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 ␥ 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 G␥1, -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, G␥5 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 G␥5, CSFL, was exchanged for that of G␥2, CAIL, G␥5 was completely processed. Conversely, G␥2-SFL was incompletely processed. Differential processing of G␥5 was found due to the presence of an aromatic amino acid in its Ca 1 a 2 X motif. Retrieving endogenous G␥ subunits from HEK293 or Neuro-2a cells with FLAG-G␤ constructs identified multiple G␥ subunits by mass spectrometry in either cell, but in both cases the most prominent one was G␥5 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.

Synthesis and Assembly of G Protein βγ Dimers: Comparison of In Vitro and In Vivo Studies

“…The C-terminus is modi fi ed with the attachment of a prenyl group (geranylgeranyl or farnesyl) by prenyl transferase at a cysteine three amino acids from the C-terminus, and then, typically, the three C-terminal residues are removed by proteolysis and the new prenylated C-terminus is carboxymethylated. This series of processing reactions is signaled by the amino acid sequence "CaaX" (where C is cysteine, "a" is usually an aliphatic and X a variable amino acid) at the end of the polypeptide, or by other residues in the CaaX sequence (Kilpatrick and Hildebrandt 2007 ) , but generally the C-terminal amino acid determines whether the G g is farnesylated or geranylgeranylated (Higgins and Casey 1996 ) .…”

A shunt pathway limits the CaaX processing of Hsp40 Ydj1p and regulates Ydj1p-dependent phenotypes

“…The α (CAMQ) and β (CVLS) subunits of phosphorylase b kinase (Phk) are farnesylated yet retain their CaaX motifs (Heilmeyer et al, 1992). The major form of Gγ5 (CSFL) is geranylgeranylated and also retains its CaaX motif (Kilpatrick and Hildebrandt, 2007). Additionally, mammalian annexin A2 (CGGDD) was identified in a screen for isoprenylated proteins.…”

The Expanding Roles of GβγSubunits in G Protein–Coupled Receptor Signaling and Drug Action

“…4). Recent reports suggest that a subpopulation of mammalian Gg subunits may escape prenylation and thus remain soluble (Cook et al, 2006;Kilpatrick and Hildebrandt, 2007). If this observation pans out, new locations for Gbg signaling may be imagined.…”