Endogenous phosphorylation of distinct gamma-aminobutyric acid type A receptor polypeptides by Ser/Thr and Tyr kinase activities associated with the purified receptor.
We have investigated the phosphorylation of gamma-aminobutyric acid type A (GABAA) receptor purified from bovine cerebral cortex in the absence of added kinases. Incubation of the affinity-purified receptor with [gamma-32P]ATP and 500 microM MnCl2 yielded incorporation of 0.45 mol of 32P/mol of muscimol binding sites within 2 h at 30 degrees C. Mn2+ was much more effective than Mg2+ as activator. Phosphorylation of the receptor was observed on at least three different polypeptides of 51, 53, and 55 kDa. It was predominant on 51- and 53-kDa polypeptides that co-migrate with the [3H]flunitrazepam photoaffinity-labeled bands, suggesting that 32P incorporation mainly occurs on alpha-subunits. A monoclonal antibody specific for alpha-subunits adsorbed the endogenously phosphorylated GABAA receptor with a stoichiometry close to 1 mol of phosphate/mol of muscimol. The phosphorylation of the 51-kDa polypeptide, corresponding to alpha 1-subunit, exhibited a micromolar affinity for ATP and sigmoid kinetics (nH = 2). Major incorporation of phosphate occurred on serine and threonine residues in roughly equimolar ratio. By enzyme-linked immunosorbent assay and immunoblotting studies we also detected a minor incorporation on tyrosine residues; this was specific for a 55-kDa polypeptide. Comparison with molecular data suggests that at least alpha 1- and alpha 2-subunits (Ser and Thr residues) and possibly gamma 2-subunits (Tyr residue) are endogenously phosphorylated by multiple kinases, with a clear preference for alpha 1-subunit. The beta-subunits were not phosphorylated in our experimental conditions. The corresponding kinase activities are closely associated to the receptor protein, indicating a new complexity in the regulation of the GABAA receptor.
Bureau
M H
MH
Laboratory of Neurochemistry, University of Liège, Belgium.
Laschet
J J
JJ
D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009118:Muscimol / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D019000:Phosphotyrosine / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011963:Receptors, GABA-A; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D009118:Muscimol; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases