Glycine, Glutamate, Thienylcyclohexylpiperidine Binding Protein (GlyBP)

GlyBP, a mutant form of the ligand binding domain of the GlyR, in which two hydrophobic loops were replaced with corresponding hydrophilic residues in AChBP. GlyBP was overexpressed in Sf9 insect cells. GlyBP was found in both cytosolic and membrane-bound fractions after subcellular fractionation. The cytosolic fraction was misfolded. In contrast, the membrane-bound form is functional as shown by its ability to reversibly bind to 2-aminostrychnine resin. After affinity purification, membrane-bound GlyBP could be isolated in an aqueous form and a membrane-associated vesicular form. Radiolabeled binding assays showed both forms of GlyBP retained abilities to bind to its ligands, with affinities comparable to those of full-length GlyR. Both forms of GlyBP are oligomeric, and are very likely pentameric.