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 can be divided into a conserved two-lobed ligand-binding domain ("S1S2") and an N-terminal approximately 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 alpha-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.
Kuusinen
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A
Viikki Biocenter, Department of Biosciences, Division of Biochemistry, P. O. Box 56, FIN-00014 University of Helsinki, Finland.
Abele
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R
Madden
D R
DR
Keinänen
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K
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D018690:Excitatory Amino Acid Agonists / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D018091:Receptors, AMPA / Q000737:chemistry*; D017470:Receptors, Glutamate / Q000737:chemistry*; D011994:Recombinant Proteins; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D018690:Excitatory Amino Acid Agonists; D010446:Peptide Fragments; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D011994:Recombinant Proteins; C109433:glutamate receptor type D; D018698:Glutamic Acid; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D005976:Glutaral