Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the β₁, γ₂S, and γ₂L subunits of the γ-aminobutyric acid type A receptor

γ-Aminobutyric acid Type A (GABA(A)) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABA(A) receptor subunits divided into five classes, α, β, γ, δ, and ρ, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABA(A) receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP- dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the β₁ subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the β₁ subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the β₁ subunit to an alanine residue eliminated the phosphorylation of the β₁ subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the γ₂S and γ₂L subunits of the GABA(A) receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the γ₂S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the γ₂S subunit fusion protein to an alanine residue eliminated the phosphorylation of the γ₂S fusion protein by PKC. The γ₂L subunit is an alternatively spliced form of the γ₂S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the γ₂S subunit. The PKC phosphorylation of the γ₂L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the γ₂L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the α₁ subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABA(A) receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABA(A) receptor function. In addition, these studies suggest that alternative splicing of the γ₂ subunit may provide a novel mechanism for the differential regulation of GABA(A) receptors by PKC.