Posttranslational modification of Gαo1 generates Gαo3, an abundant G protein in brain

G alpha(o), the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., G alpha(o1) and G alpha(o2), derived by alternative splicing of the mRNA. A third G alpha(o1)-related isoform, G alpha(o3), has been purified, representing about 30% of total G(o) in brain. Initial studies revealed distinct biochemical properties of G alpha(o3) as compared with other G alpha(o) isoforms, In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated G alpha(o1) and G alpha(o3), C-terminal peptides showed a difference of +1 Da for G alpha(o3). Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of G alpha(o3). Gel electrophoretic analysis of recombinant G alpha(o3) showed the same mobility as native G alpha(o3) but distinct to G alpha(o1). The conversion of (346)Asn-->Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5'-[gamma-thio] triphosphate binding. No cDNA coding for G alpha(o3) was detected, suggesting an enzymatic deamidation of G alpha(o1) by a yet-unidentified activity, Therefore, G alpha heterogeneity is generated not only at the DNA or RNA levels, but also at the protein level. The relative amount of G alpha(o1) and G alpha(o3) differed from cell type to cell type, indicating an additional principle of G protein regulation.