Yo-Yo IR1 whilst moderately improving 30-m sprint performances in non-previously resistance trained male soccer players. Critical Velocity can be recommended to coaches as an additional valid testing tool in soccer.     

F2L, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating Fpr2, the low-affinity N-formylpeptide receptor

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC(50) approximately 400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC(50) values similar to those found for Fpr2-expressing cell lines ( approximately 500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.     

Estimation of peak vertical velocity and relative load changes by subjective measures in weightlifting movements

To investigate the ability of the OMNI-RES (0–10) scale to estimate velocity and loading changes during sets to failure in the hang power clean (HPC) exercise. Eleven recreationally resistance-trained males (28.5 ± 3.5 years) with an average one-repetition maximum (1RM) value of 1.1 ± 0.07 kg body mass^-1 in HPC, were assessed on five separate days with 48 hours of rest between sessions. After determining the 1RM value, participants performed four sets to self-determined failure with the following relative loading ranges: 60% < 70%, 70 < 80%, 80 < 90% and > 90%. The peak vertical velocity (PVV), and Rating of Perceived Exertion (RPE) were measured for every repetition of each set. The RPE expressed after the first repetition (RPE-1) and when the highest value of PVV was achieved during the set (RPE-max) were similar and significantly lower than the RPE associated with a 5% (RPE-5%) and 10% (RPE-10%) drop in PVV. In addition, the RPE produced at failure was similar to RPE-5% only for the heaviest range (≥ 90%). Furthermore, RPE-1 was useful to distinguish loading zones between the four assessed ranges (60 < 70%, vs. 70 < 80%, vs. 80 < 90%, vs. ≥ 90%). The RPE seems to be useful to identify PVV changes (maximal, 5% and 10% drop) during continuous sets to self-determined failure and to distinguish 10% loading zone increments, from 60 to 100% of 1RM in the HPC exercise.     

Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP

Neuronal presynaptic terminals contain hundreds of neurotransmitter‐filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well‐known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5‐dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release.     

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

We investigated the regulatory effects of GRK2 on D₂ dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D₂ receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D₂ receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D₂ receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγ binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.     

A maximal isokinetic pedalling exercise for EMG normalization in cycling

An isometric maximal voluntary contraction (iMVC) is mostly used for the purpose of EMG normalization, a procedure described in the scientific literature in order to compare muscle activity among different muscles and subjects. However, the use of iMVC has certain limitations. The aims of the present study were therefore to propose a new method for the purpose of EMG amplitude normalization in cycling and assess its reliability. Twenty-three cyclists performed 10 trials of a maximal isokinetic protocol (MIP) on a cycle ergometer, then another four sub-maximal trials, whilst the EMG activity of four lower limbs muscles was registered. During the 10 trials power output (CV = 2.19) and EMG activity (CV between 4.46 and 8.70) were quite steady. Furthermore, their maximal values were reached within the 4th trial. In sub-maximal protocol EMG activity exhibited an increase as a function of exercise intensity.MIP entails a maximal dynamic contraction of the muscles involved in the pedalling action and the normalization session is performed under the same biomechanical conditions as the following test session. Thus, it is highly cycling-specific.MIP has good logical validity and within-subject reproducibility. Three trials are enough for the purpose of EMG normalization in cycling.     

Quantitative Trait Loci Controlling Vegetative Growth Rate in the Edible Basidiomycete Pleurotus ostreatus

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.     

Dopamine D2 receptors form higher order oligomers at physiological expression levels

G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.