Journal: Open Biology

Article Title: Structure of astrotactin-2: a conserved vertebrate-specific and perforin-like membrane protein involved in neuronal development

doi: 10.1098/rsob.160053

Figure Lengend Snippet: Inositol phosphate binding by ASTN-2. ( a ) Close-up of the inositol triphosphate modelled into extra density, one phosphate in electrostatic interaction with arginine 1124, a position occupied by a threonine in ASTN-1 and the inositol ring π-stacked with tryptophan 1259. ( b ) Surface charge representation of the inositol triphosphate binding pocket. ( c ) Wild-type (WT) ASTN-2 and an R1124T mutant interacting with immobilized inositol diphosphates, as measured by surface plasmon resonance (SPR). ( d ) Wild-type ASTN-2 and an R1124T mutant binding to immobilized inositol triphosphate, again measured by SPR. ( e ) A competition assay for the binding of ASTN-2 to the triphosphorylated inositide alone and with competition from Ins(1,3,4,5)P 4 (IP4) and mannose-6-phosphate (M6P) in solution. The affinity of ASTN-2 for the immobilized Ins(3,4,5)P 3 is much less affected by the presence of mannose-6-phosphate in solution than by the presence of free Ins(1,3,4,5)P 4 .

Article Snippet: The SPR experiments were performed using a Biacore T200 machine (GE Healthcare Life Sciences) at 20°C in 10 mM HEPES, pH 7.5, 150 mM NaCl, PtdIns(3,5)P 2 (C-35B6a), PtdIns(4,5)P 2 (C-45B6a), PtdIns(3,4,5)P 3 (C-39B6a) and Ins(1,3,4,5)P 4 (Q-1345) were purchased from Echelon Biosciences.

Techniques: Binding Assay, Mutagenesis, SPR Assay, Competitive Binding Assay

Journal: The Journal of Neuroscience

Article Title: Activation of Group III Metabotropic Glutamate Receptors Attenuates Rotenone Toxicity on Dopaminergic Neurons through a Microtubule-Dependent Mechanism

doi: 10.1523/JNEUROSCI.0118-06.2006

Figure Lengend Snippet: mGluRIII-induced ERK activation is mediated by Src, β-arrestin 2, and dynamin. A, Midbrain neuronal cultures were treated with the selective Src kinase inhibitors PP1 and PP2 or their inactive homolog PP3 (all at 20 μm) for 30 min before and during l-AP-4 treatment (100 μm for 3 min). Total cell lysates were immunoblotted with anti-phospho-ERK1/2. l-AP-4-induced ERK activation was blocked by Src inhibitors, but not their inactive homolog. B, C, Total cell lysates from midbrain neuronal cultures treated with l-AP-4 were blotted with anti-phospho-Src (top) or anti-Src (bottom). l-AP-4 induced rapid and transient activation of Src in the same time (B) and dose (C) courses as those of ERK1/2. D, Midbrain neuronal cultures were transfected with siRNA of β-arrestin 1 or β-arrestin 2 and treated without or with l-AP-4 (100 μm for 3 min). Total cell lysates were blotted with antibodies against β-arrestin 1, β-arrestin 2, phospho-ERK1/2 or phospho-Src, respectively. Knockdown of β-arrestin 2 but not β-arrestin 1 blocked l-AP-4-induced activation of ERK and Src. Similar results were obtained from four independent experiments. E, Midbrain neuronal cultures were treated without or with l-AP-4 (100 μm for 3 min). Myristoylated dynamin inhibitory peptide P4 or unmodified P4 (both at 25 μm) was added 30 min before and during the l-AP-4 treatment. Total cell lysates were blotted with antibodies against phospho-ERK1/2. l-AP-4-induced ERK activation was blocked by myristoylated dynamin inhibitory peptide, but not by the unmodified P4 peptide, which is membrane impermeable. Similar results were obtained from at least three independent experiments.

Article Snippet: 4-Amino-1- tert -butyl-3-(1′-naphthyl)pyrazolo[3,4- d ]pyrimidine (PP1), 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4- d ]pyrimidine (PP2), and 4-amino-7-phenylpyrazol[3,4- d ]pyrimidine (PP3) were purchased from Calbiochem (La Jolla, CA). l -(+)-2-Amino-4-phosphonobutyric ( l -AP-4), O -phospho- l -serine ( l -SOP), ( RS )-α-cyclopropyl-4-phosphonophenylglycine (CPPG), ( RS )-α-methylserine- O -phosphate (MSOP), trans- azetidine-2,4-dicarboxylic acid (tADA), ( RS )-3,5-dihydroxyphenylglycine (DHPG), (2 R ,4 R )-4-aminopyrrolidine-2,4-dicarboxylate (APDC), (2 S ,1′ S ,2′ S )-2-(carboxycyclopropyl)glycine (LCCG), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), (2 S )-α-ethylglutamic acid (EGLU), ( RS )-1-amino-5-phosphonoindan-1-carboxylic acid (APICA), NMDA, AMPA, Gln-Val-Pro-Ser-Arg-Pro-Asn-Arg-Ala-Pro (dynamin inhibitory peptide, P4), and myristoylated P4 (myr-P4) were obtained from Tocris (Ellisville, MO).

Techniques: Activation Assay, Transfection, Membrane