Oligomerization and Ligand-binding Properties of the Ectodomain of the α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunit GluRD

• Published in: The Journal of biological chemistry Vol. 274; no. 41; pp. 28937 - 28943
• Main Authors: Kuusinen, Arja, Abele, Rupert, Madden, Dean R., Keinänen, Kari
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.10.1999
• Subjects: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid - metabolism; Animals; Antibodies, Monoclonal; Binding Sites; Centrifugation, Density Gradient; Chromatography, Gel; Cross-Linking Reagents - chemistry; Dimerization; Excitatory Amino Acid Agonists - metabolism; Glutamic Acid - metabolism; Glutaral - chemistry; Kinetics; Peptide Fragments - chemistry; Peptide Fragments - immunology; Precipitin Tests; Protein Binding; Protein Conformation; Rats; Receptors, AMPA - chemistry; Receptors, Glutamate - chemistry; Recombinant Proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.41.28937

The extracellular part of ionotropic glutamate receptor (iGluR) subunits can be divided into a conserved two-lobed ligand-binding domain (“S1S2”) and an N-terminal ∼400-residue segment of unknown function (“X domain”) which shows high sequence variation among subunits. To investigate the structure and properties of the N-terminal domain, we have now produced affinity-tagged recombinant fragments which represent the X domain of the GluRD subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptors either alone or covalently linked to the ligand-binding domain (“XS1S2”). These fragments were expressed in insect cells as secreted soluble proteins and were recognized by a conformation-specific anti-GluRD monoclonal antibody. A hydrodynamic analysis of the purified fragments revealed them to be dimers, in contrast to the S1S2 ligand-binding domain which is monomeric. The X domain did not bind radiolabeled AMPA or glutamate nor did its presence affect the ligand binding properties of the S1S2 domain. Our findings demonstrate that the N-terminal domain of AMPA receptor can be expressed as a soluble polypeptide and suggest that subunit interactions in iGluR may involve the extracellular domains.