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. 1990 Jun 1;110(6):2043–2048. doi: 10.1083/jcb.110.6.2043

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

PMCID: PMC2116146  PMID: 1693621

Abstract

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma- aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.

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Selected References

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  1. Adelman J. P., Hayflick J. S., Vasser M., Seeburg P. H. In vitro deletional mutagenesis for bacterial production of the 20,000-dalton form of human pituitary growth hormone. DNA. 1983;2(3):183–193. doi: 10.1089/dna.1983.2.183. [DOI] [PubMed] [Google Scholar]
  2. Becker C. M., Hoch W., Betz H. Glycine receptor heterogeneity in rat spinal cord during postnatal development. EMBO J. 1988 Dec 1;7(12):3717–3726. doi: 10.1002/j.1460-2075.1988.tb03255.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bureau M., Olsen R. W. gamma-Aminobutyric acid/benzodiazepine receptor protein carries binding sites for both ligands on both two major peptide subunits. Biochem Biophys Res Commun. 1988 Jun 30;153(3):1006–1011. doi: 10.1016/s0006-291x(88)81328-7. [DOI] [PubMed] [Google Scholar]
  4. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fuchs K., Möhler H., Sieghart W. Various proteins from rat brain, specifically and irreversibly labeled by [3H]flunitrazepam, are distinct alpha-subunits of the GABA-benzodiazepine receptor complex. Neurosci Lett. 1988 Aug 1;90(3):314–319. doi: 10.1016/0304-3940(88)90208-x. [DOI] [PubMed] [Google Scholar]
  6. Gorman C. M., Gies D., McCray G., Huang M. The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology. 1989 Aug;171(2):377–385. doi: 10.1016/0042-6822(89)90605-3. [DOI] [PubMed] [Google Scholar]
  7. Grenningloh G., Rienitz A., Schmitt B., Methfessel C., Zensen M., Beyreuther K., Gundelfinger E. D., Betz H. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature. 1987 Jul 16;328(6127):215–220. doi: 10.1038/328215a0. [DOI] [PubMed] [Google Scholar]
  8. Houser C. R., Olsen R. W., Richards J. G., Möhler H. Immunohistochemical localization of benzodiazepine/GABAA receptors in the human hippocampal formation. J Neurosci. 1988 Apr;8(4):1370–1383. doi: 10.1523/JNEUROSCI.08-04-01370.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hurtley S. M., Helenius A. Protein oligomerization in the endoplasmic reticulum. Annu Rev Cell Biol. 1989;5:277–307. doi: 10.1146/annurev.cb.05.110189.001425. [DOI] [PubMed] [Google Scholar]
  10. Häring P., Stähli C., Schoch P., Takács B., Staehelin T., Möhler H. Monoclonal antibodies reveal structural homogeneity of gamma-aminobutyric acid/benzodiazepine receptors in different brain areas. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4837–4841. doi: 10.1073/pnas.82.14.4837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khrestchatisky M., MacLennan A. J., Chiang M. Y., Xu W. T., Jackson M. B., Brecha N., Sternini C., Olsen R. W., Tobin A. J. A novel alpha subunit in rat brain GABAA receptors. Neuron. 1989 Dec;3(6):745–753. doi: 10.1016/0896-6273(89)90243-2. [DOI] [PubMed] [Google Scholar]
  12. Levitan E. S., Schofield P. R., Burt D. R., Rhee L. M., Wisden W., Köhler M., Fujita N., Rodriguez H. F., Stephenson A., Darlison M. G. Structural and functional basis for GABAA receptor heterogeneity. Nature. 1988 Sep 1;335(6185):76–79. doi: 10.1038/335076a0. [DOI] [PubMed] [Google Scholar]
  13. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Möhler H., Battersby M. K., Richards J. G. Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1666–1670. doi: 10.1073/pnas.77.3.1666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pfeiffer F., Simler R., Grenningloh G., Betz H. Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7224–7227. doi: 10.1073/pnas.81.22.7224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Pritchett D. B., Lüddens H., Seeburg P. H. Type I and type II GABAA-benzodiazepine receptors produced in transfected cells. Science. 1989 Sep 22;245(4924):1389–1392. doi: 10.1126/science.2551039. [DOI] [PubMed] [Google Scholar]
  17. Pritchett D. B., Sontheimer H., Gorman C. M., Kettenmann H., Seeburg P. H., Schofield P. R. Transient expression shows ligand gating and allosteric potentiation of GABAA receptor subunits. Science. 1988 Dec 2;242(4883):1306–1308. doi: 10.1126/science.2848320. [DOI] [PubMed] [Google Scholar]
  18. Pritchett D. B., Sontheimer H., Shivers B. D., Ymer S., Kettenmann H., Schofield P. R., Seeburg P. H. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature. 1989 Apr 13;338(6216):582–585. doi: 10.1038/338582a0. [DOI] [PubMed] [Google Scholar]
  19. Richards J. G., Schoch P., Häring P., Takacs B., Möhler H. Resolving GABAA/benzodiazepine receptors: cellular and subcellular localization in the CNS with monoclonal antibodies. J Neurosci. 1987 Jun;7(6):1866–1886. doi: 10.1523/JNEUROSCI.07-06-01866.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schoch P., Häring P., Takacs B., Stähli C., Möhler H. A GABA/benzodiazepine receptor complex from bovine brain: purification, reconstitution and immunological characterization. J Recept Res. 1984;4(1-6):189–200. doi: 10.3109/10799898409042549. [DOI] [PubMed] [Google Scholar]
  22. Schoch P., Richards J. G., Häring P., Takacs B., Stähli C., Staehelin T., Haefely W., Möhler H. Co-localization of GABA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies. Nature. 1985 Mar 14;314(6007):168–171. doi: 10.1038/314168a0. [DOI] [PubMed] [Google Scholar]
  23. Schofield P. R., Darlison M. G., Fujita N., Burt D. R., Stephenson F. A., Rodriguez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A. Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature. 1987 Jul 16;328(6127):221–227. doi: 10.1038/328221a0. [DOI] [PubMed] [Google Scholar]
  24. Schofield P. R., Pritchett D. B., Sontheimer H., Kettenmann H., Seeburg P. H. Sequence and expression of human GABAA receptor alpha 1 and beta 1 subunits. FEBS Lett. 1989 Feb 27;244(2):361–364. doi: 10.1016/0014-5793(89)80563-0. [DOI] [PubMed] [Google Scholar]
  25. Shivers B. D., Killisch I., Sprengel R., Sontheimer H., Köhler M., Schofield P. R., Seeburg P. H. Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron. 1989 Sep;3(3):327–337. doi: 10.1016/0896-6273(89)90257-2. [DOI] [PubMed] [Google Scholar]
  26. Sieghart W., Eichinger A., Richards J. G., Möhler H. Photoaffinity labeling of benzodiazepine receptor proteins with the partial inverse agonist [3H]Ro 15-4513: a biochemical and autoradiographic study. J Neurochem. 1987 Jan;48(1):46–52. doi: 10.1111/j.1471-4159.1987.tb13125.x. [DOI] [PubMed] [Google Scholar]
  27. Sigel E., Stephenson F. A., Mamalaki C., Barnard E. A. A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex. J Biol Chem. 1983 Jun 10;258(11):6965–6971. [PubMed] [Google Scholar]
  28. Somogyi P., Takagi H., Richards J. G., Mohler H. Subcellular localization of benzodiazepine/GABAA receptors in the cerebellum of rat, cat, and monkey using monoclonal antibodies. J Neurosci. 1989 Jun;9(6):2197–2209. doi: 10.1523/JNEUROSCI.09-06-02197.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stephenson F. A., Casalotti S. O., Mamalaki C., Barnard E. A. Antibodies recognising the GABAA/benzodiazepine receptor including its regulatory sites. J Neurochem. 1986 Mar;46(3):854–861. doi: 10.1111/j.1471-4159.1986.tb13050.x. [DOI] [PubMed] [Google Scholar]
  30. Sweetnam P., Nestler E., Gallombardo P., Brown S., Duman R., Bracha H. S., Tallman J. Comparison of the molecular structure of GABA/benzodiazepine receptors purified from rat and human cerebellum. Brain Res. 1987 Sep;388(3):223–233. doi: 10.1016/0169-328x(87)90029-5. [DOI] [PubMed] [Google Scholar]
  31. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Unwin N. The structure of ion channels in membranes of excitable cells. Neuron. 1989 Dec;3(6):665–676. doi: 10.1016/0896-6273(89)90235-3. [DOI] [PubMed] [Google Scholar]
  33. Ymer S., Draguhn A., Köhler M., Schofield P. R., Seeburg P. H. Sequence and expression of a novel GABAA receptor alpha subunit. FEBS Lett. 1989 Nov 20;258(1):119–122. doi: 10.1016/0014-5793(89)81630-8. [DOI] [PubMed] [Google Scholar]
  34. Ymer S., Schofield P. R., Draguhn A., Werner P., Köhler M., Seeburg P. H. GABAA receptor beta subunit heterogeneity: functional expression of cloned cDNAs. EMBO J. 1989 Jun;8(6):1665–1670. doi: 10.1002/j.1460-2075.1989.tb03557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. de Blas A. L., Vitorica J., Friedrich P. Localization of the GABAA receptor in the rat brain with a monoclonal antibody to the 57,000 Mr peptide of the GABAA receptor/benzodiazepine receptor/Cl- channel complex. J Neurosci. 1988 Feb;8(2):602–614. doi: 10.1523/JNEUROSCI.08-02-00602.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

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