A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function.

The high affinity transporters for the monoamine neurotransmitters, dopamine, norepinephrine, and serotonin, play a key role in controlling monoaminergic neurotransmission. It is believed that the transporters (DAT, NET and SERT, respectively) are subject to tight regulation by the cellular signaling machinery to maintain monoaminergic homeostasis. Kinases constitute a pivotal role in cellular signaling, however, the regulation of monoamine transporters by the entire ensemble of kinases is unknown. Here, we perform a whole human kinome RNA interference screen to identify novel kinases involved in regulation of monoamine transporter function and surface expression. A primary screen in HEK 293 cells stably expressing DAT or SERT with siRNAs against 573 human kinases revealed 93 kinases putatively regulating transporter function. All 93 hits, which also included kinases previously implicated in monoamine transporter regulation, such as Protein kinase B (Akt) and mitogen-activated protein kinases (MAPK), were validated with a new set of siRNAs in a secondary screen. In this screen we assessed both changes in uptake and surface expression leading to selection of 11 kinases for further evaluation in HEK 293 cells transiently expressing DAT, SERT or NET. Subsequently, three kinases; salt inducible kinase 3 (SIK3), cAMP-dependent protein kinase catalytic subunit alpha (PKA C-α) and protein kinase X-linked (PrKX); were selected for additional exploration in catecholaminergic CATH.a differentiated cells (CAD) and rat chromocytoma (PC12) cells. Whereas SIK3 likely transcriptionally regulated expression of the three transfected transporters, depletion of PKA C-α was shown to decrease SERT function. Depletion of PrKX caused decreased surface expression and function of DAT without changing protein levels, suggesting that PrKX stabilizes the transporter at the cell surface. Summarized, our data provide novel insight into kinome regulation of the monoamine transporters and identifies PrKX as a yet unappreciated possible regulator of monoamine transporter function.

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini.

The norepinephrine transporter (NET) mediates reuptake of synaptically released norepinephrine in central and peripheral noradrenergic neurons. The molecular processes governing availability of NET in the plasma membrane are poorly understood. Here we use the fluorescent cocaine analogue JHC 1-64, as well as several other approaches, to investigate the trafficking itinerary of NET in live noradrenergic neurons. Confocal imaging revealed extensive constitutive internalization of JHC 1-64-labeled NET in the neuronal somata, proximal extensions and presynaptic boutons. Phorbol 12-myristate 13-acetate increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the "long loop" recycling marker Rab11, whereas less overlap was seen with the "short loop" recycling marker Rab4 and the late endosomal marker Rab7. Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements in the intracellular N terminus. We conclude that NET displays a distinct trafficking itinerary characterized by continuous shuffling between the plasma membrane and the Rab11 recycling compartment and that the functional integrity of the Rab11 compartment is critical for maintaining proper presynaptic NET function.

Quantitative determination of α(2B)-adrenoceptor-evoked myosin light chain phosphorylation in vascular smooth muscle cells.

INTRODUCTION: Phosphorylation of myosin light chains is a biochemical readout of smooth muscle cell contraction. α2-Adrenoceptor agonists and antagonists may have important applications in cardiovascular drug development. To assess α2-adrenoceptor-mediated drug effects on vascular smooth muscle contraction, we developed a cell-based assay for the quantitative determination of myosin light chain phosphorylation (pMLC20) in cultured A7r5 smooth muscle cells from rat aorta, transfected to express the human α2B-adrenoceptor (A7r5-α2B cell line). METHODS: In a 96-well format, confluent and serum-starved cells (+/- inhibitor preincubation) were treated with receptor ligands for 5-120 s and the evoked pMLC20 response was monitored with a quantitative in-cell immunoassay, employing time-resolved fluorescence technology. Western blotting, immunofluorescent labelling and intracellular calcium concentration measurements were used for assay validation. RESULTS: The α2-adrenoceptor agonist dexmedetomidine induced rapid, transient and dose-dependent (EC50 30-65 nM) myosin light chain phosphorylation, peaking at 20-45 s with an Emax value of approximately 60% over vehicle control. The endogenous agonist arginine vasopressin produced responses that were comparable to those evoked by dexmedetomidine. Blockers of α2-adrenoceptors, myosin light chain kinase, Gi-proteins, Gßγ subunits, L-type calcium channels and phospholipase C antagonized the dexmedetomidine-evoked myosin light chain phosphorylation, whereas blockers of protein kinase C and protein kinase A potentiated the response to dexmedetomidine. DISCUSSION: The novel method is suitable as a ligand profiling tool to assess the capacity of ligands to evoke or inhibit vascular smooth muscle cell contraction and for investigating the intracellular pathways involved in this process. The assay now allows the quantitative determination of pMLC20 signal induction or inhibition in vascular smooth muscle cells and is superior to conventional Western blotting due to the reduced number of cells required and the potential for measurement of detailed time curves, multiple treatments and replicates on each plate.

Octopamine receptors from the barnacle balanus improvisus are activated by the alpha2-adrenoceptor agonist medetomidine.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The alpha(2)-adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOctalpha) belongs to the alpha-adrenoceptor-like subfamily, and the other four (BiOctbeta-R1, BiOctbeta-R2, BiOctbeta-R3, and BiOctbeta-R4) belong to the beta-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of beta-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.