Conformational transition of FGFR kinase activation revealed by site-­specific unnatural amino acid reporter and single molecule FRET

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo

Excitatory amino acidergic pathways and receptors in the basal ganglia

The striatum receives the majority of excitatory amino acidergic input to the basal ganglia from neocortical and allocortical sources. The subthalamic nucleus and the substantia nigra also receive excitatory amino acidergic inputs from neocortex. The subthalamic nucleus, which has prominent projections to the pallidum and nigra, is the only known intrinsic excitatory amino acidergic component of the basal ganglia. Possible excitatory amino acidergic inputs reach the basal ganglia from the intralaminar thalamic nuclei and the pedunculo-pontine nucleus. The striatum is richly endowed with all subtypes of excitatory amino acid receptors and these appear to be inhomogeneously distributed within the striatal complex. The non-striatal nuclei contain lesser levels of excitatory amino acid receptors and the relative proportion of these receptors varies between nuclei. The presence of high densities of excitatory amino acid receptors is a phylogenetically conserved feature of the striatum and its non-mammalian homologues. In Huntington's disease, there is substantial depletion of α -amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-D-aspartate, and kainate receptors within the striatum. In Parkinson's disease substantia nigra, there is significant loss of N-methyl-D-aspartate and α -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41734/1/726_2004_Article_BF00814003.pd

Modulation of the acoustic startle response by the level of arousal: comparison of clonidine and modafinil in healthy volunteers

A sudden loud sound evokes an electromyographic (EMG) response from the orbicularis oculi muscle in humans together with an auditory evoked potential (AEP) and an increase in skin conductance (SC). Startle responses are inhibited by weak prepulses (prepulse inhibition, (PPI)) and may also be modified by the level of alertness. We compared the sedative drug clonidine and the alerting drug modafinil on sound-evoked EMG, AEP, and SC responses, on the PPI of these responses and on level of arousal and autonomic functions. Sixteen healthy male volunteers participated in four weekly sessions (clonidine 0.2 mg, modafinil 400 mg, their combination, placebo) in a double-blind, cross-over, balanced design. Responses were evoked by sound pulses of 115 and 85 dB (PPI) for 40 ms and recorded conventionally. Level of alertness, autonomic functions (pupil diameter, blood pressure, heart rate, salivation, temperature) and the plasma levels of the hormones prolactin, thyroid-stimulating hormone and growth hormone were also measured. Data were analyzed with analysis of variance with multiple comparisons. Both prepulses and clonidine attenuated all three startle responses and modafinil antagonized clonidine's effects on the EMG and AEP responses. None of the drugs affected PPI. Clonidine showed sedative and sympatholytic effects, and modafinil showed alerting and sympathomimetic effects. In conclusion, startle responses were susceptible not only to PPI but also to the level of arousal