title: Overexpression and functional characterization of the extracellular domain of the human alpha1 glycine receptor.
abstract: A novel truncated form (residues 1-214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human alpha1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized, and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus, GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homologue of the ECD of GlyR.
authors: Center for Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors: Zhenyu
authors: Z
authors: Liu
authors: Gomathi
authors: G
authors: Ramanoudjame
authors: Deqian
authors: D
authors: Liu
authors: Robert O
authors: RO
authors: Fox
authors: Vasanthi
authors: V
authors: Jayaraman
authors: Maria
authors: M
authors: Kurnikova
authors: Michael
authors: M
authors: Cascio
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D018009:Receptors, Glycine