Sequence dependence and differential expression of Gγ5 subunit isoforms of the heterotrimeric G proteins variably processed after prenylation in mammalian cells

Abstract

Between 1 and 2% of proteins coded for in the human genome, including all G protein γ 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 anti-cancer drug development. Understanding the enzymology of this pathway will be key to success for this strategy. When Gγ1, -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, Gγ5 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 1a2X motif of Gγ5, CSFL, was exchanged for that of Gγ2, CAIL, Gγ5 was completely processed. Conversely, Gγ2-SFL was incompletely processed. Differential processing of Gγ5 was found due to the presence of an aromatic amino acid in its Ca1a2X motif. Retrieving endogenous Gγ subunits from HEK293 or Neuro-2a cells with FLAG-Gβ constructs identified multiple Gγ subunits by mass spectrometry in either cell, but in both cases the most prominent one was Gγ5 expressed without C-terminal processing after prenylation. This work indicates that post-prenylation reactions can generate multiple products determined by the C-terminal Ca1a2X motif. Within the human genome 10% of predicted prenylated proteins have aromatic amino acids in their Ca1a2X sequence and would likely generate the prenylation pattern described here. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.