Antibodies to the human gamma 2 subunit of the gamma-aminobutyric acidA/benzodiazepine receptor.
A gamma-aminobutyric acidA (GABAA) receptor (GABAAR) gamma 2 subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage lambda gt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors lambda gt11 and pGEX-3X. Both beta-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the gamma 2IL were purified, and a rabbit antibody to the LacZ-gamma 2IL was made. The antibody reacted with the gamma 2IL of both LacZ and GST fusion proteins and immunoprecipitated the GABAAR/benzodiazepine receptor (GABAAR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABAAR/BZDR immunoblots with a wide peptide band of 44,000-49,000 M(r). Immunoprecipitation studies with the anti-gamma 2IL antibody suggest that in the cerebral cortex, 87% of the GABAARs with high affinity for benzodiazepines and 70% of the GABAARs with high affinity for muscimol contain at least a gamma subunit, probably a gamma 2. These results indicate that there are [3H]muscimol binding GABAARs that do not bind [3H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABAAR/BZDR antibodies indicate that the most abundant combination of GABAAR subunits in the cerebral cortex involves alpha 1, gamma 2 (or other gamma), and beta 2 and/or beta 3 subunits. These subunits coexist in > 60% of the GABAAR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20-25%) of the cerebellar GABAAR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain alpha 6, also contain gamma 2 (or other gamma) subunit(s). The alpha 1 and beta 2 or beta 3 subunits are also frequently associated with gamma 2 (or other gamma) and alpha 6 in these cerebellar receptors.
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Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499.
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Escribá
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Mallet
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Miralles
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D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D011233:Precipitin Tests; D011963:Receptors, GABA-A / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D007106:Immune Sera; D015345:Oligonucleotide Probes; D011963:Receptors, GABA-A