Identification and characterization of proteins interacting with metabotropic glutamate receptor subtype 8

Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. To elucidate the function and modulation of mGluR8, yeast two-hybrid screens of an adult rat brain cDNA library were performed with the CTDs of mGluR8a and 8b (mGluR8-C) as baits. Different components of the sumoylation cascade (ube2a, sumo-1, Pias1, Pias gamma and Pias xbeta) and some other proteins were identified as mGluR8 interacting proteins. Binding assays using recombinant GST-fusion proteins confirmed that Pias1 interacts not only with mGluR8-C, but all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminally to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine K882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo-1 and enzymes of the sumoylation cascade into HEK 293 cells showed that mGluR8a-C can be sumoylated in cells. Arginine substitution of lysine K882 within the consensus sumoylation motif, but not of other conserved lysines within the CTD, abolished in vivo sumoylation. The results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.Die presynaptischen metabotropen Glutamat-Rezeptoren der Gruppe III (mGluRs) spielen eine zentrale Rolle in der Regulation presynaptischer Aktivität über G-Protein-Effekte auf Ionenkanäle und signalübertragende Enzyme. Wie alle G-Protein-gekoppelten Rezeptoren der Klasse C hat auch mGluR8 eine verlängerte intrazelluläre C-terminale Domäne (CTD), die vermutlich die Modulation nachgeordneter Signale erlaubt. In einem Hefe-Zwei-Hybrid-Screen einer cDNA-Bibliothek aus adultem Rattenhirn, in welchem CTDs vom mGluR8a und 8b (mGluR8-C) als “Köder” verwendet wurden, konnten neben anderen Proteinen verschiedene Komponenten der Sumoylierungskaskade (ube2a, sumo-1, Pias1, Pias gamma, Pias xbeta) als Interaktionspartner identifizieret werden. Bindungsexperimente mit rekombinanten GST-Fusionsproteinen bestätigten, daß Pias1 nicht nur mit mGluR8-C, sondern mit allen Gruppe III mGluR CTDs interagiert. Die Pias1-Bindung an mGluR8-C benötigt eine N-terminal des Sumoylierungs-Konsensusmotivs liegende Region und wird nicht beeinträchtigt durch Arginin-Austausch des innerhalb dieser Region liegenden, konservierten Lysin-Restes K882. Kotransfektionsexperimente mit fluoreszenzmarkiertem mGluR8a-C, Sumo1 und Enzymen der Sumoylierungskaskade in HEK293-Zellen zeigten, daß mGluR8a-C in vivo sumoyliert werden kann. Der Arginin-Austausch des Lysin-Restes K882, jedoch nicht Austausche von anderen konservierten Lysin-Resten innerhalb der CTD-Domäne, verhinderte die in vivo Sumoylierung. Unsere Ergebnisse deuten darauf hin, daß die posttranslationale Sumoylierung einen neuen Mechanismus der Gluppe III mGluR Regulation darstellt

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

β1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected α5β1- and αVβ3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on β1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the αVβ3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of α5β1-integrin, while reducing surface expression of αVβ3-integrin. Mechanistically, Brag2-mediated αVβ3-integrin-recycling and β1-integrin endocytosis and specifically of the active/matrix-bound α5β1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via β1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, β1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating β1-integrin internalization and link for the first time the process of β1-integrin endocytosis with angiogenesis.Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre. (SFB/TR23)Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre. (Project A2)Deutsche Forschungsgemeinschaft. Transregional Collaborative Research Centre.(Project Z5)Else Kroner-Fresenius-Stiftung (2013_A2

Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3β phosphorylation

Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3β (GSK3β) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine–induced Parkinson’s dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3β phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC–PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions