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 Glu R1 glutamate receptor subunit artificial stop codons have been introdu ced into the N-terminal domain at amino acid positions 442, 510 and 56 3, namely just before and spanning the proposed first two transmembran e regions. The resultant truncated N-terminal fragments of GluR1, term ed NTI, NT2 and NT3 respectively were expressed in Cos-7 cells and the ir cellular distribution and cell-surface expression analysed using an N-terminal antibody to GluR1. All the fragments were fully glycosylat ed and were found to be associated with cell membranes but none was se creted. Differential extraction of the cell membranes indicated that b oth NT1 and NT2 behave as peripheral membrane proteins. In contrast NT 3, like the full subunit, has integral membrane protein properties. Fu rthermore only NT3 is expressed at the cell surface as determined by i mmunofluorescence and cell-surface biotinylation. Protease protection assays indicated that only NT3 had a cytoplasmic tail. Binding studies using the selective ligand pha-amino-3-hydroxy-5-methyl-4-isoxazolepr opionate ([H-3]AMPA) demonstrated that NT3 does not bind ligand. Toget her these results indicate that the first transmembrane domain of the GluR1 subunit lies between residues 509 and 562, that the N-terminal d omain alone cannot form a functional ligand-binding site and that this domain can be targeted to the cell surface provided that it has a tra nsmembrane-spanning region.