The most abundant inhibitory neurotransmitter in the central nervous system, gamma-aminobutyric acid (GABA), exerts its main effects via a GABAA receptor that gates a chloride channel in the subsynaptic membrane. These receptors can contain a modulatory unit, the benzodiazepine receptor, through which ligands of different chemical classes can increase or decrease GABAA receptor function. We have now visualized a GABAA receptor in mammalian brain using monoclonal antibodies. The protein complex recognized by the antibodies contained high- and low-affinity binding sites for GABA as well as binding sites for benzodiazepines, indicative of a GABAA receptor functionally associated with benzodiazepine receptors. As the pattern of brain immunoreactivity corresponds to the autoradiographical distribution of benzodiazepine binding sites, most benzodiazepine receptors seem to be part of GABAA receptors. Two constituent proteins were identified immunologically. Because the monoclonal antibodies cross-react with human brain, they provide a means for elucidating those CNS disorders which may be linked to a dysfunction of a GABAA receptor.

No abstract

Monoclonal antibodies (mAb) against a yaminobuyric acid/benzodiazepine receptor complex (GABAA/ BZR) were produced by using spleen cells from a mouse immunized with GABAA/BZR purified from bovine cerebral cortex. The mAb, most of which were of the IgG1 isotype could be divided into four groups (I-IV) specifying different antigenic structures. On immunoblots, group I mAb recognized exdusively the Mr 55,000 (3-subunit, while groups II and IV mAb recognized the Mr 50,000 a-subunit of bovine GABAA/BZR.Three of the four groups of mAb (I, m, and IV) crossreacted with both human and rat GABAA/BZR with the same subunit specificity as in bovine brain; the fourth group (II) crossreacted with human but not with the rat receptor. The binding sites for benzodiazepines as well as the high and low affinity GABA sites reside on the same structural complex as shown by immunoprecipitation. Ligand binding to these sites was not inhibited by mAb. Since quantitative immunoprecipitation of GABAA/BZR was achieved with mAb selective for either the a-or (3-subunit, both subunits occur in each individual receptor complex. The pattern of immunoblot staining suggests that the smaller a-subunit is not a processing product of the larger (3-subunit. Both a-and (3-subunits were present in all brain areas and species tested (rat cerebral cortex, cerebellum, and hippocampus; bovine cerebral cortex and cerebellum; human cerebral cortex). This suggests a uniform subunit composition of the receptor throughout the brain in contrast to earlier evidence for a heterogeneous subunit composition based on photoaffmnity labeling.

A monoclonal antibody directed against eukaryotic mRNA 5'-cap-binding protein (anti-CBP antibody) was used to localize cap-binding protein (CBP) in BHK-21 baby hamster kidney cells by immunofluorescence microscopy. It was found that the antibody reacts with a fibrous network extending through the cytoplasm in a radial arrangement. The network behaves like intermediate filaments in colchicine-treated cells, suggesting a direct or indirect linkage of CBP with intermediate filaments. The association of CBP with a cytoskeletal element was further confirmed by isolation of proteins from Triton X-100-extracted cells and identification of CBP in the cytoskeletal fraction with anti-CBP antibody. The major polypeptide reacting with anti-CBP antibody is a Mr 50,000 component. Tryptic peptide mapping showed that this polypeptide is related to a Mr 24,000 polypeptide identified as CBP in earlier experiments [Sonenberg, N., Morgan, M.

No abstract