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 glutamate 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 periplasmic binding proteins (Kuusinen, A., Arvola, M., and Keinänen, K., EMBO J. 14, 6327-6332). In an attempt to further characterize the structural determinants for ligand binding, we have now expressed the ligand-binding sites of GluR-B and GluR-D subunits in Escherichia coli as soluble periplasmic proteins. The bacterially expressed S1-S2 fusion proteins bound [3H]AMPA with a high affinity (Kd of 12 nM for GluR-B, Kd 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 flop splice variants of the GluR-D S1-S2 fusion protein bound [3H]AMPA with equal affinities, whereas deletion of the C-terminal one-third of the S2 segment including the flip/flop sequence resulted in a loss of binding activity. Our results highlight the potential of bacterial expression for the analysis of the binding site and support a close structural similarity between glutamate receptors and bacterial proteins.
Arvola
M
M
VTT Biotechnology and Food Research, P. O. Box 1500 Biologinkuja 1, FIN-02044 VTT, Espoo, Finland.
Keinänen
K
K
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D003854:Deoxyribonucleotides / Q000737:chemistry; D004926:Escherichia coli; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018091:Receptors, AMPA / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D003854:Deoxyribonucleotides; D008024:Ligands; D018091:Receptors, AMPA; D011993:Recombinant Fusion Proteins