gamma-Aminobutyric acid-A (GABAA) agonists down-regulate GABAA/benzodiazepine receptor polypeptides from the surface of chick cortical neurons.
The impermeant SH-cleavable reagent, 125I-labeled 3,3'-dithiopropionyl 1-sulfosuccinimidyl 1'-glycyltyrosine (DPSgt) and specific immunoprecipitation were used to quantitate gamma-aminobutyric acid-A (GABAA) receptor polypeptides with domains exposed on the surface of chick cortical neurons. The 125I label incorporated into 50- and 53-kDa receptor subunits was completely removed by washing the cells with glutathione (GSH) buffer. A single dose of 100 microns GABA or GABA agonist was added to the neurons, and after 5 days in culture the cells were washed and labeled with 125I-DPSgt. This treatment with GABA or isoguvacine reduced the level of the receptor 125I-labeled subunits by 50-60%, while the reduction by 4,5,6,7-tetrahydroisoazolo[5,4-c]pyridin-3-ol was less than 17%. The subunit down-regulation by agonist was prevented by the GABAA antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R5135). Direct iodination of membranes isolated from treated cells revealed a similar loss of subunits, indicating that the down-regulated polypeptides are not retained intracellularly. Furthermore, the level of intracellular [3H]flunitrazepam binding (per mg of protein) did not change significantly during chronic GABA treatment, while the fraction of intracellular binding rose from 7% to 15% of the total, owing to a decline in surface binding. The fate of the surface subunits during acute agonist exposure was examined by labeling intact neurons with 125I-DPSgt at 0 degrees C, incubating with GABA for 2-4 h at 37 degrees C and then washing with GSH buffer. Of the 50-53-kDa receptor peptides originally on the surface, 16 +/- 2% became protected from GSH during the 2-h GABA treatment. This sequestration was not found in cells incubated without GABA, with GABA + R5135, or with GABA at 0 degree C. A consistently lower level of sequestered subunits was recovered after a 4- versus 2-h GABA treatment at 37 degrees C, suggesting polypeptide degradation. Overall, the results indicate that GABAA receptor sequestration and subsequent degradation play an important role in agonist-dependent down-regulation.
Calkin
P A
PA
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Barnes
E M
EM
D000731:Androstanes / Q000494:pharmacology; D000818:Animals; D001378:Azasteroids / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D002642:Chick Embryo; D015536:Down-Regulation / Q000187:drug effects*; D066298:In Vitro Techniques; D007539:Isonicotinic Acids / Q000494:pharmacology; D007555:Isoxazoles / Q000494:pharmacology; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D000731:Androstanes; D001378:Azasteroids; D007539:Isonicotinic Acids; D007555:Isoxazoles; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; C015542:gaboxadol; C032061:RU 5135; C015300:isoguvacine