Oligomerization and ligand-binding properties of the ectodomain of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluRD

The extracellular part of ionotropic glutamate receptor (iGluR) subunits ca n be divided into a conserved two-lobed ligand-binding domain ("S1S2") and an N-terminal similar to 400-residue segment of unknown function ("X domain ") which shows high sequence variation among subunits. To investigate the s tructure and properties of the N-terminal domain, we have now produced affi nity-tagged recombinant fragments which represent the X domain of the GluRD subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA )-selective glutamate receptors either alone or covalently linked to the li gand-binding domain ("XS1S2"). These fragments were expressed in insect cel ls as secreted soluble proteins and were recognized by a conformation-speci fic anti-GluRD monoclonal antibody. A hydrodynamic analysis of the purified fragments revealed them to be dimers, in contrast to the S1S2 ligand-bindi ng domain which is monomeric. The X domain did not bind radiolabeled AMPA o r glutamate nor did its presence affect the ligand binding properties of th e 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.