Binding of EphrinA5 to RET receptor tyrosine kinase : An in vitro study

Eph/Ephrin signaling pathways are crucial in regulating a large variety of physiological processes during development, such as cell morphology, proliferation, migration and axonal guidance. EphrinA (efn-A) ligands, in particular, can be activated by EphA receptors at cellcell interfaces and have been proposed to cause reverse signaling via RET receptor tyrosine kinase. Such association has been reported to mediate spinal motor axon navigation, but conservation of the interactive signaling pathway and the molecular mechanism of the interaction are unclear. Here, we found Danio rerio efn-A5b bound to Mus musculus EphA4 with high affinity, revealing structurally and functionally conserved EphA/efn-A signaling. Interestingly, we observed no interaction between efn-A5b and RET from zebrafish, unlike earlier cell-based assays. Their lack of association indicates how complex efn-A signaling is and suggests that there may be other molecules involved in efn-A5-induced RET signaling.Peer reviewe

Zebrafish GDNF and its co-receptor GFR alpha 1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro

Glial cell line-derived neurotrophic factor ( GDNF) is a ligand that activates, through coreceptor GDNF family receptor alpha-1 (GFR alpha 1) and receptor tyrosine kinase "RET ", several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRa1 and GDNF, and Drosophila melanogaster RET and two isoforms of coreceptor GDNF receptor-like. Our results report high-level insect cell expression of posttranslationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRa1 and GDNF are comparably active as mammalian cell- produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFR alpha 1-GDNF binding RET is 5.9 mu M. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRa1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRa1 and GDNF cannot bind and activate non-mammalian RET and vice versa.Peer reviewe

Overview Of Data Over Digital Subscrieber Line In Czech Republic.

Import 22/07/2015Tato bakalářská práce je zaměřena na rozbor technologie ADSL a to, jak je využívána a nabízena vybranými poskytovateli v České republice k užívání veřejným sektorem. V teoretické části pojednává o technologii ADSL a jejích vývojových verzích. Dále se zabývá řešením přípojky a vlivů rušení na vedení těchto přípojek. Hlavní částí práce je část praktická, ta se zabývá průzkumem poskytovatelů v České republice. Konkrétně využitím digitálních účastnických smyček poskytovateli, jejich nabídkou ADSL služeb pevného připojení k internetu a také doplňkových služeb a poplatků s pevným připojením spojených. Poslední částí práce je analýza možností připojení těchto služeb na konkrétní přípojku. Obsah této práce má napomoci běžným uživatelům k pochopení dané problematiky spojené se zřízením a výběrem ADSL služeb. Prostředky k řešení praktické části práce jsou především komunikace s poskytovateli ADSL služeb a získávání informací.This bachelor thesis is focused on the analysis of the technology ADSL as it is used in the public sector and offered by chosen providers in Czech Republic. In the theoretical part, it refers to the technology ADSL and its developmental versions. Furthermore, it examines the connection solution and the influence of interruption on conducting these connections. The main part of the thesis is the practical part which undertakes the research of providers in Czech Republic. Namely, it explores use of digital subscriber loops by providers, theirs ADSL service offers of the stable internet connection and its complementary services and charges. The final part of the thesis is the connection analysis of these complementary services on the specific connection. The content of this thesis should provide help to common users to understand given issues related to the selection and setting up ADSL services. Tools used for the practical part are particularly communication with ADSL service providers and acquiring appropriate information.440 - Katedra telekomunikační technikyvýborn

Pull-down assays of zefn-A5^ECD, mEphA4^ECD, mEphA4^LBD and zRET^ECD.

<p>(A) Anti-Flag resin pull down of mEphA4^LBD and untagged zRET^ECD with immobilized zefn-A5^ECD. The black arrows in Lanes 3 and 8 point to the mEphA4^LBD pulled down by zefn-A5^ECD. (B) Protein G bead pull down of zRET^ECD and zefn-A5^ECD with immobilized mEphA4^LBD. The black arrows in Lanes 1 and 4 mark the zefn-A5^ECD pulled-down by mEphA4. Molecular weight standards: PageRuler Plus standard protein ladder in Lanes 1(A) and 2(B). PD: samples eluted from the beads after pull down. Lanes without the PD label contain input protein samples as indicated.</p

Coomassie-stained Native PAGE of zGDNF/zGFRα1^ECD/zRET^ECD and mEphA4^LBD/zefn-A5^ECD complexes.

<p>(A) BN PAGE image of zGDNF/zGFRα1^ECD/zRET^ECD complex. A molar ratio of 1:1:1 based on the monomeric forms of the proteins was used for zGDNF, zGFRα1^ECD and zRET^ECD. zRET^ECD and zefn-A5^ECD were incubated with a molar ratio of 1:2. The formation of zGDNF₂/zGFRα1₂^ECD complex was shown in Lane 3 (black arrow). The band corresponding to the ternary zGDNF₂/zGFRα1^ECD/zRET^ECD complex is shown in Lanes 4 and 5 (black arrows). Lanes 4 and 5 are duplicates. The band highlighted with solid rectangle (Lane 4) was analyzed using mass spectrometry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.s004" target="_blank">S1 Table</a>). (B) Clear Native (CN) PAGE of the mEphA4^LBD/zefn-A5^ECD complex. mEphA4^LBD/zefn-A5^ECD were incubated with a molar ratio of 1:2. The formation of mEphA4^LBD/zefn-A5^ECD complex is marked with a black arrow. (C) Anti-hlgG1-Fc Western blotting showing the mEphA4^LBD/zefn-A5^ECD complex formation in Lane 4 (black arrow).</p

Schematic model of RET signaling.

<p>EphrinAs, GFRα1, GDNF and RET may interact together with Celsr3/Fzd3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref014" target="_blank">14</a>] to transduce efn-As reverse signaling. β integrins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref009" target="_blank">9</a>] as well as leucine-rich repeat and immunoglobulin (LRIG) family proteins, such as LRIG1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref048" target="_blank">48</a>] and Linx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref049" target="_blank">49</a>], may play a role in RET-mediated efn-As signaling. NCAM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref050" target="_blank">50</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref051" target="_blank">51</a>]: neural cell adhesion molecule; LRIG1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref048" target="_blank">48</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198291#pone.0198291.ref052" target="_blank">52</a>]: leucine-rich repeats and immunoglobulin-like domains; TrkB: tropomyosin receptor kinase B.</p

Measurement of the affinity of zefn-A5^ECD for mEphA4^LBD and zRET^ECD.

<p>(A) BLItz sensorgrams showing mEphA4^LBD binding to immobilized zefn-A5^ECD at the indicated concentrations. (B) Saturation binding curve fitted with 1:1 binding model, using GraphPad Prism 6, showing the binding of mEphA4^LBD to immobilized zefn-A5^ECD. The binding model allowed non-specific binding, which occurs as shown by the fact that the baseline is not horizontal. The x-axis represents the concentration of mEphA4^LBD. (C) Sensorgram showing untagged zRET^ECD binding to immobilized zefn-A5^ECD at a concentration of 110 μM. The binding profile was derived from three independent experiments and plotted with mean value against running time. (D) Sensorgram showing untagged zefn-A5^ECD binding to immobilized zRET^ECD at two concentrations of 104 and 160 μM.</p

Zebrafish GDNF alone and with zGFRα1 potently activated the human RET MAPK signaling.

<p>(A) luciferase activity readouts comparing different concentrations of zGDNF, using hGDNF^BV, hGDNF^Ec and hGDNF^IC as positive controls. Yellow bar: hGDNF^Ec, green bar: hGDNF^BV; red bar: hGDNF^IC; black bar: zGDNF. (B) luciferase activity readouts comparing zGDNF and zGFRα1 activation at different concentrations on the MG87 cell-line expressing RET only. Media, zGDNF and His-tagged zGFRα1 individually were used as negative controls. N = 3–4 repeats per experiment; statistically different from control <i>*p</i>< 0.01; ** <i>p</i><0.0001. Error bars indicate SEM.</p

zGDNF supports the survival of cultured mammalian dopaminergic neurons.

<p>(A) the counted number of embryonic dopaminergic neurons in the presence of zGDNF and hGDNF^IC versus negative control (media), (B) immunofluorescent staining of midbrain culture on 5DIV with a marker for DA neurons: TH (red) and nuclear dye DAPI (blue) comparing 0.27 nM of hGDNF^IC, zGDNF and no GFL (control). Black bars: zGDNF; red bars: hGDNF^IC; white bar: negative control. N = 4 indiviaul experiments per condition; statistically different from control ***p< 0.0007, **p< 0.003, *p<0.03.</p

Biolayer inferometry (BLItz) of zebrafish proteins.

<p>Scatchard plot showing the binding of zRET^ecd and zGDNF to pre-incubated zGFRα1 using BLItz. The x-axis shows the concentration of zGDNF and zRET^ecd. The dashed line indicates the nonlinear curve fit plotted by GraphPad Prism 6 for single-site binding.</p