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 bee n unexplored. The influence of cholesterol enrichment on G-protein exp ression was examined in cultured smooth muscle cells that resemble hum an atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, w hich 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 suppo rts the premise that the mechanism by which this occurs is due to redu ced isoprenylation of the G gamma-subunit. First, the inhibitory effec t of cholesterol enrichment on the membrane content of G alpha i subun its was found to be post-transcriptional, since the mRNA steady-state levels of G alpha i(1-3) were unchanged following cholesterol enrichme nt. Second, the membrane expression of alpha and beta subunits was mim icked 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 me mbrane-associated G alpha i subunits. Fifth, cholesterol enrichment al so reduced membrane expression of G gamma-5, G gamma-7(upper); these g amma 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 al ter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect i n Gy subunit expression. Fifth, cholesterol enrichment also reduced th e membrane content of p21ras (a low molecular weight G-protein requiri ng farnesylation for membrane targeting) but did not alter the membran e content of the two proteins that do not require isoprenylation for m embrane association-PDGF-receptor or p60-src. Reduced G-protein conten t in cholesterol-laden cells was reflected by reduced G-protein-mediat ed signaling events, including ATP-induced GTPase activity, thrombin-i nduced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment r educes 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 chole sterol-enriched cells.