DIFFERENTIAL PHOSPHORYLATION OF INTRACELLULAR DOMAINS OF GAMMA-AMINOBUTYRIC-ACID TYPE-A RECEPTOR SUBUNITS BY CALCIUM CALMODULIN TYPE 2-DEPENDENT PROTEIN-KINASE AND CGMP-DEPENDENT PROTEIN-KINASE/

gamma-Aminobutyric acid type A receptor subunits (GABA(A)) can be divi ded into five classes, alpha, beta, gamma, delta, and rho, based on se quence homology. We have used purified fusion proteins of the major in tracellular domain of GABA(A) receptor subunits produced in Escherichi a coli to examine the phosphorylation of these subunits by cGMP-depend ent protein kinase (PKG) and multifunctional calcium/calmodulin-depend ent protein kinase (CAM KII). Both PKG and CAM KII phosphorylated a pu rified beta(1) subunit fusion. Both of these kinases phosphorylated se rine 409 within the beta(1) subunit; in addition, CAM KII also phospho rylated serine 384 as determined by site-specific mutagenesis. Fusion proteins of the major intracellular domains of the gamma(2)S and gamma (2)L subunits were produced. These proteins differ by 8 amino acids (L LRMFSFK). Both the gamma(2)L and gamma(2)S fusion proteins were excell ent substrates of CAM KII. However, the gamma(2)L fusion protein was p hosphorylated to higher stoichiometry due to the phosphorylation of se rine 343 within this 8 amino acid insertion. Both the gamma(2)L and ga mma(2)S subunits were phosphorylated on common residues by CAM KII ide ntified as serine 348 and threonine 350. These results identify specif ic sites of phosphorylation for CAM KII and PKG within GABA(A) recepto r subunits, suggesting a role for these two kinases in modulating GABA (A) receptor function in vivo.