Regional distribution and developmental changes of GluR1-flop protein revealed by monoclonal antibody in rat brain

From immunizations of mice with a glutathione S-transferase fusion protein containing residues 724-781 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit GluR1-flop, two monoclonal antibodies (mAbs) were developed that differed widely in their ranges of specificity. In immunocytochemical and immunoblotting assays performed on COS-7 cells transfected with one of the eight GluR1-4-flip/flop forms, mAb 19B10 recognized all eight forms, whereas mAb 8E11 was specific for GluR1-flop. By means of synthetic peptides, the epitopes were determined to be NKWWYDKG (GluR1-flop760-767) for mAb 19B10 but GSALRNPVN (GluR1-flop740-748) plus a partial epitope, QGLL (GluR1-flop757-760), for mAb 8E11. Further analysis on synthetic peptides pointed to a potential cross-reactivity of mAb 8E11 with GluR2-4-flop variants that lacked editing at the R/G site. The contribution of such cross-reactivities in histoblot labeling patterns on adult rat brain material, however, was judged to be negligible. Histoblot patterns with mAb 8E11 were dominated by strong immunoreactivity in CA1 strata radiatum and oriens and in the dentate molecular layer, whereas the CA3 region was virtually free of labeling. This pattern and those observed at different stages of postnatal development were generally similar to regional distribution patterns previously reported in the literature for GluR1-flop transcripts. Key Words: Glutamate receptors-AMPA receptor subunits-Alternative splicing-Developmentally regulated expression-R/G site.