G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells .1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras

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 G alpha i-1, G alpha i-2, G alpha i-3, Gq/11, and Gas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the G gamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of G alpha i subunits was found to be post-transcriptional, since the mRNA steady-state levels of G alpha i(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol both of which inhibit HMG-CoA reductase. Third, inhibition of G alpha i and G beta 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 G alpha i subunits. Fifth, cholesterol enrichment also reduced membrane expression of G gamma-5, G gamma-7(upper); these gamma 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-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Gy 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 association-PDGF-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.