CHARACTERIZATION OF THE LIGAND-BINDING DOMAINS OF GLUTAMATE-RECEPTOR (GLUR)-B AND GLUR-D SUBUNITS EXPRESSED IN ESCHERICHIA-COLI AS PERIPLASMIC PROTEINS

We recently reported that a functional ligand-binding site of an alpha -amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)-selective glut amate receptor (GluR)-D subunit can be expressed in insect cells as a soluble, N-glycosylated fusion protein consisting of two segments (S1 and S2) that are related by amino acid sequence to bacterial periplasm ic binding proteins (Kuusinen, A., Arvola, M., and Keinanen, K., EMBO J. 14, 6327-6332). In an attempt to further characterize the structura l determinants for ligand binding, we have now expressed the ligand-bi nding sites of GluR-B and GluR-D subunits in Escherichia coli as solub le periplasmic proteins. The bacterially expressed S1-S2 fusion protei ns bound [H-3]AMPA with a high affinity (K-d of 12 nM for GluR-B, K-d of 60 nM for GluR-D) and with a ligand pharmacology typical of native AMPA receptors, indicating that N-linked glycosylation is not required for the formation or the maintenance of the ligand-binding site. The flip and Bop splice variants of the GluR-D S1-S2 fusion protein bound [H-3]AMPA with equal affinities, whereas deletion of the C-terminal on e-third of the S2 segment including the flip/flop sequence resulted in a loss of binding activity, Our results highlight the potential of ba cterial expression for the analysis of the binding site and support a close structural similarity between glutamate receptors and bacterial proteins.