Identification of phosphatidic acid phosphatase type 2 (PAP-2) as an androgen-regulated gene in the human prostatic adenocarcinoma cell line LNCaP : W. Ulrix, J. Swinnen, W. Heyns, and G. Verhoeven. Laboratory for Experimental Medicine and Endocrinology, Faculty of Medicine, Onderwijs en Navorsing, Gasthuisberg, Catholic University of Leuven, Leuven, Belgium

Abstract

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)Nature342, 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 ofsolubilized α-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 α-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 α-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-tenninal 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 GluRl 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. Immunoreactivity was found in cell bodies and processes of neurons and also in tanicytes, a specialized glial cell type of the hypothalamus.The CA1 region of the hippocampus was particularly rich in GluR1 subunit. Electron-microscopic examination of the CA1 area revealed that immunoreactivity for the GluR1 subunit was present within the endoplasmic reticulum of neurons, at a majority of type I synapses established on dendritic spines, and also at extrasynaptic sites along the dendritic membrane of the pyramidal cells. Immunoreactivity obtained with the antiserum to the N-terminal part (253–267) detected epitopes at the extracellular face of plasma membrane, whereas the antiserum raised against the C-terminal part of the GluR1 subunit (877–889) recognized epitopes at the intracellular face of postsynaptic membranes.The results demonstrate that the antisera selectively react with GluR1 subunit(s) of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor. This subunit is distributed in an area and cell type specific manner in the forebrain and is present at synapses assumed to use excitatory amino acids as transmitters. Immunocytochemistry at the subcellular level suggests that the C-terminus of the subunit is located at the cytoplasmic face of the synaptic membrane contrary to the currently held model.