Abstract
We characterized the variable processing of the G protein gamma subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication. Ggamma subunits were isolated from purified brain G proteins and characterized by Edman sequencing, by MALDI MS, by chemical and/or enzymatic fragmentation assayed by MALDI MS, and by MS/MS fragmentation and sequencing. Multiple forms of six different Ggamma isoforms were detected. Significant variation in processing was found at both the amino termini and particularly the carboxyl termini of the proteins. All Ggamma isoforms contain a carboxyl-terminal CAAX motif for prenylation, carboxyl-terminal proteolysis, and carboxymethylation. Characterization of these proteins indicates significant variability in the normal processing of all of these steps in the prenylation reaction, including a new variation of prenyl processing resulting from cysteinylation of the carboxyl terminus. These results have multiple implications for intracellular signaling mechanisms by G proteins, for the role of prenyl processing variation in cell signaling, and for the site of action and consequences of drugs that target the prenylation modification.
Highlights
We characterized the variable processing of the G protein ␥ subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication
These include a broad range of proteins, many of which are involved in cell signaling and growth regulation, for example the Ras proto-oncogene products and the ␥ subunits of the heterotrimeric G proteins [20, 22,23,24]
Geranylgeranyl is added if this residue is a Leu or Phe, whereas Farnesyl is added if it is a Ser, Met, Gln, Cys, or Ala [19, 25]
Summary
Purification of Bovine Brain G Protein Heterotrimers—G proteins were purified from bovine brain cortex as described previously [46, 47]. In many preparations proteins were eluted from the column in line with a Finnigan LCQ mass spectrometer, and fractions were identified by the presence of previously characterized intact G␥ subunit masses. Protein Sequencing—Edman sequencing of 50-l aliquots of HPLC fractions was performed in the Medical University of South Carolina Proteogenomics Facility on a Procise 494 Applied Biosystems instrument as described previously [45]. A t test was used to evaluate consistency of observed masses with predicted masses compiled in a table of all possible combinations of plausible modifications of all known G protein ␥ subunits. This table contained about 2,500 total entries. Our assignments are consistent with only one possible structure of about 2,500 possible structures considered
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