Posttranslational modification of Gα(o1) generates Gα(o3), an abundant G protein in brain

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