Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling.
The binding of HDL to scavenger receptor-BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-beta-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-beta-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-beta-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.
Assanasen
Chatchawin
C
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Mineo
Chieko
C
Seetharam
Divya
D
Yuhanna
Ivan S
IS
Marcel
Yves L
YL
Connelly
Margery A
MA
Williams
David L
DL
de la Llera-Moya
Margarita
M
Shaul
Philip W
PW
Silver
David L
DL
D000818:Animals; D018955:CD36 Antigens / Q000378:metabolism; D019556:COS Cells; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D008076:Cholesterol, HDL / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051116:Receptors, Scavenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051122:Scavenger Receptors, Class B; D015398:Signal Transduction / Q000502:physiology*; D047392:beta-Cyclodextrins / Q000378:metabolism
D018955:CD36 Antigens; D008076:Cholesterol, HDL; D011971:Receptors, Immunologic; D051116:Receptors, Scavenger; D011993:Recombinant Fusion Proteins; C495124:SCARB1 protein, human; D051122:Scavenger Receptors, Class B; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse