The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1

In the absence of Cbk1 phosphorylation Ssd1-associated mRNAs are redirected from sites of polarized cell growth to stress granules and P-bodies

Differential Modulation of Beta-Adrenergic Receptor Signaling by Trace Amine-Associated Receptor 1 Agonists

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes

Functional characterization of amino acids that could be involved in zinc(II)-binding.

<p>For cell surface expression studies COS-7 cells were transiently transfected with the empty expression vector pcDps (mock), <i>Gpr83</i> wild type or <i>Gpr83</i> mutants. HEK293 cells were used for functional characterization. Data were evaluated from three or four independent experiments, each performed at least in triplicates. IP₃ accumulation performed as reporter gene assay was calculated fold over the basal mock transfection with 24718.3 ± 3958.7 relative light units, set to 1. The hTSHR stimulated with 100 mU/ml bTSH functioned as control for Gq/11 activation [data not shown, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-VanSande1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Allgeier1" target="_blank">[32]</a>]. Shown data represent mean ± SEM. The mutated aminoacid residues are grouped into extracellular located Ds (Asp) and És (Glu), Hs (His) and one C (Cys) that could be involved in Zn(II)-binding. Asteriks indicate significant higher basal activity in comparison to wild type. ** p<0.01, *** p<0.001 (unpaired t-test, two-tailed); Ntt – N-terminal tail.</p

Structural mGPR83 homology model with sensitive positions for constitutive receptor activation and zinc(II)-stimulation.

<p>This structual GPR83 homology model is based on the crystal structure of rhodopsin in the inactive state. Highlighted wild type amino acids were depicted from this model for experimental approaches, because they are located extracellularly at the ECLs or at the extracellular ends of the TMHs and those amino acids are known as putative determinants of metal-ion binding motifs: histidine, glutamate, asparagine or cysteine. Arranged in defined spatial arrangements they can interact e.g. with zinc(II)-ions. Interestingly, six side-chain substitutions abolished stimulation by zinc(II) (green sticks) and five substitutions at different positions expressed an increase in constitutive signaling activity of mGPR83 (red sticks). The are spatially clustered in two different regions – red cycle (CAMs) and green (zinc(II)-binding) full cycle. In summary, they are indicating the extracellular region of the mGPR83 as highly sensitive for activation. Cysteine 304 (blue stick) at TMH6 is one of the highly conserved family A GPCR residues and it was reported for several receptors that mutations here are leading almost always to constitutive receptor activation. Indeed, also the mGPR83 Cys304Trp mutation causes a slight ligand independent (constitutive) activation of Gq/11 mediated signaling pathways.</p

Concentration-response curve of zinc(II)- stimulation at mGPR83.

<p>HEK293 cells were transiently transfected with the empty expression vector pcDps (mock) or pcDps encoding the wild type <i>Gpr83</i>. Two days after transfection, stimulation with 1 nM – 1 mM ZnCl₂ was carried out, cells were lysed and IP₃-accumulation was measured in a reporter gene assay. The hTSHR stimulated with 100 mU/ml bTSH functioned as assay control [data not shown, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-VanSande1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Allgeier1" target="_blank">[32]</a>. The EC₅₀ value of the wild type mGPR83 is 10.2 ± 1.4 µM zinc(II) and was obtained from the concentration-response curve (1 nM – 1 mM Zn(II)) using GraphPad Prism. First asterisk indicates a significant increase in IP₃ formation in comparison to basal wild type. Second asterisks indicate significance in comparison to the first asterisk. Data were evaluated from a minimum of 3 independent experiments, each performed at least in triplicates and calculated fold over the mock transfection, with 24718.3 ± 3958.7 relative light units, set to 1. Shown data represent mean ± SEM. * p<0.05, ** p<0.01 (unpaired t-test, two-tailed).</p

mGPR83 signals via the Gq/11 pathway revealed by basal signaling activity, CAMs and zinc(II)-stimulation.

<p>HEK293 cells were transiently transfected with the empty expression vector pcDps (mock) or pcDps carrying the wild type <i>Gpr83</i> or the <i>Gpr83 C304W</i> mutant, respectively. Two days after transfection, stimulation with ZnCl₂ (1 nM – 1 mM; stimulation curve in Fig. 2; black column: 100 µM) was carried out and cells were lysed. IP₃-accumulation was measured in a reporter gene assay. The hTSHR stimulated with 100 mU/ml bTSH functions as assay control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-VanSande1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Allgeier1" target="_blank">[32]</a>. Data were assessed from a minimum of 3 independent experiments, each performed at least in triplicates and represent mean ± SEM calculated fold over basal mock transfection with 19775.4 ± 2259.9 relative light units, set to 1. *** p<0.001 (unpaired t-test, two-tailed).</p

Homodimerization of mGPR83.

<p>Dimerization studies were performed using sandwich ELISA. COS-7 cells were transiently transfected. As negative control serves the NHA-hGHSR (white column) and as positive control the co-transfection of NHA-tagged hMC3R and FLAG-tagged hGHSR (black column, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053347#pone.0053347-Rediger1" target="_blank">[28]</a>). The light grey column represents the average of the HA- respectively FLAG-tagged mGPR83 in combination with the correspondent tagged rM3R. The dark grey column represents co-transfection of HA-tagged mGPR83 and FLAG-tagged mGPR83. Dimerization was measured via the HA epitope. The mean absorption (492 nm/620 nm) is calculated per 1 mg/ml of protein and shown as percentage of the hMC3R/hGHSR heterodimer (absorption (492/620)/ 1mg/ml of protein: 0.3 ± 0.04). Data were assessed from 3 independent experiments, each performed in triplicates and represent mean ± SEM.</p

The orphan receptor Gpr83 regulates systemic energy metabolism via ghrelin-dependent and ghrelin-independent mechanisms

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways