Biochemical and immunocytochemical characterization of antipeptide antibodies to a cloned GluR1 glutamate receptor subunit: cellular and subcellular distribution in the rat forebrain.

Antibodies were made to synthetic peptides corresponding to residues 253-367, 757-771 and 877-889 of the published amino acid sequence of the rat brain glutamate receptor GluR1 subunit [Hollmann et al. (1989) Nature 342, 643-648]. The peptides were synthesized both as multiple copies on a branching lysyl matrix (multiple antigenic peptides) and conventional linear peptides using solid-phase synthesis. Rabbits were immunized with these peptides either without conjugation (multiple antigenic peptides) or following coupling to ovalbumin with glutaraldehyde (monomeric peptides). The antibodies from immune sera were then purified by affinity chromatography using reactigel coupled monomeric peptides. All the rabbits produced good antipeptide responses, and were characterized by immunoprecipitation of solubilized alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainate binding activity and by their staining patterns on immunoblots. Antibody to peptide 253-267 specifically immunoprecipitated 12 +/- 3, 50 +/- 3 and 44 +/- 4% of solubilized alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding activity from cortex, hippocampus and cerebellum, respectively. Under identical conditions, antibody against the 877-889 peptide removed 23 +/- 4, 9 +/- 4 and 15 +/- 9% of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites from these areas. On immunoblots of rat brain membrane samples separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, antibodies labelled a 105,000 mol. wt immunoreactive band. GluR1 was immunoaffinity-purified using subunit-specific antibodies against both N-terminal (253-267) and C-terminal (877-889) residues, covalently attached to protein A-agarose. Analysis of the purified product from each column showed a major immunoreactive band, recognized by both sera at 105,000 mol. wt and silver staining identified the same major protein. After exhaustive immunoprecipitation of solubilized membrane samples with antibody against the C-terminal of the subunit, a subpopulation of GluR1 was labelled with antibodies specific for the N-terminal part of the receptor. These observations suggest that the GluR1 subunit consists of at least two isoforms possessing a common N-terminal region but a distinct C-terminus. Immunocytochemistry, using immunoperoxidase staining, was performed for the GluR1 subunit in rat forebrain with antisera raised against the N-terminal (253-267) and the C-terminal parts (877-889) of the molecule. Both antisera gave a similar distribution of immunoreactivity at the light-microscopic level. Immunoreactivity for the GluR1 subunit was selectively distributed throughout the rat forebrain. The hippocampus, septum, amygdala and olfactory bulb exhibited the strongest immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)