Sequence dependence and differential expression of Ggamma5 subunit isoforms of the heterotrimeric G proteins variably processed after prenylation in mammalian cells

Between 1 and 2% of proteins coded for in the human genome, including all G protein gamma subunits, are predicted to be prenylated. Subsequently, prenylated proteins are proteolytically cleaved at the C terminus and carboxymethylated. These reactions are generally obligatory events required for functional expression of prenylated proteins. The biological role of prenyl substrates has made these reactions significant targets for anticancer drug development. Understanding the enzymology of this pathway will be key to success for this strategy. When Ggamma1, -2, -4, -10, -11, -12, and -13 were expressed in HEK293 cells they were completely processed according to the current understanding of the prenylation reaction. In contrast, Ggamma5 was processed to two forms; a minor one, fully processed as predicted, and a major one that was prenylated without further processing. When the Ca(1)a(2)X motif of Ggamma5, CSFL, was exchanged for that of Ggamma2, CAIL, Ggamma5 was completely processed. Conversely, Ggamma2-SFL was incompletely processed. Differential processing of Ggamma5 was found due to the presence of an aromatic amino acid in its Ca(1)a(2)X motif. Retrieving endogenous Ggamma subunits from HEK293 or Neuro-2a cells with FLAG-Gbeta constructs identified multiple Ggamma subunits by mass spectrometry in either cell, but in both cases the most prominent one was Ggamma5 expressed without C-terminal processing after prenylation. This work indicates that post-prenylation reactions can generate multiple products determined by the C-terminal Ca(1)a(2)X motif. Within the human genome 10% of predicted prenylated proteins have aromatic amino acids in their Ca(1)a(2)X sequence and would likely generate the prenylation pattern described here.