Lipoprotein promotes caveolin-1 and Ras translocation to caveolae: role of cholesterol in endothelial signaling.
To explore the role of LDL in caveolin-Ras regulation in human endothelial cells (ECs), we incubated confluent human umbilical vein endothelial cells (HUVECs) with LDL. This resulted in a high steady-state caveolin-1 (Cav-1) expression at both the mRNA and protein levels. LDL exposure appeared not to regulate the abundance of Cav-1. Immunofluorescence staining showed that Cav-1 protein migrated from the cytoplasm to the cell membrane after LDL exposure. Cav-1 protein and cholesterol partitioned mainly into the caveola fractions, and LDL increased both Cav-1 and cholesterol in these fractions. Ras protein in caveola fractions was also increased by LDL. Increased Ras was detected in Cav-1 immunoprecipitated samples, and conversely, increased Cav-1 was found in Ras-immunoprecipitated samples. We also demonstrated LDL-increased Ras activity in HUVECs by measuring the GTP/GTP+GDP ratio of Ras with [(32)P]orthophosphate labeling in the cells. Finally, we determined the binding of [(3)H]-labeled free cholesterol and recombinant H-Ras to Cav-1 fusion proteins in vitro. Both cholesterol and Ras bound to full-length GST-Cav-1, scaffolding domain (61-101), and C-terminal (135-178) Cav-1 fusion peptides. Addition of cholesterol enhanced Ras binding to the full-length and scaffolding domain of Cav-1 but not to the C-terminal Cav-1. These findings strongly suggest a role for Cav-1 in cholesterol trafficking and cholesterol-mediated intracellular signaling, which may mediate EC activation by LDL.
Zhu
Y
Y
Division of Biomedical Sciences, University of California, Riverside, USA. yi.zhu@ucr.edu
Liao
H L
HL
Wang
N
N
Yuan
Y
Y
Ma
K S
KS
Verna
L
L
Stemerman
M B
MB
D001692:Biological Transport; D051242:Caveolin 1; D022461:Caveolins / Q000096:biosynthesis / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000097:blood / Q000502:physiology*; D008078:Cholesterol, LDL / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008074:Lipoproteins / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014471:Umbilical Veins
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D008078:Cholesterol, LDL; D008074:Lipoproteins; D002784:Cholesterol; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)