In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex.

作者：Joyce B. Higgins, Patrick J. Casey · 发表于：Journal of Biological Chemistry · 年份：1994 · DOI：10.1016/s0021-9258(17)37077-1 · 被引用次数：122 · 研究领域：Neuropeptides and Animal Physiology、Chemical Synthesis and Analysis、Peptidase Inhibition and Analysis

The gamma subunits of the heterotrimeric G proteins (G gamma) are subject to carboxyl-terminal processing. This processing involves prenylation of a cysteine residue initially 4 amino acids from the carboxyl terminus, endoproteolytic truncation of the 3 terminal amino acids, and methylation of the now carboxyl-terminal prenylcysteine residue. The significance of each of these modifications in the ultimate properties of G proteins is not yet clear. We have developed in vitro systems for the production of the three processing intermediates (unmodified, prenylated, and truncated-prenylated) for two G gamma subunits, one which is subject to farnesylation (G gamma 1) and one which is geranylgeranylated (G gamma 2). Assessment of the functional consequences of the processing of G gamma was found to require reconstitution of the polypeptides with a G protein beta subunit (G beta). The ability of recombinant G beta, produced in Sf9 cells, to assemble into stable beta gamma complexes (G beta gamma) with each of the G gamma processing intermediates was assessed. Both prenylated and unprenylated G gamma subunits formed stable complexes with G beta, but surprisingly, neither of the truncated-prenylated G gamma subunits were competent for this assembly. The G beta gamma complexes which were formed were examined for their ability to interact with a G protein alpha subunit (G alpha). Only those G beta gamma complexes containing a prenylated G gamma subunit were functional in this assay. The...