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 Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(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 Galphai and Gbeta 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 Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; 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 Ggamma 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&sbd;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.
Pomerantz
K B
KB
Department of Medicine, Cornell University Medical College, 1300 York Avenue, New York, New York 10021, USA.
Lander
H M
HM
Summers
B
B
Robishaw
J D
JD
Balcueva
E
E
Hajjar
D P
DP
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D017368:Protein Prenylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D015398:Signal Transduction; D013256:Steroids / Q000378:metabolism
D012333:RNA, Messenger; D013256:Steroids; D002784:Cholesterol; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D015688:Oncogene Protein pp60(v-src); D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D010100:Oxygen