The gamma subunit composition of the major bovine brain Go and Gi proteins (GOA, GOB, GOC, Gi1, and Gi2) was characterized using antibodies against specific gamma isoforms. Each of the purified G protein heterotrimers contained a heterogeneous population of gamma subunits, and the profiles of the gamma subunits found with Gi1, Gi2, and GOA were similar. In contrast, each GO isoform had a distinct pattern of associated gamma subunits. These differences were surprising given that all three alpha O isoforms are thought to share a common amino-terminal sequence important for the binding of beta gamma dimers and that the alpha OA and alpha OC proteins may come from the same alpha O1 mRNA. The free alpha OA and alpha OC subunits had unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and gamma subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits.
Mechanisms contributing to altered heterotrimeric G-protein
expression and subsequent signaling
events during cholesterol accretion have been unexplored. The
influence of cholesterol enrichment on
G-protein expression was examined in cultured smooth muscle cells that
resemble human atherosclerotic
cells by exposure to cationized LDL (cLDL). cLDL, which increases
cellular free and esterified cholesterol
2-fold and 10-fold, respectively, reduced the cell membrane content of
Gαi-1, Gαi-2, Gαi-3, Gq/11, and
Gαs. The following evidence supports the premise that the
mechanism by which this occurs is due to
reduced isoprenylation of the Gγ-subunit. First, the inhibitory
effect of cholesterol enrichment on the
membrane content of Gαi subunits was found to be
post-transcriptional, since the mRNA steady-state
levels of Gαi(1−3) were unchanged following cholesterol
enrichment. Second, the membrane expression
of α and β subunits was mimicked by cholesterol and
17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Gαi and Gβ expression in
cholesterol-enriched cells was overcome
by mevalonate, the immediate product of HMG-CoA reductase. Fourth,
pulse-chase experiments revealed
that cholesterol enrichment did not reduce the degradation rate of
membrane-associated Gαi subunits.
Fifth, cholesterol enrichment also reduced membrane expression of
Gγ-5, Gγ-7upper; these γ subunits are
responsible for trafficking of the heterotrimeric G-protein complex to
the cell membrane as a result of
HMG-CoA reductase-dependent post-translational lipid modification
(geranylgeranylation) and subsequent
membrane association. Cholesterol enrichment did not alter
expression of G-γ-5 mRNA, as assessed by
reverse transcriptase polymerase chain reaction, supporting a
post-transcriptional defect in Gγ subunit
expression. Fifth, cholesterol enrichment also reduced the
membrane content of p21ras (a low molecular
weight G-protein requiring farnesylation for membrane targeting) but
did not alter the membrane content
of the two proteins that do not require isoprenylation for membrane
associationPDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was
reflected by reduced G-protein-mediated
signaling events, including ATP-induced GTPase activity,
thrombin-induced inhibition of cyclic AMP
accumulation, and MAP kinase activity. Collectively, these results
demonstrate that cholesterol enrichment
reduces G-protein expression and signaling by inhibiting isoprenylation
and subsequent membrane targeting.
These results provide a molecular basis for altered
G-protein-mediated cell signaling processes in
cholesterol-enriched cells.
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