Conserved phosphorylation of the intracellular domains of GABA(A) receptor beta2 and beta3 subunits by cAMP-dependent protein kinase, cGMP-dependent protein kinase protein kinase C and Ca2+/calmodulin type II-dependent protein kinase.
All mammalian GABA(A) receptor beta subunits contain a conserved consensus site for phosphorylation by a number of serine/threonine protein kinases. This site corresponds to Serine 410 of the beta2 subunit and Serine 409 of the beta3 subunit, each of which lies within the conserved sequence R-R-R-X-S-L-Q-K, where X = A (beta1, beta2 and beta4) or S (beta3). We have analysed the phosphorylation of the beta2 and beta3 subunits of the murine GABA(A) receptor by expressing the large intracellular domains of these subunits as soluble fusion proteins in E. coli. The intracellular domain of the beta2 subunit was phosphorylated to high stoichiometry by both cAMP- and cGMP-dependent protein kinases, protein kinase C and Ca2+/calmodulin type II-dependent protein kinase in vitro. Site-directed mutagenesis identified Serine 410 as the single site within the beta2 subunit phosphorylated by these four protein kinases. Using similar methodologies, Serine 409 of the beta3 subunit was shown to be a substrate for phosphorylation by these protein kinases. Serine 408 was also seen to be phosphorylated by protein kinase C and Serine 383 was phosphorylated by Ca2+/calmodulin type II-dependent protein kinase. Since beta subunits are believed to be essential for robust GABA(A) receptor expression, these results suggest a critical role for conserved phosphorylated amino acids within the beta subunits in coordinating cellular regulation of GABA(A) receptors via multiple protein kinases.
McDonald
B J
BJ
Department of Pharmacology, University College London, UK.
Moss
S J
SJ
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D051379:Mice; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011963:Receptors, GABA-A; D011494:Protein Kinases; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases