Conserved phosphorylation of the intracellular domains of GABA(A) receptor beta 2 and beta 3 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 beta 2 subunit and Serine 409 of the beta 3 subunit, each of which Lies within the conserved sequence R-R-R-X-S-L-Q-K, where X = A (beta 1, beta 2 and beta 4) or S (beta 3). We have analysed the phosphorylation of the beta 2 and beta 3 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 beta 2 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 beta 2 subunit phosphorylated by these four protein kinases. Using similar methodologies, Serine 409 of the beta 3 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 GABAA receptors via multiple protein kinases. (C) 1997 Published by Elsevier Science Ltd.