Characterization, cell-surface expression and ligand-binding properties of different truncated N-terminal extracellular domains of the ionotropic glutamate receptor subunit GluR1.
To identify the location of the first transmembrane segment of the GluR1 glutamate receptor subunit artificial stop codons have been introduced into the N-terminal domain at amino acid positions 442, 510, and 563, namely just before and spanning the proposed first two transmembrane regions. The resultant truncated N-terminal fragments of GluR1, termed NT1, NT2, and NT3 respectively were expressed in Cos-7 cells and their cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All of the fragments were fully glycosylated and were found to be associated with cell membranes but none was secreted. Differential extraction of the cell membranes indicated that both NT1 and NT2 behave as peripheral membrane proteins. In contrast NT3, like the full subunit, has integral membrane protein properties. Furthermore only NT3 is expressed at the cell surface as determined by immunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand [(3)H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ([(3)H]AMPA) demonstrated that NT3 does not bind ligand. Together these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal domain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a transmembrane-spanning region.
McIlhinney
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RA
Medical Research Council, Anatomical Neuropharmacology Unit, University of Oxford, Oxford, U.K.
Molnár
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E
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003062:Codon; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D017470:Receptors, Glutamate / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D003062:Codon; D008024:Ligands; D046911:Macromolecular Substances; D008565:Membrane Proteins; D017470:Receptors, Glutamate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid