Endogenous phosphorylation of the GABA(A) receptor protein is counteracted by a membrane-associated phosphatase.
Incubation of bovine brain membranes with [gamma-33P]ATP phosphorylated mainly a 51-kDa band. Electrophoretic co-migration was observed for 33P- and [3H]flunitrazepam-labeled bands in both membrane fractions and in affinity-purified GABA(A) receptor (GABAA-R) preparations. An alpha-subunit monoclonal antibody adsorbed most of the radiolabeled-band, suggesting that the labeled-membrane polypeptide corresponds to the GABA(A)-R alpha1-subunit, which is the only GABA(A)-R subunit with a molecular weight of 51 kDa. The phosphorylation rate was much faster in membranes than in purified receptor. Dephosphorylation was detected in membranes only. The membrane-bound phosphatase was potently inhibited by vanadate and Zn2+>>Mn2+ , but was insensitive to okadaic acid (a phosphatase 1, 2 and 2B inhibitor), cyclosporin (specific calcineurin inhibitor) and phosphatase-1 inhibitor. Endogenous kinase was activated by divalent cations including calcium (Mg2- > Mn2+ > Ca2+), whilst dephosphorylation did not require the presence of Ca2+ ions. This suggests that at least one membrane-bound phosphatase counteracts the endogenous phosphorylation of the GABA(A)-R: the lack of dephosphorylation in the purified receptor preparation indicates that, in contrast to the endogenous kinase, no phosphatase is closely associated with the receptor protein complex.
Minier
F
F
Laboratory of Neurosciences, University of Rennes-1, France. laboneur@univ-rennes1.fr
Laschet
J J
JJ
Evrard
B
B
Bureau
M H
MH
D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D002417:Cattle; D005260:Female; D008565:Membrane Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D054645:Protein Phosphatase 1; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000378:metabolism*
D008565:Membrane Proteins; D011963:Receptors, GABA-A; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1